Deficiency of MecA in Streptococcus mutans Causes Major Defects in Cell Envelope Biogenesis, Cell Division, and Biofilm Formation.

Arpan De,Wandy L Beatty,Jose Lemos,Zezhang T Wen,Ashton N Jorgensen

doi:10.3389/fmicb.2018.02130

Abstract

MecA is an adaptor protein that guides the ClpC/P-mediated proteolysis. A S. mutans MecA-deficient mutant was constructed by double-crossover allelic exchange and analyzed for the effects of such a deficiency on cell biology and biofilm formation. Unlike the wild-type, UA159, the mecA mutant, TW416, formed mucoid and smooth colonies, severely clumped in broth and had a reduced growth rate. Transmission electron microscopy analysis revealed that TW416 grows primarily in chains of giant “swollen” cells with multiple asymmetric septa, unlike the coccoid form of UA159. As compared to UA159, biofilm formation by TW416 was significantly reduced regardless of the carbohydrate sources used for growth (P < 0.001). Western blot analysis of TW416 whole cell lysates showed a reduced expression of the glucosyltransferase GtfC and GtfB, as well as the P1 and WapA adhesins providing an explanation for the defective biofilm formation of TW416. When analyzed by a colorimetric assay, the cell wall phosphate of the mutant murein sacculi was almost 20-fold lower than the parent strain (P < 0.001). Interestingly, however, when analyzed using immunoblotting of the murein sacculi preps with UA159 whole cell antiserum as a probe, TW416 was shown to possess significantly higher signal intensity as compared to the wild-type. There is also evidence that MecA in S. mutans is more than an adaptor protein, although how it modulates the bacterial pathophysiology, including cell envelope biogenesis, cell division, and biofilm formation awaits further investigation.

Highlights

Streptococcus mutans, a common inhabitant of the tooth surface, is considered as a primary causative agent of human dental caries
As predicted (Ajdic et al, 2002) (Figure 1A), the results of RT-PCR (Figure 1B) revealed that mecA (SMU.233) in S. mutans is co-transcribed as a polycistronic operon with downstream rgpG (SMU.234), which encodes the first enzyme of the rhamnose-glucose polymer (RGP) biosynthesis pathway (Yamashita et al, 1999)
When luciferase reporter assays were used to examine the mecA/rgpG, the results showed no significant differences between TW416 and the wildtype, UA159

Summary

Introduction

Streptococcus mutans, a common inhabitant of the tooth surface, is considered as a primary causative agent of human dental caries. One of the consequences of exposure to environmental stresses is the accumulation of abnormal proteins due to increased errors in transcription and translation (Lemos and Burne, 2008) Molecular chaperones, such as DnaK, DnaJ, and GroEL, are required for proper folding and assembly of those aberrant proteins (Lemos et al, 2007). Proteases, such as Clp proteases, are involved in the degradation of the proteins, under stress conditions, and under normal growth conditions (Lemos and Burne, 2002; Chattoraj et al, 2010; Kajfasz et al, 2011). It is central for bacterial viability, persistence and growth to maintain protein homeostasis by stabilizing proteins that perform essential functions and by refolding or degrading misfolded or aberrant proteins (Lemos et al, 2007)

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