MUTACIN IV PRODUCTION IN STREPTOCOCCUS MUTANS UA159: CHARACTERIZATION, REGULATION, AND MECHANISM OF SELF-IMMUNITY

Streptococcus mutans, an oral pathogen, is considered as a principle etiological agent of dental caries. To colonize successfully in the oral cavity, this organism encounters fierce competition with other oral microbiota and this process is facilitated by ribosomally synthesized small cationic peptides called mutacins (bacteriocins) secreted by S. mutans. Although for long time mutacins are regarded as important virulence factors, it is not clear how these peptides work, what is the mechanism of self-immunity, and how they are regulated. In this study, we have characterized the activity, mechanism of self-immunity, and regulation of expression of a well-known bacteriocin, mutacin IV. To clarify the activity of mutacin IV, we studied the contribution of NlmA and NlmB peptides to mutacin IV activity. Using genetic and biochemical approaches, we showed that the presence of both genes is required for optimum mutacin IV activity. We also showed that mutacin IV is active against multiple Streptococcus species. Our studies revealed that mutacin IV has comparatively broader inhibitory spectrum than mutacin V; and both of them can work synergistically to inhibit various microorganisms. Next, we investigated the immunity activity of a putative immunity protein, SMU.152, which lies just downstream of the nlmAB operon. We observed that heterologous expression of SMU.152 in two sensitive strains converted the strains to become resistant to mutacin IV. We identified that C-terminal charged residues of SMU.152 is indispensible for conferring immunity to susceptible cells. In addition, our studies demonstrated that in absence of cognate immunity protein, S. mutans can employ other paralogous proteins to prevent the self-toxicity, which implies the presence of cross-immunity or immune redundancy in S. mutans UA159. To get a clear understanding on regulation of mutacin IV production, we carried out pGh9:ISS1-mediated random transposon mutagenesis in a reporter strain containing the nlmAB promoter with a promoter less gusA (glucuronidase) gene. Mapping the position of transposon insertion sites revealed 22 unique genes of diverse cellular activities, which are involved in regulation of mutacins production. Highlight of our finding is that an extracellular ATP-independent Lon-like protease functions specifically on maturation of peptide pheromone, CSP. Here, we show that functional form of CSP (18-amino acids) is generated by a specific post export cleavage with this Lon-like protease. Our study suggests that ComDE two-component system is primarily involved in regulation of mutacins production in S. mutans UA159. We also show that both the peptide pheromone (CSP) and mutacins use the same ABC transporter, NlmTE, for secretion. During the course of this study, we discovered two more novel regulators for gene expression in this bacterium. We show that a small hypothetical protein, SMU.2137, is required for normal cellular processes in S. mutans UA159 and a protein, SMU.832, required for glucose side chain formation, can also regulate mutacin production in this organism. Collectively, our data encourage us to propose a new model for regulation of mutacins production in S. mutans UA159. According to our model, peptide pheromone (BipC) is secreted as 21-amino acids and a specific Lon-like protease cleaves the C-terminal three amino acids to generate functional peptide pheromone (18-residue). BipC activates a two-component signal transduction system (BipDE), which in turn stimulates the expression of mutacins encoding genes (such as nlmAB and nlmC). A common ABC transporter, BipTE (NlmTE), is required for both mutacins and BipC secretion and proteolytic cleavage of leader peptide after GG-motif. In sum, our study has unraveled new pathways of gene regulation in S. mutans and strengthened our understanding on overall biology of mutacins production in S. mutans