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Biochemical Analysis of CagE: A VirB4 Homologue of Helicobacter pylori Cag-T4SS.

Shariq M, Kumar N, Kumari R, Kumar A, Subbarao N, Mukhopadhyay G - PLoS ONE (2015)

Bottom Line: Here we have characterised the protein biochemically, genetically, and microscopically and report that CagE is an inner membrane associated active NTPase and has multiple interacting partners including the inner membrane proteins CagV and Cagβ.These results thus suggest the importance of CagE in Cag-T4SS functions.In future it may help in deciphering the mechanism of substrate translocation by the system.

View Article: PubMed Central - PubMed

Affiliation: Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.

ABSTRACT
Helicobacter pylori are among the most successful human pathogens that harbour a distinct genomic segment called cag Pathogenicity Island (cag-PAI). This genomic segment codes for a type IV secretion system (Cag-T4SS) related to the prototypical VirB/D4 system of Agrobacterium tumefaciens (Ag), a plant pathogen. Some of the components of Cag-T4SS share homology to that of VirB proteins including putative energy providing CagE (HP0544), the largest VirB4 homologue. In Ag, VirB4 is required for the assembly of the system, substrate translocation and pilus formation, however, very little is known about CagE. Here we have characterised the protein biochemically, genetically, and microscopically and report that CagE is an inner membrane associated active NTPase and has multiple interacting partners including the inner membrane proteins CagV and Cagβ. Through CagV it is connected to the outer membrane sub-complex proteins. Stability of CagE is not dependent on several of the cag-PAI proteins tested. However, localisation and stability of the pilus associated CagI, CagL and surface associated CagH are affected in its absence. Stability of the inner membrane associated energetic component Cagβ, a VirD4 homologue seems to be partially affected in its absence. Additionally, CagA failed to cross the membrane barriers in its absence and no IL-8 induction is observed under infection condition. These results thus suggest the importance of CagE in Cag-T4SS functions. In future it may help in deciphering the mechanism of substrate translocation by the system.

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Immunogold electron microscopy showing localisation of CagA in wild-type and 26695ΔcagE strains.Ultra thin sections of wild-type, 26695ΔcagE and 26695ΔcagA mutant cells were immunostained with anti-CagA and gold-labelled secondary antibody. 26695ΔcagA strain was used as a negative control for CagA antibody. Localisation of CagT and CagZ were used as a control for surface exposed and inner membrane proteins. Scale bar indicates 100 nm. Arrowheads indicate location of gold-labelled secondary antibody.
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pone.0142606.g007: Immunogold electron microscopy showing localisation of CagA in wild-type and 26695ΔcagE strains.Ultra thin sections of wild-type, 26695ΔcagE and 26695ΔcagA mutant cells were immunostained with anti-CagA and gold-labelled secondary antibody. 26695ΔcagA strain was used as a negative control for CagA antibody. Localisation of CagT and CagZ were used as a control for surface exposed and inner membrane proteins. Scale bar indicates 100 nm. Arrowheads indicate location of gold-labelled secondary antibody.

Mentions: Earlier it was reported that the VirB4 homologue in Hp, CagE is required for CagA translocation through Cag-T4SS and also needed for the secretion of IL-8 by the host gastric epithelial cells [37]. The prototypical VirB4 in Ag is also known to require for substrate translocation and pilus formation [40,42]. We therefore wanted to test its substrate translocation function and IL-8 induction in isogenic cagE deletion mutant strains 26695ΔcagE and P12ΔcagE respectively in our hand. In this regard, we first studied CagA surface localisation on isogenic 26695ΔcagE and P12ΔcagE strains by IFM. Wild-type strains 26695 and P12 were used as a positive control. As shown in Fig 6, unlike wild-type 26695 and P12 strains no CagA specific signal was detected on 26695ΔcagE and P12ΔcagE strains under non-permeabilised condition. However, when cagE function in P12ΔcagE was complemented with wild-type cagE allele in P12ΔcagE/cagE CagA signal was re-appeared on the bacterial cell surface under non-permeabilised condition (Fig 6). Similarly CagT and CagZ specific signals which do not depend on cagE function were also visualised under similar condition as controls for surface exposed protein and inner membrane protein respectively [31,32]. Further, we also studied surface localisation of CagA by TEM in wild-type 26695 and 26695ΔcagE strains. Unlike in wild-type strain, CagA specific signal was found to be absent on the cell surface in cagE deficient strain as reported earlier (Fig 7) [41,43]. CagT and CagZ specific signals were used as controls for outer and inner membrane proteins respectively [31,32]. As mentioned previously we employed P12ΔcagE strain instead of 26695ΔcagE for better transformability of the former strain [32]. To complement the cagE function another Hp strain was constructed where a wild-type cagE gene under cagA promoter was inserted into the P12ΔcagE chromosome in recA locus [32].


Biochemical Analysis of CagE: A VirB4 Homologue of Helicobacter pylori Cag-T4SS.

Shariq M, Kumar N, Kumari R, Kumar A, Subbarao N, Mukhopadhyay G - PLoS ONE (2015)

Immunogold electron microscopy showing localisation of CagA in wild-type and 26695ΔcagE strains.Ultra thin sections of wild-type, 26695ΔcagE and 26695ΔcagA mutant cells were immunostained with anti-CagA and gold-labelled secondary antibody. 26695ΔcagA strain was used as a negative control for CagA antibody. Localisation of CagT and CagZ were used as a control for surface exposed and inner membrane proteins. Scale bar indicates 100 nm. Arrowheads indicate location of gold-labelled secondary antibody.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643968&req=5

pone.0142606.g007: Immunogold electron microscopy showing localisation of CagA in wild-type and 26695ΔcagE strains.Ultra thin sections of wild-type, 26695ΔcagE and 26695ΔcagA mutant cells were immunostained with anti-CagA and gold-labelled secondary antibody. 26695ΔcagA strain was used as a negative control for CagA antibody. Localisation of CagT and CagZ were used as a control for surface exposed and inner membrane proteins. Scale bar indicates 100 nm. Arrowheads indicate location of gold-labelled secondary antibody.
Mentions: Earlier it was reported that the VirB4 homologue in Hp, CagE is required for CagA translocation through Cag-T4SS and also needed for the secretion of IL-8 by the host gastric epithelial cells [37]. The prototypical VirB4 in Ag is also known to require for substrate translocation and pilus formation [40,42]. We therefore wanted to test its substrate translocation function and IL-8 induction in isogenic cagE deletion mutant strains 26695ΔcagE and P12ΔcagE respectively in our hand. In this regard, we first studied CagA surface localisation on isogenic 26695ΔcagE and P12ΔcagE strains by IFM. Wild-type strains 26695 and P12 were used as a positive control. As shown in Fig 6, unlike wild-type 26695 and P12 strains no CagA specific signal was detected on 26695ΔcagE and P12ΔcagE strains under non-permeabilised condition. However, when cagE function in P12ΔcagE was complemented with wild-type cagE allele in P12ΔcagE/cagE CagA signal was re-appeared on the bacterial cell surface under non-permeabilised condition (Fig 6). Similarly CagT and CagZ specific signals which do not depend on cagE function were also visualised under similar condition as controls for surface exposed protein and inner membrane protein respectively [31,32]. Further, we also studied surface localisation of CagA by TEM in wild-type 26695 and 26695ΔcagE strains. Unlike in wild-type strain, CagA specific signal was found to be absent on the cell surface in cagE deficient strain as reported earlier (Fig 7) [41,43]. CagT and CagZ specific signals were used as controls for outer and inner membrane proteins respectively [31,32]. As mentioned previously we employed P12ΔcagE strain instead of 26695ΔcagE for better transformability of the former strain [32]. To complement the cagE function another Hp strain was constructed where a wild-type cagE gene under cagA promoter was inserted into the P12ΔcagE chromosome in recA locus [32].

Bottom Line: Here we have characterised the protein biochemically, genetically, and microscopically and report that CagE is an inner membrane associated active NTPase and has multiple interacting partners including the inner membrane proteins CagV and Cagβ.These results thus suggest the importance of CagE in Cag-T4SS functions.In future it may help in deciphering the mechanism of substrate translocation by the system.

View Article: PubMed Central - PubMed

Affiliation: Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.

ABSTRACT
Helicobacter pylori are among the most successful human pathogens that harbour a distinct genomic segment called cag Pathogenicity Island (cag-PAI). This genomic segment codes for a type IV secretion system (Cag-T4SS) related to the prototypical VirB/D4 system of Agrobacterium tumefaciens (Ag), a plant pathogen. Some of the components of Cag-T4SS share homology to that of VirB proteins including putative energy providing CagE (HP0544), the largest VirB4 homologue. In Ag, VirB4 is required for the assembly of the system, substrate translocation and pilus formation, however, very little is known about CagE. Here we have characterised the protein biochemically, genetically, and microscopically and report that CagE is an inner membrane associated active NTPase and has multiple interacting partners including the inner membrane proteins CagV and Cagβ. Through CagV it is connected to the outer membrane sub-complex proteins. Stability of CagE is not dependent on several of the cag-PAI proteins tested. However, localisation and stability of the pilus associated CagI, CagL and surface associated CagH are affected in its absence. Stability of the inner membrane associated energetic component Cagβ, a VirD4 homologue seems to be partially affected in its absence. Additionally, CagA failed to cross the membrane barriers in its absence and no IL-8 induction is observed under infection condition. These results thus suggest the importance of CagE in Cag-T4SS functions. In future it may help in deciphering the mechanism of substrate translocation by the system.

Show MeSH
Related in: MedlinePlus