Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.
ABSTRACT: Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.
Project description:Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries. Overall design: Profiling of host and pathogen transcriptome for zebrafish larvae infected with Shigella sonnei at 24 hours post infection and comparison to transcriptome of control injected larvae and control bacteria grown in liquid culture
Project description:GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.
Project description:Background:Shigella spp. are Gram-negative intracellular pathogenic bacteria belonging to the family Enterobacteriaceae and can cause bacterial dysentery, a severe diarrheal disease. The pathophysiological impact of the Gram-negative bacteria is highly related to the composition and structural variability of lipopolysaccharides, the major lipoid components of the outer membrane. Out of the 114 genes involved in the lipopolysaccharide biosynthesis pathway, 47 genes are specific to Shigella spp. Changes in the specific genes can lead to loss of the O polysaccharide side chain, resulting in rough (R) type bacteria with increased sensitivity to temperature, or hydrophobic antibiotics. The formation of various different lipopolysaccharides or lipooligosaccharides has been observed previously in a mutant line showing altered biological properties, but the genetic background has not been investigated in detail. Results:The parental strain of the mutant line, Shigella sonnei 4303, was subjected to whole genome sequencing to gain a better insight into the structure and biosynthesis of lipopolysaccharides. The sequencing revealed a 4,546,505 bp long genome including chromosomal and plasmid DNA, and the lipopolysaccharide biosynthesis genes were also identified. A comparison of the genome was performed with the phylogenetically closely related, wild type, well characterized, highly virulent strain, S. sonnei 53G. Conclusion:Analysis of the lipopolysaccharide biosynthetic genes helped us to get more insight into the pathogenicity and virulence of the bacteria. The genome revealed high similarities with S. sonnei 53G, which can be used as a standard in characterizing the S. sonnei 4303's R-type isogenic derivatives.
Project description:Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.
Project description:Shigella sonnei UCN59, isolated during an outbreak of S. sonnei in January 2007, was resistant to azithromycin (MIC 64 mg/L). The isolate contained a plasmid-borne mph(A) gene encoding a macrolide 2 -phosphotransferase that inactivates macrolides. Emergence of the mph(A) gene in S. sonnei may limit usefulness of azithromycin for treatment of shigellosis.
Project description:Two Shigella species, Shigella flexneri and Shigella sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle- and high-income countries. S. flexneri is a prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species.IMPORTANCE Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.
Project description:Shigella sonnei is the emerging pathogen globally, as it is the second common infectious species of shigellosis (bloody diarrhoea) in low- and middle-income countries (LMICs) and the leading one in developed world. The multifactorial processes and novel mechanisms have been identified in S. sonnei, that are collectively playing apart a substantial role in increasing its prevalence, while replacing the S. flexneri and other Gram-negative gut pathogens niche occupancy. Recently, studies suggest that due to improvement in sanitation S. sonnei has reduced cross-immunization from Plesiomonas shigelliodes (having same O-antigen as S. sonnei) and also found to outcompete the two major species of Enterobacteriaceae family (Shigella flexneri and Escherichia coli), due to encoding of type VI secretion system (T6SS). This review aimed to highlight S. sonnei as an emerging pathogen in the light of recent research with pondering aspects on its epidemiology, transmission, and pathogenic mechanisms. Additionally, this paper aimed to review S. sonnei disease pattern and related complications, symptoms, and laboratory diagnostic techniques. Furthermore, the available treatment reigns and antibiotic-resistance patterns of S. sonnei are also discussed, as the ciprofloxacin and fluoroquinolone-resistant S. sonnei has already intensified the global spread and burden of antimicrobial resistance. In last, prevention and controlling strategies are briefed to limit and tackle S. sonnei and possible future areas are also explored that needed more research to unravel the hidden mysteries surrounding S. sonnei.
Project description:Shigella flexneri 2a and Shigella sonnei were genetically modified to shed large quantities of outer membrane blebs. The blebs, called Generalized Modules for Membrane Antigens (GMMA), were purified and the protein content was estimated using the label-free iBAQ procedure. There were 2308 proteins identified, 660 in GMMA and 2239 in bacteria, of which 288 (GMMA) and 1695 (bacteria) were common to both S. flexneri 2a and S. sonnei. Protein abundances were classified according to the predicted localization. Predicted outer membrane or periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process.
Project description:OBJECTIVES:Shigella is a human pathogen that causes shigellosis, an acute invasive intestinal infection. Recent studies in the model bacterium Escherichia coli (E. coli) provided evidence that small regulatory RNAs (sRNAs) can contribute to antimicrobial resistance or susceptibility. One of the sRNAs is SdsR, which increases sensitivity of E. coli against fluoroquinolone by repressing the drug efflux pump, TolC. However, no reports exist about the effect of SdsR on fluoroquinolone resistance in Shigella sonnei (S. sonnei). In this study, we established the effect of SdsR on the sensitivity of S. sonnei to norfloxacin. DATA DESCRIPTION:We tested the effects of SdsR and SdsRv2 on fluoroquinolone resistance in S. sonnei in vivo. SdsRv2 is a synthetic version which promotes higher binding stability to tolC mRNA. Overexpression of either SdsR or SdsRv2 lowers the expression of tolC mRNA. Interestingly, SdsR and SdsRv2 promote the growth of S. sonnei in the presence of a sub-inhibitory concentration of norfloxacin. Mutant carrying SdsRv2 showed the highest growth advantage. This phenotype is opposite to the effect of SdsR reported in E. coli. This study is an example that demonstrates the difference in the phenotypic effect of a highly conserved sRNA in two closely related bacteria.
Project description:Shigella sonnei contains numerous IS1 elements. The existence of polymorphisms in the length of the inter-IS1 spacer is a basis for the development of a PCR-based method for the subtyping of S. sonnei strains. The usefulness of inter-IS1 spacer typing (IST) was evaluated and compared with that of pulsed-field gel electrophoresis (PFGE) by characterization of S. sonnei isolates from epidemiologically nonrelated cases and outbreaks and of isolates that were indistinguishable by PFGE and that were collected from independent infection events. IST was less discriminatory than PFGE, with discriminatory indices of 0.96 and 0.63, respectively, but was able to compensate for the drawbacks of PFGE. PFGE exhibited a high level of discriminatory power for S. sonnei isolates; however, PFGE was also, at times, too discriminatory, which was a disadvantage in constructing the clonal relationships among strains circulating over a period of months or years. Furthermore, IST provided greater subtyping information for isolates indistinguishable by PFGE. The present study indicates that IST is more useful than PFGE for investigating the genetic relationships among S. sonnei strains circulating over a longer time span and also for discriminating certain strains which are indistinguishable by PFGE.