In Depth Analysis of the Helicobacter pylori cag Pathogenicity Island Transcriptional Responses Andrea Vannini, Davide Roncarati, Marco Spinsanti ¤ , Vincenzo Scarlato*, Alberto Danielli* Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy Abstract The severity of symptoms elicited by the widespread human pathogen Helicobacter pylori is strongly influenced by the genetic diversity of the infecting strain. Among the most important pathogen factors that carry an increased risk for gastric cancer are specific genotypes of the cag pathogenicity island (cag-PAI), encoding a type IV secretion system (T4SS) responsible for the translocation of the CagA effector oncoprotein. To date, little is known about the regulatory events important for the expression of a functional cag-T4SS. Here we demonstrate that the cag-PAI cistrons are subjected to a complex network of direct and indirect transcriptional regulations. We show that promoters of cag operons encoding structural T4SS components display homogeneous transcript levels, while promoters of cag operons encoding accessory factors vary considerably in their basal transcription levels and responses. Most cag promoters are transcriptionally responsive to growth-phase, pH and other stress-factors, although in many cases in a pleiotropic fashion. Interestingly, transcription from the Pcagf promoter controlling the expression of transglycolase and T4SS stabilizing factors, is triggered by co-culture with a gastric cell line, providing an explanation for the increased formation of the secretion system observed upon bacterial contact with host cells. Finally, we demonstrate that the highly transcribed cagA oncogene is repressed by iron limitation through a direct apo-Fur regulation mechanism. Together the results shed light on regulatory aspects of the cag-PAI, which may be involved in relevant molecular and etiological aspects of H. pylori pathogenesis. Citation: Vannini A, Roncarati D, Spinsanti M, Scarlato V, Danielli A (2014) In Depth Analysis of the Helicobacter pylori cag Pathogenicity Island Transcriptional Responses. PLoS ONE 9(6): e98416. doi:10.1371/journal.pone.0098416 Editor: Ivo G. Boneca, Institut Pasteur Paris, France Received January 15, 2014; Accepted May 1, 2014; Published June 3, 2014 Copyright: ß 2014 Vannini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants from the Italian Ministry of Instruction University and Research (grant 2007LHN9JL to AD and grant 2010P3S8BR to VS), by Fondazione del Monte di Bologna (FDM756) to AD, and by the University of Bologna to VS. AV was the recipient of a fellowship from the PhD program in Functional Biology of Molecular and Cellular Systems of the University of Bologna. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: In regard to the potential conflict of interest caused by a collaboration of VS with Novartis Vaccines Diagnostics: this is an unpaid counseling activity and does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. * E-mail: vincenzo.scarlato@unibo.it (VS); alberto.danielli@unibo.it (AD) ¤ Current address: Novartis Vaccines and Diagnostics, Siena, Italy Introduction Helicobacter pylori is a major human pathogen that colonizes the gastric epithelium of more than half of humankind, worldwide. Etiology of H. pylori infections ranges from mild to acute symptoms, including gastric inflammations and duodenal diseases. While symptomatic infections can be successfully treated with antimicrobial drugs, the untreated and asymptomatic infections persist over decades, promoting the long term chronic inflamma- tion and insurgence of peptic ulcers and gastric cancer [1,2]. The severity of symptoms largely depends on the genetic diversity of the infecting strain [2], and particularly on specific genotypes of virulence-associated genes, such as the cag pathoge- nicity island (cag-PAI). The latter is a 38 kb multi-operon locus coding for 28 putative ORFs, six of which were identified as homologues of the basic type IV secretion system (T4SS) represented by the Agrobacterium tumefaciens virB operon [3]. The H. pylori cag-T4SS promotes injection of the CagA bacterial oncoprotein into host cells [4], as well as a CagA-independent induction of interleukin-8 secretion via the host AP-1 and NF-kB signaling pathways [5]. Notably, chemokine induction requires direct contact of the bacteria with epithelial host cells [6]. Host integrin receptors are engaged by the CagL protein to promote CagA translocation across the host cell membrane and to activate the Src tyrosine kinase for CagA phosphorylation [7], resulting in cytoskeletal rearrangements of the gastric epithelium [8]. Seven- teen genes of the cag-PAI are essential for CagA translocation, including three genes HP0524 (virD homologue), HP0526, and HP0540 that are dispensable for IL-8 induction [9]. Moreover, several genes of the cag-PAI are not necessary for either CagA translocation or IL-8 induction, and their role in Cag-T4SS assembly and virulence remains to be elucidated [10,11]. Proper stoichiometric ratio of gene products and appropriate expression in time and space in the host allows the assembly of functional secretion systems [12]. Polarity effects modulating transcription and translation can contribute to this regulation, but in many bacteria the assembly requires finely tuned regulatory mechanisms. For example, it has been shown that dedicated transcriptional regulators are frequently encompassed within the PAIs of type III and type IV secretion systems. Transcriptional regulators such as Fur, HilA, HilC, HilD, InvF and ExsA, controlling Salmonella enterica pathogenicity island (SPI1) and Pseudomonas aeruginosa T3SS gene expression, respectively, are informative examples to date [13–15]. H. pylori is unusual in this respect. In fact, despite multiple operons with oppositely phased ORFs and frequent intergenic PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e98416