Abstract
Cyclic dimeric adenosine 3′,5′-monophosphate (c-di-AMP), a recently identified secondary messenger in bacteria, plays a role in several bacterial processes, including biofilm formation. It is enzymatically produced by diadenylate cyclase and cleaved by c-di-AMP phosphodiesterase. c-di-AMP is believed to be essential for the viability of bacterial cells that produce it. In the current study, the biochemical and biological roles of GdpP (SMU_2140c) and DhhP (SMU_1297), two distinct Streptococcus mutans phosphodiesterases involved in the pathway producing AMP from c-di-AMP, were investigated. Liquid chromatography-tandem mass spectrometry revealed that c-di-AMP was degraded to phosphoadenylyl adenosine (pApA) by truncated recombinant GdpP, and pApA was cleaved by recombinant DhhP to yield AMP. In-frame deletion mutants lacking the dhhP gene (ΔdhhP) and both the gdpP and dhhP genes (ΔgdpPΔdhhP) displayed significantly more biofilm formation than the wild-type and a mutant strain lacking the gdpP gene (ΔgdpP; p < 0.01). Furthermore, biofilm formation was restored to the level of the wild type strain upon complementation with dhhP. Optical and electron microscopy observations revealed that ΔdhhP and ΔgdpPΔdhhP mutants self-aggregated into large cell clumps, correlated with increased biofilm formation, but cell clumps were not observed in cultures of wild-type, ΔgdpP, or strains complemented with gdpP and dhhP. Thus, deletion of dhhP presumably leads to the formation of bacterial cell aggregates and a subsequent increase in biofilm production.
Highlights
Streptococcus mutans is a major etiological agent of dental caries in humans (Hamada and Slade, 1980), a biofilm-associated infectious disease and important health problem worldwide (Thenisch et al, 2006)
The secondary messenger c-di-Adenosine monophosphate (AMP) was recently identified in bacteria and appears to be required for cell viability (Song et al, 2005; Woodward et al, 2010; Corrigan et al, 2011; Luo and Helmann, 2012; Mehne et al, 2013). c-di-AMP is synthesized by most of Gram-positive bacteria (Woodward et al, 2010; Corrigan et al, 2011; Kamegaya et al, 2011; Bai et al, 2013; Du et al, 2014), whereas the molecule is present in some Gram-negative pathogens (Barker et al, 2013) and Spirochaetae (Ye et al, 2014)
It is probable that in S. mutans, c-di-AMP is degraded to the pApA intermediate by GdpP, and this is subsequently degraded to AMP by DhhP
Summary
Streptococcus mutans is a major etiological agent of dental caries in humans (Hamada and Slade, 1980), a biofilm-associated infectious disease and important health problem worldwide (Thenisch et al, 2006). C-di-AMP has been found to regulate many important function in bacteria, including the homeostasis of cell wall peptidoglycan architecture (Luo and Helmann, 2012), size and envelope stress (Corrigan et al, 2011), biofilm formation (Corrigan et al, 2011; Diethmaier et al, 2011; Du et al, 2014; Peng et al, 2016), potassium uptake (Bai et al, 2014), metabolic enzyme function (Sureka et al, 2014), and drug resistance (Banerjee et al, 2010; Corrigan et al, 2011; Griffiths and O’Neill, 2012; Luo and Helmann, 2012; Cho and Kang, 2013) This molecule reportedly couples DNA integrity with the sporulation progression, cell growth, competence development, and spore resurrection (Oppenheimer-Shaanan et al, 2011). This molecule appears to trigger the cytosolic pathway of innate immunity (Woodward et al, 2010; Parvatiyar et al, 2012; Dey et al, 2015)
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