Rapid generation of Shigella flexneri GMMA displaying natural or new and cross-reactive O-Antigens.
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ABSTRACT:
SUBMITTER: Gasperini G
PROVIDER: S-EPMC9243986 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC4820968 | biostudies-literature
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.
2016-01-12 | PXD002517 | Pride
Project description:Shigella infections are one of the top causes of diarrhea throughout the world, with Shigella flexneri being predominant in developing countries. Currently, no vaccines are widely available and increasing levels of multidrug-resistance make Shigella a high priority for vaccine development. The serotype-specific O-antigen moiety of Shigella lipopolysaccharide has been recognized as a key target for protective immunity, and many O-antigen based candidate vaccines are in development. Recently, the Generalized Modules for Membrane Antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Here, these two technologies are compared for a vaccine against S. flexneri serotype 6. Genetic strategies for GMMA production, conjugation approaches for linkage of the O-antigen to CRM197 carrier protein, and a large panel of analytical methods for full vaccine characterization have been put in place. In a head-to-head immunogenicity study in mice, GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel. When formulated on Alhydrogel, GMMA and glycoconjugate elicited similar levels of persistent anti-O-antigen IgG with bactericidal activity. Glycoconjugates are a well-established bacterial vaccine approach, but can be costly, particularly when multicomponent preparations are required. With similar immunogenicity and a simpler manufacturing process, GMMA are a promising strategy for the development of a vaccine against Shigella.
| S-EPMC7349896 | biostudies-literature
Project description:BACKGROUND: Shigella bacteria cause dysentery, which remains a significant threat to public health. Shigella flexneri is the most common species in both developing and developed countries. Five Shigella genomes have been sequenced, revealing dynamic and diverse features. To investigate the intra-species diversity of S. flexneri genomes further, we have sequenced the complete genome of S. flexneri 5b strain 8401 (abbreviated Sf8401) and compared it with S. flexneri 2a (Sf301). RESULTS: The Sf8401 chromosome is 4.5-Mb in size, a little smaller than that of Sf301, mainly because the former lacks the SHI-1 pathogenicity island (PAI). Compared with Sf301, there are 6 inversions and one translocation in Sf8401, which are probably mediated by insertion sequences (IS). There are clear differences in the known PAIs between these two genomes. The bacteriophage SfV segment remaining in SHI-O of Sf8401 is clearly larger than the remnants of bacteriophage SfII in Sf301. SHI-1 is absent from Sf8401 but a specific related protein is found next to the pheV locus. SHI-2 is involved in one intra-replichore inversion near the origin of replication, which may change the expression of iut/iuc genes. Moreover, genes related to the glycine-betaine biosynthesis pathway are present only in Sf8401 among the known Shigella genomes. CONCLUSION: Our data show that the two S. flexneri genomes are very similar, which suggests a high level of structural and functional conservation between the two serotypes. The differences reflect different selection pressures during evolution. The ancestor of S. flexneri probably acquired SHI-1 and SHI-2 before SHI-O was integrated and the serotypes diverged. SHI-1 was subsequently deleted from the S. flexneri 5b genome by recombination, but stabilized in the S. flexneri 2a genome. These events may have contributed to the differences in pathogenicity and epidemicity between the two serotypes of S. flexneri.
| S-EPMC1550401 | biostudies-literature
Project description:BackgroundThe spectrin cytoskeleton is emerging as an important host cell target of enteric bacterial pathogens. Recent studies have identified a crucial role for spectrin and its associated proteins during key pathogenic processes of Listeria monocytogenes and Salmonella Typhimurium infections. Here we investigate the involvement of spectrin cytoskeletal components during the pathogenesis of the invasive pathogen Shigella flexneri.ResultsImmunofluorescent microscopy reveals that protein 4.1 (p4.1), but not adducin or spectrin, is robustly recruited to sites of S. flexneri membrane ruffling during epithelial cell invasion. Through siRNA-mediated knockdowns, we identify an important role for spectrin and the associated proteins adducin and p4.1 during S. flexneri invasion. Following internalization, all three proteins are recruited to the internalized bacteria, however upon generation of actin-rich comet tails, we observed spectrin recruitment to those structures in the absence of adducin or p4.1.ConclusionThese findings highlight the importance of the spectrin cytoskeletal network during S. flexneri pathogenesis and further demonstrate that pathogenic events that were once thought to exclusively recruit the actin cytoskeletal system require additional cytoskeletal networks.
| S-EPMC3384245 | biostudies-literature
Project description:Recently, generalized modules for membrane antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Saccharide length is a well-known parameter that can impact the immune response induced by glycoconjugates both in terms of magnitude and quality. However, the criticality of O-antigen length on the immune response induced by GMMA-based vaccines has not been fully elucidated. Here, Shigella and Salmonella GMMA-producing strains were further mutated in order to display homogeneous polysaccharide populations of different sizes on a GMMA surface. Resulting GMMA were compared in mice immunization studies. Athymic nude mice were also used to investigate the involvement of T-cells in the immune response elicited. In contrast with what has been reported for traditional glycoconjugate vaccines and independent of the pathogen and the sugar structural characteristics, O-antigen length did not result in being a critical parameter for GMMA immunogenicity. This work supports the identification of critical quality attributes to optimize GMMA vaccine design and improve vaccine efficacy and gives insights on the nature of the immune response induced by GMMA.
| S-EPMC7865430 | biostudies-literature
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.
| S-EPMC7806729 | biostudies-literature
Project description:Shigellosis remains a major public health problem around the world; it is one of the leading causes of diarrhoeal disease in low- and middle-income countries, particularly in young children. The increasing reports of Shigella cases associated with anti-microbial resistance are an additional element of concern. Currently, there are no licensed vaccines widely available against Shigella, but several vaccine candidates are in development. It has been demonstrated that the incidence of disease decreases following a prior Shigella infection and that serum and mucosal antibody responses are predominantly directed against the serotype-specific Shigella O-antigen portion of lipopolysaccharide membrane molecules. Many Shigella vaccine candidates are indeed O-antigen-based. Here we present the journey towards the development of a potential low-cost four-component Shigella vaccine, eliciting broad protection against the most prevalent Shigella serotypes, that makes use of the GMMA (Generalized Modules for Membrane Antigens) technology, a novel platform based on bacterial outer membranes for delivery of the O-antigen to the immune system.
| S-EPMC8880054 | biostudies-literature
Project description:An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.
| S-EPMC205806 | biostudies-other
Project description:1. Smooth to rough mutation has the same biochemical basis in Shigella as in Salmonella. It is the result of enzyme defects blocking the incorporation of the O-specific side chains that characterize the smooth lipopolysaccharide with the consequent exposure of the underlying basal structures that determine ;rough'-specificity. 2. The Shigella flexneri basal structure resembles its Salmonella analogue in that it has the same qualitative sugar composition, and enzyme defects in its biosynthetic pathway give rise to ;rough'-lipopolysaccharides that are indistinguishable from those of Salmonella chemotypes Ra, Rb, Rc and Rd. However, the Salmonella and Shigella basal structures are not identical as judged by quantitative analysis and the absence of serological cross-reaction. 3. The Sh. flexneri basal structure side chain has been isolated and characterized as an alpha-N-acetylglucosaminyl-(1-->4)-galactosyl-(1-->3)-glucose sequence with alpha-glucosyl radicals substituted on the 3- and 4-positions of the galactose and glucose respectively. The different sugar types in this side chain are incorporated into the growing molecule in the same order as in Salmonella, which explains why the enzyme defects associated with smooth to rough mutation produce the same series of R-chemotypes from both genera. The terminal alpha-glucosyl and alpha-N-acetylglucosaminyl-(1-->4)-galactosyl residues of the Sh. flexneri basal structure are sufficiently different from the terminal alpha-galactosyl and alpha-N-acetylglucosaminylglucosyl residues of the Salmonella analogue that they offer an explanation for the absence of serological cross-reaction between these two basal structures.
| S-EPMC1198276 | biostudies-other