The polymeric immunoglobulin receptor (pIgR) plays a crucial role in mucosal immunity against microbial infection by transporting polymeric immunoglobulins (pIg) across the mucosal epithelium. We report here that the human pIgR (hpIgR) can bind to a major pneumococcal adhesin, CbpA. Expression of hpIgR in human nasopharyngeal cells and MDCK cells greatly enhanced pneumococcal adherence and invasion. The hpIgR-mediated bacterial adherence and invasion were abolished by either insertional knockout of cbpA or antibodies against either hpIgR or CbpA. In contrast, rabbit pIgR (rpIgR) did not bind to CbpA and its expression in MDCK cells did not enhance pneumococcal adherence and invasion. These results suggest that pneumococci are a novel example of a pathogen co-opting the pIg transcytosis machinery to promote translocation across a mucosal barrier.
Genome‐wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae. However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn‐seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high‐content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi‐based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp‐proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP‐dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.
Streptococcus pneumoniae, a human pathogen, is naturally capable of colonizing the upper airway and sometimes disseminating to remote tissue sites. Previous studies have shown that S. pneumoniae is able to evade complement-mediated innate immunity by recruiting complement factor H (FH), a complement alternative pathway inhibitor. Pneumococcal binding to FH has been attributed to choline-binding protein A (CbpA) of S. pneumoniae and its allelic variants, all of which are surface-exposed proteins. In this study, we sought to determine the molecular basis of the CbpA-FH binding interaction. Initial deletional analysis of the CbpA protein in strain D39 (capsular serotype 2) revealed that the N-terminal region of 89 amino acids in the mature CbpA protein is required for FH binding. Immunofluorescence microscopy analysis showed that this region of CbpA is also necessary for FH deposition to the surface of the intact pneumococci. Moreover, recombinant proteins representing the 104 amino acids of the N-terminal CbpA alone was sufficient for high affinity binding to FH (K D < 1 nM). The FH binding activity was finally localized to a 12-amino acid motif in the N-terminal CbpA by peptide mapping. Further kinetic analysis suggested that additional amino acids downstream of the 12-amino acid motif provide necessary structural or conformational support for the CbpA-FH interaction. The 12-amino acid motif and its adjacent regions contain highly conserved residues among various CbpA alleles, suggesting that this region may mediate FH binding in multiple pneumococcal strains.Streptococcus pneumoniae, a Gram-positive bacterium, is a natural resident of the human upper airway. It is also an important human pathogen that causes a wide spectrum of bacterial infections including community-acquired pneumonia, otitis media, meningitis, bacteremia, and sinusitis (1). In healthy human adults, pneumococcal colonization can last weeks or months without apparent illness (2). Epidemiological studies suggest that 20 -50% of healthy individuals may carry pneumococci at any given time (2), demonstrating that S. pneumoniae is capable of withstanding host defense mechanisms. Both innate and adaptive immune responses are critical for limiting and clearing pneumococcal infections (1). Complement-mediated phagocytosis is a major innate immune mechanism against pneumococci (3, 4). The products of activated complement bound on pneumococci enhance phagocytosis by binding to complement receptors on professional phagocytes (5). Internalized pneumococci within phagocytes are killed in phagolysosomes (6). Consistent with the key role of the complement system in bacterial clearance, recent studies have indicated that S. pneumoniae is able to evade complement activation. PspA (pneumococcal surface protein A) has been shown to inhibit complement activation in mouse models, probably by blocking recruitment of the alternative pathway (4). Mice infected with PspA mutants showed significantly attenuated levels of colonization and bacteremia (7). Pneumolysin, a ma...
SummaryPhosphorylcholine is an important bioactive adduct to the teichoic acid (TA) and lipoteichoic acid (LTA) of the surface of Streptococcus pneumoniae. We have identi®ed and characterized a genetic locus lic that is required for phosphorylcholine metabolism in S. pneumoniae.
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