ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae

Abstract

The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComED58E) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level.

SPD_1495 Contributes to Capsular Polysaccharide Synthesis and Virulence in Streptococcus pneumoniae

“…4C). A previous study by Zheng et al identified the transcriptional regulator in the upstream promoter region of cps by DNA-pulldown but did not find SPD_1495 (9). Therefore, we deduce that SPD_1495 may negatively regulate CPS formation indirectly.…”

“…The identification results showed that SPD_1495 can interact with several proteins, including RpsC, ComE, SPD_1495, RecA, CshB, and CoaC (Table 2). Notably, ComE exists in two forms (nonphosphorylated and phosphorylated) in S. pneumoniae, and only phosphorylated form ComE negatively regulates the expression of the cps gene cluster via an interaction with the upstream promoter of cps (9). Thus, to further uncover the function of SPD_1495 in the formation of CPS, a phosphomimetic form (ComE D58E ) was expressed and purified to check its interaction with SPD_1495 by surface plasmon resonance (SPR).…”

“…A phosphomimetic mutant, ComE D58E , was constructed by site-directed mutagenesis of comE to exhibit the activity of ComE protein in vitro. Both wild-type ComE and ComE D58E were expressed and purified as described previously (9,33,34). The primers are listed in Table 4 (ComE-F to 16S rRNA-R).…”

“…When S. pneumoniae escapes the nasopharynx, it invades the lungs and subsequently invades the bloodstream. During invasion, CPS is highly expressed to mask the surface antigens in order to reduce complement deposition and protect the bacterium against opsonophagocytosis (4,8,9).…”

“…(10,(13)(14)(15). Further, several possible transcriptional regulators of the cps locus, such as RegM (16), ComX (17), CopY (18), CpsR (19), GlnR (20), RitR (21), and ComE (9), have also been reported to regulate CPS formation. In particular, ComE exists in S. pneumoniae in nonphosphorylated and phosphorylated forms, and only the latter form can negatively regulate the expression of the cps gene cluster via interaction with the upstream promoter of cps in vivo (9).…”