Biochemical Characterization of the Helicobacter pylori Cag-T 4 SS Unique Component

Many strains of Helicobacter pylori encode a protein secreting type IV secretion system called the Cag-T4SS. Here, we characterize one of the Cag-T4SS specific components, CagU (HP0531). We report that CagU is a bacterial inner membrane-associated protein, partially processed at the C-terminus, and that it interacts with the VirB6 and VirB8 homologues CagW and CagV, respectively. The level of expression of CagU is partially affected in the absence of cagX, cagW and cagV. Deletion of cagU aborts surface localization of CagA and affects the expression levels of CagI and CagH, which are involved in the Cag-T4SS pilus formation. Complementation of the cagU null mutation by wild-type cagU restores all these functions, suggesting its importance for Cag-T4SS function. Introduction: Helicobacter pylori (H. pylori) is one of the most successful human pathogens and is assumed to be evolved along with it [1]. It is a Gram-negative, helical shaped and flagellated micro-aerophilic organism, which mainly resides in the antral region of the stomach. The most virulent strains of H. pylori are associated with severe forms of gastric maladies, including chronic gastritis, peptic ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma and even gastric cancer [2, 3]. Molecular mechanisms associated with the initiation, progression and development of these diseases are poorly understood [4]. However, factors of both the host and the bacterium seem to be involved in the pathogenesis and disease outcome of H. pylori [4]. Many strains of H. pylori carry a ~37 kb long, relatively A/T rich genomic sequence, known as the cag Pathogenicity Island (cag-PAI) [5, 6]. The cag-PAI encodes 27 putative genes, that constitute an effector translocating type IV secretion system (T4SS), named the Cag-T4SS [5, 7]. In addition to the Cag-T4SS, H. pylori also encodes two other T4SS namely the highly conserved ComB system and less conserved tfs3 [8-11]. A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. T4SS are transporters of macromolecules widely present in both Gram-negative and Grampositive bacteria and are composed of multiple proteins [12, 13]. These systems are ancestrally related to bacterial conjugation systems [14, 15]. The prototypical VirB/VirD4 T4SS is encoded by the plant pathogen Agrobacterium tumefaciens (A. tumefaciens) and is composed of 11 VirB (VirB1–VirB11) and VirD4 proteins [16]. Although the structural arrangement of the Cag-T4SS components, inside the bacterial cell is assumed to be similar to that of prototypical T4SS, the H. pylori secretion system contains additional components [13]. Sequence analysis of the Cag proteins has revealed that only a few of them share similarity to their counterparts, like CagY to VirB10, CagX to VirB9, Cagα to VirB11 and CagE to VirB4 [17, 18]. However, a large number of other Cag-T4SS components are H. pylori specific or unique in nature such as Cagδ, CagF, CagZ, CagU, CagI, CagH, CagG, CagS, CagQ, CagM, CagN and CagD [19]. Systematic mutational analysis of the individual cag-PAI genes and subsequent functional studies of individual deletion mutants have assigned functional roles to a number of Cag-T4SS components [20]. Over the recent years, functional roles of some of the unique components are also being studied, namely Cagδ, CagF, CagZ, CagI, CagD and CagN [21-28]. However, very little is known about the unique component CagU, except its requirement for translocating CagA into the host epithelial cells as well as stimulation of IL-8 secretion by the infected host [20]. In order to understand the function of CagU in the Cag-T4SS of H. pylori 26695, we have characterized the protein using genetic, biochemical and microscopic methods. Here we report that it is an inner membrane associated protein, and is partially processed at the C-terminal end. We also provide evidence that it interacts with putative inner membrane proteins CagW, and CagV and affects the expression of CagI and CagH, which are reported to be involved in CagT4SS pilus formation.