Infection with Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations, and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we showed that cholesterol extraction by methyl-␤-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with the raft marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were cofractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, the distribution shifted to non-DRMs. Accordingly, the CagA-induced hummingbird phenotype and interleukin-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. Together, these results suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated in a cholesterol-dependent manner.

A vacuolating cytotoxin encoded by vacA in Helicobacter pylori is known as a potential virulent determinant. The relationship between different vacA alleles, vacuolating ability, and H. pylori-related diseases was investigated. Genetic analysis of 119 isolates from Taiwanese patients revealed that 104 strains were s1a/m2, 13 strains were characterized as the s1a/m1T type, which was more homologous to the s1a/m1 strains, and 2 were characterized as the s1a/m1Tm2 chimeric type. Production of high-grade cytotoxin among 11 strains with s1a/m1T was higher (72.7%) than among 66 strains with s1a/m2 (21.2%) (P < .01). Peptic ulcer occurred in 76.9% of 13 patients with s1a/m1T strains compared with 46.2% of 104 patients with s1a/m2 strains (P < .05). These results suggest that s1a/m1T strains are associated with increased cytotoxic activity and higher ulcer prevalence than are s1a/m2 strains.

SummaryHelicobacter pylori infection is an aetiological cause of gastric disorders worldwide. H. pylori has been shown to assimilate and convert host cholesterol into cholesteryl glucosides (CGs) by cholesterol-aglucosyltransferase encoded by capJ. Here, we show that CapJ-deficient (DcapJ) H. pylori resulted in greatly reduced type IV secretion system (TFSS)-associated activities, including the hummingbird phenotype of AGS cells, IL-8 production, CagA translocation/phosphorylation and CagA-mediated signalling events. Complementation of the DcapJ mutation with wild type cagJ or by adding CGs-containing lysates or exogenous fluorophore-tagged CGs reversed the mutant phenotypes. We also show that the wild-type but not DcapJ H. pylori recruited raft-associated components to sites of bacterial attachment. Fluorescence recovery after photobleaching (FRAP) analysis of AGS cells treated with fluorescence-tagged cholesterol/CGs revealed that there was a higher proportion of CGs associated with immobile fractions. CGs-associated membranes were also more resistant to a cold detergent extraction. Thus, we propose that CGs synthesized by H. pylori around host-pathogen contact sites partition in detergent-resistant membranes (DRMs), alters lateralphase segregation in membrane and reorganizes membrane architecture. These processes together promote the formation of a functional TFSS and H. pylori infection.

The Helicobacter pylori VacA causes large intracellular vacuoles in epithelial cells such as HeLa or RK13 cells. Two major VacA forms, m1 and m2, divergent in an approximately 300 amino acid segment within the cell binding domain P58, display distinct cell-type specificity. Sequence analysis of four vacA alleles showed that a m1-like allele (61) and two m2 alleles (62 and v226) mainly differed in the midregion and that v225, a m1m2 chimera, was a natural double crossover from v226 and another allele. Each of these alleles was expressed as a soluble GST-VacA fusion that did not form a large oligomer. The recombinant VacA portion nevertheless assembled into higher ordered structures and possessed biological binding activity similar to that of the native VacA. A direct comparison of fusion-cell binding activity showed that m1 > m1m2 > m2 in HeLa cells, whereas there were more similar activities in RK13 cells. Vacuolating analyses of three forms revealed a positive correlation between cell binding activity and vacuolating activity. Moreover, the m1-type N-terminal half portion of the midregion was crucial for HeLa cell cytotoxicity. Kinetic, Scatchard, and inhibition analyses suggested the presence of at least two receptors: a m1-type specific high-affinity receptor (K(d) = approximately 5 nM) and a common VacA receptor interacting similarly with m1, m1m2, and m2 via a lower affinity (K(d) = 45-67 nM). Expression of mainly the m1-type receptor on HeLa cells whereas both receptors on RK13 cells may account for distinct cell binding activity and therefore for cell-type specificity.

JMJD5, a JmjC-domain containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxia-induced cell proliferation. JMJD5 interacts directly with PKM2, pyruvate kinase M2, to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes HIF-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α are all recruited to the HRE (hypoxia response element) site in the LDHA and PKM2 loci, and mediates the recruitment of the latter two proteins. Our data uncovers a new mechanism whereby PKM2 can be regulated by factor-binding induced homo/hetero-oligomeric restructuring, paving the way to cell metabolic reprogram. Citation Format: Hung-Jung Wang, Ya-Ju Hsieh, Wen-Chi Cheng, Chun-Pu Lin, Yu-Shan Lin, So-Fang Yang, Chung-Ching Chen, Yoshihiro Izumiya, Jau-Song Yu, Hsing-Jien Kung, Wen-Ching Wang. JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1α-mediated glucose metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3359. doi:10.1158/1538-7445.AM2014-3359