(CFUs)
Work on staphylococcal diseases in our laboratory was supported by grants from the National Institute of Allergy and Infectious Diseases (NIAID), Infectious Diseases Branch (AI038897, AI052474, AI075258). The authors acknowledge membership within and support from the Region V “Great Lakes” Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Consortium (NIH Award 1-U54-AI-057153).
Competing interests: The authors declare a conflict of interests as inventors of patent applications that are related to the development of Staphylococcus aureus vaccines and are currently under commercial license.
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BMC Veterinary Research volume 20 , Article number: 399 ( 2024 ) Cite this article
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Klebsiella pneumoniae (KP), responsible for acute lung injury (ALI) and inflammation of the gastrointestinal tract, is a zoonotic pathogen that poses a threat to livestock farming worldwide. Nevertheless, there is currently no validated vaccine to prevent KP infection. The development of mucosal vaccines against KP using Lactobacillus plantarum ( L. plantarum ) is an effective strategy.
Firstly, the L. plantarum strains NC8-pSIP409-aCD11c’ and NC8-pLc23-aCD11c were constructed via homologous recombination to express the aCD11c protein either inducibly or constitutively. Both NC8-pSIP409-aCD11c’ and NC8-pLc23-aCD11c strains could enhance the adhesion and invasion of L. plantarum on bone marrow-derived dendritic cells (BMDCs), and stimulate the activation of BMDCs compared to the control strain NC8-pSIP409 in vitro. Following oral immunization of mice with NC8-pSIP409-aCD11c’ and NC8-pLc23-aCD11c, the cellular, humoral, and mucosal immunity were significantly improved, as evidenced by the increased expression of CD4 + IL-4 + T cells in the spleen, IgG in serum, and secretory IgA (sIgA) in the intestinal lavage fluid (ILF). Furthermore, the protective effects of L. plantarum against inflammatory damage caused by KP infection were confirmed by assessing the bacterial loads in various tissues, lung wet/dry ratio (W/D), levels of inflammatory cytokines, and histological evaluation, which influenced T helper 17 (Th17) and regulatory T (Treg) cells in peripheral blood and lung.
Both the inducible and constitutive L. plantarum strains NC8-pSIP409-aCD11c’ and NC8-pLc23-aCD11c have been found to stimulate cellular and humoral immunity levels and alleviate the inflammatory response caused by KP infection. These findings have provided a basis for the development of a novel vaccine against KP.
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Klebsiella pneumoniae (KP) is a common zoonotic pathogen located at the respiratory and gastrointestinal tracts, leading to acute lung injury (ALI) and gastrointestinal infections [ 1 , 2 , 3 , 4 ]. Since the initial isolation of which in airway secretions in 1875, KP has exhibited an increasing resistance to the external environment, which results in challenges due to its drug resistance and virulence [ 5 ]. With the increasing isolation rate of KP in the global livestock farming industry, which has raised increasing concerns regarding the food safety and economic implications [ 6 , 7 ]. Bacterial vaccines have shown efficacy in reducing pathogenic bacterial infections, while the complex structural composition and numerous serotypes of KP have hindered the development of targeted commercial vaccines [ 8 , 9 , 10 ]. As one of the most predominant conditional pathogens, KP primarily causes disease through the mucosal route of infection, particularly in instances of compromised host immunity [ 11 ]. Consequently, oral vaccinations are feasible to enhancing the mucosal immunity and preventing pathogens such as KP [ 12 ].
Lactobacillus , an edible beneficial microorganism, is involved in regulating the dynamic equilibrium of intestinal flora and promoting the proliferation of immunological cells to modulate the immune response [ 13 ]. Research on Lactobacillus -based live vector vaccines has advanced significantly across various pathogens. In recent years, different expression systems have been widely utilized in Lactobacillus spp., including the constitutive or inducible expression vectors. For example, the constitutive expression vector pOri23, which was based on the P23 promoter modification, and the vector pSIP409, which was based on the sppK, sppR inducible expression system, have facilitated precise manipulation of the expressed target genes [ 14 ]. Therefore, we replaced the inducible expression system in the pSIP409 vector with the P23 promoter to construct the constitutive expression vector pLc23. Since the crucial role of antigen-presenting cells (APCs), such as dendritic cells (DCs), in antigen presentation and uptake, the use of recombinant vectors fused with receptor molecules targeting DCs can significantly enhance their effectiveness in order to enhance the uptake of antigens by APCs during immunization [ 15 ]. In a previous study, Lactobacillus plantarum ( L. plantarum ) strain NC8-pSIP409-aCD11c was employed to expresses a single-chain antibody against CD11c (scFv-CD11c, aCD11c), which efficiently bound DCs, induced DC maturation, promoted T cell differentiation, and enhanced B cell production in vivo [ 16 ].
In the development of L. plantarum vaccines, it is imperative to thoroughly understand the immunomodulatory mechanisms of L. plantarum . In case studies related to respiratory diseases, changes in T helper 17 (Th17) and regulatory T (Treg) cell numbers are critical for disease progression and are linked to COPD, lung cancer, and tuberculosis [ 17 , 18 , 19 ]. The stability and suppression of CD4 + CD25 + Treg cells depend on FOXP3, a transcription factor, and FOXP3 expression and regulation require phosphorylated STAT5 (p-STAT5) [ 20 ]. Consequently, this pathway regulates the prevention of Treg’s immunological inflammation. It has been shown that the STAT5/FOXP3 signaling pathway was dramatically suppressed in a mouse model of asthma, increasing Th17 cells and decreasing Treg cells in the bronchoalveolar lavage fluid (BALF) [ 21 , 22 , 23 ]. Therefore, in this study, the inducible and constitutive L. plantarum expressing aCD11c (NC8-pSIP409-aCD11c’ and NC8-pLc23-aCD11c) were used to investigate the effect of aCD11c expression on DCs activation, and to elucidate the relationship between the immunomodulatory effects of L. plantarum on KP infection and the activation of the STAT5/FOXP3 signaling pathway.
Strains, plasmids, and primers.
Table 1 lists the strains, plasmids, and primers used in this study. L. plantarum NC8 (CCUG 61730) [ 24 ] was kindly provided by Prof. Chunfeng Wang (Jilin Agricultural University, China). The NC8 strains were quiescently grown in anaerobic conditions at 37 °C in an MRS medium containing erythromycin (Em) (10 µg/mL). E. coli Top10 strain was cultured in Luria–Bertani (LB) broth under shaking conditions at 37 °C (200 µg /mL Em). The K. pneumoniae HRB2020005 strain isolated from swine was kindly provided by Prof. Liancheng Lei (Jilin University, China) and identified via 16s rDNA (GeneBank OQ674507).
The plasmid extracted from Top10-pSIP409-aCD11c was utilized as a template. The aCD11c fragment was amplified using two primer pairs, 409-a’-F/409-a’-R and pLc23-a-F/409-a’-R (Table 1 ), respectively. Subsequently, the fragments were ligated with vectors pSIP409 ( Hind III) and pLc23 ( Hind III) using the Seamless Assembly Cloning Kit (Clone Smarter Technologies, China) to generate the recombinant plasmids pSIP409-aCD11c’ and pLc23-aCD11c. After sequencing and verification by Biocorp (Tsingke Biotechnology, China), the recombinant plasmids were electro-transformed into L. plantarum NC8 using an electroporation gene introducer (Bio-Rad, USA) with parameters set at 2.5 KV, 400 Ω, and 25 µF. This resulted in the generation of recombinant L. plantarum strains NC8-pSIP409-aCD11c’ and NC8-pLc23-aCD11c, abbreviated as 409-a and pLc23-a, respectively.
The recombinant strains were inoculated in an MRS medium for anaerobic culture. SppIP (50 ng/mL, sakacin P) was added to the NC8-pSIP409 (409) and 409-a to induce protein expression, except the strain pLc23-a. After incubation, the above three strains were harvested via centrifugation, and protein samples were obtained as previously described [ 16 ]. The samples were evaluated by western blotting using an HA-labeled primary antibody (1:1000, Beyotime, China), followed by an HRP-conjugated goat anti-mouse IgG (1:5000, Solarbio, China) as the secondary antibody. Detection was performed using a chemiluminescence imager.
C57BL/6 mice (5–6 weeks old) were obtained from the Experimental Animal Center of Three Gorges University, Yichang. Bone marrow-derived dendritic cells (BMDCs) were acquired according to the previously methods [ 25 ]. BMDCs were isolated from the tibiae and fibulae of mice. The culture medium of BMDCs was supplemented with 20 ng /mL GM-CSF and 10 ng /mL IL-4 (PeproTech, USA). On the 8th day, cells were harvested and placed in 24-well culture dishes at a density of 2 × 10 5 cells per well. The cells were then incubated for 24 h. Following that, the adhesion and invasion test of 409-a and pLc23-a were conducted. The strains were cultivated together with cells (MOI = 1000), and the monoclonal anti-mouse CD11c antibody (Bioss, China) was introduced. Following a two-hour period of stimulation, the cells were exposed to aseptic PBS solution containing 0.2% Triton X-100 for a duration of 10 min. After being diluted in a gradient manner, then the cells were incubated in MRS culture plates (37 °C, 10 µg/mL Em) overnight. Subsequently, the cells were counted in order to evaluate the rate of L. plantarum adhesion. Concurrently, those cells were exposed to L. plantarum stimulation for 2 h were subjected to invasion assays, after being treated with gentamicin (500 µg /mL). The adhesion or invasion ratio was computed according to the previously method [ 16 ].
After BMDCs were cultured in 24-well plates, 409, 409-a, pLc23-a, and LPS were added to each group of cells. L. plantarum was added to the cultures at an MOI of 10. Subsequent experiments were performed after overnight incubation. Cells were collected and incubated with antibodies purchased from BD, such as APC-labeled anti-mouse CD11c, FITC-labeled anti-mouse CD40, PerCP-Cy5.5-labeled anti-mouse CD80, and their respective isotype control antibodies. Then, all samples were processed by flow cytometry (FCM) (BD FACSMelody, USA) for analysis. The databases were parsed by FlowJo V10.
BALB/c mice (6–8 weeks old) were supplied by the Experimental Animal Center of Three Gorges University, Yichang, Hubei Province, China. In total, 75 same-aged mice were randomly and equally categorized into five groups, namely, PBS, KP, 409, 409-a, and pLc23-a. Mice were immunized orally twice consecutively on 1st to 3rd day and 15th to 17th day (Fig. 1 A). The L. plantarum immunization dosage was 10 9 CFU/100 µL/ mouse. Furthermore, the PBS group was fed an equal volume of PBS. One week after the immunization, 3 mice were randomly selected from the PBS and L. plantarum groups to perform FCM assays.
On the 25th day, all BALB/c mice, except those in the PBS group, were injected with 1 × 10 7 CFU KP by intraperitoneal injection. After 24 h, the lung, spleen, and liver organs were removed and minced from euthanized mice after rapid cervical dislocation. The tissue homogenates were appropriately diluted and incubated on LB agar plates in a 37 °C incubator for colony counting. Colonization was calculated as the ratio of organ colonies to inoculation colonies.
Detection of CD4 + T cells activation via FCM and antibody levels through ELISA. ( A ) Immunization and infection processes in mice and the implementation of separate assays. Flow chart ( B ) and analysis ( C ) of the expression of IFN-γ + CD4 + T cells and IL-4 + CD4 + T cells in spleen. ( D ) The levels of sIgA in ILF and IgG in the serum of mice after the second immunization. A one-way ANOVA test was performed to determine statistical significance (* P < 0.05, ** P < 0.01 and *** P < 0.001)
On the 24th day, 3 mice per group were measured by FCM. The spleens were aseptically removed and ground in RPMI 1640 medium. Furthermore, the supernatant was discarded via centrifugation and lysed by erythrocyte lysis solution, washed with PBS, and permeabilized. Subsequently, the cells were counted after re-suspension in 1 mL of culture medium. Surface and intracellular staining of T cells were separately performed. For intracellular staining, RPMI1640 complete medium (10% FBS) containing PMA, Ionomycin and Brefeldin A stimulating agent (BD Leukocyte Activation Cocktail, USA) was added into a 48-well culture plate containing 2 × 10 6 cells per well and cultured in an incubator for 5 h (37 °C, 5% CO 2 ) for stimulation. After the completion of stimulation, BV510 labeled anti-mouse CD3e and BV421 labeled anti-mouse CD4 were added for surface staining, APC labeled anti-mouse IFN-γ and PE-Cy7 labeled anti-mouse IL-4 and their respective isotype control antibodies (BD Pharmingen) were used for intracellular staining after fixation and permeabilization (BD fixation/permeabilization solution kit, USA). Lastly, the single-cell suspended solution was re-suspended in PBS for FCM assays.
Blood samples were obtained after the second immunization, and the supernatants were collected via centrifugation. Intestinal tissues were washed with pre-cooled PBS containing 1% protease inhibitor PMSF (Beyotime, China). Furthermore, the contents were obtained, and the supernatant was collected via centrifugation. Mucosal antibody secretory IgA (sIgA) in intestinal lavage fluid (ILF) was detected as per the manufacturer’s protocol (MEIMIAN, China). Concurrently, IgG levels in the serum were detected using the Cytokine ELISA Kit for Mouse IgG (MEIMIAN, China).
On the 32nd day, peripheral blood of 3 mice from each group was obtained, and one-half of the anticoagulated blood was supplemented with RPMI1640 complete medium (10% FBS) containing PMA, Ionomycin and Brefeldin A stimulating agent (BD Leukocyte Activation Cocktail, USA), which was cultured in an incubator for 5 h (37 °C, 5% CO 2 ) and homogenized every 1 h. The suspensions were incubated with BV421 labeled anti-mouse CD4, fixed, permeabilized, and incubated with PE-labeled anti-mouse IL-17 A and their respective isotype control antibodies. This was then rinsed with PBS and resuspended for FCM detection of Th17 cells. Concurrently, lungs were obtained under aseptic conditions to prepare single-cell suspensions. Tissue fragments were digested in RPMI1640 digestion solution for 30 min (10% FBS, 25 U/mL DNase I, 2.0 mg/mL collagenase IV, 1.0 mg/mL collagenase I) and mixed slowly. After digestion, the lung tissue was gently ground and filtered through a cell sieve (70 μm), centrifuged, and lysed twice by adding erythrocyte lysis solution. The lysis was terminated by PBS. The supernatant was discarded via centrifugation and suspended in RPMI 1640 complete medium to obtain single-cell suspensions. BV421-labeled anti-mouse CD4 and PE-labeled anti-mouse CD25 were first treated with cell suspensions to detect Treg cells in the other half of the unstimulated peripheral blood and lung tissues. After fixation and permeabilization, APC-labeled anti-mouse FOXP3 and their respective isotype control antibodies were added for incubation and used for FCM detection.
On the 7th day after the KP infection, lungs were removed from mice sacrificed by cervical dislocation, washed with sterile PBS, and wet weights were obtained. Lungs were then dried in a thermostat at 65 °C for 24 h, removed, and weighed. The W/D ratio was calculated to estimate the effect of recombinant L. plantarum on pulmonary edema.
The lung and intestinal tissues of mice were obtained, washed with aseptic PBS, and immobilized in 4% paraformaldehyde solution. Furthermore, tissues were treated with dehydration and inserted in paraffin wax, sectioned, colored with hematoxylin and eosin (H&E), and observed using a microscope (Leica, Germany).
Lysis buffer RIPA (Beyotime, USA) consisting of proteinase and phosphatase inhibitor compounds was added to the samples. The mixture was homogenized and the supernatant was obtained via centrifugation. For the western blotting assay, primary antibodies included STAT5, p-STAT5, FOXP3, and GAPDH (ABclonal, China), whereas secondary antibodies included HRP-conjugated goat anti-rabbit IgG (1:5000, Solarbio, China). A chemiluminescence imager was used for detection. The results were analyzed using Image J.
The expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β) mRNA from lung tissue was detected using qRT-PCR. Total RNA was extracted using an RNA extraction kit (Tsingke, China), and reversal transcription was performed by cDNA Synthesis Kit (Vazyme, China). PCR was performed as per the manufacturer’s protocol in a reaction mixture containing 2×Universal SYBR green qPCR mix (ABclonal, China). Transcripts of the indicated genes were detected on a 7500 Real-Time PCR system Real-Time PCR system (Thermo Fisher, USA). Amplifications were processed with gene-targeted primers as follows: β-actin (AY618569), forward primer (Fw) 5’- AATCGTGCGTGACATCAAAG-3’ and reverse primer (Rv) 5’- AAGAAGGAAGGCTGGAAAAGAG-3’. TNF-α (NM_013693), Fw 5’- CAGAAAGCATGATCCGCGAC-3’ and Rv 5’-TCTGAGTGTGAGGGTCTGGG − 3’. TGF-β (M13177), Fw 5’-GCTGAACCAAGGAGACGGAA-3’ and Rv 5’- GTTGGTATCCAGGGCTCTCC-3’. IL-1β (NM_008361), Fw 5’- ATGAAAGACGGCACACCCAC-3’ and Rv 5’-GCTTGTGCTCTGCTTGTGAG-3’. The conserved gene β-actin was used as an internal control.
The GraphPad Prism 6.01 software was used for statistical analysis. Data are presented as the mean ± standard error of the mean (S.E.M.) and were assessed through one-way ANOVA (Dunnett’s multiple comparison test) in at least three independent experiments. P < 0.05 was considered statistically significant.
The plasmid pSIP409-aCD11c’ added only the HA-tag as a flag after the aCD11c sequence to allow the detection of protein expression, while the cell wall fractions were collected to detect the expression of the aCD11c protein of strains 409-a and pLc23-a. The results showed that both different types of vectors successfully expressed aCD11c protein with the same size (39 kDa) (Fig. 2 C), indicating that aCD11c was expressed by both strains. Furthermore, the expression of aCD11c protein was higher in the inducible strain 409-a than in the constitutive strain pLc23-a under the same treatment conditions.
Structural diagrams of pSIP409-aCD11c’ and pLc23-aCD11c plasmids and the detection of aCD11c expression. ( A ) Plasmid profile of pSIP409-aCD11c’. pSIP409 is a shuttle vector that can be activated by the addition of SppIP, with the sequence MAGNSSNFIHKIKQIFTHR. ( B ) Plasmid profile of pLc23-aCD11c. pLc23 is a modified version of pSIP409 that employs a continuous expression system (pOri23) to substitute for the inducible expression system (sppK, sppR). ( C ) The expression of aCD11c. M: Low molecular weight protein standard; Lane 1: NC8-pSIP409-aCD11c’; Lane 2: NC8-pLc23-aCD11c; Lane 3: empty vector NC8-pSIP409
The recombinant strains expressing aCD11c protein improved the adhesion and invasion efficiency of BMDCs. Furthermore, the inducible expression of strain 409-a was more effective than the constitutive expression of strain pLc23-a in adhesion ( P < 0.01) (Fig. 3 A) and invasion rate ( P < 0.05) (Fig. 3 A). The increased adhesion rates were distinctly decreased during the competitive assay, wherein the anti-CD11c antibody was used before co-incubation with BMDCs ( P < 0.001) (Fig. 3 A). This showed that the cellular adhesion was improved via the expression of aCD11c on the surface of the strains. Similar results were observed in the invasion study ( P < 0.001) (Fig. 3 A). This suggests that the expression of aCD11c significantly increased the numbers of bacteria in BMDCs, whereas the presence of anti-CD11c antibody decreased these results. A FACS assay was performed to further analyze the activation of targeting strains to BMDCs. Both targeting strains effectively promoted the expression of CD40 ( P < 0.05, P < 0.01) and CD80 ( P < 0.001, P < 0.05) in BMDCs compared with the 409 strain as control (Fig. 3 B). These results showed that aCD11c protein promoted the activation of BMDCs in vitro.
The adhesion and invasion of aCD11c-expression strains and activation for BMDCs. ( A ) Adhesion and invasion of targeting L. plantarum to BMDCs. ( B ) The expression of CD11c + CD40 + and CD11c + CD80 + for BMDCs were analyzed by FCM. LPS was used as a positive control. A one-way ANOVA test was performed to determine statistical significance (* P < 0.05, ** P < 0.01 and *** P < 0.001)
In the mouse model, to further determine the T cell response induced by the strains 409-a and pLc23-a, we determined the interferon-γ (IFN-γ) and interleukin-4 (IL-4)-producing T cells in the spleen (Fig. 1 B). The results indicated that the expression of CD4 + IL-4 + T cells from the 409-a group was upregulated compared with the 409 control group after immunization ( P < 0.05). However, the expression of IFN-γ was not considerably changed (Fig. 1 C), indicating that immunization can activate CD4 + T cells and stimulate the differentiation of CD4 + T cells toward Th2 subtypes. Further, the sIgA assay was performed to evaluate the ability of gastrointestinal mucosa to resist bacterial and viral adhesion. After oral immunization with L. plantarum , sIgA levels in ILF were significantly increased in both the 409-a ( P < 0.001) and pLc23-a ( P < 0.01) groups compared with the PBS group. Furthermore, the 409-a was significantly increased compared with the 409 groups ( P < 0.01) (Fig. 1 D). IgG, which is the main antibody in the serum that exerts antibacterial activity, was measured to determine the immune response of the body. The IgG expression in serum was increased in both the 409-a ( P < 0.01) and pLc23-a ( P < 0.05) groups compared with the 409 groups (Fig. 1 D), indicating that oral immunization could effectively induce the mucosal immunity and humoral immunity.
To verify the ability of mice immunized with L. plantarum to defend against KP infection, the amounts of bacteria in the lung, liver, and spleen of different groups of mice were measured on the 7th day after KP infection. The amounts of bacteria in the lungs, livers, and spleens of mice immunized with strains 409-a and pLc23-a were significantly decreased compared with those in the control KP group ( P < 0.01) (Fig. 4 ), especially in lung tissue ( P < 0.001) (Fig. 4 A). This showed that mice immunized with L. plantarum could efficiently alleviate the colonization ability of KP in different tissues, in particularly L. plantarum expressing aCD11c could efficiently alleviate the colonization ability of KP in lung.
Detection of bacterial loads of different organs after KP infection for 24 h in mice., Bacterial loads of lung ( A ), liver ( B ), and spleen ( C ) organs. A one-way ANOVA test was performed to determine statistical significance (* P < 0.05, ** P < 0.01 and *** P < 0.001)
To better evaluate the changes in the Th17 cells and Treg cells following KP infection in the mouse model, the levels of Th17 in peripheral blood and Treg cells in lung were detected 3 days after KP infection. The levels of CD4 + IL-17 A + Th17 cells in peripheral blood were significantly lower in the aCD11c-expressing group compared with those in the 409 groups ( P < 0.01) (Fig. 5 A). The 409-a + KP group and pLc23-a + KP exhibited substantially higher levels of CD4 + CD25 + FOXP3 + Treg cells than the 409 + KP group in lung ( P < 0.01 and P < 0.05) (Fig. 5 B). Furthermore, there was no significant difference in Th17 cells and Treg cells levels between the two groups 409-a and pLc23-a (Fig. 5 A and B), indicating that the expression of aCD11c reduced the Th17 cells in peripheral blood and improved Treg cells expression levels in lung.
Detection of the expression of CD4 + IL-17 A + Th17 cells and CD4 + CD25 + FOXP3 + Treg cells after KP infection. ( A ) Flow chart and analysis of the expression of CD4 + IL-17 + Th17 cells in peripheral blood. ( B ) Flow chart and analysis of the expression of CD4 + CD25 + FOXP3 + Treg cells in lung by FCM. A one-way ANOVA was performed to determine statistical significance (* P < 0.05, ** P < 0.01 and *** P < 0.001)
Following intraperitoneal injection of KP, mice in the experimental groups, as compared to the PBS group, exhibited symptoms such as a disheveled coat, restlessness, decreased appetite, reduced motility, and increased fecal excretion within 24 h. On the 7th day post-KP infection, three mice from each group were sampled, and their pulmonary tissue wet/dry (W/D) ratios were recorded. The results indicated that the W/D ratios decreased in the L. plantarum -immunized groups compared to the KP group ( P < 0.01) (Fig. 6 A), in particularly L. plantarum expressing aCD11c groups. This suggests that L. plantarum expressing aCD11c can alleviate pulmonary edema induced by KP infection. The tissues showed varying degrees of inflammatory pathological alterations, with altered lung tissue structural deformation, and inflammatory cells and exudates flooding the alveolar space. In the intestine, the intestinal villi were separated, the epithelial cells were morphologically aberrant, the intestinal mucosa was injured, and the tissue mucosa or muscle layer was destroyed. Mice in the immunized L. plantarum groups exhibited significant relief of pathological symptoms (Fig. 6 B).
Evaluation of inflammatory response after KP infection. ( A ) W/D ratios of pulmonary tissue in each group were recorded. A one-way ANOVA test was applied to estimate statistical significance (* P < 0.05, ** P < 0.01 and *** P < 0.001). ( B ) Lung and intestinal samples from different KP-infected groups were acquired, fixed, and embedded in paraffin. Sections were stained through H&E (magnification, ×200). Scale bar, 200 μm
KP infection can affect the inflammatory process in the lungs via NF-κB and other validation-related signaling pathways. The question arises whether targeting L. plantarum can influence the expression of Treg cells through the modulation of the STAT5/FOXP3 signaling pathway, as evidenced by the subsequent findings. The results showed an upregulation of p-STAT5 levels in the lung tissues of the 409-a and pLc23-a groups compared to the 409 group. The upregulation was notably more significant in the 409-a group after KP infection ( P < 0.001) (Fig. 7 B). Furthermore, there was significantly higher in FOXP3 expression in the 409-a ( P < 0.01) and pLc23-a ( P < 0.01) groups when compared with the 409 control group (Fig. 7 C). Analyzing the FCM results, it was observed that the levels of CD4 + CD25 + FOXP3 + Treg cells were significantly higher in lung tissue in the aCD11c-expressing groups compared to the 409 group ( P < 0.01) (Fig. 7 B). This indicates that aCD11c-expressing strains have the potential to improve Treg cell expression by regulating the STAT5/FOXP3 signaling pathway in the lung.
Activation of the STAT5/FOXP3 signaling pathway and expression of inflammatory factors in the lung after KP infection. ( A ) Expression of STAT5, p-STAT5, FOXP3, and GAPDH in the lungs of different groups of mice immunized with L. plantarum after KP infection. ( B and C ) The analysis of STAT5 phosphorylation level and evaluation of FOXP3 expression. ( D-F ) Gene expression of IL-1β, TNF-α and TGF-β in the lung. The housekeeping gene β-actin was selected as an internal control for normalization. A one-way ANOVA test was performed to determine the statistical significance (* P < 0.05, ** P < 0.01 and *** P < 0.001)
KP can cause pathogenic changes in lung tissue by activating several inflammatory signaling pathways and impairing the production of cytokines involved in inflammation. Upon assessing the expression levels of IL-1β, TGF-β, and TNF-α in lung tissue, the findings revealed that both the 409-a and pLc23-a groups significantly reduced the expression of KP-induced inflammatory cytokines IL-1β ( P < 0.001) (Fig. 7 D), TGF-β ( P < 0.01) (Fig. 7 E) and TNF-α ( P < 0.001) (Fig. 7 F) compared with that in the 409 group. This indicates that vaccination with L. plantarum expressing aCD11c can effectively prevent the production of inflammatory cytokines due to KP.
Lactic acid bacteria (LAB), as a representative intestinal probiotic, exhibit the ability for stable colonization in the gastrointestinal tract. LAB can stimulate the mucosal immune response by maintaining the equilibrium of intestinal microorganisms. Moreover, LAB functions as an effective oral vaccine delivery vehicle, offering immunological defense against pathogenic bacterial infections [ 13 ]. There is an important connection between the gut microbiome and the lung, known as the “gut-lung axis” (GLA). Disruptions to intestinal and pulmonary homeostasis can lead to allergic or inflammatory reactions. However, probiotics such as LAB can help regulate the microbiota and alleviate these conditions [ 26 , 27 , 28 ]. Despite the sophisticated nature of the pathogenesis and influencing factors involved in gastrointestinal and respiratory diseases, probiotics can effectively alleviate allergic or inflammatory reactions. This is accomplished by regulating microbiota when there is a disruption in intestinal and pulmonary homeostasis [ 29 ]. Probiotics can stimulate the body’s immune response, strengthen the protective function of the mucosal barrier, inhibit the invasion of pathogenic bacteria, and decrease the morbidity of respiratory or gastrointestinal diseases to some extent [ 30 , 31 , 32 ]. Specifically, the intestinal microbiota can mediate distal immune regulation in the lungs through the gut-lung axis [ 33 ]. In this study, oral administration of L. plantarum expressing aCD11c was found to have an immunoprotective effect against KP lung infection in mice. Furthermore, the investigation into the state of cellular and humoral immunity during immune protection and the regulatory mechanisms laid the foundation for the future development of KP vaccines.
Although KP infections are currently not major issues in livestock, the increasing detection of drug-resistant KP strains is concerning, especially with the growing integration of pets into human life [ 34 , 35 ]. Existing KP vaccines have limited efficacy due to the complex and variable serotypes of the pathogen [ 3 , 36 , 37 ]. Currently, the efficacy of pertinent vaccines cannot be assured, and they struggle to address the complex and variable serotypes of KP [ 8 , 38 ]. L. plantarum is a promising probiotic-based vaccine candidate. It can efficiently suppress harmful bacteria through metabolite production [ 39 , 40 ] and can effectively colonize both the gut and respiratory tract [ 41 ]. In the mouse model, L. plantarum immunization decreased organ bacterial loads, demonstrating its potential as a KP vaccine (Fig. 1 ).
Under normal conditions, the intestinal and pulmonary microbiomes maintain a dynamic equilibrium. The intestinal microbiota can control the threshold of immune activation and influence the systemic immune response. The mucosal immune system, which includes sIgA, acts as a key defense against inhaled pathogens in the respiratory and gastrointestinal tracts [ 42 ]. In this study, KP infection damaged the intestinal and lung mucosal barriers, while oral administration of L. plantarum was able to increase sIgA levels in ILF (Fig. 4 D). Ultimately, the mucosal integrity of the intestinal and lung tissues was superior to the control group, as observed in histopathological sections after KP infection in mice (Fig. 6 B).
DCs play a crucial role in antigen processing and T cell activation [ 15 , 43 , 44 ], when activated by foreign pathogens, they travel to nearby lymph nodes to transmit antigens to T cells, mobilizing them to promote the acquired immune response [ 45 ]. Recently, therapies targeting APCs have shown promise. The DC-SIGN, FcR, and CD11c receptors enable DCs to process foreign antigens and identify pathogens involved in innate immunity. As part of the host immune response, immature DCs acquire and internalize specific antigens, express costimulatory molecules, mature, and transport processed antigens to drive T-cell differentiation and B-cell generation [ 46 , 47 ]. In this study, L. plantarum strains expressing the aCD11c protein were able to more effectively target and activate DCs in vitro (Fig. 4 ). Analysis of the T-cell response showed that immunization with the L. plantarum strains NC8-pSIP409-aCD11c’and NC8-pLc23-aCD11c triggered a Th2-skewed immune response, with increased IL-4 production and B-cell antibody generation, rather than a Th1 IFN-γ response (Fig. 1 B and C). This Th2 response is more effective against extracellular pathogens such as bacteria, and is mediated by IL-4, which drives the maturation of B cells into plasma cells and increases antibody production [ 48 ]. This finding is consistent with the increased IgG level in humoral immunity (Fig. 4 D).
When immunity is compromised, the lung barrier cannot withstand external bacterial infestation or infection, and the absence of effective antibiotics against KP can complicate treatments. KP induces severe acute lung inflammation, such as ALI, causing respiratory failure or mortality [ 4 , 49 ]. The primary pathogenic mechanisms of ALI are triggered by inflammatory responses, oxidative stress, and apoptosis [ 50 ]. Th17/Treg is closely linked with the immunopathogenesis of prevalent clinical lung diseases such as tuberculosis and asthma [ 51 , 52 , 53 , 54 ]. Tregs can suppress the non-specific immunological effects of immune effector cells via direct contact, killing immune effector cells, or indirectly triggering apoptosis [ 55 , 56 ]. Furthermore, Tregs have the ability to inhibit the synthesis of inflammatory molecules by expressing high levels of galectin-1, eliminating Pseudomonas aeruginosa and KP [ 57 ]. The study found that immunization with aCD11c-expression L. plantarum led to a decrease in CD4 + IL-17 + Th17 cells in the peripheral blood (Fig. 5 ).
STAT5/FOXP3 signaling pathway is crucial for Treg cells evolution and function [ 20 , 57 , 58 ]. The study revealed that the group treated with aCD11c-expressing L. plantarum had significantly greater levels of phosphorylated STAT5 (p-STAT5) and the transcription factor FOXP3 than did the KP infection group. This finding suggested that L. plantarum intervention activated the STAT5/FOXP3 pathway under inflammatory conditions, leading to increased FOXP3 expression and enhanced Treg function (Fig. 7 A and B). However, the expression levels of p-STAT5 and FOXP3 were not positively correlated in the KP infection group compared to the control group. This may be because the KP infection affected the production or expression of other signaling pathways and cytokines, such as the PI3K/Akt/mTOR pathway and TGF-β, TLR, and hemoglobin, which can also contribute to FOXP3 expression through compensatory mechanisms [ 59 , 60 ]. Bacterial translocation and intrapulmonary immune response mobilization induce inflammatory factors such as IL-1β and TNF-α to damage lung tissue and trigger oxidative stress causing subsequent infection [ 61 , 62 ]. However, IL-1β, TNF-α, and TGF-β expression were lower in the aCD11c groups than that in the KP group. Furthermore, inflammatory pathological changes in the lung and gut decreased, indicating that L. plantarum intervention could reduce KP-induced inflammation (Fig. 7 C). Although TNF-α production in lung tissues was higher in the 409 + KP group than in the KP group, it probably due to the fact that L. plantarum 409 induced the production of some inflammatory factors as an exogenous stimulus or immunogen [ 63 ], the side effect was alleviated by L. plantarum 409-a and pLc23-a, which express the aCD11c protein, by stimulating the DCs to better regulate cellular immunity and humoral immunity.
In conclusion, this study revealed that the targeting of the L. plantarum strains NC8-409-aCD11c ’ and NC8-pLc23-aCD11c, which exhibit induced expression of aCD11c in the former and constitutive expression in the latter, could effectively improve the adhesion, invasion, and activation of BMDCs in vitro. L. plantarum strains expressing aCD11c were able to enhance cellular, humoral, and mucosal immunity in mice after oral immunization. Furthermore, L. plantarum -induced immunity was able to reduce inflammatory pathological changes in tissues by activating the STAT5/FOXP3 signaling pathway. This led to an increase in CD4 + CD25 + FOXP3 + Treg cells from the lungs and a reduction in Th17 cells from the peripheral blood after infection. No significant differences were found between the two L. plantarum strains in the experiments, except for protein expression. The study then determined the relationship between targeting L. plantarum , dendritic cells, and KP based on the improved protein expression of the strain with constitutive aCD11c expression.
Data is provided within the manuscript.
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The authors would like to thank Prof. Chunfeng Wang in Jilin Agricultural University from China, for providing Lactobacillus plantarum NC8 and Prof. Liancheng Lei in Jilin University from China, for providing Klebsiella pneumoniae HRB2020005 strain.
This work was supported by Natural Science Foundation of Hubei Province (2021CFB173).
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College of Animal Science and Technology, Yangtze University, Jingzhou, 434025, China
Yang Zeng, Tiantian Li, Xueyang Chen, Chun Fang, Xiongyan Liang, Jing Liu & Yuying Yang
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YZ and JL conceived and designed research. YZ, TTL, XYC, XWF conducted experiments. YZ and JL analyzed data. CF, XYL, YYY contributed to the text editing. YZ wrote the manuscript and JL reviewed the manuscript. All authors all read and approved the manuscript.
Correspondence to Jing Liu or Yuying Yang .
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We obtained informed consent from the Experimental Animal Center of Three Gorges University to use the animals in this study. In the experiments, we collected blood, spleen, intestinal lavage fluid, internal organs, liver and lung. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed (People’s Republic of China Ministry of Health, document NO.55, 2001), and all procedures were approved by the Ethics Committee of Health Science Center, Yangtze University (202401002). This study was conducted in accordance with the ARRIVE guidelines.
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Zeng, Y., Li, T., Chen, X. et al. Oral administration of Lactobacillus plantarum expressing aCD11c modulates cellular immunity alleviating inflammatory injury due to Klebsiella pneumoniae infection. BMC Vet Res 20 , 399 (2024). https://doi.org/10.1186/s12917-024-04248-9
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Volume 29, Number 4—April 2023
Experimental infection of north american deer mice with clade i and ii monkeypox virus isolates.
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The global spread of monkeypox virus has raised concerns over the establishment of novel enzootic reservoirs in expanded geographic regions. We demonstrate that although deer mice are permissive to experimental infection with clade I and II monkeypox viruses, the infection is short-lived and has limited capability for active transmission.
Monkeypox virus (MPXV; genus Orthopoxvirus , Poxviridae ), which causes mpox disease, is a zoonotic pathogen that is endemic in Central Africa (clade I) and Western Africa (clade II) ( 1 ). In mid-May 2022, the World Health Organization first reported an increasing number of mpox cases in nonendemic countries, most of which had no established travel links to endemic regions ( 2 ). By October 2022, the outbreak encompassed >100 countries with reported confirmed mpox cases ( 3 ).
The global spread of MPXV outside of regions in which this virus was known to be endemic raises concerns over reverse zoonotic events resulting in the establishment of novel wildlife reservoirs. Small mammals, including rodents, have previously been implicated as enzootic reservoirs of MPXV. In North America, studies have shown that prairie dogs are susceptible to MPXV infection and may serve as a potential reservoir, but data on other wild rodents are limited ( 4 ). Peromyscus species rodents have an extensive and geographically diverse host range spanning most regions across North America and are well-established reservoirs for several zoonotic pathogens ( 5 ).
We evaluated the competency of deer mice ( Peromyscus maniculatus rufinus ) as a potential zoonotic reservoir for MPXV by using representative isolates from both clades. We infected groups of 12 adult (>6 weeks of age) deer mice with 1 of 3 MPXV isolates through intranasal instillation. The isolates included a clade II human isolate from the 2022 outbreak (MPXV/SP2833) (challenge dose 10 6 PFU); a second clade II virus isolated directly from a North American prairie dog (USA-2003) (challenge dose 10 6 PFU); and a historical clade I isolate (MPXV/V79-1-005) (challenge dose 10 4 PFU). For each virus preparation, we administered the maximum challenge dose based on titration on Vero cells. On days 4 and 10 postinfection, we euthanized 3 male and 3 female mice and collected selected solid organs for analysis of viral titers using molecular assays targeting of envelope protein gene (B6R) ( 6 ) and infectious viral quantification assays. In addition, we collected oral and rectal swab specimens and tested them similarly to assess the potential for shedding.
We conducted animal studies in accordance with the Canadian Council of Animal Care guidelines and following an animal use document approved by an institutional Animal Care and Use Committee, in a Biosafety Level 4 laboratory of the Public Health Agency of Canada. We conducted fully validated molecular assays in accordance with Public Health Agency of Canada special pathogens diagnostic procedures.
Figure . Monkeypox virus infectious titers from lung and nasal turbinate samples from experimentally infected deer mice. Groups of 12 deer mice (6 male, 6 female) were experimentally infected with monkeypox virus...
Throughout the course of the study, we observed no obvious signs of disease in any of the infected deer mice. We did not record daily weights because of the requirement for anesthetizing animals before any hands-on manipulation. Analysis of tissue samples from mice infected with the 2022 Canada isolate (MPXV/SP2833) revealed limited and sporadic spread of MPXV beyond the sites of inoculation (nasal turbinates and lungs) ( Table ). By comparison, USA-2003 appeared to disseminate beyond the respiratory tract, resulting in uniform detection of MPXV DNA in liver and spleen specimens collected at 4 days postinfection (dpi). The clade I virus (MPXV/V79-1-005) yielded results more similar to those for USA-2003; nasal turbinate, lung, liver and spleen samples were positive at 4 dpi. By day 10 dpi, organ specimens from most mice across the 3 infection groups were trending toward clearance ( Table ). Infectious titers conducted on lung and nasal turbinate specimens collected at both timepoints from the 3 challenge groups corroborated these findings and demonstrated decreasing viral titers between the 2 timepoints ( Figure ).
Of note, the clade I virus did not achieve high titers in either organ, even when analyzed at 4 dpi. Although this finding may suggest the MPXV/V79-1-005 isolate does not replicate as efficiently in deer mice, the apparent low viral titers observed may be attributable to the lower inoculum dose. A similar challenge dose of this strain resulted in lethal infection in CAST/EiJ mice ( 7 ). Further, subsequent cell culture propagations of MPXV/V79-1-005 resulted in similar titers as the clade II isolates used previously, suggesting that all 3 replicate to a similar extent on Vero cells. Nevertheless, follow-up studies with other clade I viruses are warranted.
We collected oral and rectal swab specimens to assess shedding and the potential for transmission of MPXV from infected deer mice. Overall, shedding, as suggested by the presence of MPXV DNA in swab extracts, was readily detectable in deer mice inoculated with either clade II virus at day 4, but we noted decreasing levels of positivity by day 10. Shedding of MPXV/V79-1-005 (clade 1) was far less than that of either of the clade II viruses we evaluated ( Table ).
Our study suggests that these rodents may support a short-term but abortive infection with at least clade II MPXV isolates, although with limited capacity to spread. Given the short duration of infection, these animals probably do not represent a viable enzootic reservoir for MPXV. Further studies should be conducted on other rodents in North America and Europe to assess their competency as vectors or reservoirs of MPXV. Particular interest should be given to Rattus species rodents that may frequently come into contact with medical waste containing viable MPXV.
Mr. Deschambault is a senior laboratory technician in the Special Pathogens Program of the Public Health Agency of Canada. His research interests include disease modeling and vaccine development for emerging and high - consequence viral pathogens.
This work was funded by the Public Health Agency of Canada. We obtained the MPXV USA-2003 reagent NR-2500 through the BEI Resources Repository, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
All authors declare no conflict of interest.
DOI: 10.3201/eid2904.221594
Original Publication Date: March 06, 2023
Table of Contents – Volume 29, Number 4—April 2023
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EID | Deschambault Y, Klassen L, Soule G, Tierney K, Azaransky K, Sloan A, et al. Experimental Infection of North American Deer Mice with Clade I and II Monkeypox Virus Isolates. Emerg Infect Dis. 2023;29(4):858-860. https://doi.org/10.3201/eid2904.221594 |
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AMA | Deschambault Y, Klassen L, Soule G, et al. Experimental Infection of North American Deer Mice with Clade I and II Monkeypox Virus Isolates. . 2023;29(4):858-860. doi:10.3201/eid2904.221594. |
APA | Deschambault, Y., Klassen, L., Soule, G., Tierney, K., Azaransky, K., Sloan, A....Safronetz, D. (2023). Experimental Infection of North American Deer Mice with Clade I and II Monkeypox Virus Isolates. , (4), 858-860. https://doi.org/10.3201/eid2904.221594. |
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Microfilariae of Onchocerca lienalis were obtained from the umbilical skin of naturally infected cattle and were injected into mice. Maximum numbers of microfilariae were recovered from the skin and ears of mice when using the subcutaneous route of inoculation. Microfilariae were distributed throughout the pinna of the ear but were concentrated towards the tip where histological sections showed them to be in the dermis and adipose tissue. Using the number of parasites recovered from the ears as an index of the intensity of infection it was found that inbred CBA/H T6T6 mice were one of the most susceptible of 11 strains of mice examined. No difference in susceptibility was found between male and female CBA mice of the same age, but marked differences were demonstrated between male CBA mice of different ages. After infection with 5 000 microfilariae the recovery of parasites from the ears increased rapidly to a peak at day 35 when 10% of the inoculum was recovered, and thereafter declined up to day 242. Over a range of inoculation doses examined it was found that there was a direct, linear relationship between the number of microfilariae recovered from the ears and the number in the inoculated dose. CBA mice showed marked resistance to reinfection with microfilariae. Six days after challenge with a secondary infection recoveries of microfilariae from the ears were only 26% of the level in challenge controls and fell to 3% of the level of controls by day 35. It is concluded that the model of O. lienalis microfilariae in CBA mice shows considerable promise as a tool for research into immunological responses to skin-dwelling microfilariae, which are the principal cause of pathology in onchocerciasis.
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Nature Communications volume 15 , Article number: 7711 ( 2024 ) Cite this article
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Baculovirus is an obligate parasitic virus of the phylum Arthropoda . Baculovirus including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used in the laboratory and industrial preparation of proteins or protein complexes. Due to its large packaging capacity and non-replicative and non-integrative natures in mammals, baculovirus has been proposed as a gene therapy vector for transgene delivery. However, the mechanism of baculovirus transduction in mammalian cells has not been fully illustrated. Here, we employed a cell surface protein-focused CRISPR screen to identify host dependency factors for baculovirus transduction in mammalian cells. The screening experiment uncovered a series of baculovirus host factors in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and NPC intracellular cholesterol transporter 1 (NPC1). Further investigation illustrated that EXTL3 affected baculovirus attachment and entry by participating in heparan sulfate biosynthesis. In addition, NPC1 promoted baculovirus transduction by mediating membrane fusion and endosomal escape. Moreover, in vivo, baculovirus transduction in Npc1 −/+ mice showed that disruption of Npc1 gene significantly reduced baculovirus transduction in mouse liver. In summary, our study revealed the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape in mammalian cells, which is useful for understanding baculovirus transduction in human cells.
Introduction.
Baculoviruses are enveloped, double-strand DNA viruses comprising large genomes ranging from 80 to 180 kilobase pairs 1 . As the first baculovirus with full sequence information, AcMNPV has a genome of 133,894 nucleotides in size that contains 154 predicted non-overlapping open reading frames 2 . In nature, AcMNPV produces two structurally and functionally distinct virion forms, budded virions (BVs) and occlusion-derived virions (ODVs) 3 . BVs occur primarily in a loose viral envelope containing a single nucleocapsid, while ODVs have more than one nucleocapsid in a single envelope 4 . Although the two virion forms may share some common nucleocapsid proteins such as P39, the envelope components are largely different. BVs have envelope glycoproteins GP64, whereas ODVs contain polyhedral envelope protein P32-34, polyhedrin P29 4 , 5 , and per os infectivity factors (PIFs) that are required for the infection in insect gut 6 , 7 . BVs capture cell membranes as their envelopes and transmit within the hosts. By contrast, ODVs acquire envelopes from cell nuclear membrane and transmit between hosts 8 , 9 . BVs enter cells through receptor-mediated endocytosis 10 , 11 , while ODVs appear to fuse directly with the plasma membrane 12 , 13 .
Like other enveloped viruses, the interactions between envelope glycoproteins and host cell receptors are important for baculovirus entry. GP64 and F protein are the major envelope glycoproteins of lepidopteran nucleopolyhedrovirus (NPVs) 14 . In group I NPVs such as AcMNPV, GP64 functions as a fusion protein mediating viral entry into host cells 10 , while the F protein Ac23 seemed dispensable for infection in insect cells 14 , 15 . Different from group I NPVs, group II NPVs such as Spodoptera exigua MNPV and Lymantria dispar MNPV do not have GP64 protein and utilize F protein as the fusion protein for virus entry 16 , 17 , 18 .
As a class III fusion protein, AcMNPV GP64 protein has 512 amino acids 19 , 20 constituting five domains or structurally distinct regions, among which domain I contains fusion peptide and receptor binding peptide 10 , 19 . Different from class I and class II fusion proteins, GP64 does not undergo proteolytic cleavage 20 , 21 . The membrane fusion process of AcMNPV is triggered by a reversible, pH-dependent conformational change of GP64 22 , which remains trimeric in pre-fusion and post-fusion states 19 . Despite the existing studies in insect cells, the cellular receptor for GP64 binding in mammalian cells has not been reported.
AcMNPV is one of the most widely used baculoviruses in laboratory research and industry 23 . Baculovirus expression vector system (BEVS) was developed in the 1980s by using BVs as a transgene delivery vector to insect cells 24 . BEVS has now become one of the most widely used expression systems in the laboratory and industry for production of recombinant proteins 25 , virus-like particles 26 , 27 , and baculovirus-based vaccines 28 , 29 , 30 , 31 , 32 , 33 .
In addition to gene transfer into insect cells, baculovirus has been employed as gene delivery vectors in mammalian cells 34 , 35 , 36 . Baculovirus is capable of delivering a variety of genome editing tools into human cells, including zinc finger nucleases (ZFNs) 37 , 38 , 39 , TALENs 40 , CRISPR-Cas 41 , and CRISPR-based prime editors 42 . Genome editing in primary neurons and induced pluripotent stem cells (iPSCs) has also been achieved by baculovirus delivery of CRISPR-Cas9. Compared to other viral vectors 43 , 44 , the non-replicative and non-integrative natures render baculovirus a potentially safe gene therapy vector. Another notable advantage of baculovirus as a gene transfer vector lies in its large packaging capacity, which enables the delivery of large genome-editing tools such as prime editors in a single virion 42 .
More importantly, baculovirus has been exploited for in vivo gene delivery in mammals. Direct injection of baculovirus in mouse and rat brains could efficiently transduce neural cells 45 . Intravitreal baculovirus injection mediated gene transfer in mouse and rabbit eyes 46 , 47 . In cancer mouse models, intratumoral baculovirus injection was shown to be capable of delivering antiangiogenic proteins for cancer therapy 48 , 49 . Engineering efforts have shown that the transduction efficiency of baculovirus in mammalian cells in vitro and in vivo could be enhanced 50 , 51 , 52 , 53 .
Despite the widespread applications, the mechanism of baculovirus transduction in mammalian cells is yet poorly understood. Previous studies have shown that polybrene and heparin can inhibit baculovirus transduction in mammalian cells, suggesting an important role of electrostatic interactions 54 , 55 . Syndecan-1 (SDC-1) was also found to be important for baculovirus binding on mammalian cells 56 . Other studies suggested that baculovirus attachment on mammalian cells depended on cell surface phospholipids 57 and cholesterol 58 , 59 . Meanwhile, restriction factors that limit baculovirus transduction in mammalian cells have also been identified 60 . Nevertheless, there is yet no systematic investigation on baculovirus host factors in mammalian cells.
Genetic screen using clustered regularly interspaced short palindromic repeats (CRISPR) has been widely used in the dissection of host-pathogen interactions 61 . In a previous study, we established a cell surface protein-focused CRISPR library, so-called surfaceome CRISPR (SfCRISPR), and showed that this library could efficiently identify rhinovirus host factors 62 . In the present study, we employed this sfCRISPR library to perform forward genetic screen on a recombinant baculovirus carrying EGFP transgene (Bac-EGFP). Two rounds of negative selection identified several candidate host dependency factors for baculovirus transduction in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and Niemann-Pick C1 (NPC1). In-depth analyses illustrated the role of EXTL3 and NPC1 for the in vitro and in vivo baculovirus transduction in mammalian cells.
To identify cell surface host factors of baculovirus in mammalian cells, we performed genetic screen using a previously described cell surfaceome CRISPR library (SfCRISPR) that contained sgRNAs targeting to 1344 cell surface protein-coding genes in human genome 62 . We examined several cell lines for baculovirus transduction and found that HEK-293T cells exhibited highest transduction rate (Supplementary Fig. 1a ), which was important for reducing the background in the negative EGFP selection (Fig. 1a ). HEK-293T cells were transduced with lentivirus (LV) carrying the SfCRISPR library. Next-generation sequencing (NGS) analysis showed that this cell library had full coverage of sgRNAs (Supplementary Fig. 1b ) with a uniform distribution as predicted (Supplementary Fig. 1c ). In addition, we analyzed the shift of sgRNAs targeting essential and nonessential genes by comparing the sgRNAs at 5 days and 12 days after LV transduction and found that sgRNAs targeting essential genes were significantly depleted (Supplementary Fig. 1d ) as described 63 . These results suggested that the library was constructed successfully and was qualified for downstream phenotypic screen.
a Flowchart showing the negative selection approach for baculovirus host factor identification using surfaceome CRISPR screen. Candidate gene hits identified from the first ( b ) and second ( c ) rounds of selection. d The effects of knockout of the seven candidate genes on Bac-EGFP transduction, as determined by flow cytometry analyses. The p -value between Nontarget and EXTL3 -KO-1, EXTL3 -KO-2, NPC1 -KO-1, NPC1 -KO-2, MBTPS2 -KO-1 and MBTPS2 -KO-2 groups are 1e-5, 1e-5, 6e-6, 1e-5, 4e-5 and 4e-5, respectively. e Fluorescence imaging of Bac-EGFP infected cells. The experiment is repeated three times independently with similar results. f RT-qPCR quantification of EGFP mRNA in the lysate of EXTL3 and NPC1 knockout cells at 48 h post-Bac-EGFP transduction. β-actin is used as an internal control. g Evaluation of the effects of EXTL3 and NPC1 knockdown on Bac-EGFP transduction. The p- value between SiRNA-nontarget and SiRNA- NPC1 -2 groups is 1e-5. h Evaluation of the effects of NPC1 inhibitor U18666A on Bac-EGFP transduction. For ( d , f , and g ), Data are presented as mean ± SD ( n = 3) from three biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t- test. Mock group is the PBS treatment without baculovirus. Source data are provided as a Source Data file.
The validated cell library containing cell surface protein knockouts was then infected with baculovirus harboring enhanced green fluorescent protein transgene (Bac-EGFP). The Bac-EGFP-transduced cells were sorted by flow cytometry for enriching EGFP negative cells (Fig. 1a ). This negative selection approach allowed enrichment of cells deficient in baculovirus host factors. Two rounds of negative selection were performed and sgRNA enrichment was observed (Supplementary Fig. 1e ). With the process of negative selection, we observed an increased frequency of EGFP negative cells in round 2 selection, suggesting of successfully imposed selection pressure (Supplementary Fig. 2a, b ).
The cells after round 1 and round 2 selection were collected and the genomic DNA was extracted and analyzed by NGS. The genes with enriched sgRNAs were determined using modified robust rank aggregation (a-RRA) analyses in the MAGeCK pipeline (MAGeCK score) and candidate gene hits with an FDR cutoff of 0.05 were displayed (Fig. 1b, c and Supplementary Data File 1 ). Seven candidate hits were consistently enriched in both rounds of selection, including exostosin-like glycosyltransferase 3 (EXTL3), Niemann-Pick C1 (NPC1), SREBP cleavage activating protein (SCAP), FAM20B glycosaminoglycan xylosylkinase (FAM20B), membrane-bound transcription factor site-2 protease (MBTPS2), glucuronic acid epimerase (GLCE) and transmembrane 9 superfamily member 3 (TM9SF3). For each candidate gene, two sgRNAs were designed to construct knockout cells in HEK-293T cells (Supplementary Fig. 3a–g ).
It was found that the knockout of EXTL3 , NPC1 , SCAP , MBTPS2 and GCLE with both sgRNAs significantly reduced Bac-EGFP transduction (Fig. 1d ). Importantly, EXTL3 and NPC1 knockout reduced EGFP-positive cells to less than 50% of that in the non-targeting sgRNA group (Fig. 1d ). Thus, subsequent experiments were focused on studying the function of EXTL3 and NPC1. EXTL3 contains a glycosyltransferase domain and plays a critical role in the biosynthesis of heparan sulfate, which can facilitate viral attachment and entry 64 , 65 , 66 . NPC1 is a known host factor for various clinically important viruses 67 , 68 . Therefore, in the subsequent experiments, we focused our investigation on the functions and mechanisms of EXTL3 and NPC1 in baculovirus transduction.
In consistency with the flow cytometry experiments, fluorescent microscopy imaging showed that EXTL3 or NPC1 knockout reduced Bac-EGFP transduction (Fig. 1e ). Moreover, RT-qPCR analysis revealed decreased mRNA expression of EGFP transgene in EXTL3 and NPC1 knockout cells (Fig. 1f ). Similar to Bac-EGFP transduction, baculovirus carrying tdTomato (Bac-tdTomato) also exhibited reduced transduction efficiency in EXTL3 and NPC1 knockout HEK-293T cells (Supplementary Fig. 4a, b ). In addition, we designed EXTL3 - and NPC1 -targeting siRNAs (Supplementary Fig. 4c, d ) and found that knockdown of EXTL3 and NPC1 could also suppress baculovirus transduction albeit with lesser impact than EXTL3 and NPC1 knockout (Fig. 1g ). The function of NPC1 in baculovirus transduction was further validated by U18666A, a small molecule inhibitor of NPC1. It was found that U18666A inhibited Bac-EGFP transduction in a dose-dependent manner in HEK-293T cells (Fig. 1h ). Collectively, these results demonstrated that EXTL3 and NPC1 were host dependency factors for baculovirus transduction in human cells.
Similar to the results in HEK-293T cells, EXTL3 and NPC1 knockout in HeLa cells (Supplementary Fig. 5a ) inhibited Bac-EGFP transduction (Fig. 2a and Supplementary Fig. 5b ). The NPC1 inhibitor U18666A reduced EGFP fluorescence in Bac-EGFP-transduced HeLa cells (Fig. 2b and Supplementary Fig. 5c ). Next we constructed single clones of HeLa cells for EXTL3 and NPC1 knockouts respectively. These single clones were confirmed to contain genomic modifications at both alleles of EXTL3 and NPC1 (Supplementary Fig. 5d ). Consistent with the above results, EXTL3 −/− and NPC1 −/− clones exhibited reduced Bac-EGFP transduction, as determined by EGFP expression at both protein (Fig. 2c and Supplementary Fig. 5e ) and mRNA (Fig. 2d ) levels. Moreover, rescue experiments by overexpressing EXTL3 and NPC1 in corresponding knockout single clones (Supplementary Fig. 5f, g ) restored baculovirus transduction (Fig. 2c, d and Supplementary Fig. 5h ).
a Evaluation of the effects of EXTL3 and NPC1 knockouts on Bac-EGFP transduction, as determined by flow cytometry analyses. The p- value between Nontarget and NPC1 -KO-2 groups is 2e-5. b Evaluation of the effects of NPC1 inhibitor U18666A on Bac-EGFP transduction. Evaluation of the effects of EXTL3 and NPC1 knockouts and overexpression rescue on Bac-EGFP transduction, as determined by flow cytometry (EGFP protein) ( c ) and RT-qPCR ( EGFP mRNA) ( d ). β-actin is used as an internal control for RT-qPCR. For ( c ), the p -values between Nontarget and EXTL3 −/− and NPC1 −/− groups are 5e-8 and 5e-6, respectively. The p -value between EXTL3 −/− and EXTL3 rescued groups is 2e-5. The p -value between NPC1 −/− and NPC1 rescued groups is 1e-5. For ( d ), the p- value between Nontarget and EXTL3 −/ − and NPC1 −/− groups are 4e-7 and 7e-8, respectively. Evaluation of the effects of EXTL3 or NPC1 knockout and overexpression rescue on Bac-EGFP attachment ( e ) and entry ( f ). g qPCR quantification of the intracellular baculovirus genome GP64 gene after Bac-EGFP transduction in non-targeting sgRNA, NPC1 −/− and NPC1 rescued HeLa cells. Bac-EGFP is incubated with cells at an MOI of 2 for 1 h and then removed from the medium. The total GP64 in each well is quantified. The p- values between NPC1 −/− and Nontarget and NPC1 rescued group at 47 h are 5e-5 and 3e-5, respectively. For ( a ), ( c – g ), Data are presented as mean ± SD ( n = 3) from three biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t- test. Mock group is the PBS treatment without baculovirus. Source data are provided as a Source Data file.
To determine the roles of EXTL3 and NPC1 during baculovirus transduction, we investigated their functions in viral attachment and entry. For viral attachment assay, Bac-EGFP was incubated with cells at 4 °C for 1 h. The cells with attached baculovirus were harvested and lysed for qPCR quantification of viral loads. For virus entry assay, Bac-EGFP was incubated with cells first at 4 °C for 1 h and then at 37 °C for 45 min to initiate the internalization of Bac-EGFP. Surface-bound Bac-EGFP was removed by trypsin treatment and the internalized virus was quantified by qPCR. It was found that EXTL3 −/− cells significantly reduced baculovirus attachment and entry, which could be restored by EXTL3 overexpression (Fig. 2e, f ). By contrast, NPC1 knockout or overexpression rescue did not significantly affect baculovirus attachment or entry (Fig. 2e, f ). Taken together, these results suggested that EXTL3, but not NPC1, was involved in baculovirus attachment and entry.
To explore whether NPC1 participated in any transduction process following attachment and entry, we sought to analyze the function of NPC1 on endosomal escape. It is known that baculovirus does not replicate in mammalian cells 69 , thus we monitored the decay rate of intracellular baculovirus genome in an approach similar to a previous study 62 . Non-targeting sgRNA-treated, NPC1 −/− and NPC1 rescued HeLa cells were transduced with Bac-EGFP for 1 h and then the virus-containing medium was replaced with fresh medium. The total GP64 quantity in each well was monitored over a course of 47 h after the removal of virus. It was found that baculovirus DNA decreased in a time-dependent manner (Fig. 2g ), suggesting that the cellular DNA degradation machinery was active toward baculovirus genome. Importantly, NPC1 −/− cells retained significantly higher level of baculovirus genome than non-targeting sgRNA-treated cells starting from 8 h after removal of virus (Fig. 2g ). Consistently, NPC1 overexpression rescue significantly reduced baculoviral genome quantity at 35 h and 47 h in comparison to NPC1 −/− cells. To exclude the possibility that the difference in intracellular viral genome was due to differential cell proliferation, we examined the proliferation of non-targeting sgRNA, NPC1 −/− and NPC1 rescued cells and observed minor difference between each group (Supplementary Fig. 5i ). These data showed that knockout of NPC1 could extend the persistence of intracellular baculovirus genome and thus indicated a potential role of NPC1 in baculovirus endosomal escape.
The above results prompted us to expand the investigation on the mechanism of retention of Bac genome. Bafilomycin A1 (BafA1) is a specific inhibitor of V-type ATPases 70 and has been reported to be capable of inhibiting endosomal escape of viruses by suppressing endosomal acidification 71 , 72 . In the present study, it was found that pre-treatment of cells with BafA1 could prolong the retention of intracellular Bac genome (Supplementary Fig. 6a ), similar to the effects of NPC1 knockout. In addition, BafA1 treatment had minor impact on cell proliferation (Supplementary Fig. 6b ), suggesting that the above-observed effects of BafA1 on intracellular Bac genome were unlikely due to its side effects on cells. Collectively, these data established a link between NPC1-mediated endosomal escape and the exposure and decay of Bac genome in cytoplasm.
EXTL3 (Exostosin-like protein 3) belongs to EXT protein family, which includes EXT1, EXT2, EXTL1, EXTL2, and EXTL3 73 . EXTL3 encodes a glycosyltransferase responsible for the biosynthesis of heparan sulfate (HS). Two glycosyltransferase domains, GT47 and GT64, of EXTL3 in the Golgi luminal region are the functional domains for HS biosynthesis 74 (Fig. 3a ). We constructed EXTL3 −/− HEK-293T cells (Supplementary Fig. 7a ) to explore the function of EXTL3-mediated HS biosynthesis in baculovirus transduction. It was found that baculovirus attachment and entry in EXTL3 −/− cells were markedly reduced as compared to non-targeting sgRNA cells (Fig. 3b ). These results were consistent with those in HeLa cells and illustrated an important role of EXTL3 in baculovirus attachment and entry.
a Structural organization of EXTL3 protein. b Evaluation of baculovirus attachment and entry in EXTL3 −/− cells. GP64 gene was quantified by qPCR. The p- value between Nontarget and EXTL3 −/− in entry groups is 4e-5. c Western blotting detection of heparan sulfate proteoglycan 2 (HSPG2) in wild-type and EXTL3 −/− cells. The experiment is repeated three times independently with similar results. d Analysis of the effects of heparin sodium on Bac-EGFP transduction, as determined by flow cytometry quantification of EGFP-positive cells. e Analysis of the effects of heparin sodium on Bac-EGFP attachment and entry, as determined by qPCR quantification of attached or internalized baculovirus GP64 gene. f Evaluation of the effects of HPSE on baculovirus transduction. The p -value between Nontarget and Nontarget+HPSE groups is 2e-5. g Schematic presentation of HSPG biosynthesis. h Evaluation of the effects of EXT2 knockout on baculovirus transduction. The p- value between Nontarget and EXT2 -KO groups is 7e-6. i Investigation of the relationship between EXTL3 and NPC1 functions in baculovirus transduction. The p- value between No heparin sodium and With heparin sodium in NPC1 −/− groups is 1e-6. The p- value between No heparin sodium and With heparin sodium in NPC1 rescued groups is 2e-6. For ( b , e , f , h , and i ), Data are presented as mean ± SD ( n = 3) from three biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t- test. Mock group is the PBS treatment without baculovirus. Source data are provided as a Source Data file.
Given that HS is a known baculovirus attachment and entry factor in mammalian cells 54 , 55 , 56 , we sought to explore whether EXTL3 affected baculovirus attachment and entry by affecting HS biosynthesis. We thus examined the expression of heparan sulfate proteoglycan 2 (HSPG2) protein in EXTL3 −/− HEK-293T cells and found that EXTL3 knockout abolished the production of HSPG2 (Fig. 3c ). In addition, we found that Bac-EGFP transduction could be inhibited by heparin sodium in a dose-dependent manner (Fig. 3d and Supplementary Fig. 7b ), which is a competitive inhibitor for cellular HS 65 , 75 . Similarly, 2 μg/μL heparin sodium could significantly inhibit baculovirus attachment and entry (Fig. 3e ). Moreover, 24 h pretreatment with heparanase (HPSE) could significantly reduce baculovirus transduction in non-targeting sgRNA control cells or in overexpression-rescued EXTL3 −/− cells (Fig. 3f ). These results collectively suggested that HS was important for baculovirus attachment and entry and that EXTL3 promoted baculovirus transduction by involving in HS biosynthesis.
Next, we sought to investigate whether other members in the EXT protein family have similar roles in baculovirus transduction. HS biosynthesis is initiated by the attachment of xylose to specific serine residues in HSPG core proteins, followed by the formation of a linkage tetrasaccharide, glucuronic acid-galactose-galactose-xylose (GlcA-Gal-Gal-Xyl). EXTL3 links the first N-acetyl-D-glucosamine (GlcNAc) residue to GlcA and an enzyme complex composed of EXT1 and EXT2 adds GlcA-GlcNAc disaccharide repeats to the nascent chain, followed by a series of processing reactions 76 on the chain (Fig. 3g ). We constructed EXT2 knockout HEK-293T cells (Supplementary Fig. 7c ) and found that EXT2 knockout could also affect baculovirus transduction (Fig. 3h ) though the degree of inhibition was not as prominent as those with EXTL3 knockout. These results suggested that different moieties or forms of HSPG may have differential effects on baculovirus transduction.
Next, we sought to explore the relationship between EXTL3 and NPC1 functions in baculovirus transduction. We found that in the presence of heparin sodium, the efficiency of baculovirus transduction in NPC1 −/− HeLa cells was further reduced (Fig. 3i and Supplementary Fig. 7d ). In addition, NPC1 overexpression rescue did not eliminate the inhibitory activity of heparin sodium on baculovirus transduction (Fig. 3i and Supplementary Fig. 7d ). These results suggested that NPC1 could affect baculovirus transduction through a EXTL3-independent pathway and that the functions of NPC1 and EXTL3 in baculovirus transduction were additive to each other.
To elucidate the mechanism of action of NPC1 in baculovirus endosomal escape, we first used DiOC18 dye to label baculovirus particles (DiOC18-Bac-EGFP). The fluorescence of DiOC18 is self-quenched in labeled viruses and dequenched when membrane fusion occurs 77 , 78 . DiOC18-staining experiments showed that the membrane fusion of baculovirus was significantly suppressed in NPC1 −/− HeLa cells which could be restored by NPC1 overexpression, as analyzed by confocal microscopy and flow cytometry experiments (Fig. 4a, b ). These results are consistent with the above findings and strongly suggested that NPC1 promoted baculovirus endosomal escape by involving in membrane fusion.
DiOC18-staining experiment for illustration of NPC1 function in baculovirus membrane fusion, as analyzed by confocal microscopy ( a ) or flow cytometry analyses ( b ). The p- value between DiOC18 only and Nontarget groups is 1e-5. The p -value between NPC1 −/− and NPC1 rescued groups is 4e-6. c The structural organization of NPC1. d Design of NPC1 constructs. Analyses of the interactions between HA-tagged GP64 and Myc-tagged full-length NPC1, NPC1-A, NPC1-C, NPC1-I ( e ), NPC1-ΔA, NPC1-ΔC or NPC1-ΔI ( f ). g Investigation of the effects of overexpression rescue with full-length or truncation constructs of NPC1 on baculovirus transduction, as determined by the EGFP-positive cells via flow cytometry. Mock, PBS treatment without baculovirus. The p -values between NPC1 −/− and Nontarget and NPC1 rescued groups are 8e-8 and 1e-7, respectively. h Neutralization of Bac-EGFP transduction in HEK-293T cells by recombinant protein of NPC1 C domain, as determined by flow cytometry analyses. i ESI-MS confirmation of purified GST-I protein ( n = 1). j Far-western analysis of the interaction between GP64 and GST-I. For ( b , g , and h ), Data are presented as mean ± SD ( n = 3) from three biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t- test. For ( e , f , and j ), The experiment is repeated three times independently with similar results. Source data are provided as a Source Data file.
NPC1 contains 13 transmembrane helices (TM) and 3 distinct lumenal domains A, C, and I 79 (Fig. 4c ). It has been reported that NPC1 affects the endosomal escape of Ebola virus through its C domain 67 , 80 . To assess which luminal domain of NPC1 interacts with GP64, we performed co-immunoprecipitation experiments. We designed constructs encoding individual domains of A, C, and I (NPC1-A, NPC1-C, and NPC1-I), and constructs with deletions of individual domain (NPC1-ΔA, NPC1-ΔC, and NPC1-ΔI) (Fig. 4d ). Co-IP analyses of the interactions between GP64 and NPC1-A, NPC1-C or NPC1-I showed that both NPC1-C and NPC1-I could interact with GP64 (Fig. 4e ). Additional Co-IP analyses showed that GP64 could interact with full-length NPC1 and truncation constructs NPC1-ΔA, NPC1-ΔC and NPC1-ΔI (Fig. 4f ), suggesting that the A, C or I domains might have redundant functions for GP64 binding. Collectively, these Co-IP experiments suggested that the C and I domains of NPC1 could both involve in the interactions with GP64.
To understand the roles of individual NPC1 domains in supporting baculovirus transduction, we performed a rescue experiment by generating stable cell lines harboring full-length NPC1 or truncation constructs on the basis of NPC1 −/− HeLa cells (Supplementary Fig. 8a ). It was found that the truncation construct NPC1-ΔA could partly restore the susceptibility of cells to baculovirus transduction whereas NPC1-ΔC or NPC1-ΔI did not significantly restore baculovirus transduction (Fig. 4g and Supplementary Fig. 8b ). These results were consistent with the Co-IP results in that C and I domains play more important roles in baculovirus transduction than A domain. In addition, we noted that while full-length NPC1 could restore baculovirus transduction to a level similar to that in non-targeting sgRNA group, none of the truncation constructs could achieve a comparable rescuing efficiency (Fig. 4g ). This suggested that the intact structural organization of NPC1 could be important for GP64 binding and baculovirus transduction. Collectively, the above results suggested that C and I domains played predominant roles in NPC1 binding with GP64 while the intact structure of NPC1 might be also important.
Next, we sought to investigate whether purified proteins of NPC1 C and I domains could interact with baculovirus or baculovirus proteins. We first performed a neutralization experiment with a commercial recombinant protein of NPC1 C domain (residues R372 to F622). It was found that NPC1 C domain could inhibit Bac-EGFP transduction in a dose-dependent manner in HEK-293T cells (Fig. 4h ). Because the function of NPC1-I domain was rarely reported in viral infection and no commercial source could be found, we expressed and purified GST tagged NPC1-I domain protein from BL21 (DE3) Escherichia coli cells. Despite of an unknown impurity band (Supplementary Fig. 8c ), the identity of the target protein band was confirmed by mass spectrometry (Fig. 4i ). We set a far WB assay to investigate the interaction between NPC-I and GP64, where GST-tagged NPC1-I domain was resolved by SDS-PAGE, transferred to a PVDF membrane and then probed with purified GP64 protein. It was found that NPC1-I domain could directly interact with GP64 in vitro (Fig. 4j ). Interestingly, we found that NPC1-C and NPC1-I shared 30% protein sequence similarity as analyzed by ClustalW (Supplementary Fig. 8d ). It was thus likely that NPC1-I resembled NPC1-C for virus interaction and endosomal escape, as reported in Ebola virus 67 , 80 , 81 .
To understand baculovirus transduction in mice, we first examined the toxicity of intravenously administrated baculovirus. We administrated Bac-EGFP to C57BL/6J mice via tail vein and found that Bac-EGFP resulted in a transient loss of body weight in the first four days post injection (Supplementary Fig. 9a ), which was eventually recovered. Similarly, a transient change of blood routine was observed in Bac-EGFP-treated mice (Supplementary Fig. 9b ).
We then analyzed the vulnerability of different tissues to baculovirus transduction in wild-type mice and found that liver displayed highest degree of Bac-EGFP transduction (Fig. 5a, d ). It was noted that wild-type mice liver exhibited highest expression of Npc1 (Fig. 5b ). To investigate whether baculovirus transduction in mouse liver was related with Npc1 expression, we sought to construct Npc1 knockout mice. Unfortunately, Npc1 −/− C57BL/6J mice showed notably reduced birth rate and body weight (Supplementary Fig. 9c ). A previous study also showed that homozygous Npc1 knockout in mice resulted in reduced general health and pathologic changes in multiple organs 82 . By contrast, Npc1 −/+ mice did not show defects in birth rate or body weight (Supplementary Fig. 9c ), nor did liver show pathologic changes as compared to wild-type mice (Supplementary Fig. 9d ). Therefore, we used Npc1 −/+ heterozygous C57BL/6J mice for subsequent analyses. The gene disruption of Npc1 was validated in the mRNA levels (Fig. 5b ). Importantly, Bac-EGFP transduction in liver tissues of Npc1 −/+ mice was significantly reduced (Fig. 5c ) as compared to that in wild-type mice, suggesting that baculovirus transduction in mouse liver was dependent on Npc1 expression. It was also noted that in Npc1 −/+ mice, EGFP mRNA could not be detected in organs other than liver (Fig. 5d ).
a Investigation of the tissue tropism of Bac-EGFP in wild-type mice at 72 h post intravenous injection, as determined by EGFP expression at protein ( n = 3). The fluorescence images are acquired using confocal microscopy on flash-frozen tissues that are fixed with optimal cutting temperature compound (OCT). b RT-qPCR quantification of NPC1 mRNA in the lysate of liver, kidney, lung, brain, and spleen in wild-type and Npc1 −/+ mice. The p- value between WT and Npc1 −/+ in spleen groups is 1e-8. RT-qPCR quantification of EGFP mRNA in the lysate of liver ( c ) and other tissues including kidney, lung, brain, and spleen ( d ) in wild-type and heterozygous Npc1 −/+ knockout mice at 72 h post-Bac-EGFP injection. The EGFP mRNA in the lysates of kidney, lung, brain, and spleen ( d ) in heterozygous Npc1 −/+ knockout mice is below the limit of detection. For ( b – d ), β-actin is used as an internal control. The data in this figure are from independent biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t- test. For ( b and d ), Data are presented as mean ± SD of biological replicates ( n = 5 for ( b ) and = 3 for ( d )). For c , Data are collected from nine biological replicates ( n = 9). The thick horizontal dashed line represents the median and thin horizontal dashed line indicates interquartile range between 25th and 75th percentile. Source data are provided as a Source Data file.
The large packaging capacity allows baculovirus to carry multiple transgene cassettes for the expression of large protein complexes in insect cells 83 , 84 , 85 . For the same reason, baculovirus has been an attractive gene therapy vector for delivering large or multiple gene cassettes in single viral particles, which can overcome the packaging limitation associated with conventional viral vectors. However, despite of the existing studies on baculovirus entry mechanism in mammalian cells 54 , 55 , 57 , 58 , 59 , there has been no systematic investigation on baculovirus host factors in mammalian cells. In the present study, we performed a forward genetic screen using the SfCRISPR library that carries approximately 1400 cell surface proteins. Two proteins, EXTL3 and NPC1, were identified as host dependency factors and were shown to involve in baculovirus attachment and entry, and endosomal escape processes respectively.
Investigation of EXTL3 suggested that it promoted baculovirus attachment and entry by mediating heparan sulfate (HS) biosynthesis. EXT2, another enzyme involved in HSPG biosynthesis, was also shown to affect baculovirus transduction. These results were consistent with the previous findings 54 , 55 and confirmed the importance of HS for baculovirus transduction. As HS is widely distributed across different cell types and tissues, we did not further explore the dependency of baculovirus transduction on HS. However, it is likely that different cells or tissues display different expression profiles of EXT family proteins or proteins related to HS biosynthesis, which can be important determinants for baculovirus transduction.
Moreover, it was found in the present study that NPC1 participated in baculovirus membrane fusion and endosomal escape, but not entry or attachment. These findings were consistent with the function of NPC1 in infection of filoviruses 86 and African swine fever virus (ASFV) 87 . Unlike the case in EBOV where the C domain of NPC1 interacted with EBOV glycoprotein 86 , it was found in the present study that both the C and I domains of NPC1 interacted with baculovirus GP64 protein. Additionally, an NPC1 inhibitor U18666A that can block EBOV infection was found to be efficient in inhibiting baculovirus transduction in mammalian cells. The structural basis of baculovirus GP64 interaction with NPC1-C and -I domains and the mechanism of U18666A-mediated inhibition of baculovirus transduction require further investigation. In consistency with these observations in mammalian cells, previous studies suggested that Bombyx mori NPC1 and NPC2 proteins promoted Bombyx mori nucleopolyhedrovirus (BmNPV) infection in insect cells by facilitating membrane fusion and endosomal escape 88 , 89 . More interestingly, BafA1 treatment experiments suggested the NPC1-mediated endosomal escape could release Bac genome into cytoplasm for transgene expression, the process of which will also accelerate Bac genome degradation.
One major discovery in the present study is that partial knockout of Npc1 reduced baculovirus transduction in mouse liver. To the best of our knowledge, this was the first study revealing host factors for in vivo baculovirus transduction in mammals. This result implied that the expression of NPC1 and its function in endosomal escape could be a critical determinant for baculovirus transduction in mammals. This knowledge can be exploited to design novel strategies of targeted baculovirus delivery for tissues or cells with high NPC1 expression. In addition, our study shed light on engineering baculovirus GP64 protein for enhanced interactions with mammalian NPC1. Nevertheless, although the present and previous studies revealed general safety of baculovirus administration in mice, the safety of baculovirus in humans remains unknown and should be carefully addressed in future studies.
In addition to EXTL3 and NPC1 , our studies revealed several other genes that might involve in baculovirus transduction in HEK-293T cells. Among these genes, MBTPS2 seemed to have a major impact on the transduction efficiency. MBTPS2 encodes site-2 protease (S2P), which is a hydrophobic zinc metalloprotease. S2P can cleave transmembrane proteins to release their nucleus-localizing components that regulate the transcription of genes involved in lipid biosynthesis 90 and ER stress response 91 . It has been reported that S2P can mediate the activation of antiviral proteins through regulated intramembrane proteolysis (RIP) process during HCV infection 92 . Thus, it could be interesting to investigate in future studies whether MBTPS2 plays similar functions during baculovirus transduction in mammalian cells.
One limitation in our study was that the HEK-293T cells used in the screening process were deficient of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. While the high baculovirus transduction efficiency in HEK-293T could reduce the background signal in the negative EGFP selection, the lack of cGAS-STING pathway eliminated the possibility of uncovering host factors involved in cGAS-STING signaling Recent studies showed that cGAS-STING signaling could impede baculovirus transduction in mammalian cells by affecting interferon (IFN) production 60 . Interestingly, cGAS-STING-mediated IFN production in mammalian cells could be inhibited by AcMNPV P26 protein 93 . In future studies, it would be interesting to perform CRISPR screen on cGAS-STING-competent cells to uncover baculovirus host factors that act in the context of IFN signaling.
HeLa cells were obtained from the American Type Culture Collection (ATCC). HEK-293T, U87-MG, Club, HK-2, U251, and A549 cells were obtained from the Cell Bank of Shanghai Institutes for Biological Science (SIBS). All the cells used in this study were validated by VivaCell Biosciences (Shanghai, China). All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Thermo, Pittsburgh, USA) supplemented with 10% fetal bovine serum (FBS, Thermo) and 1% penicillin-streptomycin (Thermo) and maintained at 37 °C in a fully humidified incubator containing 5% CO 2. All cells were confirmed by PCR to be free of mycoplasma contamination.
To produce lentivirus (LVs), HEK-293T cells at a confluence of 70–90% were transfected with LV packaging plasmid pMD2.G, envelope plasmid psPAX, and transfer plasmid pLentiCRISPR-v2 that carried Cas9 gene and a single sgRNA or pooled sgRNA plasmid library with a mass ratio of 1: 1.5: 2 using Lipofectamine 3000 (Thermo). In the case of overexpression, the transfer plasmids were pLenti-EF1α-IRES-NPC1-Bsd, plenti-EF1α-IRES-EXTL3-Bsd, pLenti-EF1α-IRES-NPC1ΔA-Bsd, pLenti-EF1α-IRES-NPC1ΔC-Bsd and pLenti-EF1α-IRES-NPC1ΔI-Bsd). At 6 h after transfection, the medium was replaced with fresh medium. The medium supernatant containing LVs was harvested at 48–60 h post-transfection by centrifugation at 2000 rpm (420 × g ) for 10 min, filtrated through a 0.45 μm filter (Merck, Darmstadt, Germany) and stored at −80 °C.
HeLa cells and HEK-293T cells were transduced with LVs in the presence of polybrene (10 μg/mL, Merck) using spinfection through centrifugation at 2000 rpm (420 × g ) for 2 h. At 24 h post-transduction, LV-containing medium was removed and cells were cultured in fresh medium in the presence of 1–2 μg/mL puromycin for 3 to 5 days or 2–5 μg/mL blasticidin (Thermo) for 7–10 days to remove empty cells containing no LVs. Finally, survived cells were collected, aliquoted, and stored in liquid nitrogen.
Bac-EGFP recombinant viruses were generated using Bac-to-Bac baculoviral expression system (Thermo, Cat. No. A11101). Transgene plasmid pFastBac carrying EGFP gene was transformed into DH10Bac E. coli competent cells that harbored the parental bacmid to form a recombinant bacmid encoding the EGFP transgene. The recombinant bacmid was then transfected into insect cells for production of recombinant baculovirus particles. At 72 h post-transfection, the culture supernatant containing baculovirus particles was harvested and concentrated by Optima XPN-100 Ultracentrifuge (Beckman Coulter, California, USA). Virus titer was determined using the 50% tissue culture infectious dose (TCID 50 ) assay 94 .
For baculovirus transduction, HEK-293T or HeLa cells were seeded at a cell density of 40%. At 24 h after seeding, the cells were transduced with baculovirus at an MOI of 1 or 2 for HEK-293T or HeLa respectively. MOIs other than 1 and 2 were indicated in figure legends. At 24 h after baculovirus transduction, baculovirus-containing medium was removed and the cells were incubated in DMEM (Thermo) supplemented with 10% FBS (Thermo) for another 24 h. Thereafter, the cells were collected and the efficiency of baculovirus transduction was quantified by flow cytometry (CytoFLEX, Beckman Coulter, California, USA) and visualized by fluorescence microscope (EVOS M5000, Thermo). The expression of EGFP mRNA was quantified by RT-qPCR using specific primers (Supplementary Table 2 ).
The human surfaceome CRISPR library was described in our previous work 62 , which contained 16,975 sgRNAs targeting 1314 surface protein genes and 1000 non-targeting sgRNAs. To construct surfaceome CRISPR library in HEK-293T, the cells were transduced with LV library at an MOI of 0.3 using spinfection as described above. Cells of more than 500-fold coverage of the library size were collected, aliquoted, and stored in liquid nitrogen.
The HEK-293T cell library of 1 × 10 7 cells was seeded onto 15 cm petri dishes. At 24 h after seeding, the cells were incubated with recombinant baculovirus carrying an EGFP transgene (Bac-EGFP) of 3 × 10 7 infective units (IU) for 24 h. The medium containing virus was then removed and cells washed with phosphate-buffered saline (PBS) for three times and then cultured in fresh medium for another 24 h. Then the treated cells were harvested and sorted using flow cytometry (Moflo, Beckman Coulter, California, USA) for EGFP-negative cells. Genomic DNA of the sorted cells was extracted using phenol: chloroform: isoamyl alcohol (v/v/v, 25:24:1) and then purified using ethanol precipitation. Genome-integrated sgRNAs were amplified from the collected genomic DNA by PCR using primers containing Illumina adapters (Supplementary Table 1 ). PCR amplicons were analyzed by Genewiz (Suzhou, Jiangsu, China) using next-generation sequencing (NGS) on Illumina HiSeq 3000 platform. After removing the adapters, the 20 bp sgRNA was mapped to the reference sgRNA with 1 bp mismatch allowed. The raw read counts were subjected to MAGeCK analyses to determine the enriched sgRNA and gene knockouts. A false discovery rate (FDR) of less than 0.05 was applied to identify significantly enriched sgRNAs and candidate gene knockouts.
The LV transfer plasmids pLentiCRISPR-v2-sgRNA carrying single sgRNAs (Supplementary Table 3 ) for knockout cell line construction were generated as previously described above 62 . The LVs were packaged and transduced onto cells as described above. To evaluate the knockout efficiency, the genomic DNA of knockout cells was extracted using Quick Extraction kit (Lucigen, Wisconsin, USA) and the modified genomic sites were PCR amplified using corresponding primers (Supplementary Table 4 ). The PCR amplicons were sequenced by Sanger sequencing (Genewiz) and gene disruption efficiency was analyzed by TIDE website ( https://tide.nki.nl/ ) 95 . Single clones were obtained by cell sorting using flow cytometry (Moflo, Beckman) and genotyped by Sanger sequencing to determine the mutations at each allele.
Total RNA from cultured cells was extracted using TRIzol (Thermo), chloroform (Sinopharm, Ningbo, China) and purified using isopropanol precipitation. RNA was then reverse transcribed into cDNA by PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan). The mRNA levels were determined by reverse transcription quantitative PCR (RT-qPCR) using SYBR green dye on Applied Biosystems Q6 Real-Time PCR cycler (Thermo) and specific primers (Supplementary Table 2 ). All SYBR Green primers were validated with dissociation curves. The expression of genes was normalized to β-actin or RPLP0 .
For RT-qPCR on mouse tissues, the tissue samples were collected, frozen in liquid nitrogen, and homogenized using grinding beads through tissue grinding device (Jingxin, Shanghai, China) in TRIzol reagent (Thermo). The extraction and quantification processes in tissue RNA were the same to that in cell culture samples as described above.
Genomic DNA of baculovirus or baculovirus-containing cells was extracted using phenol: chloroform: isoamyl alcohol (v/v/v, 25: 24: 1) and then purified using ethanol precipitation. The total DNA concentration was determined for each sample. For quantitative PCR (qPCR) reaction, 2 ng total DNA was added into each reaction as the template. GP64 DNA, which was used as indicator of viral genome content, was quantified by qPCR using SYBR green dye on Applied Biosystems Q6 Real-Time PCR cycler (Thermo) and specific primers (Supplementary Table 2 ). All SYBR Green primers were validated with dissociation curves. The genomic DNA level was normalized to β-actin.
HEK-293T cells were seeded onto 6-well plates with a density of 5 × 10 5 cells per well. At 24 h after seeding, cells were transfected with 100 pmol siRNA (Genepharma, Shanghai, China) (Supplementary Table 5 ) using 7.5 μL Lipofectamine 2000 (Thermo) for 6 h, then washed with PBS and cultured in fresh DMEM (Thermo) supplemented with 10% fetal bovine serum (FBS, Thermo). At 48 h post-transfection, cells were infected with baculovirus at an MOI of 1 for 24 h, then washed with PBS for three times and cultured in fresh medium for 24 h. The cell samples were harvested and lysed for total RNA extraction, and the mRNA levels of EGFP , EXTL3 , and NPC1 in cell lysate were determined using RT-qPCR as described above.
Virus attachment and entry assays were performed as described 96 with minor modifications. HEK-293T cells or HeLa cells were seeded onto 12-well plates at a density of 200,000 cells per well and incubated overnight. For virus attachment assay, cells were incubated with baculovirus at an MOI of 20 in cold medium without FBS on ice for 60 min, then washed with cold Dulbecco’s phosphate-buffered saline (DPBS) for three times and harvested. The baculovirus genomic DNA was extracted from the cells containing attached virus and quantified by qPCR as described above. For virus entry assay, cells were incubated with baculovirus at an MOI of 20 in cold medium on ice for 60 min, washed by cold DPBS for three times, treated with pre-warmed medium containing FBS, and then incubated at 37 °C for 40 min. The treated cells were washed with PBS for three times and then treated with 0.25% trypsin (Thermo) for 30 s to remove surface-bound baculovirus particles. The genomic DNA of internalized baculovirus was extracted and quantified by qPCR as described above.
EXTL3 and NPC1 genes were codon-optimized for expression in human cells and synthesized by Genewiz (Supplementary Tables 6 , 7 ). The 20 bp sgRNA-targeting sites and PAM sequences were mutated with silent mutations. Myc and FLAG tags were added to the C-terminus of these genes for WB detection. These genes were cloned into the XbaI and BamH1 sites of pLV-EF1α-IRES-Bsd plasmid. The lentiviral overexpression plasmids of NPC1 truncation mutants (NPC1-ΔA, NPC1-ΔC, and NPC1-ΔI) were constructed from the full-length gene in pLV-EF1ɑ-IRES-NPC1-Bsd. For LV packaging, the transgene plasmid containing EXTL3, NPC1, NPC1ΔA, NPC1ΔC or NPC1ΔI was co-transfected into HEK-293T cells with helper plasmids pMD2.G and psPAX as described above. The transgene-containing LVs were transduced into EXTL3 −/− or NPC1 −/− cells, and the cells were subjected to selection with 2 μg/mL blasticidin (Thermo) for 7 to 10 days to purge empty cells containing no LVs. Finally, survived cells were collected, aliquoted, and stored in liquid nitrogen.
The experiment was performed in an approach similar to a previous study 62 . Non-targeting sgRNA-treated, NPC1 −/− and NPC1 overexpression rescued HeLa cells were seeded onto 24-well plates at a density of 80,000 cells per well and incubated overnight. The next day the cells were incubated with Bac-EGFP at an MOI of 2 for 1 h. The virus-containing medium was removed and the cells were washed with PBS for three times and then treated with 0.25% trypsin (Thermo) for 30 s to remove surface-bound baculovirus particles. These treated cells were supplemented with pre-warmed DMEM medium containing FBS and then incubated at 37 °C for indicated time. Intracellular baculovirus genomic DNA was extracted from each well. The total baculovirus GP64 amount in each well was determined by qPCR and then normalized to the zero time point.
Non-targeting sgRNA, NPC1 −/− , and NPC1 rescued HeLa cells were seeded onto 96-well plates at a density of 10,000 cells per well and incubated at 37 °C overnight. The cells were incubated with Bac-EGFP at an MOI of 2 for 1 h, washed with PBS for three times and then treated with 0.25% trypsin (Thermo) for 30 s to remove surface-bound baculovirus particles. At indicated time points, the supernatant was removed and cells incubated with complete medium containing 10% CCK-8 reagent (MeilunBio, Liaoning, China) at 37 °C for 1 h. Cell proliferation was quantified by measuring absorbance at 450 nm using microplate reader (SpectraMax iD3, Molecular Devices, Shanghai, China). The background signal in empty wells without cells was subtracted from each sample.
Wild-type HeLa cells were seeded on to 24-well plates at a density of 80,000 cells per well and incubated overnight. The next day the cells were pre-incubated with 100 nM BafA1 (MedChemExpress, New Jersey, USA, Cat. No. HY-100558) for 1 h, followed by transduction with Bac-EGFP at an MOI of 2 for 1 h in the presence of 100 nM BafA1. The virus-containing medium was then removed, the cells washed three times with PBS and then treated with 0.25% trypsin (Thermo) for 30 s to remove surface-bound baculovirus particles. These cells were incubated in DMEM medium containing FBS and 10 nM BafA1 for indicated time. The genomic DNA of intracellular baculovirus was extracted from cells and quantified by qPCR as described above.
For CCK-8 assay, cells were seeded on to 96-well plates at a density of 10,000 cells per well. The next day, the cells were pre-incubated with 100 nM BafA1 and transduced with Bac-EGFP to mimic the treatment as described above. At 48 h after removal of virus, cell proliferation was quantified by CCK-8 as described above at indicated time points.
For WB analyses, cells were lysed with RIPA buffer (Beyotime Biotechnology, Beijing, China) on ice for 10 min and centrifuged at 12,000 rpm (13,523 × g ) at 4 °C for 10 min to remove cell debris. The total protein concentration in cell lysate was determined using the BCA Protein Assay Kit (Thermo). Cell lysate was mixed with SDS-PAGE loading buffer (Takara) containing 200 mM dithiothreitol (DTT), incubated at 95 °C for 10 min, and resolved on 4–12% PAGE gels (GenScript, Nanjing, China). Protein samples were transferred onto nitrocellulose membranes (Merck) using an iBlot gel transfer system (Thermo). The following primary and secondary antibodies were used in WB including anti-Myc rabbit antibody (CST, Cat. No. 2272S), anti-HA rabbit antibody (CST, Cat. No. 3724S), anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody (Abcam, Cambridge, UK, Cat. No. ab255829) and HRP-conjugated anti-rabbit IgG (CST, Cat. No. 5127S). Anti-β actin antibody conjugated with HRP (CST, Cat. No. 5125S) was used as an internal control.
HEK-293T cells were seeded onto 6-well plates at a density of 1 × 10 6 cells per well. After the confluency reached 70% to 80%, plasmid was transfected into cells. Plasmids containing Myc-labeled NPC1 or NPC1 mutant and plasmids containing HA-labeled GP64 (Supplementary Table 8 ) were co-transfected into HEK-293T cells by Lipofectamine 3000 (Thermo). At 24 h after transfection, cells were resuspended with co-IP lysis buffer. One-half of the lysate was used to incubate with magnetic beads (Thermo) containing anti-HA or anti-Myc antibodies at room temperature for 1 h. Magnetic beads were collected with a magnetic rack (Thermo), then proteins bound to the magnetic beads were eluted by 1x SDS-PAGE loading buffer containing DTT. Finally, total proteins and co-immunoprecipitated proteins were detected by WB as described above.
HEK-293T cells were seeded on to 48-well plates at a density of 1 × 10 5 cells per well. After Bac-EGFP incubation with recombinant protein of NPC1 C domain (residues R372-F622) (SinoBiological, Beijing, China, Cat. No. 16499-H32H) at indicated concentrations at 4 °C for 30 min. Thereafter, HEK-293T cells were transduced with Bac-EGFP at an MOI of 1 for 24 h in the presence of NPC1-C domain protein. Then baculovirus-containing medium was removed and the cells were incubated in DMEM (Thermo) supplemented with 10% FBS (Thermo) for 24 h and then harvested. The efficiency of baculovirus transduction was quantified by flow cytometry (CytoFLEX, Beckman Coulter, California, USA).
NPC1-I domain was codon-optimized for expression in Escherichia coli, synthesized by Genewiz, and cloned into pGEX4T-1-GST plasmid. The recombinant pGEX4T-1-GST-NPC1-I plasmid was transformed into chemically competent BL21 (DE3) E. coli cells. After overnight culturing, single colonies were selected and amplified in Luria–Bertani (LB) medium containing (100 mg/mL) ampicillin overnight. The next day, the culture was inoculated into fresh LB medium at 1: 100 dilution and cultured at 37 °C for about 3 h until OD 600 reached 0.6–0.8. Protein expression was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22 °C overnight. The cells were then collected by centrifugation at 4,000 rpm (1681 × g ) for 15 min at 4 °C.
For protein purification, the cell pellet was resuspended with PBS and then lysed by sonication. The cell lysate was centrifuged at 12,000 rpm (13,523 × g ) for 30 min at 4 °C and the supernatant was isolated. The supernatant was run through a glutathione resin affinity chromatography column (Merck) for protein capture. The resin was sufficiently washed with PBS and the protein was eluted with wash buffer supplemented with reduced glutathione. The eluted proteins were concentrated and further purified by size exclusion chromatography (Superdex 200 Increase 10/300GL) with a buffer containing 20 mM Tris pH 8.0, 250 mM NaCl. The fractions containing target proteins were pooled and concentrated for mass spectrometry analysis.
The purified NPC1-I protein was validated by Orbitrap Fusion MS (Thermo Fisher, San Jose, CA) using Proteome Discoverer 2.2 and Xcalibur analysis software at the Analytical Chemistry Platform at SIAIS, ShanghaiTech University. The entire gels were rinsed with water for 3–4 h, then the bands of GST (control, n = 1) and NPC-I (sample, n = 1) were excised with a clean scalpel. The two excised bands were cut into cubes (1 mm × 1 mm) separately. The gel slices were transferred into two microcentrifuge tubes and spun down by a microcentrifuge. For in-gel reduction, alkylation, and de-staining, the gels were incubated with 100 μL de-staining solution (100 mM ammonium bicarbonate buffer containing 50% acetonitrile, vol/vol) for 30 min at 37 °C, and then incubated with 500 μL neat acetonitrile for 10 min until gel slices shrank and became opaque and stiff. These gel slices were span to remove all liquid and 40 μL of 10 mM DTT solution was added to cover gel slices for 30 min at 56 °C in an air thermostat. The tubes were chilled down to room temperature, and incubated with 500 μL acetonitrile for 10 min and then all liquid was removed. The gels were incubated with 40 μL of 55 mM iodoacetamide solution for 20 min at room temperature in the dark, treated with acetonitrile, and then all liquid removed. The dry gels were incubated with 70 μL trypsin buffer containing 13 μg/mL trypsin for 100 min at 4 °C and then incubated with 20 μL 100 mM ammonium bicarbonate containing 10% (vol/vol) acetonitrile at 37 °C overnight. The next day the samples were incubated with 200 μL extraction buffer (1 vol: 2 vol of 5% formic acid: acetonitrile) for 15 min at 37 °C in a shaker. Then the supernatant was collected into a PCR tube, dried in a vacuum centrifuge, and stored at 4 °C for further analysis.
For liquid chromatography (LC) analysis, the above samples were supplemented with 20 μL of 0.1% (vol/vol) formic acid, vortexed, and centrifuged for 5 min at 12,000 × g . These treated peptides were loaded onto an analytical column (Ionopticks, AUR2-25075C18A, 25 cm × 75 μm, C18, 1.6 μm) connected to an Easy-nLC1200 UHPLC-Orbitrap Fusion (Thermo Fisher Scientific). The elution gradient and mobile phase constitution used for peptide separation were set as follows: 0–60 min, 5–30% buffer B; 60–70 min, 30–45% buffer B; 70–75 min, 45–100% buffer B; 75–80 min, 100% buffer B at a flow rate of 300 nL/min. Mobile phase in buffer A was 0.1% formic acid in water and mobile phase in buffer B was 0.1% formic acid in 80% acetonitrile.
Peptides eluted from the LC column were directly electro-sprayed into the mass spectrometer with the application of a distal 2.1 kV spray voltage. Survey full-scan mass spectra (from m/z 350 to 1800) were acquired in the Orbitrap analyzer (Orbitrap Fusion, Thermo Fisher Scientific) with resolution r = 60,000 at m/z 400. The cycle time of the MS-MS2 events was 3 s, sequentially generated and selected from the full mass spectrum at a 32% normalized collision energy. The dynamic exclusion time was set to 10 s. The acquired MS/MS data were analyzed using the AA sequence of protein GST, using Protein Discoverer 2.2 with the parameter settings as follows: precursor and fragment mass tolerance of 10 ppm and 0.02 Da and dynamic modifications of + 15.995 Da for Oxidation (Met) and + 42.011 Da for Acetyl (N-terminus), and + 57.021 Da for Carbamidomethyl (C terminus) as static modifications. To accurately estimate peptide probabilities and filter the false discovery, the fixed value PSM validator node was used and set to have a maximum delta Cn of 0.05. Trypsin was defined as cleavage enzyme and the maximal number of missed cleavage sites was set to two.
Far western blotting was performed as previously described 97 . Briefly, 0.2–1 μg purified NPC1-I protein (with an unknown impurity band) or a control GST protein was mixed with SDS-PAGE loading buffer (Takara) containing 200 mM DTT, incubated at 95 °C for 10 min and resolved on 4–12% PAGE gels (GenScript, Nanjing, China). Protein samples were transferred onto nitrocellulose (PVDF) membranes (Merck) using an iBlot gel transfer system (Thermo). PVDF membrane was treated with PBS solution containing gradient concentrations of guanidine hydrochloride (GuHCl) solution (Solarbio, China) for protein unfolding and refolding. The GuHCl gradient was decreased from 6 M to 3 M and then 0.1 M with the membrane being treated with each gradient for 30 min. Finally, the membrane was treated with PBS without GuHCl at 4 °C overnight. The membrane was then blocked with PBS containing 5% milk (w/v) and incubated with 1–10 μg GP64 protein (SinoBiological, Beijing, China) overnight at 4 °C. The membrane was then treated with an anti-GP64 mouse antibody (Abcam, Cat. No. ab91214) as the primary antibody and an HRP-conjugated anti-mouse antibody (R&D, Cat. No. HAF007) as the secondary antibody. The bait protein could be detected at the location of the prey protein on the membrane if they interact to form a complex.
Baculovirus was labeled with DiOC18 (Thermo) at a final concentration of 2 μM in PBS. The solution was rotated at room temperature in the dark for 1 h and then filtered through a 0.22 μm pore size syringe filter (Merck) to remove unbound dyes and aggregates. Cells were infected with DiOC18-labeled baculovirus at 37 °C for 3 h, then washed with PBS three times and treated with trypsin briefly to remove free and surface-bound viruses. Thereafter, the cells were fixed with PFA and analyzed by flow cytometry (CytoFLEX, Beckman Coulter, California, USA) and confocal microscopy (LSM 710, Zeiss, Oberkochen, Germany). A control group with DiOC18 dye alone, without the addition of baculovirus, was prepared following the same filtration process as described above to assess the background fluorescence labeling of cells by free dyes in the DiOC18-Bac-EGFP-treated groups.
Npc1 −/+ and wild-type C57BL/6 mice were purchased from, bred, and raised in GemPharmatech (Nanjing, China). All mice were housed in animal facility with ambient room temperature of 20–26 °C, humidity of 50–70%, and dark/light cycle of 12 h. Baculovirus-related animal experiments were performed in OBiO-tech corporation (Shanghai, China). For baculovirus, transduction, 6–8-week-old male Npc1 −/+ and wild-type C57BL/6 mice were used. The mice were maintained within a Specific Pathogen-Free (SPF) facility with free access to water and food. The housing facility for mice was under a 12:12 h light: dark cycle at temperatures 20–26 °C, humidity 40–70%. For transduction experiments, mice were randomly grouped ( n = 3 to 9 per group as indicated) and injected with baculoviruses through tail vein with a dose of 4.4 × 10 4 IU per gram body weight. Animals were sacrificed under an anesthetic condition at the indicated time post-baculovirus transduction. The tissues including liver, brain, spleen, lung, and kidney were collected and flash-frozen in liquid nitrogen or fixed in PFA buffer for further analyses.
For tissue tropism of Bac-EGFP transduction, Bac-EGFP-transduced tissues were flash-frozen and fixed with optimal cutting temperature compound (OCT). The fixed tissues were sectioned at 5 μm, and the sections were fixed with PFA and stained with Hoechst (Thermo). The fluorescence images were acquired using confocal microscopy (LSM 710, Zeiss).
All data were the results from at least three biological replicates and were shown as mean ± standard deviation unless noted otherwise. All experiments were repeated three times independently (biological replicates) unless noted otherwise. No data were excluded for analyses. Statistical analyses and graphing were performed with GraphPad Prism 7.0. The P -values were determined using two-tailed unpaired Student’s t-test unless otherwise noted.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
The authors declare that all data supporting the findings of this study are available in the article, its Supplementary Data, its Source Data, or from the corresponding authors upon request. Source data are provided with this paper. The NGS data generated in this study have been deposited in the SRA database under accession code PRJNA1131913 . Source data are provided with this paper.
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We thank Lishuang Zhang and Pengwei Zhang from the Discovery Technology Platform of SIAIS for the support of flow cytometry experiments, Wei Zhu from the Analytical Chemistry Platform for assistance with mass spectrometry analysis, and the Biomedical Big Data Platform for the design of sgRNA libraries and MAGeCK analyses. This research was supported by the Science and Technology Commission of Shanghai Municipality (23ZR1442100 to J.L.), the Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University (2022A0301-417-01 to J.L.), and the Postgraduate Scientific Research Innovation Project of Central South University (2023ZZTS0542 to Y.H.).
These authors contributed equally: Yuege Huang, Hong Mei.
Furong Laboratory, Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
Yuege Huang & Jia-Da Li
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China
Hong Mei, Chunchen Deng, Wei Wang, Chao Yuan, Yan Nie & Jia Liu
School of Life Science and Technology, ShanghaiTech University, Shanghai, China
Chunchen Deng, Chao Yuan & Jia Liu
Hunan Key Laboratory of Animal Models for Human Diseases, Changsha, Hunan, China
Shanghai Clinical Research and Trial Center, Shanghai, China
Guangzhou Laboratory, Guangzhou International Bio Island, Guangzhou, Guangdong, China
Shanghai Asiflyerbio Biotechnology, Shanghai, China
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J.L., J.D.L., and H.M. conceived this study, J.L., H.M., Y.H., and Y.N. designed the experiments, and Y.H., H.M., C.D., and C.Y. carried out the experiments and analyzed the data. W.W., H.M., and L.J. designed the sgRNA libraries, and Y.H., H.M., J.L, and J.D.L. wrote the manuscript. All authors contributed to the manuscript and approved the submission.
Correspondence to Hong Mei , Jia-Da Li or Jia Liu .
Competing interests.
J. Liu is the founder and shareholder of Shanghai AsiFlyer Biotechnology. The remaining authors declare no competing interests.
All study protocols involving mice were approved by the ethical committee of ShanghaiTech University (approval number: 20230821001) and conducted in accordance with regulatory policies in China for the care and use of animals.
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Huang, Y., Mei, H., Deng, C. et al. EXTL3 and NPC1 are mammalian host factors for Autographa californica multiple nucleopolyhedrovirus infection. Nat Commun 15 , 7711 (2024). https://doi.org/10.1038/s41467-024-52193-w
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DOI : https://doi.org/10.1038/s41467-024-52193-w
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Mouse models have been a particularly important resource for studying infection by a wide range of human pathogens and are widely used for preclinical screening of vaccines/therapies due to their reproducibility, low cost, and ease of experimental manipulation. Although mouse models are not appropriate for studying some aspects of pathogen ...
Erwinia persicinus (E. persicina) is a plant pathogenic bacterial species that was previously isolated from a case of human infection. This study aimed to create an experimental infection protocol for E. persicina in laboratory mice. Seventy-two adult mice were divided into four groups (18 animal/group): the control group (G1), the group ...
In-bred strains of mice are commonly used to model pathogenic infections due to their cost and utility. In order to understand better the nature of experimental tuberculosis in mice, we infected BALB/c mice with a virulent field isolate of Mycobacterium bovis. Mice were sacrificed at intervals in order to visualise the pathological lesions in ...
Erwinia persicinus (E. persicina) is a plant pathogenic bacterial species that was previously isolated from a case of human infection.This study aimed to create an experimental infection protocol for E. persicina in laboratory mice. Seventy-two adult mice were divided into four groups (18 animal/group): the control group (G1), the group infected with E. persicina (G2), the group immune ...
The experimental infection with the isolated infectious LSDV could serve as a platform for future vaccine evaluation study using an LSDV challenge model. Lumpy skin disease (LSD) is one of the ...
Inflammatory and pathological changes in Escherichia coli ...
In conclusion, it is clear that the mouse genetic background influences the Leishmania infection outcome and this feature must be taken into account when designing experiments. Although BALB/c mice will not die of VL (differently from untreated humans), they can be used to study the immunopathology changes occurring during VL.
The objective of this study was to test the ability of bovine viral diarrhea virus (BVDV) to infect mice. Two mice each were either mock infected or inoculated with one of three BVDV strains by the intraperitoneal (IP) (n = 8) or intranasal (IN) (n = 8) route. All mice were euthanized at day 7 postinfection (p.i.).
(1) Background. A definition of healthcare-associated infections is essential also for the attribution of the restorative burden to healthcare facilities in case of harm and for clinical risk management strategies. Regarding M. chimaera infections, there remains several issues on the ecosystem and pathogenesis. We aim to review the scientific evidence on M. chimaera beyond cardiac surgery, and ...
Staphylococcus aureus infections of mice. When designed to recapitulate human disease, animal studies with infectious agents aim to provide experimental proof for the molecular basis of pathogenesis, the establishment of protective immunity and the molecular mechanisms whereby immunity is achieved (33-35).Over the past forty years, infectious diseases research championed the mouse as a model ...
Detection of CD4 + T cells activation via FCM and antibody levels through ELISA. (A) Immunization and infection processes in mice and the implementation of separate assays.Flow chart (B) and analysis (C) of the expression of IFN-γ + CD4 + T cells and IL-4 + CD4 + T cells in spleen.(D) The levels of sIgA in ILF and IgG in the serum of mice after the second immunization.
The global spread of monkeypox virus has raised concerns over the establishment of novel enzootic reservoirs in expanded geographic regions. We demonstrate that although deer mice are permissive to experimental infection with clade I and II monkeypox viruses, the infection is short-lived and has limited capability for active transmission.
The most common cause of ARDS is pulmonary infection, ... T. & Braun, A. Noninvasive measurement of pulmonary function in experimental mouse models of airway disease. Lung 199, 255-261.
Assistant professor of pediatrics - infectious disease Dr. Katherine King and postdoctoral fellow Dr. Katie Matatall discuss their work revealing that long-l...
Low-dose EBV infection of humanized mice results in asymptomatic, persistent infection. ... Experimental infection of NOD/SCID mice reconstituted with human CD34+ cells with Epstein-Barr virus.
The present study was undertaken to compare the parasitemia of B. microti infection in BALB/c and F1 (B10 x CBA) mice by two different methods: intraperitoneal injection of parasites or infection by the oral route. In both groups, experimental mice were inoculated with 5 x 10 (7) infected erythrocytes in 100 microliters of blood.
Methodology in experimental infections of mice with Hymenolepis nana. "natural" infection technique based on the uptake of eggs with feed is described. The ratio of the number of parasites recovered 14 days post infection to the number of eggs given (q ratio) is close to 1 if few eggs are given (= 5 per mouse).
The central and necessary role of IL-10 in protecting against severe inflammatory pathology has been clearly shown in many models of experimental infection with intracellular pathogens using Il-10 ...
Virulence assessment of six major pathogenic Candida ...
Experimental infection of mice with the microfilariae of Onchocerca lienalis Parasitology. 1982 Oct:85 (Pt 2):283-93. ... but marked differences were demonstrated between male CBA mice of different ages. After infection with 5 000 microfilariae the recovery of parasites from the ears increased rapidly to a peak at day 35 when 10% of the ...
For transduction experiments, mice were randomly grouped (n = 3 to 9 per group as indicated) and injected with baculoviruses through tail vein with a dose of 4.4 × 10 4 IU per gram body weight ...