Birgit Brenneke

Identification of Burkholderia pseudomallei Genes Required for the Intracellular Life Cycle and In Vivo Virulence

ABSTRACT The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

BackgroundCampylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans.ResultsHere, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified.ConclusionsThe genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments.

Intestinal Microbiota Composition of Interleukin-10 Deficient C57BL/6J Mice and Susceptibility to Helicobacter hepaticus-Induced Colitis

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10−/− mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10−/− mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.

Inhibitory Effect of Enterohepatic Helicobacter hepaticus on Innate Immune Responses of Mouse Intestinal Epithelial Cells

ABSTRACT Enterohepatic Helicobacter species infect the intestinal tracts and biliary trees of various mammals, including mice and humans, and are associated with chronic inflammatory diseases of the intestine, gallstone formation, and malignant transformation. The recent analysis of the whole genome sequence of the mouse enterohepatic species Helicobacter hepaticus allowed us to perform a functional analysis of bacterial factors that may play a role in these diseases. We tested the hypothesis that H. hepaticus suppresses or evades innate immune responses of mouse intestinal epithelial cells, which allows this pathogen to induce or contribute to chronic inflammatory disease. We demonstrated in the present study that the innate immune responses of intestinal epithelial cells to lipopolysaccharide (LPS) via Toll-like receptor 4 (TLR4) and to flagellin-mediated activation via TLR5 are reduced by H. hepaticus infection through soluble bacterial factors. In particular, H. hepaticus lysate and the soluble component LPS antagonized TLR4- and TLR5-mediated immune responses of intestinal epithelial cells. H. hepaticus lysate and LPS inhibited development of endotoxin tolerance to Escherichia coli LPS. Suppression of innate immune responses by H. hepaticus LPS thus may affect intestinal responses to the resident microbial flora, epithelial homeostasis, and intestinal inflammatory conditions.

In Vivo Sequence Variation in HopZ, a Phase-Variable Outer Membrane Protein of Helicobacter pylori

ABSTRACT The Helicobacter pylori outer membrane protein HopZ is regulated by a phase-variable CT repeat and occurs in two distinct allelic variants. Whole-genome comparisons of isolates from one human volunteer recently provided evidence for in vivo selection for the hopZ ON status. We explored the frequency of sequence variation in hopZ during acute and chronic human infection and studied the association of hopZ with the phylogeographic population structure of H. pylori. hopZ ON variants were cultured from 24 out of 33 volunteers challenged with the hopZ OFF strain BCS 100. Transmission of H. pylori within families was also frequently associated with a status change of hopZ. In contrast, hopZ sequences obtained from 26 sets of sequential isolates from chronically infected individuals showed no changes of status, suggesting that the hopZ status selected during early infection is subsequently stable. Mutations leading to amino acid changes in HopZ occurred more frequently in ON than in OFF status isolates during chronic infection, indicating that sequence changes are more likely the result of positive selection in ON isolates than of a loss of negative selection pressure in OFF isolates. Analysis of 63 isolates from chronically infected individuals revealed no significant correlation of hopZ status with chronic atrophic gastritis. hopZ sequences were obtained from a globally representative collection of 54 H. pylori strains. All H. pylori populations contained hopZ-positive isolates. The data suggest that hopZ has been acquired and split into the two variants before the human migration out of Africa.

Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential

The enterohepatic Epsilonproteobacterium Helicobacter hepaticus persistently colonizes the intestine of mice and causes chronic inflammatory symptoms in susceptible mouse strains. The bacterial factors causing intestinal inflammation are poorly characterized. A large genomic pathogenicity island, HHGI1, which encodes components of a type VI secretion system (T6SS), was previously shown to contribute to the colitogenic potential of H. hepaticus. We have now characterized the T6SS components Hcp, VgrG1, VgrG2 and VgrG3, encoded on HHGI1, including the potential impact of the T6SS on intestinal inflammation in a mouse T‐cell transfer model. The H. hepaticus T6SS components were expressed during the infection and secreted in a T6SS‐dependent manner, when the bacteria were cultured either in the presence or in the absence of mouse intestinal epithelial cells. Mutants deficient in VgrG1 displayed a significantly lower colitogenic potential in T‐cell‐transferred C57BL/6 Rag2−/− mice, despite an unaltered ability to colonize mice persistently. Intestinal microbiota analyses demonstrated only minor changes in mice infected with wild‐typeH. hepaticus as compared with mice infected with VgrG1‐deficient isogenic bacteria. In addition, competitive assays between both wild‐type and T6SS‐deficient H. hepaticus, and between wild‐type H. hepaticus and Campylobacter jejuni or Enterobacteriaceae species did not show an effect of the T6SS on interbacterial competitiveness. Therefore, we suggest that microbiota alterations did not play a major role in the changes of pro‐inflammatory potential mediated by the T6SS. Cellular innate pro‐inflammatory responses were increased by the secreted T6SS proteins VgrG1 and VgrG2. We therefore concluded that the type VI secretion component VgrG1 can modulate and specifically exacerbate the innate pro‐inflammatory effect of the chronic H. hepaticus infection.

Role of the Helicobacter hepaticus Flagellar Sigma Factor FliA in Gene Regulation and Murine Colonization

The enterohepatic Helicobacter species Helicobacter hepaticus colonizes the murine intestinal and hepatobiliary tract and is associated with chronic intestinal inflammation, gall stone formation, hepatitis, and hepatocellular carcinoma. Thus far, the role of H. hepaticus motility and flagella in intestinal colonization is unknown. In other, closely related bacteria, late flagellar genes are mainly regulated by the sigma factor FliA (sigma(28)). We investigated the function of the H. hepaticus FliA in gene regulation, flagellar biosynthesis, motility, and murine colonization. Competitive microarray analysis of the wild type versus an isogenic fliA mutant revealed that 11 genes were significantly more highly expressed in wild-type bacteria and 2 genes were significantly more highly expressed in the fliA mutant. Most of these were flagellar genes, but four novel FliA-regulated genes of unknown function were identified. H. hepaticus possesses two identical copies of the gene encoding the FliA-dependent major flagellin subunit FlaA (open reading frames HH1364 and HH1653). We characterized the phenotypes of mutants in which fliA or one or both copies of the flaA gene were knocked out. flaA_1 flaA_2 double mutants and fliA mutants did not synthesize detectable amounts of FlaA and possessed severely truncated flagella. Also, both mutants were nonmotile and unable to colonize mice. Mutants with either flaA gene knocked out produced flagella morphologically similar to those of wild-type bacteria and expressed FlaA and FlaB. flaA_1 mutants which had flagella but displayed reduced motility did not colonize mice, indicating that motility is required for intestinal colonization by H. hepaticus and that the presence of flagella alone is not sufficient.

Role of Energy Sensor TlpD of Helicobacter pylori in Gerbil Colonization and Genome Analyses after Adaptation in the Gerbil

ABSTRACT Helicobacter pylori maintains colonization in its human host using a limited set of taxis sensors. TlpD is a proposed energy taxis sensor of H. pylori and dominant under environmental conditions of low bacterial energy yield. We studied the impact of H. pylori TlpD on colonization in vivo using a gerbil infection model which closely mimics the gastric physiology of humans. A gerbil-adapted H. pylori strain, HP87 P7, showed energy-dependent behavior, while its isogenic tlpD mutant lost it. A TlpD-complemented strain regained the wild-type phenotype. Infection of gerbils with the complemented strain demonstrated that TlpD is important for persistent infection in the antrum and corpus and suggested a role of TlpD in horizontal navigation and persistent corpus colonization. As a part of the full characterization of the model and to gain insight into the genetic basis of H. pylori adaptation to the gerbil, we determined the complete genome sequences of the gerbil-adapted strain HP87 P7, two HP87 P7 tlpD mutants before and after gerbil passage, and the original human isolate, HP87. The integrity of the genome, including that of a functional cag pathogenicity island, was maintained after gerbil adaptation. Genetic and phenotypic differences between the strains were observed. Major differences between the gerbil-adapted strain and the human isolate emerged, including evidence of recent recombination. Passage of the tlpD mutant through the gerbil selected for gain-of-function variation in a fucosyltransferase gene, futC (HP0093). In conclusion, a gerbil-adapted H. pylori strain with a stable genome has helped to establish that TlpD has important functions for persistent colonization in the stomach.