The copYAZ Operon Functions in Copper Efflux, Biofilm Formation, Genetic Transformation, and Stress Tolerance in Streptococcus mutans

Abstract

In bacteria, copper homeostasis is closely monitored to ensure proper cellular functions while avoiding cell damage. Most Gram-positive bacteria utilize the copYABZ operon for copper homeostasis, where copA and copB encode copper-transporting P-type ATPases, whereas copY and copZ regulate the expression of the cop operon. Streptococcus mutans is a biofilm-forming oral pathogen that harbors a putative copper-transporting copYAZ operon. Here, we characterized the role of copYAZ operon in the physiology of S. mutans and delineated the mechanisms of copper-induced toxicity in this bacterium. We observed that copper induced toxicity in S. mutans cells by generating oxidative stress and disrupting their membrane potential. Deletion of the copYAZ operon in S. mutans strain UA159 resulted in reduced cell viability under copper, acid, and oxidative stress relative to the viability of the wild type under these conditions. Furthermore, the ability of S. mutans to form biofilms and develop genetic competence was impaired under copper stress. Briefly, copper stress significantly reduced cell adherence and total biofilm biomass, concomitantly repressing the transcription of the gtfB, gtfC, gtfD, gbpB, and gbpC genes, whose products have roles in maintaining the structural and/or functional integrity of the S. mutans biofilm. Furthermore, supplementation with copper or loss of copYAZ resulted in significant reductions in transformability and in the transcription of competence-associated genes. Copper transport assays revealed that the ⌬copYAZ strain accrued significantly large amounts of intracellular copper compared with the amount of copper accumulation in the wild-type strain, thereby demonstrating a role for CopYAZ in the copper efflux of S. mutans. The complementation of the CopYAZ system restored copper expulsion, membrane potential, and stress tolerance in the copYAZ-null mutant. Taking these results collectively, we have established the function of the S. mutans CopYAZ system in copper export and have further expanded knowledge on the importance of copper homeostasis and the CopYAZ system in modulating streptococcal physiology, including stress tolerance, membrane potential, genetic competence, and biofilm formation. IMPORTANCE S. mutans is best known for its role in the initiation and progression of human dental caries, one of the most common chronic diseases worldwide. S. mutans is also implicated in bacterial endocarditis, a life-threatening inflammation of the heart valve. The core virulence factors of S. mutans include its ability to produce and sustain acidic conditions and to form a polysaccharide-encased biofilm that provides protection against environmental insults. Here, we demonstrate that the addition of copper and/or deletion of copYAZ (the copper homeostasis system) have serious implications in modulating biofilm formation, stress tolerance, and genetic transformation in S. mutans. Manipulating the pathways affected by copper and the copYAZ system may help to develop potential therapeutics to prev...

Pleiotropic Regulation of Virulence Genes in Streptococcus mutans by the Conserved Small Protein SprV

“…This dysregulation of the biofilm formation pathway in S. mutans suggested the differential expression of several genes of the pathway upon sprV inactivation. Several factors that affect biofilm formation in S. mutans are also known to impact its ability to tolerate environmental stresses and uptake DNA naturally (18,(24)(25)(26). As expected, our experiments showed not only that the sprV mutant was susceptible to various stresses and antibiotics ( Fig.…”

“…Furthermore, the function of CopZ, a small copper chaperone protein, has been recently characterized in S. mutans. However, it is unclear how the deletion of CopZ affects biofilm formation and bacteriocin production (18).…”

Streptococcus mutans copper chaperone, CopZ, is critical for biofilm formation and competitiveness

“…Beyond copper transport, copZ has been shown to affect biofilm detachment but to be unnecessary for biofilm formation in oral commensal S. gordonii (Mitrakul et al ., ). In S. mutans , the copYAZ operon has been implicated in oxidative stress and gtfB transcription (Singh et al ., ). In this study, we demonstrate that CopZ is critical for biofilm formation by Gtfs and competitiveness by mutacins.…”

Two-component signal transduction systems in oral bacteria

“…In addition, VicRK Spn regulates pspA [105], which encodes a species-specific surface protein (PspA) involved in complement evasion [128]. In S. mutans , VicR Sm also directly represses genes involved in competence and competence-induced bacteriocin ( comCDE and nlmC ) [113], and copY [117], part of the copYAZ operon required for copper homeostasis and regulation of membrane potential, competence, and biofilm formation [129]. In S. sanguinis , VicRK Ss does not regulate gtfP , a unique gene encoding a glucosyltransferase for the synthesis of soluble glucan [90] but does regulate spxB encoding pyruvate oxidase, required for conversion of pyruvate, inorganic phosphate, and O 2 to H 2 O 2 , CO 2 , and AcP [130].…”