Antonello Covacci

Coxsackie B4 virus infection of β cells and natural killer cell insulitis in recent-onset type 1 diabetic patients

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic β cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between β cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of β cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect β cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect β cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.

Helicobacter pylori Activates Mitogen-activated Protein Kinase Cascades and Induces Expression of the Proto-oncogenes c-fos and c-jun

Helicobacter pylori is an etiological agent in the development of mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma. Patients infected with H. pylori carry a 3–6-fold increased risk of developing cancer compared with uninfected individuals. H. pylori strains expressing the cytotoxin-associated antigen A (CagA) are more frequently associated with the development of neoplasia than cagA-negative strains. However, the molecular mechanism by whichH. pylori causes neoplastic transformation remains unclear. Here we report that exposure of gastric epithelial cells to H. pylori induces activation of the transcription factor activator protein 1. Activation of the proto-oncogenes c-fos and c-jun is strongly induced. We show that H. pylori activates the ERK/MAP kinase cascade, resulting in Elk-1 phosphorylation and increased c-fos transcription. H. pylori strains that do not express CagA or that are mutated incag genes encoded by the CagI pathogenicity island do not induce activator protein 1, MAP kinase activity, or c-fosor c-jun activation. Proto-oncogene activation may represent a crucial step in the pathomechanism of H. pyloriinduced neoplasia.

Streptococcus pneumoniae pilus subunits protect mice against lethal challenge.

ABSTRACT Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that this pathogen possesses pili led us to investigate their protective abilities in a mouse model of intraperitoneal infection. Both active and passive immunization with recombinant pilus subunits afforded protection against lethal challenge with the S. pneumoniae serotype 4 strain TIGR4.

Helicobacter pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53 (ASPP2) tumor suppressor pathway of the host

Type I strains of Helicobacter pylori (Hp) possess a pathogenicity island, cag, that encodes the effector protein cytotoxin-associated gene A (CagA) and a type four secretion system. After translocation into the host cell, CagA affects cell shape, increases cell motility, abrogates junctional activity, and promotes an epithelial to mesenchymal transition-like phenotype. Transgenic expression of CagA enhances gastrointestinal and intestinal carcinomas as well as myeloid and B-cell lymphomas in mice, but the mechanism of the induced cancer formation is not fully understood. Here, we show that CagA subverts the tumor suppressor function of apoptosis-stimulating protein of p53 (ASPP2). Delivery of CagA inside the host results in its association with ASPP2. After this interaction, ASPP2 recruits its natural target p53 and inhibits its apoptotic function. CagA leads to enhanced degradation of p53 and thereby, down-regulates its activity in an ASPP2-dependent manner. Finally, Hp-infected cells treated with the p53-activating drug Doxorubicin are more resistant to apoptosis than uninfected cells, an effect that requires ASPP2. The interaction between CagA and ASPP2 and the consequent degradation of p53 are examples of a bacterial protein that subverts the p53 tumor suppressor pathway in a manner similar to DNA tumor viruses. This finding may contribute to the understanding of the increased risk of gastric cancer in patients infected with Hp CagA+ strains.

Composition and Gene Expression of the cag Pathogenicity Island in Helicobacter pylori Strains Isolated from Gastric Carcinoma and Gastritis Patients in Costa Rica

ABSTRACT The composition and in vitro expression of the cagpathogenicity island genes in a group of Helicobacter pylori strains obtained from patients suffering from chronic gastritis-associated dyspepsia (n = 26) or gastric carcinoma (n = 17) were analyzed. No significant difference in the distribution of the 10 studied regions was found between the cases and the controls. Nine strains did not harbor any of the selected regions: eight (30.8%) isolated from patients with gastritis only and one (5.9%) from a patient with gastric carcinoma. No association was found between the number of repeated sequences at the 3′ end of the cagA gene or the presence of tyrosine phosphorylation motifs and the clinical origin of the strains. ThevirB10 homolog gene was the sole gene studied to be significantly expressed more often in cancer strains than in gastritis strains (P = 0.03).

Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk

ABSTRACT The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies. IMPORTANCE Simultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease. Simultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease.