Pneumococcal surface protein C (PspC) binds to both human secretory immunoglobulin A (sIgA) and complement factor H (FH). FH, a regulator of the alternative pathway of complement, can also mediate adherence of different host cells. Since PspC contributes to adherence and invasion of host cells, we hypothesized that the interaction of PspC with FH may also mediate adherence of pneumococci to human cells. In this study, we investigated FH-and sIgA-mediated pneumococcal adherence to human cell lines in vitro. Adherence assays demonstrated that preincubation of Streptococcus pneumoniae D39 with FH increased adherence to human umbilical vein endothelial cells (HUVEC) 5-fold and to lung epithelial cells (SK-MES-1) 18-fold, relative to that of D39 without FH on the surface. The presence of sIgA enhanced adherence to SK-MES-1 6-fold and to pharyngeal epithelial cells (Detroit 562) 14-fold. Furthermore, sIgA had an additive effect on adherence to HUVEC; specifically, preincubation of D39 with both FH and sIgA led to a 21-fold increase in adherence. Finally, using a mouse model, we examined the significance of the FH-PspC interaction in pneumococcal nasal colonization and lung invasion. Mice intranasally infected with D39 preincubated with FH had increased bacteremia and lung invasion, but they had similar levels of nasopharyngeal colonization compared to that of mice challenged with D39 without FH.
PcsB of Streptococcus pneumoniae is an essential hydrolase involved in the separation of dividing cells. In this study, it was found that PcsB localizes to the plasma membrane and is released into the growth environment, yet it is detectable on the pneumococcal surface by flow cytometry analysis. High temperature and osmolarity led to upregulation of pcsB expression.Streptococcus pneumoniae colonizes the human nasopharynx and is a significant cause of morbidity (12). A DNA microarray based on the open reading frames of S. pneumoniae TIGR4 was used to measure differential gene regulation in S. pneumoniae D39 grown in subinhibitory concentrations of penicillin (Pen) or clarithromycin (Cln). One notable upregulated gene was pcsB (protein required for cell separation in group B streptococci [14]; The Institute for Genomic Research annotation SP2216). Based on the array results, expression of pcsB was upregulated 5.69-and 2.63-fold in response to the presence of 0.5ϫ MIC of Pen and Cln, respectively, relative to growth in media with no antibiotic added (8).PcsB of S. pneumoniae shows 37% identity and 50% similarity to IDG60 of Streptococcus mutans. IDG60 is not essential, and deletion mutants display pleomorphic morphology and increased sensitivity to stress conditions (2). Conditions of low pH, high salt, and high temperature upregulate IDG60 expression (3).In contrast, PcsB was found to be essential in S. pneumoniae (15). Mutants with decreased levels of PcsB could be obtained only when the native promoter was replaced with an inducible or constitutive promoter (14). PcsB is the only essential hydrolase identified at present in S. pneumoniae (15).Based on Signal P (1, 16) and PSORT (13) algorithms, PcsB has a signal peptidase I motif but no other motifs that would otherwise predict its localization (21). The purpose of this study was to determine cellular localization and confirm pcsB upregulation in response to stress.Bacterial strains and growth conditions. Pneumococcal strains D39 (serotype 2) and WU2 (serotype 3) and their respective nonencapsulated derivatives, R6 and JD908 (11), were used in this study. Pneumococci were cultured on sheep blood agar plates overnight at 37°C in 5% CO 2 or grown in ToddHewitt broth (Difco, Detroit, MI) supplemented with 0.5% yeast extract (THY) at 37°C.rPcsB. PCR was used to amplify the portion of pcsB corresponding to amino acids 28 to 392 (excluding the leader sequence) with primers 565F and 565R (Table 1). The PCR product was cloned into pET100 vector (Invitrogen, Carlsbad, CA), which incorporates an N-terminal His 6 tag to facilitate purification, and cloning success was confirmed by sequencing (SeqWright, Houston, TX). Recombinant PcsB (rPcsB) was purified and stored in phosphate-buffered saline (PBS) at Ϫ20°C until use. The recombinant protein migrated to the expected position as confirmed by Western analysis using Pierce INDIA His probe-horseradish peroxidase (data not shown).Rabbit polyclonal serum. A New Zealand White rabbit was subcutaneously immunized with a mixture o...
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