PBP1a-deficiency causes major defects in cell division, growth and biofilm formation by Streptococcus mutans.

Zezhang T Wen,Jacob P Bitoun,Sumei Liao,Bernard Beall

doi:10.1371/journal.pone.0124319

Abstract

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.

Highlights

Streptococcus mutans, a key etiological agent of dental caries, lives almost exclusively on the tooth surface in biofilms known as dental plaque
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
Like other Gram-positive bacteria, S. mutans possesses a thick layer of peptidoglycan whose homeostasis is known to be crucial in pathophysiology including cell division and growth, stress tolerance, and biofilm formation

Summary

Introduction

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 [1,2,3,4]. The adhesive glucans function as scaffold and facilitate adherence to substrata and inter-cellular interactions and biofilm accumulation via glucan-binding proteins (Gbps). Surface-associated protein P1 ( SpaP) is a high affinity adhesin that the bacterium utilizes to adhere to the tooth surface via interactions with salivary agglutinins. This bacterium 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

Methods

Results

Conclusion