ABSTRACTPrevious studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation byStreptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression ofbrpAis regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In aGalleria mellonella(wax worm) model, BrpA deficiency was shown to diminish the virulence ofS. mutansOMZ175, which, unlikeS. mutansUA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain ofS. mutans.

Background: Group A streptococcal pili are diverse. Results: Mutational analyses identified a specific transpeptidase and lysine residue of the shaft protein required for pilus assembly in a serotype M6 strain. Conclusion: Assembly and cell wall anchoring of the pili are accomplished by concerted actions of transpeptidases. Significance: Our findings lead to predictions of homologous pilus assembly mechanisms in other species.

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In this report, we define requirements for the successful translocation and functional maturation of the adhesin P1 of Streptococcus mutans. Conformational epitopes recognized by anti-P1 monoclonal antibodies (MAbs) were further characterized, thus facilitating the use of particular MAbs as tools to monitor the locations of various forms of the protein. We show that correct localization of P1 is dependent on structural features of the molecule itself, including a requisite A region-P region intramolecular interaction that occurs within the cell prior to secretion. P1 also was shown to be affected by several members of the protein-foldingsecretion-turnover apparatus. It does not achieve a fully functional form in the absence of the trigger factor PPIase homolog RopA, and its translocation is delayed when DnaK levels are limited. In addition, dnaK message levels are differentially altered in the presence of P1 lacking the alanine-rich compared to the proline-rich repeat domains. Lastly, nonsecreted P1 lacking the P region accumulates within the cell in the absence of htrA, implying an intracellular HtrA protease function in the degradation and turnover of this particular internal-deletion polypeptide. However, the opposite effect is seen for full-length P1, suggesting a sensing mechanism and substrate-dependent alteration in HtrA's function and effect that is consistent with its known ability to switch between chaperone and protease, depending on environmental perturbations.Streptococcus mutans is an oral bacterium that is a major etiologic agent of dental caries (19). The multidomain (M r , ϳ185,000) wall-associated multifunctional fibrillar cell surface protein P1 (15), encoded by spaP (28, 33), was originally identified as antigen (Ag) I/II (59). It is also called PAc (48), encoded by pac (49), and is widely understood to enable attachment of S. mutans to the acquired salivary pellicle on teeth. Ag I/II family proteins are expressed by most oral streptococci and mediate interactions with salivary constituents; host cell matrix proteins, such as fibronectin, fibrinogen, and collagen; and other oral bacteria (reviewed in reference 26). They share architectural features with a broad category of surface adhesins prevalent among streptococci and staphylococci called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), whose functions are dependent on complex structural elements (63). P1 contains a 38-residue amino-terminal signal sequence, a series of three 82-residue alanine-rich tandem repeats (A region); a 144-residue variable (V) region, where 20/36 amino acid differences between P1 and PAc are clustered (4); a central proline-rich (P) region, comprised of three 39-residue tandem repeats; and carboxy-terminal membrane-and wall-spanning regions, including the LPXTG motif characteristic of wall-anchored sortase substrates (14, 66). A schematic representation of the molecule and derivatives evaluated in this study is shown in Fig. 1.Several regions have been implicated in the ligand bindin...

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