Naomi Ohnishi

Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse

Infection with cagA-positive Helicobacter pylori is associated with gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma of B cell origin. The cagA-encoded CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system and, upon tyrosine phosphorylation by Src family kinases, specifically binds to and aberrantly activates SHP-2 tyrosine phosphatase, a bona fide oncoprotein in human malignancies. CagA also elicits junctional and polarity defects in epithelial cells by interacting with and inhibiting partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) independently of CagA tyrosine phosphorylation. Despite these CagA activities that contribute to neoplastic transformation, a causal link between CagA and in vivo oncogenesis remains unknown. Here, we generated transgenic mice expressing wild-type or phosphorylation-resistant CagA throughout the body or predominantly in the stomach. Wild-type CagA transgenic mice showed gastric epithelial hyperplasia and some of the mice developed gastric polyps and adenocarcinomas of the stomach and small intestine. Systemic expression of wild-type CagA further induced leukocytosis with IL-3/GM-CSF hypersensitivity and some mice developed myeloid leukemias and B cell lymphomas, the hematological malignancies also caused by gain-of-function SHP-2 mutations. Such pathological abnormalities were not observed in transgenic mice expressing phosphorylation-resistant CagA. These results provide first direct evidence for the role of CagA as a bacterium-derived oncoprotein (bacterial oncoprotein) that acts in mammals and further indicate the importance of CagA tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori-associated neoplasms.

Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity

Helicobacter pylori cagA-positive strains are associated with gastritis, ulcerations and gastric adenocarcinoma. CagA is delivered into gastric epithelial cells and, on tyrosine phosphorylation, specifically binds and activates the SHP2 oncoprotein, thereby inducing the formation of an elongated cell shape known as the ‘hummingbird’ phenotype. In polarized epithelial cells, CagA also disrupts the tight junction and causes loss of apical–basolateral polarity. We show here that H. pylori CagA specifically interacts with PAR1/MARK kinase, which has an essential role in epithelial cell polarity. Association of CagA inhibits PAR1 kinase activity and prevents atypical protein kinase C (aPKC)-mediated PAR1 phosphorylation, which dissociates PAR1 from the membrane, collectively causing junctional and polarity defects. Because of the multimeric nature of PAR1 (ref. 14), PAR1 also promotes CagA multimerization, which stabilizes the CagA–SHP2 interaction. Furthermore, induction of the hummingbird phenotype by CagA-activated SHP2 requires simultaneous inhibition of PAR1 kinase activity by CagA. Thus, the CagA–PAR1 interaction not only elicits the junctional and polarity defects but also promotes the morphogenetic activity of CagA. Our findings revealed that PAR1 is a key target of H. pylori CagA in the disorganization of gastric epithelial architecture underlying mucosal damage, inflammation and carcinogenesis.

Effects of Helicobacter pylori CagA protein on the growth and survival of B lymphocytes, the origin of MALT lymphoma

Helicobacter pylori (H. pylori) is a causative agent of gastrointestinal diseases such as atrophic gastritis and gastroduodenal ulcer. Infection of cagA-positive H. pylori is also associated with gastric carcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The cagA gene product CagA is directly injected into the bacteria-attached host cells via the bacterial type IV secretion system. The translocated CagA deregulates intracellular signaling pathways and thereby initiates pathogenesis. In this work, we examined the biological effects of CagA on B cells, from which MALT lymphoma arises. Ectopic expression of CagA in interleukin 3-dependent B cells inhibited cell proliferation by suppressing the JAK-STAT signaling. CagA was also capable of preventing hydroxyurea-induced B-cell apoptosis through inhibiting p53 accumulation. In contrast to the effects of CagA in gastric epithelial cells, the observed CagA activities in B cells were independent of its tyrosine phosphorylation. Our results indicate that CagA possesses both phosphorylation-dependent and -independent activities in mammalian cells and that biological impacts of CagA depend on cell-type context. As a result of B-cell growth inhibition, CagA may diminish anti-H. pylori immune responses. Furthermore, CagA may play a role in the development of MALT lymphoma by impairing p53-dependent apoptosis.

SHP2 Tyrosine Phosphatase Converts Parafibromin/Cdc73 from a Tumor Suppressor to an Oncogenic Driver

Deregulation of SHP2 is associated with malignant diseases as well as developmental disorders. Although SHP2 is required for full activation of RAS signaling, other potential roles in cell physiology have not been elucidated. Here we show that SHP2 dephosphorylates parafibromin/Cdc73, a core component of the RNA polymerase II-associated factor (PAF) complex. Parafibromin is known to act as a tumor suppressor that inhibits cyclin D1 and c-myc by recruiting SUV39H1 histone methyltransferase. However, parafibromin can also act in the opposing direction by binding β-catenin, thereby activating promitogenic/oncogenic Wnt signaling. We found that, on tyrosine dephosphorylation by SHP2, parafibromin acquires the ability to stably bind β-catenin. The parafibromin/β-catenin interaction overrides parafibromin/SUV39H1-mediated transrepression and induces expression of Wnt target genes, including cyclin D1 and c-myc. Hence, SHP2 governs the opposing functions of parafibromin, deregulation of which may cause the development of tumors or developmental malformations.

Differential oncogenic potential of geographically distinct Helicobacter pylori CagA isoforms in mice.

Infection with cagA‐positive Helicobacter pylori is associated with gastric carcinoma. The cagA‐encoded CagA protein is delivered into gastric epithelial cells and, upon tyrosine phosphorylation at the C‐terminal EPIYA segments, binds and deregulates SHP‐2 oncoprotein. On the basis of the differential alignment of the EPIYA segments, CagA can be subdivided into Western CagA, which is produced by H. pylori isolated in Western countries, and East Asian CagA, which is produced by H. pylori circulating in East Asian countries. Western CagA contains EPIYA‐A, EPIYA‐B and variable numbers of EPIYA‐C segments, whereas East Asian CagA contains EPIYA‐A, EPIYA‐B and variable numbers of EPIYA‐D segments. Upon tyrosine phosphorylation, EPIYA‐C and EPIYA‐D, respectively, serve as low‐affinity and high‐affinity SHP‐2‐binding sites. We previously reported that systemic expression of East Asian CagA (CagA‐ABDD) induces gastrointestinal and hematopoietic malignancies in mice. In this study, we generated transgenic mice that systemically express Western CagA (CagA‐ABCCC), the levels of which are comparable to those in mice expressing East Asian CagA. The mice developed gastric epithelial hypertrophy and gastrointestinal tumors and also showed lymphoid abnormality but not myeloid abnormalities such as granulocytosis and myeloid leukemia found in mice carrying East Asian CagA. The incidence of tumors in mice expressing Western CagA was significantly lower than that in mice expressing East Asian CagA. Our results indicate that Western CagA is qualitatively less oncogenic than East Asian CagA. Differential oncogenic potential of geographically distinct CagA isoforms may contribute to the differential prevalence of gastric carcinoma between East Asian countries and Western countries.

The CagA protein of Helicobacter pylori suppresses the functions of dendritic cell in mice

CagA protein is the most assessed effecter molecule of Helicobacter pylori. In this report, we demonstrate how CagA protein regulates the functions of dendritic cells (DC) against H. pylori infection. In addition, we found that CagA protein was tyrosine-phosphorylated in DC. The responses to cagA-positive H. pylori in DC were reduced in comparison to those induced by cagA-negative H. pylori. CagA-overexpressing DC also exhibited a decline in the responses against LPS stimulation and the differentiation of CD4(+) T cells toward Th1 type cells compared to wild type DC. In addition, the level of phosphorylated IRF3 decreased in CagA-overexpressing DC stimulated with LPS, indicating that activated SHP-2 suppressed the enzymatic activity of TBK1 and consequently IRF3 phosphorylation. These data suggest that CagA protein negatively regulates the functions of DC via CagA phosphorylation and that cagA-positive H. pylori strains suppress host immune responses resulting in their chronic colonization of the stomach.

Genome Sequence of a Bacillus anthracis Outbreak Strain from Zambia, 2011

ABSTRACT In August 2011, an anthrax outbreak occurred among Hippopotamus amphibius hippopotamuses and humans in Zambia. Here, we report the draft genome sequence of the Bacillus anthracis outbreak strain CZC5, isolated from tissues of H. amphibius hippopotamuses that had died in the outbreak area.

Helicobacter pylori induces IL-1β protein through the inflammasome activation in differentiated macrophagic cells

More than 50% of people in the world are infected with Helicobacter pylori (H. pylori), which induces various gastric diseases. Especially, epidemiological studies have shown that H. pylori infection is a major risk factor for gastric cancer. It has been reported that the levels of interleukin (IL)-1β are upregulated in gastric tissues of patients with H. pylori infection. In this study, we investigated the induction mechanism of IL-1β during H. pylori infection. We found that IL-1βmRNA and protein were induced in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells after H. pylori infection. This IL-1β production was inhibited by a caspase-1 inhibitor and a ROS inhibitor. Furthermore, K(+) efflux and Ca(2+) signaling were also involved in this process. These data suggest that NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) and its complex, known as NLRP3 inflammasome, are involved in IL-1β production during H. pylori infection because it is reported that NLRP3 inflammasome is activated by ROS, K(+) efflux and/or Ca(2+) signaling. These findings may provide therapeutic strategy for the control of gastric cancer in H. pylori-infected patients.

Paired-like homeodomain protein ESXR1 possesses a cleavable C-terminal region that inhibits cyclin degradation.

The eukaryotic cell cycle is regulated by sequential activation and inactivation of cyclin–cyclin-dependent kinase (Cdk) complexes. In this work, we screened human cDNAs that can rescue yeast Saccharomyces cerevisiae from lethality caused by ectopic expression of human cyclin E and isolated a cDNA encoding ESXR1, a paired-like homeodomain-containing protein with a unique C-terminal proline-rich repeat region. In adult tissues, ESXR1 is primarily expressed in the testis. We demonstrate that ESXR1 prevents degradation of ubiquitinated cyclins in human cells. Accordingly, elevation of ESXR1 level results in accumulation of cyclin A and cyclin B1 and thereby provokes M-phase arrest. In human cells, the 65-kDa full-length ESXR1 protein is capable of proteolytically processing into N-terminal 45-kDa and C-terminal 20-kDa fragments. The C-terminal fragment, containing a proline-rich repeat region, is localized to the cytoplasm and displays the ability to inhibit cyclin degradation. In contrast, the N-terminal fragment, containing a paired-like homeodomain, is localized exclusively in the nucleus, suggesting that it plays a role in transcription. Our results indicate that proteolytic processing of ESXR1 plays a role in concerted regulation of the cell cycle and transcription in human cells.

A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.