Dataset Information

Pneumococcal LytR Protein Is Required for the Surface Attachment of Both Capsular Polysaccharide and Teichoic Acids: Essential for Pneumococcal Virulence.

ABSTRACT: The LytR-Cps-Psr family proteins are commonly present in Gram-positive bacteria, which have been shown to implicate in anchoring cell wall-related glycopolymers to the peptidoglycan. Here, we report the cellular function of SPD_1741 (LytR) in Streptococcus pneumoniae and its role in virulence of pneumococci. Pneumococcal ?lytR mutants have been successfully constructed by replacing the lytR gene with erm cassette. The role of LytR in pneumococcal growth was determined by growth experiments, and surface accessibility of the LytR protein was analyzed using flow cytometry. Transmission electron microscopy (TEM) and immunoblotting were used to reveal the changes in capsular polysaccharide (CPS). Dot blot and ELISA were used to quantify the amount of teichoic acids (TAs). The contribution of LytR on bacterial virulence was assessed using in vitro phagocytosis assays and infection experiments. Compared to the wild-type strain, the ?lytR mutant showed a defect in growth which merely grew to a maximal OD₆₂₀ of 0.2 in the liquid medium. The growth of the ?lytR mutant could be restored by addition of recombinant ?TM-LytR protein in culture medium in a dose-dependent manner. TEM results showed that the D39?lytR mutant was impaired in the surface attachment of CPS. Deletion of lytR gene also impaired the retention of TAs on the surface of pneumococci. The reduction of CPS and TAs on the pneumocccal cells were confirmed using Dot blot and ELISA assays. Compared to wild-type D39, the ?lytR mutant was more susceptible to the phagocytosis. Animal studies showed that the ability to colonize the nasophaynx and virulence of pneumococci were affected by impairment of the lytR gene. Collectively, these results suggest that pneumococcal LytR is involved in anchoring both the CPS and TAs to cell wall, which is important for virulence of pneumococci.

SUBMITTER: Ye W

PROVIDER: S-EPMC6008509 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Pneumococcal LytR Protein Is Required for the Surface Attachment of Both Capsular Polysaccharide and Teichoic Acids: Essential for Pneumococcal Virulence.

Ye Weijie W Zhang Jinghui J Shu Zhaoche Z Yin Yibing Y Zhang Xuemei X Wu Kaifeng K

Frontiers in microbiology 20180613

The LytR-Cps-Psr family proteins are commonly present in Gram-positive bacteria, which have been shown to implicate in anchoring cell wall-related glycopolymers to the peptidoglycan. Here, we report the cellular function of SPD_1741 (LytR) in Streptococcus pneumoniae and its role in virulence of pneumococci. Pneumococcal ΔlytR mutants have been successfully constructed by replacing the lytR gene with erm cassette. The role of LytR in pneumococcal growth was determined ...[more]

PMID: 29951042

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Project description:UnlabelledThe use of pneumococcal capsular polysaccharide (PPS)-based vaccines has resulted in a substantial reduction in invasive pneumococcal disease. However, much remains to be learned about vaccine-mediated immunity, as seven-valent PPS-protein conjugate vaccine use in children has been associated with nonvaccine serotype replacement and 23-valent vaccine use in adults has not prevented pneumococcal pneumonia. In this report, we demonstrate that certain PPS-specific monoclonal antibodies (MAbs) enhance the transformation frequency of two different Streptococcus pneumoniae serotypes. This phenomenon was mediated by PPS-specific MAbs that agglutinate but do not promote opsonic effector cell killing of the homologous serotype in vitro. Compared to the autoinducer, competence-stimulating peptide (CSP) alone, transcriptional profiling of pneumococcal gene expression after incubation with CSP and one such MAb to the PPS of serotype 3 revealed changes in the expression of competence (com)-related and bacteriocin-like peptide (blp) genes involved in pneumococcal quorum sensing. This MAb was also found to induce a nearly 2-fold increase in CSP2-mediated bacterial killing or fratricide. These observations reveal a novel, direct effect of PPS-binding MAbs on pneumococcal biology that has important implications for antibody immunity to pneumococcus in the pneumococcal vaccine era. Taken together, our data suggest heretofore unsuspected mechanisms by which PPS-specific antibodies could affect genetic exchange and bacterial viability in the absence of host cells.ImportanceCurrent thought holds that pneumococcal capsular polysaccharide (PPS)-binding antibodies protect against pneumococcus by inducing effector cell opsonic killing of the homologous serotype. While such antibodies are an important part of how pneumococcal vaccines protect against pneumococcal disease, PPS-specific antibodies that do not exhibit this activity but are highly protective against pneumococcus in mice have been identified. This article examines the effect of nonopsonic PPS-specific monoclonal antibodies (MAbs) on the biology of Streptococcus pneumoniae. The results showed that in the presence of a competence-stimulating peptide (CSP), such MAbs increase the frequency of pneumococcal transformation. Further studies with one such MAb showed that it altered the expression of genes involved in quorum sensing and increased competence-induced killing or fratricide. These findings reveal a novel, previously unsuspected mechanism by which certain PPS-specific antibodies exert a direct effect on pneumococcal biology that has broad implications for bacterial clearance, genetic exchange, and antibody immunity to pneumococcus.

Project description:The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively. Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively. Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B. Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

Dataset Information

Pneumococcal LytR Protein Is Required for the Surface Attachment of Both Capsular Polysaccharide and Teichoic Acids: Essential for Pneumococcal Virulence.

Publications

Pneumococcal LytR Protein Is Required for the Surface Attachment of Both Capsular Polysaccharide and Teichoic Acids: Essential for Pneumococcal Virulence.

Similar Datasets

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