Clinical isolates of Helicobacter pylori show marked diversity, which may derive from genomic changes that occur during the often lifelong association of the bacterium with its human host. We used the rhesus macaque model, together with DNA microarrays, to examine genomic changes in H. pylori that occur early during experimental infection. Microarray analysis showed that H. pylori recovered from challenged macaques had deleted babA, a member of a large family of paralogous outer membrane proteins (OMPs) that mediates attachment of H. pylori to the Lewis B blood group antigen on gastric epithelium. In some cases the babA gene was replaced by babB, an uncharacterized OMP that is closely related to babA. In other cases the babA gene was present but was not expressed because of alteration in dinucleotide CT repeats in the 5 coding region. In either case, strains lacking babA did not adhere to Lewis B, which is expressed on macaque gastric epithelium. Absence of babA and duplication of babB was also seen in H. pylori isolates derived from human clinical samples, suggesting that this gene conversion event is not unique to experimentally infected rhesus monkeys. These results demonstrate in real time with a relevant animal model that H. pylori regulates OMP expression in vivo by using both antigenic variation and phase variation. We suggest that changes in babA and babB after experimental infection of macaques represent a dynamic response in the H. pylori outer membrane that facilitates adherence to the gastric epithelium and promotes chronic infection.

Bartonella quintana is a fastidious, gram-negative, rod-shaped bacterium that causes prolonged bacteremia in immunocompetent humans and severe infections in immunocompromised individuals. We sought to define the outer membrane subproteome of B. quintana in order to obtain insight into the biology and pathogenesis of this emerging pathogen and to identify the predominant B. quintana antigens targeted by the human immune system during infection. We isolated the total membrane proteins of B. quintana and identified 60 proteins by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass fingerprinting. Using the newly constructed proteome map, we then utilized two-dimensional immunoblotting with sera from 21 B. quintana-infected patients to identify 24 consistently recognized, immunoreactive B. quintana antigens that have potential relevance for pathogenesis and diagnosis. Among the outer membrane proteins, the variably expressed outer membrane protein adhesins (VompA and VompB), peptidyl-prolyl cis-trans-isomerase (PpI), and hemin-binding protein E (HbpE) were recognized most frequently by sera from patients, which is consistent with surface expression of these virulence factors during human infection.Bartonella quintana, the agent of trench fever, is a fastidious, gram-negative, rod-shaped organism that can cause prolonged bacteremia in immunocompetent humans and severe infections in immunocompromised individuals. Humans are the only known reservoir for B. quintana (12), and the vector for transmission is the human body louse, Pediculus humanus corporis (38). B. quintana infections have occurred worldwide, and severe, potentially lethal complications, such as endocarditis and bacillary angiomatosis, can develop in immunocompromised patients with AIDS, cancer, and organ transplants. However, little is known about the pathogenesis of B. quintana, and diagnosis of human infection remains extremely challenging. To address this paucity of knowledge, we sought to identify potential membrane-associated virulence factors, as well as protective and diagnostically relevant B. quintana antigens, by characterizing the total membrane fraction and immunome of B. quintana.Bacterial outer membrane proteins (OMP) can be important virulence factors, playing a critical role in adherence, invasion, and immune evasion during infection of the host, as well as during transmission via arthropod vectors. Many outer membrane-associated proteins that are important for pathogenesis also are consistent targets for the host immune system after infection. Workers in our lab previously identified a family of variably expressed outer membrane proteins (Vomp) that play a role in adhesion and autoaggregation (45). To initially identify the Vomp family, we used two-dimensional (2D) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to visualize changes in expression of membrane proteins in sequential isolates from animals experimentally infected with B. quintana.To identify additional membrane pr...

We used a quantitative real-time reverse transcriptase PCR assay to measure the transcript abundance of 46 known and putative Helicobacter pylori virulence genes, including 24 genes on the Cag pathogenicity island. The expression profile of H. pylori cells grown in vitro was also compared to expression in vivo after experimental infection of rhesus macaques. Transcript abundance in vitro (mid-log phase) ranged from about 0.004 (feoB and hpaA) to 20 (ureAB, napA, and cag25) copies/cell. Expression of most genes was repressed during the transition from logarithmic-to stationary-phase growth, but several well-characterized H. pylori virulence genes (katA, napA, vacA, and cagA) were induced. Comparison of results in the rhesus macaque with similar data from humans showed a strong correlation (r ‫؍‬ 0.89). The relative in vivo expression in the rhesus monkey was highly correlated with in vitro expression during mid-log (r ‫؍‬ 0.89)-and stationary (r ‫؍‬ 0.88)-phase growth. Transcript abundance was on average three-to fourfold reduced in vivo compared to in vitro during mid-log phase. However, when compared to stationary phase, increased expression in vivo was observed for 6 of 7 genes on a contiguous portion of the pathogenicity island, several of which are thought to encode the H. pylori type IV structural pilus and its accessory proteins. These results suggest the possibility that some genes encoding the H. pylori type IV structural pilus and accessory proteins may form an operon that is induced during growth in vivo.

qRT-PCR is a powerful tool to measure gene expression in human or animal tissue that contains minute amounts of microbial mRNA, and the results reflect on the physiology of the pathogen in its natural host.