Role of Intraspecies Recombination in the Spread of Pathogenicity Islands within the Escherichia coli Species

Horizontal gene transfer is a key step in the evolution of bacterial pathogens. Besides phages and plasmids, pathogenicity islands (PAIs) are subjected to horizontal transfer. The transfer mechanisms of PAIs within a certain bacterial species or between different species are still not well understood. This study is focused on the High-Pathogenicity Island (HPI), which is a PAI widely spread among extraintestinal pathogenic Escherichia coli and serves as a model for horizontal transfer of PAIs in general. We applied a phylogenetic approach using multilocus sequence typing on HPI-positive and -negative natural E. coli isolates representative of the species diversity to infer the mechanism of horizontal HPI transfer within the E. coli species. In each strain, the partial nucleotide sequences of 6 HPI–encoded genes and 6 housekeeping genes of the genomic backbone, as well as DNA fragments immediately upstream and downstream of the HPI were compared. This revealed that the HPI is not solely vertically transmitted, but that recombination of large DNA fragments beyond the HPI plays a major role in the spread of the HPI within E. coli species. In support of the results of the phylogenetic analyses, we experimentally demonstrated that HPI can be transferred between different E. coli strains by F-plasmid mediated mobilization. Sequencing of the chromosomal DNA regions immediately upstream and downstream of the HPI in the recipient strain indicated that the HPI was transferred and integrated together with HPI–flanking DNA regions of the donor strain. The results of this study demonstrate for the first time that conjugative transfer and homologous DNA recombination play a major role in horizontal transfer of a pathogenicity island within the species E. coli

Helicobacter pylori Impairs Murine Dendritic Cell Responses to Infection

International audienceBACKGROUND: Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs) within autophagosomes. METHODOLOGY/PRINCIPAL FINDINGS: A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection. CONCLUSIONS/SIGNIFICANCE: H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host

Polyamines Are Required for Virulence in Salmonella enterica Serovar Typhimurium

Sensing and responding to environmental cues is a fundamental characteristic of bacterial physiology and virulence. Here we identify polyamines as novel environmental signals essential for virulence of Salmonella enterica serovar Typhimurium, a major intracellular pathogen and a model organism for studying typhoid fever. Central to its virulence are two major virulence loci Salmonella Pathogenicity Island 1 and 2 (SPI1 and SPI2). SPI1 promotes invasion of epithelial cells, whereas SPI2 enables S. Typhimurium to survive and proliferate within specialized compartments inside host cells. In this study, we show that an S. Typhimurium polyamine mutant is defective for invasion, intracellular survival, killing of the nematode Caenorhabditis elegans and systemic infection of the mouse model of typhoid fever. Virulence of the mutant could be restored by genetic complementation, and invasion and intracellular survival could, as well, be complemented by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection. Interestingly, intracellular survival of the polyamine mutant was significantly enhanced above the wild type level by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection, indicating that these polyamines function as an environmental signal that primes S. Typhimurium for intracellular survival. Accordingly, experiments addressed at elucidating the roles of these polyamines in infection revealed that expression of genes from both of the major virulence loci SPI1 and SPI2 responded to exogenous polyamines and was reduced in the polyamine mutant. Together our data demonstrate that putrescine and spermidine play a critical role in controlling virulence in S. Typhimurium most likely through stimulation of expression of essential virulence loci. Moreover, our data implicate these polyamines as key signals in S. Typhimurium virulence

Conjugative Plasmids of Neisseria gonorrhoeae

Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker

Intraspecies Variation in the Emergence of Hyperinfectious Bacterial Strains in Nature

Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection. However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in virulence during the infective process- a prospect that has significant implications for human and animal health. Our findings indicate that the capacity to become hypervirulent (100-fold decreased LD50) was much more evident in certain S. enterica strains than others. Hyperinfectious salmonellae were among the most virulent of this species; restricted to certain serotypes; and more capable of killing vaccinated animals. Such strains exhibited rapid (and rapidly reversible) switching to a less-virulent state accompanied by more competitive growth ex vivo that may contribute to maintenance in nature. The hypervirulent phenotype was associated with increased microbial pathogenicity (colonization; cytotoxin production; cytocidal activity), coupled with an altered innate immune cytokine response within infected cells (IFN-β; IL-1β; IL-6; IL-10). Gene expression analysis revealed that hyperinfectious strains display altered transcription of genes within the PhoP/PhoQ, PhoR/PhoB and ArgR regulons, conferring changes in the expression of classical virulence functions (e.g., SPI-1; SPI-2 effectors) and those involved in cellular physiology/metabolism (nutrient/acid stress). As hyperinfectious strains pose a potential risk to human and animal health, efforts toward mitigation of these potential food-borne contaminants may avert negative public health impacts and industry-associated losses