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Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo.

Chimalapati S, Cohen JM, Camberlein E, MacDonald N, Durmort C, Vernet T, Hermans PW, Mitchell T, Brown JS - PLoS ONE (2012)

Bottom Line: Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface.The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations.These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium.

View Article: PubMed Central - PubMed

Affiliation: Centre for Respiratory Research, Department of Medicine, Royal Free and University College Medical School, Rayne Institute, London, United Kingdom.

ABSTRACT
Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection.

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Effect of the Δlgt mutation on surface accessability of S. pneumoniae lipoproteins.(A) Flow cytometry analysis of the mean proportion of bacteria positive for IgG binding after incubation with immune sera containing high antibody titres towards lipoproteins of S. pneumoniae. Black columns represent wild-type strain and clear columns represent the Δlgt strain. Error bars represent SDs and P values were obtained using multiple ANOVA test with post-hoc analysis. (B) Immunofluorescence of the S. pneumoniae wild-type (WT), Δlgt and ΔPpmA strains using anti- PpmA antibody and FITC conjugated secondary antibody. (C) Immunofluorescence of the S. pneumoniae wild-type (WT), Δlgt and ΔPhtD strains using anti-PhtD antibody and Cy2 conjugated secondary antibody.
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pone-0041393-g003: Effect of the Δlgt mutation on surface accessability of S. pneumoniae lipoproteins.(A) Flow cytometry analysis of the mean proportion of bacteria positive for IgG binding after incubation with immune sera containing high antibody titres towards lipoproteins of S. pneumoniae. Black columns represent wild-type strain and clear columns represent the Δlgt strain. Error bars represent SDs and P values were obtained using multiple ANOVA test with post-hoc analysis. (B) Immunofluorescence of the S. pneumoniae wild-type (WT), Δlgt and ΔPpmA strains using anti- PpmA antibody and FITC conjugated secondary antibody. (C) Immunofluorescence of the S. pneumoniae wild-type (WT), Δlgt and ΔPhtD strains using anti-PhtD antibody and Cy2 conjugated secondary antibody.

Mentions: To further confirm the reduced cell surface location of lipoproteins in the Δlgt strain, IgG binding to live S. pneumoniae wild-type and Δlgt strain bacteria after incubation in polyclonal mouse sera from mice vaccinated with a S. pneumoniae Δpab strain [46] was assessed using flow cytometry. This sera contains high IgG antibody titres to the lipoproteins PsaA and PpmA as well several non-lipoprotein antigens [46]. IgG binding to the Δlgt strain was significantly reduced compared to IgG binding to the wild-type strain, compatible with reduced IgG recognition of lipoproteins in the Δlgt strain due to their loss from the bacterial surface (Fig. 3A). Furthermore, immuno-fluorescence microscopy using polyclonal antibodies to PpmA identified significant fluorescence with wild-type S. pneumoniae but much reduced fluorescence for the Δlgt strain and no fluorescence for the negative control ΔppmA strain (Fig. 3B). In contrast, immunoflorescence microscopy using polyclonal antibodies to the cell wall protein PhtD was not affected by in the Δlgt strain (Fig. 3C). Taken together, the immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy demonstrate that the quantity of lipoproteins localised to the cell membrane and available for interactions with external agents is greatly reduced in the Δlgt strain.


Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo.

Chimalapati S, Cohen JM, Camberlein E, MacDonald N, Durmort C, Vernet T, Hermans PW, Mitchell T, Brown JS - PLoS ONE (2012)

Effect of the Δlgt mutation on surface accessability of S. pneumoniae lipoproteins.(A) Flow cytometry analysis of the mean proportion of bacteria positive for IgG binding after incubation with immune sera containing high antibody titres towards lipoproteins of S. pneumoniae. Black columns represent wild-type strain and clear columns represent the Δlgt strain. Error bars represent SDs and P values were obtained using multiple ANOVA test with post-hoc analysis. (B) Immunofluorescence of the S. pneumoniae wild-type (WT), Δlgt and ΔPpmA strains using anti- PpmA antibody and FITC conjugated secondary antibody. (C) Immunofluorescence of the S. pneumoniae wild-type (WT), Δlgt and ΔPhtD strains using anti-PhtD antibody and Cy2 conjugated secondary antibody.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3404074&req=5

pone-0041393-g003: Effect of the Δlgt mutation on surface accessability of S. pneumoniae lipoproteins.(A) Flow cytometry analysis of the mean proportion of bacteria positive for IgG binding after incubation with immune sera containing high antibody titres towards lipoproteins of S. pneumoniae. Black columns represent wild-type strain and clear columns represent the Δlgt strain. Error bars represent SDs and P values were obtained using multiple ANOVA test with post-hoc analysis. (B) Immunofluorescence of the S. pneumoniae wild-type (WT), Δlgt and ΔPpmA strains using anti- PpmA antibody and FITC conjugated secondary antibody. (C) Immunofluorescence of the S. pneumoniae wild-type (WT), Δlgt and ΔPhtD strains using anti-PhtD antibody and Cy2 conjugated secondary antibody.
Mentions: To further confirm the reduced cell surface location of lipoproteins in the Δlgt strain, IgG binding to live S. pneumoniae wild-type and Δlgt strain bacteria after incubation in polyclonal mouse sera from mice vaccinated with a S. pneumoniae Δpab strain [46] was assessed using flow cytometry. This sera contains high IgG antibody titres to the lipoproteins PsaA and PpmA as well several non-lipoprotein antigens [46]. IgG binding to the Δlgt strain was significantly reduced compared to IgG binding to the wild-type strain, compatible with reduced IgG recognition of lipoproteins in the Δlgt strain due to their loss from the bacterial surface (Fig. 3A). Furthermore, immuno-fluorescence microscopy using polyclonal antibodies to PpmA identified significant fluorescence with wild-type S. pneumoniae but much reduced fluorescence for the Δlgt strain and no fluorescence for the negative control ΔppmA strain (Fig. 3B). In contrast, immunoflorescence microscopy using polyclonal antibodies to the cell wall protein PhtD was not affected by in the Δlgt strain (Fig. 3C). Taken together, the immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy demonstrate that the quantity of lipoproteins localised to the cell membrane and available for interactions with external agents is greatly reduced in the Δlgt strain.

Bottom Line: Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface.The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations.These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium.

View Article: PubMed Central - PubMed

Affiliation: Centre for Respiratory Research, Department of Medicine, Royal Free and University College Medical School, Rayne Institute, London, United Kingdom.

ABSTRACT
Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection.

Show MeSH
Related in: MedlinePlus