Loss of very-long O-antigen chains optimizes capsule-mediated immune evasion by Salmonella enterica serovar Typhi.

UnlabelledExpression of capsular polysaccharides is a variable trait often associated with more-virulent forms of a bacterial species. For example, typhoid fever is caused by the capsulated Salmonella enterica serovar Typhi, while nontyphoidal Salmonella serovars associated with gastroenteritis are noncapsulated. Here we show that optimization of the immune evasive properties conferred by the virulence-associated (Vi) capsular polysaccharide involved an additional alteration to the cell envelope of S. Typhi, namely inactivation of the fepE gene, encoding the regulator of very-long O-antigen chains. Introduction of the capsule-encoding viaB locus into the nontyphoidal S. enterica serovar Typhimurium reduced complement deposition in vitro and intestinal inflammation in a mouse colitis model. However, both phenotypes were markedly enhanced when the viaB locus was introduced into an S. Typhimurium fepE mutant, which lacks very-long O-antigen chains. Collectively, these data suggest that during the evolution of the S. Typhi lineage, loss of very-long O-antigen chains by pseudogene formation was an adaptation to maximize the anti-inflammatory properties of the Vi capsular polysaccharide.ImportanceGenomic comparison illustrates that acquisition of virulence factors by horizontal gene transfer is an important contributor to the evolution of enteric pathogens. Acquisition of complex virulence traits commonly involves horizontal transfer of a large gene cluster, and integration of the gene cluster into the host genome results in the formation of a pathogenicity island. Acquisition of the virulence-associated (Vi) capsular polysaccharide encoded by SPI7 (Salmonella pathogenicity island 7) was accompanied in the human-adapted Salmonella enterica serovar Typhi by inactivation of the fepE gene, encoding the regulator of very-long O-antigen chains. We show that the resulting loss of very-long O-antigen chains was an important mechanism for maximizing immune evasion mediated by the Vi capsular polysaccharide. These data suggest that successful incorporation of a capsular polysaccharide requires changes in the cell envelope of the hosting pathogen

Egy virulencia regulátor mutáns Salmonella enterica törzs mint kereszt-immunitást biztosító orális vakcina jelölt vizsgálata = Investigation of a virulence regulator mutant strain of Salmonella enterica as a candidate for live oral vaccine providing cross-immunity

Kutatásaink során egy Salmonella rfaH mutánst mint potenciális vakcina jelöltet vizsgáltunk több aspektusból. Mivel az RfaH fehérje egy transzkripciós regulátor, vizsgáltuk, hogy mely virulencia faktorokra kifejtett hatása okozza a vakcina törzs attenuációját. Microarray felhasználásával kimutattuk, hogy direkt módon az LPS-szintézise és a 4-es Salmonella pathogenitási szigeten (SPI-4) található sii operon áll az RfaH szabályozása alatt. Igazoltuk, hogy a SPI-4 deléciója csak mérsékelten csökkenti a Salmonella virulenciát, tehát az RfaH hiányában bekövetkező LPS-amputáció (mély R fenotípus) tehető felelőssé a nagymértékben csökkent virulenciáért. Izogén strukturális LPS mutánsokkal szemben az rfaH mutáns optimális mértékű attenuációját tudtuk kimutatni; amellett, hogy a mutáció biztonságossá tette az élő vakcina törzs alkalmazását, megmaradt a protektív immunválasz kiváltásához nélkülözhetetlen immunogenitása. Igazoltuk továbbá, hogy a bélbaktériumok felszínén található konzervált antigének immunogenitása fokozódik az LPS szintézisének csökkenése mellett. Immunológiai módszerekkel kipreparáltuk a kereszt-reaktivitásért felelős antigéneket és ezeket tömegspektroszkópiás módszerekkel azonosítottuk. Állatkísérletekkel igazoltuk, hogy az rfaH mutánsok valóban képesek kereszt-protekciót kiváltani különböző Salmonella szerotípusok között. | Vaccine potential of a Salmonella rfaH mutant as vaccine candidate was investigated from several aspects. As RfaH is a transcriptional regulator, we analysed transcriptome of the mutant (compared to the isogenic wild-type strain) on microarrays. We showed direct influence of RfaH on LPS-synthesis as well as the sii operon carried on Salmonella pathogenicity island-4 (SPI-4). By showing that a SPI-4 mutant is slightly attenuated only, we proposed that truncation of LPS (resulting in the so-called deep-rough phenotype) is responsible for the high virulence attenuation of the rfaH mutant. Attenuation and protective capacity of rfaH mutant was compared to isogenic structural LPS mutants. It was shown that - in contrast to any structural mutations - loss of RfaH results in an optimal phenotype, where high attenuation is paralleled by retained immunogenicity of the vaccine candidate. Furthermore, we proved that immunogenicity of conserved outer membrane proteins shared by members of the family Enterobacteriaceae is increased in LPS mutants (including rfaH). These cross-reactive antigens were purified by immunological techniques and identified using mass spectrometry. Animal experiments proved that rfaH mutants, indeed, generate serotype-independent immunity to Salmonella pathogens

ELMO1 has an essential role in the internalization of Salmonella Typhimurium into enteric macrophages that impacts disease outcome.

Backgrounds and aims4-6 million people die of enteric infections each year. After invading intestinal epithelial cells, enteric bacteria encounter phagocytes. However, little is known about how phagocytes internalize the bacteria to generate host responses. Previously, we have shown that BAI1 (Brain Angiogenesis Inhibitor 1) binds and internalizes Gram-negative bacteria through an ELMO1 (Engulfment and cell Motility protein 1)/Rac1-dependent mechanism. Here we delineate the role of ELMO1 in host inflammatory responses following enteric infection.MethodsELMO1-depleted murine macrophage cell lines, intestinal macrophages and ELMO1 deficient mice (total or myeloid-cell specific) was infected with Salmonella enterica serovar Typhimurium. The bacterial load, inflammatory cytokines and histopathology was evaluated in the ileum, cecum and spleen. The ELMO1 dependent host cytokines were detected by a cytokine array. ELMO1 mediated Rac1 activity was measured by pulldown assay.ResultsThe cytokine array showed reduced release of pro-inflammatory cytokines, including TNF-α and MCP-1, by ELMO1-depleted macrophages. Inhibition of ELMO1 expression in macrophages decreased Rac1 activation (~6 fold) and reduced internalization of Salmonella. ELMO1-dependent internalization was indispensable for TNF-α and MCP-1. Simultaneous inhibition of ELMO1 and Rac function virtually abrogated TNF-α responses to infection. Further, activation of NF-κB, ERK1/2 and p38 MAP kinases were impaired in ELMO1-depleted cells. Strikingly, bacterial internalization by intestinal macrophages was completely dependent on ELMO1. Salmonella infection of ELMO1-deficient mice resulted in a 90% reduction in bacterial burden and attenuated inflammatory responses in the ileum, spleen and cecum.ConclusionThese findings suggest a novel role for ELMO1 in facilitating intracellular bacterial sensing and the induction of inflammatory responses following infection with Salmonella

Host-pathogen interactions in typhoid fever: the model is the message

Spanish Ministerio de Economía y Competitividad (MINECO) and the European Regional Fund PCIN-2015-131 (Infect-ERA) and BIO2016-75235-

Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium

BACKGROUND: The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions. RESULTS: We performed a systematic analysis of the regulation of type III effectors during growth in vitro. We have tested the ability of seven regulatory genes to regulate ten effector genes. Each regulator was expressed in the absence of the other six to avoid cascade effects. Our results confirm and extend the previously reported regulation of TTSS1 and TTSS2 effectors by InvF-SicA and SsrB respectively. CONCLUSION: The set of strains constructed for this study can be used to quickly and systematically study the regulation of newly identified effector genes of Salmonella enterica. The approach we have used can also be applied to study complex regulatory cascades in other bacterial species

Very long O-antigen chains enhance fitness during Salmonella-induced colitis by increasing bile resistance.

Intestinal inflammation changes the luminal habitat for microbes through mechanisms that have not been fully resolved. We noticed that the FepE regulator of very long O-antigen chain assembly in the enteric pathogen Salmonella enterica serotype Typhimurium (S. Typhimurium) conferred a luminal fitness advantage in the mouse colitis model. However, a fepE mutant was not defective for survival in tissue, resistance to complement or resistance to polymyxin B. We performed metabolite profiling to identify changes in the luminal habitat that accompany S. Typhimurium-induced colitis. This analysis suggested that S. Typhimurium-induced colitis increased the luminal concentrations of total bile acids. A mutation in fepE significantly reduced the minimal inhibitory concentration (MIC) of S. Typhimurium for bile acids in vitro. Oral administration of the bile acid sequestrant cholestyramine resin lowered the concentrations of total bile acids in colon contents during S. Typhimurium infection and significantly reduced the luminal fitness advantage conferred by the fepE gene in the mouse colitis model. Collectively, these data suggested that very long O-antigen chains function in bile acid resistance of S. Typhimurium, a property conferring a fitness advantage during luminal growth in the inflamed intestine

Differences in Salmonella enterica serovar Typhimurium strain invasiveness are associated with heterogeneity in SPI-1 gene expression

Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE+ and SopE− S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH–gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA–gfp reporter mirrored that of PprgH–gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection