Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system

Abstract

Gram-negative bacteria Helicobacter pylori cause gastric ulcer, duodenal cancer, and found in almost half of the world's residents. The protein responsible for this disease is secreted through type IV secretion system (TFSS) of H. pylori. TFSS is encoded by 40-kb region of chromosomal DNA known as cag-pathogenicity island (PAI). TFSS comprises of three major components: cytoplasmic/inner membrane ATPase, transmembrane core-complex and outer membranous pilli, and associated subunits. Core complex consists of CagX, CagT, CagM, and Cag3(δ) proteins as per existing knowledge. In this study, we have characterized one of the important component of core-complex forming sub-unit protein, i.e., CagX. Complete ORF of CagX except signal peptide coding region was cloned and expressed in pET28a vector. Purification of CagX protein was performed, and polyclonal anti-sera against full-length recombinant CagX were raised in rabbit model. We obtained a very specific and high titer, CagX anti-sera that were utilized to characterize endogenous CagX. Surface localization of CagX was also seen by immunofluorescence microscopy. In short for the first time a full-length CagX was characterized, and we showed that CagX is the part of high molecular weight core complex, which is important for assembly and function of H. pylori TFSS.

Keywords: Cag-PAI; CagX; H. pylori; molecular characterization; type IV secretion system.

Figure 1. Induction profile of CagX protein: Total E. coli extract harboring pET-cagX plasmid was separated through 10% SDS-PAGE followed by staining with CBB stain. Induction of culture was done using 1 mM IPTG (indicated by arrow).

Figure 2. SDS-PAGE showing partial purification of recombinant CagX. Lane 1–4 represent 30% ammonium sulfate fraction, flow through, washing and NaCl eluted recombinant protein respectively. M indicates standard molecular size markers. Arrow indicates position of the recombinant protein. Gel was stained with Coomassie blue.

Figure 3. (A) western blot showing specificity of the anti-CagX antibody. Lane -1, total E. coli cell extract (uninduced), lane-2, induced E. coli extract, lane-3, total H. pylori 26695 cell extract. Blot was probed with preimmune serum (left panel) and anti-CagX serum (right panel). (B) western blot (left panel) and CBB stain (right panel) of total cell extract of H. pylori wild type and H. pylori cagX null strain to show specificity of raised CagX antisera.

Figure 4. western blot showing sub-cellular localization of CagX in wild-type Hp, HpΔcagA and HpΔcagT mutants. W, S and TM indicate whole-cell, soluble (cytoplasmic/periplasmic) and total membrane respectively. Primary antibody used in western blot is marked.

Figure 5. Visualization of CagX into wild type and null mutant of H. pylori. Fluorescence images were recorded which have showed the surface localization of CagX in H. pylori wild type strain.

Figure 6. 2-D blue native PAGE analysis of DDM solubilizes H. pylori extract. Blot was probed with anti-CagX antibody.

Figure 7. SDS PAGE showing expression profile of recombinant CagX protein conjugated with 6-His at C-terminal (left). Western blot (right panel) of MBP pull-down sample showing CagX oligomerization. Recombinant MBP tagged CagX and poly-histidine tagged CagX were mixed and subjected to pull-down analysis. MBP was used as negative control. Anti- MBP and Anti-His antibody was used to probe the pull down sample. MBP and histidine tagged proteins are indicated along with antibodies used.