Aeryun Kim

Helicobacter pylori bab Paralog Distribution and Association with cagA, vacA, and homA/B Genotypes in American and South Korean Clinical Isolates

Helicobacter pylori genetic variation is a crucial component of colonization and persistence within the inhospitable niche of the gastric mucosa. As such, numerous H. pylori genes have been shown to vary in terms of presence and genomic location within this pathogen. Among the variable factors, the Bab family of outer membrane proteins (OMPs) has been shown to differ within subsets of strains. To better understand genetic variation among the bab genes and to determine whether this variation differed among isolates obtained from different geographic locations, we characterized the distribution of the Bab family members in 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). Overall, we identified 23 different bab genotypes (19 in AH and 11 in KH), but only 5 occurred in greater than 5 isolates. Regardless of strain origin, a strain in which locus A and locus B were both occupied by a bab gene was the most common (85%); locus C was only occupied in those isolates that carried bab paralog at locus A and B. While the babA/babB/- genotype predominated in the KH (78.8%), no single genotype could account for greater than 40% in the AH collection. In addition to basic genotyping, we also identified associations between bab genotype and well known virulence factors cagA and vacA. Specifically, significant associations between babA at locus A and the cagA EPIYA-ABD motif (P<0.0001) and the vacA s1/i1/m1 allele (P<0.0001) were identified. Log-linear modeling further revealed a three-way association between bab carried at locus A, vacA, and number of OMPs from the HOM family (P<0.002). En masse this study provides a detailed characterization of the bab genotypes from two distinct populations. Our analysis suggests greater variability in the AH, perhaps due to adaptation to a more diverse host population. Furthermore, when considering the presence or absence of both the bab and homA/B paralogs at their given loci and the vacA genotype, an association was observed. Our results highlight the multifactorial nature of H. pylori mediated disease and the importance of considering how the specific combinations of H. pylori virulence genes and their multiple interactions with the host will collectively impact disease progression.

Role of bacterial γ-glutamyltranspeptidase as a novel virulence factor in bone-resorbing pathogenesis.

Mammalian γ-glutamyltranspeptidase (GGT) has been identified as a bone-resorbing factor. Since GGT of Bacillus subtilis exhibits similarity in their primary structure and enzymatic characteristics with mammalian GGTs, the bone-resorbing activity of bacterial GGT was examined in this study. Osteoclastogenesis was performed in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. A conditioned medium from GGT-overproducing B. subtilis culture showed significantly higher activity of osteoclast formation than a conditioned medium from wild-type B. subtilis culture. Recombinant GGT (rGGT) of wild-type B. subtilis and an enzymatic activity-defected rGGT of B. subtilis 2288 mutant were expressed in Escherichia coli and purified using His tag. Both purified rGGTs induced similar levels of osteoclastogenesis, suggesting that B. subtilis GGT possesses virulent bone-resorbing activity and its activity is probably independent of its enzymatic activity. Furthermore, a recombinant protein of B. subtilis GGT heavy subunit (Bs rGGT/H) showed strong activity of osteoclastogenesis while the light subunit failed to show strong activity, suggesting that the bone-resorbing activity is mainly located at the heavy subunit. More importantly, the GGT enzymatic activity may not be required for this virulence activity since the light subunit contains the catalytic pocket. In addition, B. subtilis rGGT stimulated mRNA expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and cyclooxygenase-2 (COX-2), while an osteoprotegerin inhibited the osteoclast formation induced by Bs rGGT/H. This is the first demonstration that bacterial GGT itself is sufficient to act as a bone-resorbing virulence factor via RANKL-dependent pathway. Therefore, it can be hypothesized that GGT of periodontopathic bacteria may play an important role as a virulence factor in bone destruction.

Comparative analysis of the Hom family of outer membrane proteins in isolates from two geographically distinct regions: The United States and South Korea

Helicobacter pylori encodes numerous outer membrane proteins (OMPs), but only a few have been characterized in depth. Deletion, duplication, and allelic variation of many of the H. pylori OMPs have been reported, which suggests that these proteins may play key roles in host adaptation. Herein, we characterize the variation observed within the Hom family of OMPs in H. pylori obtained from two geographically distinct populations.

Helicobacter pylori outer membrane protein, HomC, shows geographic dependent polymorphism that is influenced by the Bab family

The array of outer membrane proteins (OMPs) found in Helicobacter pylori provides a crucial component for persistent colonization within the gastric niche. Not only does H. pylori harbor a wide number of OMPs, but these OMPs often vary across strains; this likely contributes to immune evasion, adaptation during long term colonization, and potentially differential disease progression. Previous work from our group described OMP differences among the Bab family (babA, babB, and babC) and Hom family (homA and homB) from 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). In the current study, we expanded our investigation to include the less well characterized Hom family member, HomC.Overall, we identified and genotyped three homC variants: homCS, homCL, and homCM, in both populations. Similar to other polymorphic genes, the KH group showed less overall diversity, with 97.5% of strains harboring homCL. In contrast, a more heterogeneous profile was observed in strains derived from an American population; we found nearly equal distribution of homCS and homCL. Further analysis of the AH group identified associations between homC polymorphism and bab genotype; in AH strains, there was a significant association between homCL and carriage of babA at locus A. Since babA is an important virulence factor for the development of severe gastric disease, these data may suggest that homC polymorphism plays a role in H. pylori pathogenesis.

ArsRS-Dependent Regulation of homB Contributes to Helicobacter pylori Biofilm Formation

One elusive area in the Helicobacter pylori field is an understanding of why some infections result in gastric cancer, yet others persist asymptomatically for the life-span of the individual. Even before the genomic era, the high level of intraspecies diversity of H. pylori was well recognized and became an intriguing area of investigation with respect to disease progression. Of interest in this regard is the unique repertoire of over 60 outer membrane proteins (OMPs), several of which have been associated with disease outcome. Of these OMPs, the association between HomB and disease outcome varies based on the population being studied. While the molecular roles for some of the disease-associated OMPs have been evaluated, little is known about the role that HomB plays in the H. pylori lifecycle. Thus, herein we investigated homB expression, regulation, and contribution to biofilm formation. We found that in H. pylori strain G27, homB was expressed at a relatively low level until stationary phase. Furthermore, homB expression was suppressed at low pH in an ArsRS-dependent manner; mutation of arsRS resulted in increased homB transcript at all tested time-points. ArsRS regulation of homB appeared to be direct as purified ArsR was able to specifically bind to the homB promoter. This regulation, combined with our previous finding that ArsRS mutations lead to enhanced biofilm formation, led us to test the hypothesis that homB contributes to biofilm formation by H. pylori. Indeed, subsequent biofilm analysis using a crystal-violet quantification assay and scanning electron microscopy (SEM) revealed that loss of homB from hyper-biofilm forming strains resulted in reversion to a biofilm phenotype that mimicked wild-type. Furthermore, expression of homB in trans from a promoter that negated ArsRS regulation led to enhanced biofilm formation even in strains in which the chromosomal copy of homB had been deleted. Thus, homB is necessary for hyper-biofilm formation of ArsRS mutant strains and aberrant regulation of this gene is sufficient to induce a hyper-biofilm phenotype. In summary, these data suggest that the ArsRS-dependent regulation of OMPs such as HomB may be one mechanism by which ArsRS dictates biofilm development in a pH responsive manner.

Effect of Areca nut on Helicobacter pylori-induced gastric diseases in mice.

Areca nut (AN) chewing is a habit in many countries in Central, Southern, and Southeast Asia. It is strongly associated with the occurrence of oral, pharyngeal, and esophageal cancer as well as systemic inflammation. However, the association between AN intake and the development of gastric lesions has not yet been identified. The aim of this study was to investigate the effect of AN on gastric diseases using a mouse model for Helicobacter pylori infection. We studied four groups of mice: those fed a normal diet (ND), those fed a diet containing 2.5% AN (AD), those fed ND and infected with H. pylori PMSS1 strain (ND/HP), and those fed AD and infected with H. pylori PMSS1 strain (AD/HP). Food intake and body weight were monitored weekly during the experiments. At 10 weeks, the mice were sacrificed, and the stomach weight, H. pylori colonization, and gastric inflammation were evaluated. The stomach weight had increased significantly in the ND/HP and AD/HP groups along with increases in H. pylori colonization; however, there was no significant difference between these two groups with respect to stomach weight and colonization. On histological grading, mononuclear cell infiltration was severer in the AD/HP group than in the ND/HP group. These data suggest that chronic gastric inflammation was aggravated by AN treatment in the mice with H. pylori-induced gastric lesions. Furthermore, as previously suggested, this animal model is useful to determine the effect of potential carcinogens on gastric lesions induced by H. pylori infection.