Stress-responsive proteins are upregulated in Streptococcus mutans during acid tolerance

Abstract

Streptococcus mutans is an important pathogen in the initiation of dental caries as the bacterium remains metabolically active when the environment becomes acidic. The mechanisms underlying this ability to survive and proliferate at low pH remain an area of intense investigation. Differential two-dimensional electrophoretic proteome analysis of S. mutans grown at steady state in continuous culture at pH 7?0 or pH 5?0 enabled the resolution of 199 cellular and extracellular protein spots with altered levels of expression. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 167 of these protein spots. Sixty-one were associated with stress-responsive pathways involved in DNA replication, transcription, translation, protein folding and proteolysis. The 61 protein spots represented isoforms or cleavage products of 30 different proteins, of which 25 were either upregulated or uniquely expressed during acid-tolerant growth at pH 5?0. Among the unique and upregulated proteins were five that have not been previously identified as being associated with acid tolerance in S. mutans and/or which have not been studied in any detail in oral streptococci. These were the single-stranded DNA-binding protein, Ssb, the transcription elongation factor, GreA, the RNA exonuclease, polyribonucleotide nucleotidyltransferase (PnpA), and two proteinases, the ATP-binding subunit, ClpL, of the Clp family of proteinases and a proteinase encoded by the pep gene family with properties similar to the dipeptidase, PepD, of Lactobacillus helveticus. The identification of these and other differentially expressed proteins associated with an acid-tolerant-growth phenotype provides new information on targets for mutagenic studies that will allow the future assessment of their physiological significance in the survival and proliferation of S. mutans in low pH environments. INTRODUCTIONStreptococcus mutans is now well recognized as being associated with the initiation of dental caries, since its acid fermentation by-products can result in the demineralization of tooth enamel (Hamada & Slade, 1980;Harper & Loesche, 1984;Loesche, 1986;van Houte, 1994;van Ruyven et al., 2000). A key to the survival of S. mutans at low pH is its ability to maintain a transmembrane pH gradient (DpH), with the interior of the cell more alkaline. This is achieved by upregulation of a proton-translocating F 1 F 0 -ATPase that extrudes H + as the external environment becomes more acidic. This results in an increased use of ATP for H + extrusion and a consequent reduction in cell yield (Belli & Marquis, 1991;Hamilton & Buckley, 1991;Dashper & Reynolds, 1992;Quivey et al., 2001). A series of recent physiological, mutagenic and proteome studies (Quivey et al., 1995;Gutierrez et al., 1996 Gutierrez et al., , 1999Jayaraman et al., 1997; Hamilton & Svensäter, 1998;Hahn et al., 1999;Hanna et al., 2001; Kremer et al., 2001;Lemos et al., 2001;Li et al., 2002;Wilkins et al., 2002;Len et al., 2004), however, indicates that S. mutans regulates its pheno...

Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance

“…However, since an overall 2?7-fold increase in NADP-dependent glyceraldehyde-3-phosphate dehydrogenase at pH 5?0 represented nearly twice the increase in the cumulative levels of each of the other two enzymes in the alternative oxidative branch of glycolysis, this suggested that NADP-dependent glyceraldehyde-3-phosphate dehydrogenase may indeed be of prime importance in determining this direction. This would be in keeping with the high level of anabolic activity required to maintain the structural integrity of molecules such as proteins and DNA in a non-optimal acidic cytoplasm (Crow & Wittenberger, 1979;Quivey et al, 2001;Len et al, 2004). This notion was supported by the detection of a high level of transketolase (Tkt) solely on 2-DGE gels at pH 5?0.…”

“…Of these, 123 were identified by MALDI-TOF analysis and 53 found to be associated with regulatory and/or stressresponsive pathways (Len et al, 2004). The remaining 70 protein spots were associated with metabolism; the majority were associated with glycolysis, alternative acid production and branched-chain amino acid synthesis (Table 1).…”

Implication of the NorB Efflux Pump in the Adaptation of Staphylococcus aureus to Growth at Acid pH and in Resistance to Moxifloxacin

“…The acid stress response has been shown to include increased transcript levels of genes involved in utilization of urease (ureA, ureB, ureC, and ureF) and of nuoF, which encodes an NADH dehydrogenase, which can increase internal cell pH by assisting proton export from the bacterial cell (1,2,5,6,28). A decrease in transcripts encoding the F 0 F 1 ATPase was also observed, which was somewhat surprising since this F 0 F 1 ATPase is considered an important proton extrusion mechanism in several Gram-positive bacterial species, such as Streptococcus mutans (17). Although acid stress responses do not include increases in major virulence factors or biofilm formation, there were noteworthy changes in several global regulators that control the expression of virulence genes and drug efflux transporters (5), such as the SaeRS and ArlRS, two-component regulators, the Ser/Thr kinase PknB, and its phosphatase Stp1.…”

Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation

“…The aciduricity of this organism has been attributed largely to the proton-extruding F 1 F 0 -ATPase that functions well at pH 5.0 and below, allowing the organism to maintain adequate DpH when the external pH falls to 4.0 and lower (Bender et al, 1986;Cotter & Hill, 2003). Other mechanisms of acid resistance in S. mutans include induction of stress proteins (Hamilton & Svensäter, 1998;Len et al, 2004;Svensäter et al, 1997;Wilkins et al, 2002), changes in membrane-associated proteins and fatty acid composition (Fozo et al, 2007;Hasona et al, 2007), DNA repair enzymes (Hahn et al, 1999;Hanna et al, 2001) and increase in alkali production through several metabolic pathways (Burne et al, 1999;Griswold et al, 2006). Moreover, the ComCDE quorum-sensing system has been found to play a role in cell-density-dependent acid tolerance by S. mutans (Li et al, 2001b).…”