'Add, stir and reduce': Yersinia spp. as model bacteria for pathogen evolution

In the study of molecular microbiology and bacterial genetics, pathogenic species of the Yersinia genus have been pillars for research aimed at understanding how bacteria evolve into mammalian pathogens. The advent of large-scale population genomic studies has hugely accelerated progress in this field, and the pathogenic Yersinia species have re-emerged as model organisms to help shape our understanding of the evolutionary processes involved in pathogenesis. In this review, we highlight how microbial genomic studies of the yersiniae have revealed distinct features marking the evolutionary path towards pathogenesis, which are changing our understanding of pathogen evolution. As these features are also found in the genomes of other members of the Enterobacteriaceae, they provide a blueprint for the evolution of enteropathogenic bacteria

Revisiting aminocoumarins for the treatment of melioidosis.

Burkholderia pseudomallei causes melioidosis, a potentially lethal disease that can establish both chronic and acute infections in humans. It is inherently recalcitrant to many antibiotics, there is a paucity of effective treatment options and there is no vaccine. In the present study, the efficacies of selected aminocoumarin compounds, DNA gyrase inhibitors that were discovered in the 1950s but are not in clinical use for the treatment of melioidosis were investigated. Clorobiocin and coumermycin were shown to be particularly effective in treating B. pseudomallei infection in vivo. A novel formulation with dl-tryptophan or l-tyrosine was shown to further enhance aminocoumarin potency in vivo. It was demonstrated that coumermycin has superior pharmacokinetic properties compared with novobiocin, and the coumermycin in l-tyrosine formulation can be used as an effective treatment for acute respiratory melioidosis in a murine model. Repurposing of existing approved antibiotics offers new resources in a challenging era of drug development and antimicrobial resistance

A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface.

To investigate Yersinia pathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organism Caenorhabditis elegans was studied. Three strains of Yersinia pestis, including a strain lacking pMT1, caused blockage and death of C. elegans; one strain, lacking the haemin storage (hms) locus, caused no effect. Similarly, 15 strains of Yersinia enterocolitica caused no effect. Strains of Yersinia pseudotuberculosis showed different levels of pathogenicity. The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9.5 % an intermediate infection, and 9.5 % a severe infection. There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection of C. elegans and those able to form a biofilm on an abiotic surface. Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm. Seven transposon mutants of Y. pseudotuberculosis strain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis. A screen of 15 defined C. elegans mutants identified four where mutation caused (complete) resistance to infection by Y. pseudotuberculosis YPIII pIB1. These mutants, srf-2, srf-3, srf-5 and the dauer pathway gene daf-1, also exhibit altered binding of lectins to the nematode surface. This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms

Prevalence of Type VI Secretion System in Spanish Campylobacter jejuni Isolates.

Infections from Campylobacter jejuni pose a serious public health problem and are now considered the leading cause of foodborne bacterial gastroenteritis throughout the world. Sequencing of C. jejuni genomes has previously allowed a number of loci to be identified, which encode virulence factors that aid survival and pathogenicity. Recently, a Type VI secretion system (T6SS) consisting of 13 conserved genes was described in C. jejuni strains and recognised to promote pathogenicity and adaptation to the environment. In this study, we determined the presence of this T6SS in 63 Spanish C. jejuni isolates from the food chain and urban effluents using whole-genome sequencing. Our findings demonstrated that nine (14%) strains harboured the 13 ORFs found in prototype strain C. jejuni 108. Further studies will be necessary to determine the prevalence and importance of T6SS-positive C. jejuni strains

Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni

© 2015 Jervis et al. Background: Campylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backgrounds. Methods: A series of three vectors that allow integration of genes onto the C. jejuni chromosome were constructed by standard cloning techniques with expression driven from three different strong promoters. Following integration onto the C. jejuni chromosome expression levels were quantified by fluorescence measurements and cells visualized by fluorescence microscopy. Results: We have created plasmid, pCJC1, designed for recombination-mediated delivery of genes onto the C. jejuni chromosome. This plasmid contains a chloramphenicol resistance cassette (cat) with upstream and downstream restriction sites, flanked by regions of the C. jejuni pseudogene Cj0223. Cloning of genes immediately upstream or downstream of the cat gene allows their subsequent introduction onto the C. jejuni chromosome within the pseudogene. Gene expression can be driven from the native gene promoter if included, or alternatively from the cat promoter if the gene is cloned downstream of, and in the same transcriptional orientation as cat. To provide increased and variable expression of genes from the C. jejuni chromosome we modified pCJC1 through incorporation of three relatively strong promoters from the porA, ureI and flaA genes of C. jejuni, Helicobacter pylori and Helicobacter pullorum respectively. These promoters along with their associated ribosome binding sites were cloned upstream of the cat gene on pCJC1 to create plasmids pCJC2, pCJC3 and pCJC4. To test their effectiveness, a green fluorescent protein (gfp) reporter gene was inserted downstream of each of the three promoters and following integration of promoter-gene fusions onto the C. jejuni host chromosome, expression levels were quantified. Expression from the porA promoter produced the highest fluorescence, from flaA intermediate levels and from ureI the lowest. Expression of gfp from the porA promoter enabled visualization by fluorescent microscopy of intracellular C. jejuni cells following invasion of HeLa cells. Conclusions: The plasmids constructed allow stable chromosomal expression of genes in C. jejuni and, depending on the promoter used, different expression levels were obtained making these plasmids useful tools for genetic complementation and high level expression

Genomic variations define divergence of water/wildlife-associated Campylobacter jejuni niche specialists from common clonal complexes

Although the major food-borne pathogen Campylobacter jejuni has been isolated from diverse animal, human and environmental sources, our knowledge of genomic diversity in C. jejuni is based exclusively on human or human food-chain-associated isolates. Studies employing multilocus sequence typing have indicated that some clonal complexes are more commonly associated with particular sources. Using comparative genomic hybridization on a collection of 80 isolates representing diverse sources and clonal complexes, we identified a separate clade comprising a group of water/wildlife isolates of C. jejuni with multilocus sequence types uncharacteristic of human food-chain-associated isolates. By genome sequencing one representative of this diverse group (C. jejuni 1336), and a representative of the bank-vole niche specialist ST-3704 (C. jejuni 414), we identified deletions of genomic regions normally carried by human food-chain-associated C. jejuni. Several of the deleted regions included genes implicated in chicken colonization or in virulence. Novel genomic insertions contributing to the accessory genomes of strains 1336 and 414 were identified. Comparative analysis using PCR assays indicated that novel regions were common but not ubiquitous among the water/wildlife group of isolates, indicating further genomic diversity among this group, whereas all ST-3704 isolates carried the same novel accessory regions. While strain 1336 was able to colonize chicks, strain 414 was not, suggesting that regions specifically absent from the genome of strain 414 may play an important role in this common route of Campylobacter infection of humans. We suggest that the genomic divergence observed constitutes evidence of adaptation leading to niche specialization

The Antimicrobial Peptide, Bactenecin 5, Supports Cell-Mediated but Not Humoral Immunity in the Context of a Mycobacterial Antigen Vaccine Model.

Bactenecin (Bac) 5 is a bovine antimicrobial peptide (AMP) capable of killing some species of bacteria through the inhibition of protein synthesis. Bac5 and other AMPs have also been shown to have chemotactic properties and can induce inflammatory cytokine expression by innate immune cells. Recently, AMPs have begun to be investigated for their potential use as novel vaccine adjuvants. In the current work, we characterise the functionality of Bac5 in vitro using murine macrophage-like cells, ex vivo using human tonsil tissue and in vivo using a murine model of vaccination. We report the effects of the peptide in isolation and in the context of co-presentation with mycobacterial antigen and whole, inert Bacillus subtilis spore antigens. We find that Bac5 can trigger the release of nitric oxide from murine macrophages and upregulate surface marker expression including CD86, MHC-I and MHC-II, in the absence of additional agonists. When coupled with mycobacterial Ag85 and B. subtilis spores, Bac5 also enhanced IFNγ secretion. We provide evidence that B. subtilis spores, but not the Bac5 peptide, act as strong adjuvants in promoting antigen-specific immunoglobulin production in Ag85B-vaccinated mice. Our findings suggest that Bac5 is an important regulator of the early cell-mediated host immune response

Draft Genome Sequence of the Enteropathogenic Bacterium Campylobacter jejuni Strain cj255.

Published onlineJournal ArticleThe enteropathogen Campylobacter jejuni is a global health disaster, being one of the leading causes of bacterial gastroenteritis. Here, we present the draft genome sequence of C. jejuni strain cj255, isolated from a chicken source in Islamabad, Pakistan. The draft genome sequence will aid in epidemiological studies and quarantine of this broad-host-range pathogen.Higher Education Commission of Pakistan and British Counci

Delineation of the Innate and Adaptive T-Cell Immune Outcome in the Human Host in Response to Campylobacter jejuni Infection

Background: Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Despite the significant health burden this infection presents, molecular understanding of C. jejuni-mediated disease pathogenesis remains poorly defined. Here, we report the characterisation of the early, innate immune response to C. jejuni using an ex-vivo human gut model of infection. Secondly, impact of bacterial-driven dendritic cell activation on T-cell mediated immunity was also sought.Methodology: Healthy, control paediatric terminal ileum or colonic biopsy tissue was infected with C. jejuni for 8-12 hours. Bacterial colonisation was followed by confocal microscopy and mucosal innate immune responses measured by ELISA. Marked induction of IFN gamma with modest increase in IL-22 and IL-17A was noted. Increased mucosal IL-12, IL-23, IL-1 beta and IL-6 were indicative of a cytokine milieu that may modulate subsequent T-cell mediated immunity. C. jejuni-driven human monocyte-derived dendritic cell activation was followed by analyses of T cell immune responses utilising flow cytometry and ELISA. Significant increase in Th-17, Th-1 and Th-17/Th-1 double-positive cells and corresponding cytokines was observed. The ability of IFN gamma, IL-22 and IL-17 cytokines to exert host defence via modulation of C. jejuni adhesion and invasion to intestinal epithelia was measured by standard gentamicin protection assay.Conclusions: Both innate and adaptive T cell-immunity to C. jejuni infection led to the release of IFN gamma, IL-22 and IL-17A; suggesting a critical role for this cytokine triad in establishing host anti-microbial immunity during the acute and effectors phase of infection. In addition, to their known anti-microbial functions; IL-17A and IL-17F reduced the number of intracellular C. jejuni in intestinal epithelia, highlighting a novel aspect of how IL-17 family members may contribute to protective immunity against C. jejuni

Sequential Vaccination With Heterologous Acinetobacter baumannii Strains Induces Broadly Reactive Antibody Responses

Antibody therapy may be an alternative treatment option for infections caused by the multi-drug resistant (MDR) bacterium Acinetobacter baumannii. As A. baumannii has multiple capsular serotypes, a universal antibody therapy would need to target conserved protein antigens rather than the capsular polysaccharides. We have immunized mice with single or multiple A. baumannii strains to induce antibody responses to protein antigens, and then assessed whether these responses provide cross-protection against a collection of genetically diverse clinical A. baumannii isolates. Immunized mice developed antibody responses to multiple protein antigens. Flow cytometry IgG binding assays and immunoblots demonstrated improved recognition of both homologous and heterologous clinical strains in sera from mice immunized with multiple strains compared to a single strain. The capsule partially inhibited bacterial recognition by IgG and the promotion of phagocytosis by human neutrophils. However, after immunization with multiple strains, serum antibodies to protein antigens promoted neutrophil phagocytosis of heterologous A. baumannii strains. In an infection model, mice immunized with multiple strains had lower bacterial counts in the spleen and liver following challenge with a heterologous strain. These data demonstrate that antibodies targeting protein antigens can improve immune recognition and protection against diverse A. baumannii strains, providing support for their use as an antibody therapy