PBP1a-Deficiency Causes Major Defects in Cell Division, Growth and Biofilm Formation by Streptococcus mutans

April PBP1a-Deficiency Causes Major Defects in Cell Division, Growth and Biofilm Formation by Streptococcus mutans Zezhang T. Wen 0 1 2 3 Jacob P. Bitoun 0 1 2 3 Sumei Liao 0 1 2 3 0 1 Department of Comprehensive Dentistry and Biomaterials, Louisiana State University Health Sciences Center , New Orleans, LA, 70112 , United States of America, 2 Center of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center , New Orleans, LA, 70112 , United States of America, 3 Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center , New Orleans, LA, 70112 , United States of America 1 Funding: This work was supported in part by R01 grant DE19452 from the National Institute for Dental and Craniofacial Research, United States of America (http://www.nidcr.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Academic Editor: Bernard Beall, Centers for Disease Control & Prevention, UNITED STATES 3 a Current address: Department of Microbiology & Immunology, Tulane University School of Medicine , 1430 Tulane Avenue, SL-38, New Orleans, LA , United States of America b Current address: Department of Electrical and Computer Engineering, Frank Reidy Research Center for Bioelectrics, Old Dominion University , Norfolk, VA , United States of America Streptococcus mutans, a key etiological agent of human dental caries, lives almost exclusively on the tooth surface in plaque biofilms and is known for its ability to survive and respond to various environmental insults, including low pH, and antimicrobial agents from other microbes and oral care products. In this study, a penicillin-binding protein (PBP1a)deficient mutant, strain JB467, was generated by allelic replacement mutagenesis and analyzed for the effects of such a deficiency on S. mutans' stress tolerance response and biofilm formation. Our results so far have shown that PBP1a-deficiency in S. mutans affects growth of the deficient mutant, especially at acidic and alkaline pHs. As compared to the wild-type, UA159, the PBP1a-deficient mutant, JB467, had a reduced growth rate at pH 6.2 and did not grow at all at pH 8.2. Unlike the wild-type, the inclusion of paraquat in growth medium, especially at 2 mM or above, significantly reduced the growth rate of the mutant. Acid killing assays showed that the mutant was 15-fold more sensitive to pH 2.8 than the wild-type after 30 minutes. In a hydrogen peroxide killing assay, the mutant was 16-fold more susceptible to hydrogen peroxide (0.2%, w/v) after 90 minutes than the wild-type. Relative to the wild-type, the mutant also had an aberrant autolysis rate, indicative of compromises in cell envelope integrity. As analyzed using on 96-well plate model and spectrophotometry, biofilm formation by the mutant was decreased significantly, as compared to the wild-type. Consistently, Field Emission-SEM analysis also showed that the PBP1a-deficient mutant had limited capacity to form biofilms. TEM analysis showed that PBP1a mutant existed primarily in long rod-like cells and cells with multiple septa, as compared to the coccal wild-type. The results presented here highlight the importance of pbp1a in cell morphology, stress tolerance, and biofilm formation in S. mutans. - Competing Interests: The authors have declared that no competing interests exist. Streptococcus mutans, a key etiological agent of dental caries, lives almost exclusively on the tooth surface in biofilms known as dental plaque. S. mutans possesses multiple mechanisms to colonize the tooth surface [14]. S. mutans possesses at least three glucosyltransferases (Gtfs) that utilize dietary sucrose as substrate to produce high molecular weight, highly adhesive extracellular glucans (also mutans) [46]. The adhesive glucans function as scaffold and facilitate adherence to substrata and inter-cellular interactions and thus, biofilm accumulation via glucan-binding proteins (Gbps). Surface-associated protein P1 (also SpaP) is a high affinity adhesin that the bacterium utilizes to adhere to the tooth surface via interactions with salivary agglutinins. In addition, this bacterium also actively releases extracellular deoxyribonucleic acids (eDNA) that form nanofiber network facilitating cell-surface and cell-cell interactions [2]. S. mutans is known for its ability to survive and respond to various environmental insults, low pH and various toxic metabolites. The cell envelope is essential in maintenance of cell shape, cell growth and cell division, and in protection against various environmental insults, including toxic metabolites and antimicrobials. The cell envelope is also directly involved in environmental signaling and bacterial cellsurface and cell-cell interactions, and thus, bacterial colonization and biofilm formation [7, 8]. The cell envelope of Gram-positive bacteria is featured with a thick layer of peptidoglycan (PG). Efficient cell division and growth requires coordinated remodeling of the PG sacculus and requires the activities of PG hydrolases, synthases, and morphogenesis proteins [9]. Penicillin-binding proteins (PBPs) function to increase resistance to -lactams antibiotics through sequestration, although some PBPs have low-affinity for -lactams and function in maintaining proper cell morphology. These low-affinity PBPs are thought to have evolved from horizontal gene transfer with commensal streptococci, followed by recombination events [1014]. PBPs are grouped into two classes based on enzymatic activity towards peptidoglycan biosynthesis and/or maintenance [1518]. Class A PBPs (PBP1a, PBP1b, and PBP2a) have both transpeptidase and glucosyltransferase activity. Class B PBPs (PBPx, PBP2x, and PBP2b) possess only transpeptidase activity. Multiple studies have been done on PBPs concerning antibiotic resistance, cell envelope biogenesis and cell division [1924], but currently, limited information is available concerning their effects in biofilm formation, especially in S. mutans [19, 25, 26]. S. mutans possesses at least six putative PBPs homologues [27], but the function of these PBP proteins remain largely unclear. Recently, we have started to characterize the PBPs in S. mutans pathohpysiology by use functional genomics approach. Results so far have shown that S. mutans PBP1a (SMU.467c) is required for optimal growth under acidic and alkaline conditions. Also, deficiency of PBP1a causes at least a 50-fold increase in sensitivity to the redox-cycling agent paraquat. Furthermore, our results show that depletion of PBP1a leads to an increased susceptibility to both acid and hydrogen peroxide stress and major defects in biofilm formation. Lastly, PBP1a-deficiency leads to morphological changes of S. mutans cells. Bacterial strains and plasmids used in this study are listed in Table 1. S. mutans strains were maintained in Brain Heart Infusion (BHI, Difco Laboratories) medium. So (...truncated)