Anica M. Wandler

A Transgenic Drosophila Model Demonstrates That the Helicobacter pylori CagA Protein Functions as a Eukaryotic Gab Adaptor

Infection with the human gastric pathogen Helicobacter pylori is associated with a spectrum of diseases including gastritis, peptic ulcers, gastric adenocarcinoma, and gastric mucosa–associated lymphoid tissue lymphoma. The cytotoxin-associated gene A (CagA) protein of H. pylori, which is translocated into host cells via a type IV secretion system, is a major risk factor for disease development. Experiments in gastric tissue culture cells have shown that once translocated, CagA activates the phosphatase SHP-2, which is a component of receptor tyrosine kinase (RTK) pathways whose over-activation is associated with cancer formation. Based on CagAs ability to activate SHP-2, it has been proposed that CagA functions as a prokaryotic mimic of the eukaryotic Grb2-associated binder (Gab) adaptor protein, which normally activates SHP-2. We have developed a transgenic Drosophila model to test this hypothesis by investigating whether CagA can function in a well-characterized Gab-dependent process: the specification of photoreceptors cells in the Drosophila eye. We demonstrate that CagA expression is sufficient to rescue photoreceptor development in the absence of the Drosophila Gab homologue, Daughter of Sevenless (DOS). Furthermore, CagAs ability to promote photoreceptor development requires the SHP-2 phosphatase Corkscrew (CSW). These results provide the first demonstration that CagA functions as a Gab protein within the tissue of an organism and provide insight into CagAs oncogenic potential. Since many translocated bacterial proteins target highly conserved eukaryotic cellular processes, such as the RTK signaling pathway, the transgenic Drosophila model should be of general use for testing the in vivo function of bacterial effector proteins and for identifying the host genes through which they function.

Dynamic Evolution of the LPS-Detoxifying Enzyme Intestinal Alkaline Phosphatase in Zebrafish and Other Vertebrates

Alkaline phosphatases (Alps) are well-studied enzymes that remove phosphates from a variety of substrates. Alps function in diverse biological processes, including modulating host-bacterial interactions by dephosphorylating the Gram-negative bacterial cell wall component lipopolysaccharide (LPS). In animals, Alps are encoded by multiple genes characterized by either ubiquitous expression (named Alpls for their liver expression, but a key to proper bone mineralization), or their tissue-specific expression, for example in the intestine (Alpi). We previously characterized a zebrafish alpi gene (renamed here alpi.1) that is regulated by Myd88-dependent innate immune signaling and that is required to prevent a host’s excessive inflammatory reactions to its resident microbiota. Here we report the characterization of two new alp genes in zebrafish, alpi.2 and alp3. To understand their origins, we investigated the phylogenetic history of Alp genes in animals. We find that vertebrate Alp genes are organized in three clades with one of these clades missing from the mammals. We present evidence that these three clades originated during the two vertebrate genome duplications. We show that alpl is ubiquitously expressed in zebrafish, as it is in mammals, whereas the other three alps are specific to the intestine. Our phylogenetic analysis reveals that in contrast to Alpl, which has been stably maintained as a single gene throughout the vertebrates, the Alpis have been lost and duplicated multiple times independently in vertebrate lineages, likely reflecting the rapid and dynamic evolution of vertebrate gut morphologies, driven by changes in bacterial associations and diet.

Transgenic expression of the Helicobacter pylori virulence factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila.

Gastric cancer development is strongly correlated with infection by Helicobacter pylori possessing the effector protein CagA. Using a transgenic Drosophila melanogaster model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression.

Identification of genetic modifiers of CagA-induced epithelial disruption in Drosophila

Helicobacter pylori strains containing the CagA protein are associated with high risk of gastric diseases including atrophic gastritis, peptic ulcers, and gastric cancer. CagA is injected into host cells via a Type IV secretion system where it activates growth factor-like signaling, disrupts cell-cell junctions, and perturbs host cell polarity. Using a transgenic Drosophila model, we have shown that CagA expression disrupts the morphogenesis of epithelial tissues such as the adult eye. Here we describe a genetic screen to identify modifiers of CagA-induced eye defects. We determined that reducing the copy number of genes encoding components of signaling pathways known to be targeted by CagA, such as the epidermal growth factor receptor (EGFR), modified the CagA-induced eye phenotypes. In our screen of just over half the Drosophila genome, we discovered 12 genes that either suppressed or enhanced CagAs disruption of the eye epithelium. Included in this list are genes involved in epithelial integrity, intracellular trafficking, and signal transduction. We investigated the mechanism of one suppressor, encoding the epithelial polarity determinant and junction protein Coracle, which is homologous to the mammalian Protein 4.1. We found that loss of a single copy of coracle improved the organization and integrity of larval retinal epithelia expressing CagA, but did not alter CagAs localization to cell junctions. Loss of a single copy of the coracle antagonist crumbs enhanced CagA-associated disruption of the larval retinal epithelium, whereas overexpression of crumbs suppressed this phenotype. Collectively, these results point to new cellular pathways whose disruption by CagA are likely to contribute to H. pylori-associated disease pathology.

The bacterial virulence factor CagA induces microbial dysbiosis that contributes to excessive epithelial cell proliferation in the Drosophila gut

Gut microbiota facilitate many aspects of human health and development, but dysbiotic microbiota can promote hyperplasia and inflammation and contribute to human diseases such as cancer. Human patients infected with the gastric cancer-causing bacterium Helicobacter pylori have altered microbiota; however, whether dysbiosis contributes to disease in this case is unknown. Many H. pylori human disease phenotypes are associated with a potent virulence protein, CagA, which is translocated into host epithelial cells where it alters cell polarity and manipulates host-signaling pathways to promote disease. We hypothesized that CagA alone could contribute to H. pylori pathogenesis by inducing microbial dysbiosis that promotes disease. Here we use a transgenic Drosophila model of CagA expression to genetically disentangle the effects of the virulence protein CagA from that of H. pylori infection. We found that expression of CagA within Drosophila intestinal stem cells promotes excess cell proliferation and is sufficient to alter host microbiota. Rearing CagA transgenic flies germ-free revealed that the dysbiotic microbiota contributes to cell proliferation phenotypes and also elicits expression of innate immune components, Diptericin and Duox. Further investigations revealed interspecies interactions are required for this dysbiotic CagA-dependent microbiota to promote proliferation in CagA transgenic and healthy control Drosophila. Our model establishes that CagA can alter gut microbiota and exacerbate cell proliferation and immune phenotypes previously attributed to H. pylori infection. This work provides valuable new insights into the mechanisms by which interactions between a specific virulence factor and the resident microbiota can contribute to the development and progression of disease.

A Greasy Foothold for Helicobacter pylori

In this issue of Cell Host & Microbe, Murata-Kamiya and colleagues (Murata-Kamiya et al., 2010) report that the gastric pathogen Helicobacter pylori uses the plasma membrane component phosphatidylserine to facilitate translocation of the CagA virulence factor into host cells to establish a successful infection.

Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.

Mechanistic and Preclinical Insights from Mouse Models of Hematologic Cancer Characterized by Hyperactive Ras

RAS genes are mutated in 5%-40% of a spectrum of myeloid and lymphoid cancers with NRAS affected 2-3 times more often than KRAS Genomic analysis indicates that RAS mutations generally occur as secondary events in leukemogenesis, but are integral to the disease phenotype. The tractable nature of the hematopoietic system has facilitated generating accurate mouse models of hematologic malignancies characterized by hyperactive Ras signaling. These strains provide robust platforms for addressing how oncogenic Ras expression perturbs proliferation, differentiation, and self-renewal programs in stem and progenitor cell populations, for testing potential therapies, and for investigating mechanisms of drug response and resistance. This review summarizes recent insights from key studies in mouse models of hematologic cancer that are broadly relevant for understanding Ras biology and for ongoing efforts to implement rational therapeutic strategies for cancers with oncogenic RAS mutations.