The virulence of Salmonella enterica critically depends on the functions of two type III secretion systems (T3SS), with the Salmonella pathogenicity island 1 (SPI1)-encoded T3SS required for host cell invasion and the SPI2-T3SS enabling Salmonella to proliferate within host cells. A further T3SS is required for the assembly of the flagella. Most serovars of Salmonella also possess a lipopolysaccharide with a complex O-antigen (OAg) structure. The number of OAg units attached to the core polysaccharide varies between 16 and more than 100 repeats, with a trimodal distribution. This work investigated the correlation of the OAg length with the functions of the SPI1-T3SS and the SPI2-T3SS. We observed that the number of repeats of OAg units had no effect on bacterial motility. The interaction of Salmonella with epithelial cells was altered if the OAg structure was changed by mutations in regulators of OAg. Strains defective in synthesis of very long or long and very long OAg species showed increased translocation of a SPI1-T3SS effector protein and increased invasion. Invasion of a strain entirely lacking OAg was increased, but this mutant strain also showed increased adhesion. In contrast, translocation of a SPI2-T3SS effector protein and intracellular replication were not affected by modification of the OAg length. Mutant strains lacking the entire OAg or long and very long OAg were highly susceptible to complement killing. These observations indicate that the architecture of the outer membrane of Salmonella is balanced to permit sufficient T3SS function but also to confer optimal protection against antimicrobial defense mechanisms.Salmonella enterica is a remarkable pathogen with strategies for adaptation to different lifestyles in the environment as well as within various host organisms. The requirements for life within the host can vary dramatically, for example, after transition from extracellular life within the intestine to an intracellular life within a special organelle formed inside infected host cells (reviewed in reference 23). The presence of an outer membrane is an important structural feature that enables commensal as well as pathogenic bacteria to adapt to the intestine and to resist bile salts and various molecules of the host innate immune system (44). Of specific importance is the lipopolysaccharide (LPS), the major constituent of the outer leaflet of the outer membrane. LPS is composed of (i) the lipid A portion, consisting of acyl chains linked to phosphorylated N-acetylglucosamine; (ii) the inner and outer core moieties, consisting of rather conserved sugars; and (iii) a highly variable O antigen (OAg). The LPS of S. enterica serovar Typhimurium and several other serovars has special characteristics, with extreme heterogeneity in the length of the OAg repeats. LPS species with a short OAg (S-OAg) consisting of about 16 repeats of OAg units can be found. In addition, LPS species with long and very long OAg (L-OAg and VL-OAg, respectively) are present, containing about 35 and more than 100 repeat...
Reporter gene fusions are essential tools for the investigation of gene regulation. Such fusions are traditionally generated by transposon mutagenesis and identified by a suitable selection procedure. Alternatively, specific reporter fusions can be generated by cloning of DNA fragments containing promoters or other regulatory elements in reporter plasmids. Here, we describe a novel approach for the rapid generation of reporter gene fusions in single copies at defined positions in bacterial genomes. This technique utilizes the Red recombinase for the homologous recombination of PCR-generated cassettes containing various currently used reporter genes, such as those for -galactosidase, luciferase, and green fluorescent protein. The approach allows the generation of transcriptional or translational reporter fusions in a single step without the requirement for recombinant DNA constructs and is applicable to various enterobacterial species. Generation of reporter fusions by Red recombination is rapid, overcomes the current limitations of transposon mutagenesis or reporter plasmids, and offers new options for the study of bacterial gene regulation.
Recombinant engineering using Red recombinase-based approaches offers efficient and rapid approaches to deletion and modification of genes. Here we describe a novel application of Red recombinant engineering that enables direct manipulation of chromosomal loci by electroporation with short synthetic DNA molecules. We demonstrate the use of this approach for the generation of scarless in-frame deletions in chromosomal genes of Salmonella enterica. Furthermore, we describe rapid site-directed mutagenesis within bacterial chromosomes without any requirement for cloning and mutating genes in vitro or for reintroducing mutant alleles into the chromosome. This approach can be expected to facilitate mutational analysis in S. enterica and in other bacterial species able to support Red-mediated recombination.
The molecular mechanisms of virulence of the gastrointestinal pathogen Salmonella enterica are commonly studied using cell culture models of infection. In this work, we performed a direct comparison of the interaction of S. enterica serovar Typhimurium ( S. Typhimurium) with the non-polarized epithelial cell line HeLa, the polarized cell lines CaCo2, T84 and MDCK, and macrophage-like RAW264.7 cells. The ability of S. Typhimurium wild-type and previously characterized auxotrophic mutant strains to enter host cells, survive and proliferate within mammalian cells and deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) was quantified. We found that the entry of S. Typhimurium into polarized cells was much more efficient than entry into non-polarized cells or phagocytic uptake. While SPI2-T3SS dependent intracellular proliferation was observed in HeLa and RAW cells, the intracellular replication in polarized cells was highly restricted and not affected by defective SPI2-T3SS. The contribution of aromatic amino acid metabolism and purine biosynthesis to intracellular proliferation was distinct in the various cell lines investigated. These observations indicate that the virulence phenotypes of S. Typhimurium are significantly affected by the cell culture model applied.
BackgroundType III secretion systems (T3SS) are essential virulence factors of most Gram-negative bacterial pathogens. T3SS deliver effector proteins directly into the cytoplasm of eukaryotic target cells and for this function, the insertion of a subset of T3SS proteins into the target cell membrane is important. These proteins form hetero-oligomeric pores acting as translocon for the delivery of effector proteins. Salmonella enterica is a facultative intracellular pathogen that uses the Salmonella Pathogenicity Island 2 (SPI2)-encoded T3SS to manipulate host cells in order to survive and proliferate within the Salmonella-containing vacuole of host cells. Previous work showed that SPI2-encoded SseB, SseC and SseD act to form the translocon of the SPI2-T3SS.ResultsHere we investigated the structural requirements of SseB and SseD to form a functional translocon. Based on bioinformatic predictions, deletional analyses of SseB and SseD were performed and the effect on secretion by the T3SS, formation of a translocon, translocation of effector proteins and intracellular replication was investigated. Our data showed that both SseB and SseD are very sensitive towards alterations of the primary structure of the proteins. Although proteins encoded by mutant alleles were still secreted, we observed that all mutations resulted in a loss of function of the SPI2-T3SS.ConclusionThese observations indicate that translocon proteins of the SPI2-T3SS are highly evolved towards the formation of multi-subunit complex in the host cell membrane. Structural alterations are not tolerated and abrogate translocon function.
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