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 ~ Introduction
 ~  Materials and Me...
 ~ Results
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  Table of Contents  
Year : 2012  |  Volume : 30  |  Issue : 4  |  Page : 423-430

Salmonella enterica serovar Typhi plasmid pR ST98 -mediated inhibition of autophagy promotes bacterial survival in infected fibroblasts

1 Medical College of Soochow University, Suzhou; Anhui Key Laboratory for Infection and Immunity, Bengbu, China
2 Medical College of Soochow University, Suzhou, China

Date of Submission19-Mar-2012
Date of Acceptance01-May-2012
Date of Web Publication24-Nov-2012

Correspondence Address:
R Huang
Medical College of Soochow University, Suzhou
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.103763

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 ~ Abstract 

pR ST98 is a chimeric plasmid isolated from Salmonella enterica serovar typhi (S. typhi) and mediates both drug-resistance and virulence of S. typhi. Autophagy has been recently reported as an important component of the innate immune response against intracellular pathogen. In this study, we investigated the effect of pR ST98 on cellular autophagy, apoptosis and bacterial survival in infected fibroblasts. S. typhi strain ST 8 carrying pR ST98 , Salmonella typhimurium strain SR-11 carrying a 100 Kb virulent plasmid, and avirulent S. typhi strain ST 10 without plasmid were tested in this experiment. Results showed that embryonic fibroblasts infected with ST 8 containing pR ST98 had decreased autophagy accompanied by increased bacterial survival and apoptosis. Further study showed that autophagy inducer rapamycin reversed pR ST98 -mediated inhibition of autophagy and reduced apoptosis in infected fibroblasts. Our data indicate that pR ST98 can inhibit autophagy, thus facilitating S. typhi survival and promoting apoptosis of host cells. This study contributes to understanding the underlying mechanism of pR ST98 -mediated virulence in S. typhi.

Keywords: Salmonella enterica serovar typhi, autophagy, apoptosis, plasmid

How to cite this article:
Lv J, Wu S, Wei L, Li Y, He P, Huang R. Salmonella enterica serovar Typhi plasmid pR ST98 -mediated inhibition of autophagy promotes bacterial survival in infected fibroblasts. Indian J Med Microbiol 2012;30:423-30

How to cite this URL:
Lv J, Wu S, Wei L, Li Y, He P, Huang R. Salmonella enterica serovar Typhi plasmid pR ST98 -mediated inhibition of autophagy promotes bacterial survival in infected fibroblasts. Indian J Med Microbiol [serial online] 2012 [cited 2019 Jun 16];30:423-30. Available from:

 ~ Introduction Top

 Salmonella More Details enterica serovar typhi (S. typhi) is the main cause of typhoid fever, a classic systemic infection and a serious public health problem in developing countries. Recent data have shown that 22 million cases occur every year resulting in deaths predominantly in school-age children and young adults. [1],[2] Research showed that Salmonella plasmid was an important factor in salmonella virulence phenotype. [3] A pandemic outbreak of multidrug-resistant S. typhi spread to 13 provinces and cities of China in the early 1990 s. [4] In a survey of antimicrobial susceptibility, 591 strains of S. typhi were isolated from patients and examined for antimicrobial susceptibility in our lab. Results showed that more than 80% of the isolated strains were multidrug-resistant, caused by a large plasmid of 150 kilo-bases (Kb), classified to incompatibility group C. This plasmid was designated as pR ST98 and known to mediate S. typhi multidrug-resistance to many antibiotics. [4] Patients infected with S. typhi containing pR ST98 exhibited severe clinical symptoms and high mortality rates, suggesting that pR ST98 might encode not only multidrug resistance but also virulence of S. typhi. In our previous study, the genotype research on pR ST98 revealed that it carried a gene with 99.8% homology to Salmonella plasmid virulence (spv) gene, [4] which is a highly conserved region of 8 Kb in the plasmids of all other pathogenic Salmonella spp. except S. typhi and encodes virulent phenotypes. [5] Therefore, we speculated that pR ST98 might be a chimeric plasmid that not only carries multidrug resistance but also increases virulence of host bacteria.

Under normal physiological conditions, autophagy is an important catabolic process involved in maintaining cell homeostasis and remodeling cell growth and differentiation. Recent publications also report that autophagy plays critical roles in response to a variety of intracellular pathogens. [6] For example, some intracellular bacteria such as Burkholderia pseudomallei and Mycobacterium tuberculosis are targeted and effectively killed by autophagy, [7],[8] suggesting that autophagy mediates degradation of invading pathogens and maintains stability of host cells. Beside its protective role, autophagy can also interrupt the fusion of pathogen-containing vacuoles with lysosome when some pathogens, such as Legionella pneumophila and Coxiella burnetti reside within autophagosome-like vacuoles, allowing the pathogens utilize these vesicles as a niche for their growth and survival. [9],[10]

The high morbidity of Salmonella infection and emergence of multidrug-resistant S. typhi strains become a major threat to public health worldwide. Recent research suggests that autophagy is involved in the pathological process of Salmonella typhimurium (S. typhimurium) infection. Intracellular Salmonella can be recognized by autophagy; however, the consequences of autophagic recognition of Salmonella are still unclear. [11] A certain Salmonella protein could cause macrophage cells death by inducing autophagy and accelerate Salmonella infection. [12] S. typhimurium could be recognized by autophagy in vitro infection, and the consequence of autophagic recognition had a restrictive effect on intracellular bacterial replication. [13],[14] Some researchers reported that autophagy inhibitor wortmannin could enhance intracellular bacterial growth of Salmonella. [15] Therefore, autophagy plays an important role in Salmonella infection although the role of autophagy in Salmonella infection remains unclear.

We have reported that plasmid pR ST98 induces rapid apoptosis in infected macrophages and spv homologous genes present on pR ST98 , [4],[16] while the underlying mechanism remains unclear. Autophagy is a type II programmed cell death. As autophagy is considered as an important innate immunity in the resistance to invading pathogens and its intensity is closely related to the development of pathogenic infections, it is intriguing to know whether the chimeric plasmid pR ST98 has an influence on S. typhi infection through autophagy. The aim of this study was to investigate the effect of pR ST98 on cellular autophagy and subsequent infectious outcomes. Our study could help to reveal the underlying mechanism of pR ST98 -meidated virulence in S. typhi and provide experimental as well as theoretical basis for the treatment of S. typhi infection through regulating autophagy pathway.

 ~ Materials and Methods Top

Bacterial strains and culture

The multidrug-resistant S. typhi strain (ST 8 ) harbouring pR ST98 was obtained from the blood of patients during a typhoid fever outbreak in Jiangsu, China. The antibiotic-sensitive naturally plasmid free S. typhi strain (ST 10 ) was used as a negative control. The S. typhimurium strain SR-11 carrying a 100 Kb virulent plasmid was used as a positive control (kindly gifted by Professor Roy Curtiss III, the School of Life Sciences, Arizona State University, USA). Bacteria were grown at 37°C overnight to mid-logarithmic phase in Luria-Bertani (LB) broth. The bacterial cultures were centrifuged at 4,500 rpm for 5 min and suspended in Dulbecco's modified Eagle's medium (DMEM, GIBCO, USA) without antibiotics before adding to cells. The quantity of live bacteria for cells stimulation was determined by viable plate counts.

Cells strain and culture

Embryonic fibroblasts from wide-type mouse (WT-MEFs) and mouse with deficient autophagy-associated gene 5 (Atg5 -/- MEFs) were kindly provided by Life Sciences Institute, University of Science and Technology, China. Cells were maintained in DMEM supplemented with 15% heat-inactivated fetal bovine serum (FBS, GIBCO, USA) without antibiotics at 37°C in a humidified incubator containing 5% CO 2 and 95% oxygen.

Cells infection by bacteria

Cells from exponentially growing cultures were used in all experiments and seeded in 24-well tissue culture plates at 5×10 5 cells per well 16-24 h before use. Concentration of cells for transmission electron microscopy (TEM) and Western blotting was 1×10 7 /ml. Mid-logarithmic phase grown cultures of SR-11, ST 8 , and ST 10 were added to WT-MEFs and Atg5 -/- MEFs respectively at a multiplicity of infection (MOI) of 100:1. In the meanwhile, a group of WT-MEFs treated with autophagy inducer-rapamycin (RAPA, Sigma-Aldrich, St. Louis, MO, 0.2μg/ml DMEM) were set up for infection. Uninfected cells were set up as a blank control. After incubated at 37°C for 2 h (0-h time point), infected cells were washed three times with sterile phosphate-buffered saline (PBS); then DMEM complete containing amikacin (100μg/ml) was added to kill remaining extracellular bacteria. After 2 h of further incubation at 37°C, medium in the plates was replaced with DMEM containing amikacin (10μg/ml) to inhibit the propagation of possible extracellular bacteria in the medium. Cells were collected and detected at 1, 3, 5, 8, 10 h time points after infection. All assays were performed in triplicate and repeated at least three times.

Plasmid DNA extraction

Plasmid extraction was operated according to the method recommended by Takahashi et al.[17] The DNA extracts were analyzed by electrophoresis on 0.7% agarose containing ethidium bromide (1μg/ml).

Visualization of autophagic vacuoles by Fluorescence Microscopy

Cells were incubated with 50 μM monodansycadaverine (MDC, Sigma-Aldrich, St. Louis, MO) in Dulbecco's PBS (DPBS) at 37°C for 15 min at the indicated time points. After incubation, cells were rinsed three times with DPBS and immediately observed under fluorescence microscopy (Olympus, Tokyo, Japan). Excitation filter was 365 nm, beam splitter was 395 nm, and absorption filter was 420 nm.

Preparation of samples for TEM

Cells were collected and centrifuged at 1,500 rpm for 5 min. Cells were then fixed with 2.5% glutaraldehyde in 0.1 M PBS, post-fixed in 1% osmium tetroxide, and dehydrated through a series of graded acetone washes. Samples were embedded in epoxy resin, sectioned, and placed onto 200-mesh copper grids. The grids were stained with uranyl acetate and lead citrate. Finally samples were examined for autophagy using a Hitachi TEM (H-600, Japan).

Protein isolation and western blotting

Cells were lysed in the solution containing 1% Triton X-100, 1% sodium dodecylcholate, 0.1% SDS, 10 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 12,000 rpm for 10 min at 4°C, supernatant was used for analysis. Quantitation of protein was tested by the method of bicinchoninic acid (BCA). Twenty microliters of sample was loaded for each lane on SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane for 1 h in a semi-dry transfer system (Transfer buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). Membrane was dyed with 1×ponceau red dye (2.6 M ponceaus, 0.018 M trichloroacetic acid, 0.014 M sulfosalicyclic acid) for 5 min. After blocking with 5% dry milk in TBS-T (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, and 0.02% Tween-20) for 1 h, the membrane was incubated with 1:1000 polyclonal anti-LC3 antibody (MBL, Japan) at room temperature for 1 h. After 10 min washing in TBS-T and 10 min washing in TBS, the membrane was incubated with HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000, MBL, Japan) at room temperature for 1 h, and then incubated with HRP chemiluminescence reagent (Millipore, USA) for 1 min. Images were captured with the Bio-Rad VersaDoc image system (Bio-Rad, USA). For an internal control, the membrane was stripped and reprobed with monoclonal anti-GAPDH antibody (Sigma-Aldrich, USA).

Assessment of apoptosis by flow cytometry after annexin-V/propidium iodide labelling

An annexin-V (Ann V)/propidium iodide (PI) apoptosis detection kit (Biouniquer Technology CO, LTD, USA) was used to detect the apoptosis of infected cells in accordance with the manufacturer's instructions. Samples were detected by flow cytometry (FACSCalibur, BD Company, USA) within 1 h after staining. Samples were subjected to flow cytometry and results were presented in four divided areas based on Ann V/PI double fluorescent staining: Ann V - /PI - (lower left area, LL area) represents the normal living cells, Ann V + /PI - (lower right area, LR area) represents the early apoptotic cells, Ann V + /PI + (upper right area, UR area) represents the late apoptotic or secondary necrotic cells, Ann V - /PI + (upper left area, UL area) represents the mechanical injury cells. The sum of the early and late apoptotic rate was the total apoptotic rate of infected cells. Data was acquired with Cellquest software and analyzed by Modifit software (Verity Software House Company; USA).

Assessment of bacterial intracellular survival

Cells were lysed with 200μ l 0.1% Triton X-100 for 15 min, collected into sterile EP tube and serially diluted 10 times with PBS. One hundred microliter diluents from the last three dilution of each group were plated onto LB agar at 37°C for 24 h to enumerate bacterial colony-forming unit (CFU).

Statistical analysis

Data were presented as means ± standard deviations (SD). One-way ANOVA was used for analyzing the differences among groups. A P-value less than 0.05 was considered significant.

 ~ Results Top

The profile of plasmid in experimental bacteria

The plasmid from multidrug-resistant strain ST 8 was extracted and analyzed by electrophoresis and result was presented in [Figure 1]. The plasmids of E. Coli V517 and S. flexneri 24570 were loaded as size markers. The strain ST 8 carried a plasmid with a molecular mass of 150 Kb and this plasmid was designated as pR ST98 . Standard virulent strain SR-11 contained a 100-Kb virulent plasmid. The avirulent strain ST 10 did not contain any plasmid.
Figure 1: Plasmid profile of experimental bacteria. E. coli V517, plasmid size marker (54.4, 7.3, 5.6, 4.0, 2.7 and 2.1 Kb); S. flexneri 24570, plasmid size marker (159.6, 4.0 and 3.0 Kb); S. typhi strain ST10, plasmid-free; S. typhimurium strain SR-11 carrying a 100 Kb virulence plasmid; S. typhi strain ST8 carrying a 150-Kb plasmid-pRST98

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Infection of ST 8 containing pR ST98 inhibited autophagic vacuoles visualized by MDC fluorescent staining

It has been established that MDC has autofluorescent properties. MDC accumulates as a selective fluorescent marker for autophagic vacuoles under in vivo conditions by interacting with membrane lipids that are highly concentrated in the autophagic compartments. [18] MDC-positive vesicles respond to autophagy induction when this process is stimulated, both in cultured cells and in animals. In this study, MDC staining was used for identification and visualization of the autophagic process with fluorescence microscopy [Figure 2]. In the cytoplasm of WT-MEFs and RAPA-treated WT-MEFs (RAPA+WT-MEFs) infected by ST 10 at 1 h, we observed many typical autophagic vesicles [Figure 2]a, d. Whereas, only very few sporadic autophagic vesicle were observed in the cytoplasm of WT-MEFs infected by ST 8 and SR-11 at 1 h [Figure 2]b and c, suggesting that autophagy was absent in the presence of virulent strain SR-11 and ST 8 containing pR ST98 . While autophagy-inducer RAPA-treatment induced autophagic vesicles in WT-MEFs even with the infection of ST 8 and SR-11 [Figure 2]e and f. No autophagic vesicles were observed in the autophagy-dificient fibroblasts (Atg5 -/- MEFs) infected by all three bacteria [Figure 2]g-i. This result suggests that similar to virulent SR-11 strain, ST 8 strain containing pR ST98 plasmid suppresses autophagy when it infects the wild-type fibroblasts.
Figure 2: Autophagic vesicles visualized with MDC fluorescent staining. Autophagic vesicles presented in the cytoplasm of WT-MEFs and RAPA-treated WT-MEFs infected by ST10 at early stage (1 h) of infection (a). There were no autophagic vesicles in WT-MEFs infected by ST8 (b) and SR-11(c) at early time points of infection. When WT-MEFs were treated with RAPA, autophagic vesicles occurred in the cells infected by ST10 (d), ST8 (e) and SR-11(f) at early stage. Autophagic vesicles were not observed in Atg5-/-MEFs infected by any bacteria (g, h, and i). Scale bar=25 μm

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Infection of ST 8 containing pR ST98 inhibited autophagic morphology detected by TEM

Further, morphology of autophagy was assessed with TEM. Uninfected cells showed normal appearance of cytoplasm with a few vacuoles and uniform distribution of nuclear chromatin [Figure 3]a. WT-MEFs infected with ST 10 showed typical autophagic vacuole with double or multilayer membrane in the cytoplasm [[Figure 3]b, arrows]. WT-MEFs infected with ST8 showed no autophagic vacuole in cytoplasm, suggesting that infection with ST8 containing pRST98 inhibited autophagy in infected cells [Figure 3]c. Autophagy inducer RAPA-treated WT-MEFs infected by ST 8 , ST 10 and SR-11 at 1 h all displayed autophagic vacuoles [[Figure 3]d-f, arrows].
Figure 3: Morphological changes of autophagy in infected-cells visualized with TEM. The structure of uninfected cells showed intact plasma and nuclear membranes (a). Double-or multilayer-membrane of autophagic vacuole (AV) was observed in WT-MEFs infected by ST10 and ST8 (b, c). RAPA-treated WT-MEFs infected by ST8, ST10 and SR-11 at the early time points of infection (d, e and f). Black arrows show AV

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Autophagic activity is inhibited by infection of strain ST 8 containing plasmid pR ST98

Microtubule-associated protein-LC3 is a major constituent of the autophagosome. During autophagy, the cytoplasmic form (LC3-I) is processed and recruited to the autophagosome, where LC3-II is generated by site specific proteolysis and lipidation near to the C-terminus. The hallmark of autophagic activation is thus the formation of cellular autophagosome punctae containing LC3-II. As the amount of LC3-II correlates well with the number of autophagosomes, in this study, we measured the expression of LC3-II as an indicative of autophagic activity [Figure 4]. Immunoblotting of LC3 showed two bands: LC3-I (18 kDa) and LC3-II (16 kDa). Atg5-/-MEFs showed no LC3-II expression at both tested time-point, indicating no autophagy occurred in autophagy gene-deficient cells. WT-MEFs infected by avirulent ST 10 -free plasmid showed LC3-II expression, which was strengthened by RAPA-treatment. However, WT-MEFs infected by virulent SR-11 and ST 8 showed no LC3-II expression at 3 h and a little expression at 1 h [Figure 4]a and b, suggesting that similar as SR-11 infection, ST 8 infection suppressed autophagy in the wide-type fibroblasts. This infection-mediated suppression was reversed by RAPA-treatment as shown by the LC3-II expression in both RAPA-treated WT-MEFs infected by ST 8 and SR-11.
Figure 4: Expression of LC3-II in infected-cells detected by Western blotting. RAPA-treated MEFs, WT-MEFs and Atg5-/-MEFs infected by ST10, ST8 and SR-11 at 1 and 3 h. LC3-II expressed in WT-MEFs infected by ST10 and RAPA-treated WT-MEFs infected by STsub>10, ST8 as well as SR-11 at 1 h (a) and 3 h (b). The expression of LC3-II in RAPA-treated WT-MEFs infected by ST10 was stronger than in WT-MEFs infected by ST10. LC3-II did not express in pRST98-containing strain ST8-and standard virulent strain SR-11-infected WT-MEFs and Atg5-/-MEFs infected by any bacteria

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Infection of ST 8 containing pR ST98 enhanced apoptosis of host cells

The effect of ST 8 containing pR ST98 on apoptosis of infected cells was further investigated by assessing the surface expression of phosphatidylserine using annecin-V in conjunction with propidium iodide for flow cytometry analysis. Four quadrant Figures made by flow cytometry were shown in [Figure 5] and quantitative data were presented in [Figure 6]. In ST 10 -infected WT-MEFs, only 0.98% of cells were apoptotic (AnnV + /PI - ) at 3 h postinfection [Figure 5]a, and this percentage increased to 6.53% at 10 h postinfection [Figure 5]b. In contrast, virulent strain SR-11 infection caused an increase in the apoptosis rate from 6.62% at 3 h postinfection [Figure 5]c to 37.91% at 10 h postinfection [Figure 5]d, which was significantly higher than ST 10 caused apoptosis rate. WT-MEFs infected by ST 8 containing pR ST98 showed similar apoptotic rate [Figure 5]e and f as caused by virulent SR-11 (7.51% at 3 h and 36.88% at 10 h post-infection), suggesting that ST 8 strain containing pR ST98 enhanced apoptosis of host cells. The apoptotic rates in Atg5 -/- MEFs infected with ST 10 [Figure 5] g and h were lower than in SR-11 [Figure 5] I and j and ST 8 [Figure 5]k and l infected Atg5 -/- MEFs, indicating that the virulence of ST 10 was lower than SR-11 and ST 8 . RAPA-treatment significantly decreased apoptosis in WT-MEFs infected by SR-11 [Figure 5]o and p and ST 8 [Figure 5] q and r, suggesting that RAPA-induced autophagy could reverse the ST8-induced apoptosis. Interestingly, RAPA-treatment increased apoptosis in WT-MEFs infected by ST 10 [Figure 5] m and n compared with the apoptotic rate in RAPA-untreated WT-MEFs infected by ST 10 [Figure 5] a and b.
Figure 5: Assessment of infected cells with flow cytometry after Ann V/PI double staining. Figures made by flow cytometry were divided into four areas: Q1: (Lower Left area, LL area, Ann V-/PI-) represented the normal living cells; Q2: (Lower Right area, LR area, Ann V+/PI-) represented the early apoptotic cells; Q3: (Upper Right area, UR area, Ann V+PI+) represented the late apoptotic or secondary necrotic cells; Q4: (Upper Left area, UL area, Ann V-PI+) represented the mechanical injury cells

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Figure 6: Quantitative result of apoptosis. WT-MEFs infected by SR- 11 and ST8 showed similar increase, while WT-MEFs infected by ST10 showed significant decrease in apoptotic rate over time. The apoptotic rate in RAPA-treated WT-MEFs infected by SR-11 was lower than that in untreated WT-MEFs (#P<0.05). The apoptotic rate in RAPA-treated WTMEFs infected by ST8 was significant lower than that in untreated WTMEFs ( & P<0.05). After ST10 infection, compared to WT-MEFS, RAPAtreated WT-MEFs showed higher apoptotic rate over time (*P<0.05)

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Plasmid pR ST98 promoted intracellular bacterial survival

Bacterial survival in infected cells was seen in [Figure 7]. ST 10 -infected WT-MEFs had low bacterial survival (3.3×10 3 CFU at 1 h and 9.8×10 3 CFU at 10 h), while both SR-11-and ST 8 -infected WT-MEFs exhibited significant high level in bacterial survival (SR-11: 2.1×10 4 CFU at 1 h and 3.6×10 4 CFU at 10 h; ST 8 : 2×10 4 CFU at 1 h and 3.5×10 4 CFU at 10 h). RAPA-treatment significantly decreased bacterial survival in WT-MEFs infected by SR-11 (3.3×10 3 CFU at 1 h and 9.1×10 3 CFU at 10 h) and ST 8 (3.4×10 3 CFU at 1 h and 8.7×10 3 CFU at 10 h), suggesting that RAPA-induced autophagy inhibited bacterial survival in the infected fibroblasts. ST 8 infection suppressed autophagy that promoted bacterial survival. This could be the underlying mechanism of increased apoptosis of infected cells. These results suggest that deficiency of autophagy promotes bacterial survival in infected fibroblasts, while activation of autophagy could decreases bacterial growth.
Figure 7: Bacterial survival in infected cells. In cells infected by ST8, there was no difference between Atg5-/-MEFs and WT-MEFs (P> 0.05), both cells showed high bacterial survival. Bacterial survival in RAPA-treated WT-MEFs was significant lower than in Atg5-/- MEFs and WT-MEFs ( & P<0.05) at each time point of infection. The condition of bacterial survival in cells infected by strain SR-11 was similar to that of cells infected by ST8 (#P<0.05)

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 ~ Discussion Top

In this study, we assessed autophagic morphology using MDC florescent staining and TEM on mouse embryonic fibroblasts infected with SR-11, ST 10 and ST 8 . We further evaluated the level of LC3-II, a marker for autophagy, in those infected-cells. Our results showed that autophagy was inhibited in WT-MEFs infected by pR ST98 -containing strain ST 8 but not by avirulent ST 10 free-containing plasmid , Plasmid pR ST98 -mediated inhibition of autophagy promoted bacterial survival, which might contribute to the apoptosis of infected fibroblasts.

Deficient autophagy induced by pR ST98 may play a critical role in the process of S. typhi infection through promoting bacterial survival and aggravating the damage of host cells. WT-MEFs infected by ST 8 containing pR ST98 were more permissive to intracellular growth of bacteria, which resulted in a higher apoptosis of host cells. Infection-induced apoptosis might be due to the reason that cells infected by pR ST98 -containing bacteria could not effectively control the proliferation of invading pathogen when autophagy was deficient. This point was further supported by the evidence that induction of autophagy with RAPA significantly decreased apoptosis and bacterial survival in cells infected with ST 8 . This may be an underlying mechanism of how pR ST98 increases virulence of S. typhi. Autophagy inducer-RAPA could reverse the inhibition of autophagy mediated by pR ST98 and effectively control S. typhi infection, thus attenuating the damage of host cells.

WT-MEFs infected by avirulent ST 10 -free containing any virulent plasmid exhibited autophagic morphology and lower apoptotic rate compared with ST 10 infected Atg5 -/- MEFs. While RAPA-treatment increased apoptotic rate in WT-MEFS infected by ST 10 without increasing bacterial survival, suggesting that excessive autophagy caused by RAPA-treatment might result in serious damage. Therefore, autophagy might act as a double-edged sword in host cells with S. typhi infection. Moderate activation of autophagy restricts S. typhi propagation and reduces the damage to host cells. Host cells could not effectively control S. typhi propagation when autophagy is deficient. However, excessive autophagy might cause serious damage to host cells. The mechanism of excessive autophagy-caused cellular damage and corresponding signaling pathways need further studies.

pR ST98 is a large chimeric plasmid containing complex sequences of unknown functions. We have reported that spv homologous genes exist on pR ST98 suggesting it might be a virulence plasmid, while little is known about its virulent mechanism in host bacteria. [4] Spv is a highly conserved region existing on the plasmids of all other pathogenic Salmonella spp. except S. typhi and mainly encodes the virulent phenotype of plasmid. Spv consists of one regulatory gene-spvR and four structural genes including spvA, B, C, D, while spvB and spvC is the primary virulent gene segment. SpvB protein can destroy actin filaments and induce apoptosis in eukaryotic cells. Animal experiment has also revealed that deletion of spvB reduces the virulence of Salmonella infection in mice. [19],[20] Our previous study has shown that 99.8% homologous genes with spvR and spvB existed on pR ST98 , [4] and our above data suggest that pR ST98 can increase virulence of S. typhi through inhibition of autophagy, therefore, our further investigation will be carried out to manifest whether autophagy inhibition induced by pR ST98is directly correlated with spvB or a combination of others genes.

In summary, our results suggest that pR ST98 promotes bacterial survival through inhibiting autophagy and consequently enhances apoptosis of infected fibroblasts, a mechanism underlying pR ST98 -mediated virulence in S. typhi. This study also indicates that autophagy plays an important role in Salmonella infection. Moderate activation of autophagy has a beneficial effect on restricting Salmonella proliferation and reducing the injury of host cells, while its deficiency or inhibition might worse infection and result in the serious damage of host cells. This study provides an evidence to understand the mechanism of pR ST98 -mediated virulence and suggests that regulating autophagy pathway could be a potential treatment of Salmonella infection.

 ~ References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]


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2004 - Indian Journal of Medical Microbiology
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