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

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

Protective Contributions against Invasive Streptococcus pneumoniae Pneumonia of Antibody and Th17-Cell Responses to Nasopharyngeal Colonisation

The nasopharyngeal commensal bacteria Streptococcus pneumoniae is also a frequent cause of serious infections. Nasopharyngeal colonisation with S. pneumoniae inhibits subsequent re-colonisation by inducing Th17-cell adaptive responses, whereas vaccination prevents invasive infections by inducing antibodies to S. pneumoniae capsular polysaccharides. In contrast, protection against invasive infection after nasopharyngeal colonisation with mutant S. pneumoniae strains was associated with antibody responses to protein antigens. The role of colonisation-induced Th17-cell responses during subsequent invasive infections is unknown. Using mouse models, we show that previous colonisation with S. pneumoniae protects against subsequent lethal pneumonia mainly by preventing bacteraemia with a more modest effect on local control of infection within the lung. Previous colonisation resulted in CD4-dependent increased levels of Th17-cell cytokines during subsequent infectious challenge. However, mice depleted of CD4 cells prior to challenge remained protected against bacteraemia, whereas no protection was seen in antibody deficient mice and similar protection could be achieved through passive transfer of serum. Serum from colonised mice but not antibody deficient mice promoted phagocytosis of S. pneumoniae, and previously colonised mice were able to rapidly clear S. pneumoniae from the blood after intravenous inoculation. Thus, despite priming for a Th17-cell response during subsequent infection, the protective effects of prior colonisation in this model was not dependent on CD4 cells but on rapid clearance of bacteria from the blood by antibody-mediated phagocytosis. These data suggest that whilst nasopharyngeal colonisation induces a range of immune responses, the effective protective responses depend upon the site of subsequent infectio

The effects of methionine acquisition and synthesis on Streptococcus pneumoniae growth and virulence

Extent: 14 p.Bacterial pathogens need to acquire nutrients from the host, but for many nutrients their importance during infection remain poorly understood. We have investigated the importance of methionine acquisition and synthesis for Streptococcus pneumoniae growth and virulence using strains with gene deletions affecting a putative methionine ABC transporter lipoprotein (Sp_0149, metQ) and/or methionine biosynthesis enzymes (Sp_0585 - Sp_0586, metE and metF). Immunoblot analysis confirmed MetQ was a lipoprotein and present in all S. pneumoniae strains investigated. However, vaccination with MetQ did not prevent fatal S. pneumoniae infection in mice despite stimulating a strong specific IgG response. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry demonstrated that MetQ has both a high affinity and specificity for L-methionine with a KD of ~ 25 nM, and a DmetQ strain had reduced uptake of C14-methionine. Growth of the ΔmetQ/ΔmetEF strain was greatly impaired in chemically defined medium containing low concentrations of methionine and in blood but was partially restored by addition of high concentrations of exogenous methionine. Mixed infection models showed no attenuation of the ΔmetQ, ΔmetEF and ΔmetQ/DmetEF strains in their ability to colonise the mouse nasopharnyx. In a mouse model of systemic infection although significant infection was established in all mice, there were reduced spleen bacterial CFU after infection with the ΔmetQ/ΔmetEF strain compared to the wild-type strain. These data demonstrate that Sp_0149 encodes a high affinity methionine ABC transporter lipoprotein and that Sp_0585 – Sp_0586 are likely to be required for methionine synthesis. Although Sp_0149 and Sp_0585-Sp_0586 make a contribution towards full virulence, neither was essential for S. pneumoniae survival during infection.Shilpa Basavanna, Suneeta Chimalapati, Abbas Maqbool, Bruna Rubbo, Jose Yuste, Robert J. Wilson, Arthur Hosie, Abiodun D. Ogunniyi, James C. Paton, Gavin Thomas and Jeremy S. Brow

The Effects of PspC on Complement-Mediated Immunity to Streptococcus pneumoniae Vary with Strain Background and Capsular Serotype▿

Streptococcus pneumoniae may evade complement activity by binding of factor H (FH), a negative regulator of the alternative pathway, to the surface protein PspC. However, existing data on the effects of FH binding to PspC on complement activity are conflicting, and there is also considerable allelic variation in PspC structure between S. pneumoniae strains that may influence PspC-dependent effects on complement. We have investigated interactions with complement for several S. pneumoniae strains in which the gene encoding PspC has been deleted. The degree of FH binding varied between strains and was entirely dependent on PspC for seven strains. Data obtained with TIGR4 strains expressing different capsular serotypes suggest that FH binding is affected by capsular serotype. Results of immunoblot analysis for C3 degradation products and iC3b deposition assays suggested that FH bound to PspC retained functional activity, but loss of PspC had strikingly varied effects on C3b/iC3b deposition on S. pneumoniae, with large increases on serotype 4, 6A, 6B, and 9V strains but only small increases or even decreases on serotype 2, 3, 17, and 23F strains. Repeating C3b/iC3b assays with TIGR4 strains expressing different capsular serotypes suggested that differences in the effect of PspC on C3b/iC3b deposition were largely independent of capsular serotype and depend on strain background. However, data obtained from infection in complement-deficient mice demonstrated that differences between strains in the effects of PspC on complement surprisingly did not influence the development of septicemia

Immunoelectron microscopy of thin sections of the wild-type and Δ0928 strains using antibodies to the PpmA and SlrA lipoproteins

<p><b>Copyright information:</b></p><p>Taken from "Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence"</p><p></p><p>Molecular Microbiology 2007;67(3):541-557.</p><p>Published online 16 Dec 2007</p><p>PMCID:PMC2228790.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> Controls with bacteria stained with the secondary antibody alone demonstrated only one or two gold particles per cell (not shown), and controls probed with anti-Ply are included to demonstrate the pattern of protein expression for an intracellular protein. The scale bar in the top right panel is 0.2 μm long

Coomassie stained SDS-PAGE of Triton X-114 extracts from wild-type, 0928C and Δ0928 strains

<p><b>Copyright information:</b></p><p>Taken from "Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence"</p><p></p><p>Molecular Microbiology 2007;67(3):541-557.</p><p>Published online 16 Dec 2007</p><p>PMCID:PMC2228790.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> Numbered lines correspond to the band numbers shown in , and the gels are aligned so that the migration positions of molecular mass markers (marked for the Δ0928 strain gel) are identical between the three gels

Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence-9

<p><b>Copyright information:</b></p><p>Taken from "Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence"</p><p></p><p>Molecular Microbiology 2007;67(3):541-557.</p><p>Published online 16 Dec 2007</p><p>PMCID:PMC2228790.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence-8

<p><b>Copyright information:</b></p><p>Taken from "Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence"</p><p></p><p>Molecular Microbiology 2007;67(3):541-557.</p><p>Published online 16 Dec 2007</p><p>PMCID:PMC2228790.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence-4

<p><b>Copyright information:</b></p><p>Taken from "Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence"</p><p></p><p>Molecular Microbiology 2007;67(3):541-557.</p><p>Published online 16 Dec 2007</p><p>PMCID:PMC2228790.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence-0

<p><b>Copyright information:</b></p><p>Taken from "Maturation of lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence"</p><p></p><p>Molecular Microbiology 2007;67(3):541-557.</p><p>Published online 16 Dec 2007</p><p>PMCID:PMC2228790.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p