Effects of Deletion of the Streptococcus pneumoniae Lipoprotein Diacylglyceryl Transferase Gene lgt on ABC Transporter Function and on Growth In Vivo

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 Δ lgt 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 Δ lgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δ lgt 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 Δ lgt 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 Δ lgt mutant were associated with only slightly delayed growth in complete medium. However the Δ lgt 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 Δ lgt mutant from establishing invasive infection.

TH17-Mediated Protection against Pneumococcal Carriage by a Whole-Cell Vaccine Is Dependent on Toll-Like Receptor 2 and Surface Lipoproteins

“…For several Gram-positive bacteria, others demonstrated that, in the absence of functional Lgt, prolipoproteins were in decreased abundance in the cell membrane but in greater abundance in culture supernatants (11,21). This was also confirmed for a S. pneumoniae lgt deletion mutant (10). We constructed an lgt deletion mutant in our vaccine background strain, strain RM200, by insertional deletion of a spectinomycin resistance cassette in place of lgt.…”

“…To address this question, we generated an lgt deletion mutant in our colonization strain, clinical serotype 6B pneumococcus 0603. Deletion of lgt in other pneumococcal strains has been shown to attenuate the ability of the bacteria to colonize, as evidenced by earlier clearance of ⌬lgt pneumococci from nasal washes from mice following intranasal infection (10). To evaluate whether our mutant 0603 strain could successfully colonize mice, we intranasally challenged naive C57BL/6 mice with the live ⌬lgt 0603 mutant strain.…”

“…The surface expression of pneumococcal lipoproteins has been demonstrated to be critical for TLR2-dependent proinflammatory responses from macrophages stimulated in vitro and immune responses in a mouse pneumonia model (16). This requirement for surface localization of lipoproteins for pneumococcal virulence has also been suggested in models of sepsis via intraperitoneal infection and in pneumonia (10). Here we demonstrate a requirement for TLR2 in WCV-mediated immune responses and protection against NP colonization, and we use the WCV with the lgt deletion to evaluate the effects of surface localization of lipoproteins on the proinflammatory, immunogenic, and protective potential of this WCV mutant.…”

“…Extracellularly secreted pneumococcal prolipoproteins are covalently linked to the cell membrane by Lgt-mediated attachment of a membrane diacylglyceride to a cysteine residue within the lipoprotein signal peptide, via a conserved mechanism for posttranslational lipidation of prolipoproteins (8). Deletion of lgt in S. pneumoniae and other bacteria results in decreases in the lipoprotein contents of cellular membrane extracts and greater prolipoprotein contents in culture supernatants (9)(10)(11)(12). Bacterial lipoproteins are potent ligands for TLR2 (13) and are associated with proinflammatory cytokine stimulation (14,15).…”

Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence

“…S. aureus and its mutants lacking lipoteichoic acid (⌬ltaS) or D-alanylation of teichoic acids (⌬dltA) stimulated NO production in a murine macrophage cell line; however, the ⌬lgt mutant failed to induce NO production in a dose-dependent manner (36). These results suggest that not lipoteichoic acid (LTA) but Lpp of S. aureus induce NO production in murine Like many other bacteria, S. pneumoniae ⌬lgt mutants hardly can grow in blood, bronchoalveolar lavage fluid, or cation-depleted medium, and virulence is attenuated in mouse models such as nasopharyngeal colonization, where sepsis or pneumonia was significantly decreased (38). Furthermore, in ⌬lgt mutants, TLR2 signaling is significantly decreased, and it appears that the leukocyte responses to Lpp are required for TLR2-and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae (39).…”