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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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(A) Western blots showing stability of inner membrane and pilus associated Cag proteins in the absence of CagE in 26695ΔcagE.Antibodies used in Western blots are indicated. CagA and OMP are used as loading controls. (B) Western blots showing stability of pilus associated CagH, CagI and CagL in wild-type P12, P12ΔcagE and P12ΔcagE/cagE. CagT was used as a loading control. Antibodies used in Western blotting are indicated.
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pone.0142606.g004: (A) Western blots showing stability of inner membrane and pilus associated Cag proteins in the absence of CagE in 26695ΔcagE.Antibodies used in Western blots are indicated. CagA and OMP are used as loading controls. (B) Western blots showing stability of pilus associated CagH, CagI and CagL in wild-type P12, P12ΔcagE and P12ΔcagE/cagE. CagT was used as a loading control. Antibodies used in Western blotting are indicated.

Mentions: We therefore asked the question how CagE affects pilus formation. To search for answers, we first looked into the stability and localisation of known Cag-T4SS inner membrane proteins CagV, Cagβ, CagZ, CagF and pilus associated proteins CagI, CagL and its predicted regulator CagH in the absence of cagE in Hp26695ΔcagE and P12ΔcagE strains [39,41]. Cell extracts from wild-type 26695, P12, mutants 26695ΔcagE, P12ΔcagE, and cagE complemented P12ΔcagE/cagE strains were prepared, separated in SDS-PAGE and subjected to the Western blot analysis using the indicated antibodies. As shown in Fig 4A, the stability of none of the proteins were affected in the absence of CagE, except that of Cagβ in 26695ΔcagE. Stability of Cagβ is slightly reduced compared to the wild-type strain. However, unlike in 26695ΔcagE, stability of CagI, CagL and CagH were found to be affected in P12ΔcagE strain (Fig 4B). Nonetheless, complementation of the wild-type function restored back the native status in P12ΔcagE/cagE (Fig 4B). P12ΔcagE deletion mutant strain was employed here to overcome certain technical difficulties associated with the strain 26695 in gene complementation studies. It is worth mentioning at this point that Hp strain 26695 has unusually very low gene complementation ability for certain sets of gene compared to strain like P12 [32]. CagA and OMP were used as loading controls for the stability studies in 26695, while CagT was used as loading control in P12 studies. Next, we looked into the localisation of CagI, CagL and CagH in the mutant strains 26695ΔcagE and P12ΔcagE and cagE complemented strain P12ΔcagE/cagE by cell fractionation assay. As shown in Fig 5A, pilus associated CagI, CagL and surface exposed CagH were recovered in the membrane fraction only from 26695ΔcagE mutant strain. On the other hand in wild-type cell extracts these proteins were detected in both the soluble and membrane fractions. Although these proteins were found to be unstable in P12ΔcagE, still the residual proteins were detected only in the membrane fraction (Fig 5B, see arrow). Nonetheless, when the mutant gene was complemented with wild-type cagE allele in P12ΔcagE/cagE strain the wild-type status was restored back (Fig 5B). These results therefore suggest that absence of CagE made these proteins mis-localised in 26695ΔcagE while unstable and mis-localised in P12ΔcagE.


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)

(A) Western blots showing stability of inner membrane and pilus associated Cag proteins in the absence of CagE in 26695ΔcagE.Antibodies used in Western blots are indicated. CagA and OMP are used as loading controls. (B) Western blots showing stability of pilus associated CagH, CagI and CagL in wild-type P12, P12ΔcagE and P12ΔcagE/cagE. CagT was used as a loading control. Antibodies used in Western blotting are indicated.
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pone.0142606.g004: (A) Western blots showing stability of inner membrane and pilus associated Cag proteins in the absence of CagE in 26695ΔcagE.Antibodies used in Western blots are indicated. CagA and OMP are used as loading controls. (B) Western blots showing stability of pilus associated CagH, CagI and CagL in wild-type P12, P12ΔcagE and P12ΔcagE/cagE. CagT was used as a loading control. Antibodies used in Western blotting are indicated.
Mentions: We therefore asked the question how CagE affects pilus formation. To search for answers, we first looked into the stability and localisation of known Cag-T4SS inner membrane proteins CagV, Cagβ, CagZ, CagF and pilus associated proteins CagI, CagL and its predicted regulator CagH in the absence of cagE in Hp26695ΔcagE and P12ΔcagE strains [39,41]. Cell extracts from wild-type 26695, P12, mutants 26695ΔcagE, P12ΔcagE, and cagE complemented P12ΔcagE/cagE strains were prepared, separated in SDS-PAGE and subjected to the Western blot analysis using the indicated antibodies. As shown in Fig 4A, the stability of none of the proteins were affected in the absence of CagE, except that of Cagβ in 26695ΔcagE. Stability of Cagβ is slightly reduced compared to the wild-type strain. However, unlike in 26695ΔcagE, stability of CagI, CagL and CagH were found to be affected in P12ΔcagE strain (Fig 4B). Nonetheless, complementation of the wild-type function restored back the native status in P12ΔcagE/cagE (Fig 4B). P12ΔcagE deletion mutant strain was employed here to overcome certain technical difficulties associated with the strain 26695 in gene complementation studies. It is worth mentioning at this point that Hp strain 26695 has unusually very low gene complementation ability for certain sets of gene compared to strain like P12 [32]. CagA and OMP were used as loading controls for the stability studies in 26695, while CagT was used as loading control in P12 studies. Next, we looked into the localisation of CagI, CagL and CagH in the mutant strains 26695ΔcagE and P12ΔcagE and cagE complemented strain P12ΔcagE/cagE by cell fractionation assay. As shown in Fig 5A, pilus associated CagI, CagL and surface exposed CagH were recovered in the membrane fraction only from 26695ΔcagE mutant strain. On the other hand in wild-type cell extracts these proteins were detected in both the soluble and membrane fractions. Although these proteins were found to be unstable in P12ΔcagE, still the residual proteins were detected only in the membrane fraction (Fig 5B, see arrow). Nonetheless, when the mutant gene was complemented with wild-type cagE allele in P12ΔcagE/cagE strain the wild-type status was restored back (Fig 5B). These results therefore suggest that absence of CagE made these proteins mis-localised in 26695ΔcagE while unstable and mis-localised in P12ΔcagE.

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