Influence of BrpA on Critical Virulence Attributes ofStreptococcus mutans

Abstract

Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. The brpA gene codes for a predicted surfaceassociated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P < 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation in S. mutans, which are key virulence attributes of the organism.Streptococcus mutans, the primary etiological agent of human dental caries, exists almost exclusively in biofilms on tooth surfaces. The pathogenic potential of S. mutans is attributable to potent biofilm-forming abilities, a high capacity to produce acids, a high degree of acid tolerance, and the possession of high-affinity systems for the assimilation of many carbohydrate sources (9).Biofilms are surface-attached, structurally and compositionally complex bacterial communities (17,(31)(32)(33). Accumulating data suggest that bacterial cells in biofilms interact with and coordinate the expression of a wide range of genes in response to evolving environmental conditions, including pH, oxygen, carbon source and nutrient availability, cell density, and the presence of a solid surface. A series of highly coordinated physiological and biochemical functions is required for the bacteria to form mature biofilms in response to environmental cues (17,(31)(32)(33). The development of highly structured biofilms, however, gives the adherent populations the flexibility to cope with fluctuating environments and the selective advantages that surface association offers (17).Recent studies have revealed that several genetic regulatory networks in S. mutans are required for bacterial adherence, biofilm accumulation, and growth under the conditions encountered during biofilm formation. The cell-density-dependent Com system, which is known to regulate genetic competence in S. mutans and other naturally competent streptococci, plays a significant role in biofilm formation and acid tolerance (20, 21). The LuxS enzyme, which is responsible for th...

Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans

“…mutans can also function as a transcriptional regulator (Bitoun et al, 2012a(Bitoun et al, , 2013Wen et al, 2006). Previous studies showed that deficiency of BrpA caused substantial alterations in the transcriptional profile of the deficient mutant, including changes in transcripts involved in peptidoglycan biosynthesis, DNA repair and stress tolerance response, and biofilm formation (Bitoun et al, 2012a(Bitoun et al, , 2013Wen et al, 2006). Consistent with our previous findings that BrpA is a key regulator of the acid tolerance response in Strep.…”

“…Like several members of the LCP proteins (Hanson et al, 2011(Hanson et al, , 2012Lazarevic et al, 1992), BrpA in Strep. mutans can also function as a transcriptional regulator (Bitoun et al, 2012a(Bitoun et al, , 2013Wen et al, 2006). Previous studies showed that deficiency of BrpA caused substantial alterations in the transcriptional profile of the deficient mutant, including changes in transcripts involved in peptidoglycan biosynthesis, DNA repair and stress tolerance response, and biofilm formation (Bitoun et al, 2012a(Bitoun et al, , 2013Wen et al, 2006).…”

“…Different from the respective BrpB and BrpA single mutants, however, the BrpB/BrpA-deficient double mutant showed limited capacity to mount an adaptive acid tolerance response after initial incubation in low pH medium. Considering both the BrpA (Bitoun et al, 2013;Wen et al, 2006) and BrpB single mutants were still capable of launching an adaptive acid tolerance response, a logical explanation is that BrpB and BrpA double deficiency imposed an additive effect on the deficient mutant that severely damaged or destroyed the constitutive acid tolerance responses, such as those on cell envelope integrity and nucleoid structure. Even if not directly affected, the adaptive acid tolerance response in JB819 failed to sufficiently compensate the resulting defects in acid tolerance response.…”

“…1). Previous studies showed that BrpA deficiency did not result in any major effect on cell growth when cells were grown in regular BHI (Bitoun et al, 2013;Wen & Burne, 2002;Wen et al, 2006). When complemented with a wild-type copy of the brpA/brpB operon plus their cognate promoter(s) in multiple copy shuttle vector pDL:brpB/A, the complemented strain, JB819C, showed a further decreased doubling time with a level closer to the wild-type (Fig.…”

“…Relative to the wild-type, Strep. mutans strains deficient of BrpA displayed significant increases in susceptibility to cell envelope antimicrobials, and drastic reductions in acid and oxidative stress tolerance responses (Bitoun et al, 2012a;Chatfield et al, 2005;Wen et al, 2006). In addition, the deficient mutants also had an elevated autolysis rate and major defects in cell division, cell morphology and biofilm formation (Bitoun et al, 2012a(Bitoun et al, , 2013Wen et al, 2006).…”