Figure 1.
Cellular localization of CagI in H. pylori.
(A) Western blots showing sub-cellular fractionation of wild-type H. pylori. TC, S and TM indicate total-cell lysate, soluble (cytoplasmic/periplasmic) and total membrane fractions respectively. Volume of TM was adjusted corresponding to that of S fraction and then equal volume of each was loaded in to gel. (B) Western blots showing osmotic shock analysis of H. pylori. r-TM, r-OS, and Os stand for residual total membrane, residual osmotic shocked content (cytosolic contents) and osmotic shocked fraction (periplasmic contents) respectively. Antibodies used are marked. (C) Western blots showing selective biotinylation of CagI from biotin labeled wild-type H. pylori (Hp) and HpΔcagT mutant strains. TM and S stand for total membrane fraction and soluble fraction. Antibodies used are marked. M-indicates molecular size standard. Arrow indicates position of non-biotinylated DnaB. (D) Immunofluorescence microscopy showing cellular localization of CagI in wild-type H. pylori (Hp) under permeabilized (P) and non-permeabilized (NP) conditions. H. pylori cells were fixed and permeabilized with 0.2% Triton X-100 and probed with antibodies as indicated. Alexa fluor 488 (green colour) and Alexa fluor 594 (red colour) conjugated secondary antibodies were used for signal generation and finally examined by immunofluorescence microscopy. Permeabilized HpΔcagI cells are showing specificity of α-CagI antibody. Pre-immune serum was used as control antibody. Scale bars indicate 5µM.
Figure 2.
Immunogold electron microscopy (IEM) reveals pili like structure associated CagI in wild-type H. pylori (Hp).
H. pylori strains were grown on BHI agar plates and immunogold labeling of ultrathin sections was performed as described in Materials and Methods. (A, B, C, and D) Wild-type H. pylori stained with anti-CagI and gold-labeled secondary antibody. (E) Wild- type H. pylori stained with rabbit pre-immune (preimm.) serum and gold-labeled secondary antibody. (F) Isogeneic mutant Hp∆cagI stained with anti-CagI and gold-labeled secondary antibody. (G, H, and I) Isogenic Hp∆cagX, Hp∆cagV, and Hp∆cagE mutant strains showing absence of pili in TEM sections. Scale bars indicate 100 nm. Arrowheads indicate location of gold-labeled secondary antibody.
Figure 3.
Co-immunoprecipitation (Co-IP) showing interaction between native CagI and CagH in H. pylori (Hp).
(A) Western blots showing Co-IP of CagH and CagL by anti-CagI and Co-IP of CagI and CagL by anti-CagH antibody from H. pylori cell extract. (B) SDS-PAGE showing co-expression of MBP-tagged CagH and without tag CagI. (C and D) Western blots showing Co-IP of recombinant CagI (without tag) and MBP-CagH by anti-CagH and anti-CagI antibodies respectively. Antibodies used in Western blots are indicated.
Figure 4.
Co-immunoprecipitation (Co-IP) from biotinylated H. pylori extract and recombinant proteins co-expressed in E. coli showing interacting partners of CagI.
(A) Western blots showing co-immunoprecipitation of biotinylated proteins by anti-CagI antibody from biotin labeled H. pylori extract. Blots were probed with HRP-conjugated anti-biotin antibody (α-biotin) and anti-CagI antibody (α-CagI). Arrows indicate biotinylated protein bands. (B and C) Western blots showing Co-IP of recombinant Hp1489 (GST-tag) and CagI (without tag) by anti-CagI and anti-Hp1489 antibodies respectively. Antibodies used in Western blots are indicated. M-indicates standard protein size markers.
Figure 5.
Western blots showing expression of CagI and CagH in mutant H. pylori strains (cag-PAI genes) in comparison to wild-type H. pylori (Hp).
(A) Equal amounts (OD600-2.0) of all indicated Hp cells were lysed in SDS sample buffer and were separated in SDS-PAGE, transferred to PVDF membrane and Western blotted. (B) Stabilities of Cag-T4SS components CagX, CagT, CagM, CagF, and CagZ in isogenic HpΔcagI mutant strain. Antibodies used are indicated. OMP was used as loading control.
Figure 6.
Surface localization of CagA in wild-type and HpΔcagI strains.
(A) Immunofluorescence microscopy showing surface localization of CagA in wild-type and HpΔcagI strains. Wild-type and HpΔcagI cells were fixed and one set was permeabilized with 0.2% Triton X-100. M, NP and P stand for merge, non-permeabilized and permeabilized cell respectively. Primary antibodies used in IFM are indicated. Secondary antibodies used were Alexa fluor 488 (green colour) and Alexa fluor 594 (red colour) conjugated. (B and C) Immunogold electron microscopy (IEM) showing surface localization of CagA in HpΔcagI and wild-type H. pylori (Hp) strains. Immunogold labeling of H. pylori strains ultrathin sections were performed as described in Materials and Methods. (B) HpΔcagI cells were stained with anti-CagA and gold-labeled secondary antibody. (C) Wild-type H. pylori cells were stained with anti-CagA and gold-labeled secondary antibody. (D) HpΔcagA cells were stained with anti-CagA and gold-labeled secondary antibody. (E) Pre-immune (preimm.) serum was used as negative control. Scale bars indicate 100 nm. Arrowheads indicate location of gold-labeled secondary antibody.