Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression analysis of Streptococcus mutans UA159 under Hyperosmotic stress environment

ABSTRACT: Oral streptococci metabolize carbohydrate to produce organic acids, not only decrease the environmental pH, but also increase osmolality of dental plaque fluid due to tooth demineralization and consequent calcium and phosphate accumulation. Thus, to persevere in the dental plaque, acidogenic bacteria should evolve sophisticated molecular machineries to counter the detrimental effect of elevated osmolality. This study was aimed to obtain a global view on strategies taken by streptococcus mutans to deal with physiologically relevant elevated osmolality, and preserves within a cariogenic dental plaque. We investigated phenotypic change of S. mutans biofilm upon sub-lethal level of hyperosmotic challenge. We found that hyperosmotic condition was able to initiate S. mutans biofilm dispersal by reducing both microbial content and extracellular polysaccharides matrix. We then used DNA microarray with qPCR validation to systemically investigate the underlying molecular machinery of this bacteria in response to hyperosmotic stimuli. Among those identified 50 differentially regulated genes, down-regulation of gtfB and comC were believed to be responsible for the observed biofilm dispersal. Further analysis of microarray data showed significant up-regulation of genes and pathways involved in carbohydrates metabolism. Specific genes involved in heat shock response and acid tolerance were also upregulated, indicating potential cross-talk between hyperosmotic and other environmental stress. Based on the data obtained in this study, we believe that although hyperosmotic condition may induce significant stress response on S. mutans, this cariogenic bacterium has evolved sophisticated molecular machineries to counter those elicited detrimental effects. In the meantime, it will take full advantage of these environmental stimuli to better fit the fluctuating environments within oral cavity, and thus emerge as numeric-predominant bacteria under cariogenic conditions. A six-chip study using total RNA recovered from mid-logarithmic phase of S. mutans UA159 from three separate cultures of strains submitted for 15 minutes to hyperosmotic stimuli (0.4M NaCl) and three separate cultures of strains kept under no stress condition.

ORGANISM(S): Streptococcus mutans UA159

SUBMITTER: Yulong Niu

PROVIDER: E-GEOD-47170 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Lysine malonylation is an evolutionarily conserved PTM from bacteria to mammals and involves malonyl-coenzyme (CoA) as a cofactor. Since the malonyl group has an acidic carboxylic group under physiological pH, malonylated lysine is negatively charged, which might impact on protein function and enzymatic activities. Like other short-chain acyl-CoAs, malonyl-CoA can be synthesized from its corresponding short-chain acyl salt, malonate, catalyzed by malonyl-CoA synthetase (Witkowski et al., 2011). In addition, malonyl-CoA can be produced during the carboxylation of acetyl-CoA by acetyl-CoA carboxylase (ACC), the carboxylation of acetyl-CoA by propionyl-CoA carboxylase, and the β-oxidation of old-chain-length dicarboxylic acids (Peng et al., 2011). Most studies on Kmal have been performed in mammalian systems and identified thousands of malonylated sites, revealing its role in the progress of diseases like type 2 diabetes, schizophrenia, and cardiac hypertrophy (Du et al., 2015; Smith et al., 2022; Wu et al., 2022). There has been increasing interest in dissecting the regulatory roles of Kmal in several bacterial species, such as Escherichia coli (Qian et al., 2016), Bacillus amyloliquefaciens (Fan et al., 2017), Mycobacterium tuberculosis (Bi et al., 2022), and Staphylococcus aureus (Shi et al., 2021), which demonstrates that Kmal exists in diverse prokaryotic organisms and participates in the regulation of various physiological processes. Even so, whether Kmal exists and affects protein functions in streptococci remains unknown. S. mutans is considered to be the most prevalent and cariogenic species in active carious lesions of humans, residing primarily in dental plaque, which is a biofilm that forms on the tooth surfaces. During the past decades, studies of S. mutans have focused on revealing the molecular mechanisms underlying the robust biofilm formation on tooth surfaces, the metabolism of a wide variety of carbohydrates obtained from the host diet, and the adaption of numerous environmental challenges (Lemos et al., 2019). Several studies have been performed to reveal the roles of PTMs in biofilm and cariogenic virulence. Wang et al. found that the phosphorylation of VicR could inhibit the expression of the glucosyltransferases gtfBC, thereby reducing the synthesis of extracellular polysaccharides (EPS), the major component of cariogenic biofilm (Wang et al., 2021). Acetylome study on the S. mutans depicted that acetylated substrates were globally altered in the biofilm state compared to the planktonic state, and the acetylated GtfBC showed decreased activities (Lei et al., 2021; Ma et al., 2021). Our previous study revealed that the S-glutathionylation of a thioredoxin-like protein is important for interspecies competition and cariogenicity of S. mutans(Li et al., 2020). These results suggest that various PTM types exist in S. mutans and participate in diverse physiological processes. The genome of S. mutans UA159 codes an acetyl coenzyme A carboxylase (ACC), which could synthesize malonyl-CoA through the carboxylation of acetyl-CoA (Ajdić et al., 2002), implying the existence of malonylation in this bacterium. Therefore, this study aimed to confirm the existence of Kmal in S. mutans and identify the malonylated sites globally. The present findings provide a systematic view of the functional roles of Kmal in various metabolic pathways of S. mutans.

Project description:Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation pH is a major environmental factor that regulates gene expression in many bacteria. Streptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and the pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we used DNA microarray to perform genome-wide transcriptional analysis of S. mutans in response to acidic pH. The results showed that adaptation of S. mutans to pH 5.5 for 2 hrs induced differential expression of nearly 14% of genes in the genome, including 169 up-regulated genes and 108 down-regulated genes, largely categorized into six groups. Especially, we found that the genes encoding multiple two-component systems, including CiaHR, LevSR, LiaSR, ScnKR, HK/RR07 and ComDE, were up-regulated during acid adaptation. These findings were further confirmed by real time qRT-PCR and phenotypic assays of the gene deletion mutants. The results support that the multiple two-component systems are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH. Total RNAs were isolated from S. mutans UA159 cells (0.6 at OD600) grown in a TYG broth (3% tryptone, 0.3% yeast extract and 20 mM glucose) at either pH 5.5 or pH 7.5. The RNAs were treated with RNase-free DNase 1 and purified by Qiagen RNeasy mini columns. The purified RNAs were used to generate cDNA probes by an indirect labeling method based on the protocol from TIGR. The cDNAs were coupled with AlexaFluor 555 or AlexaFluor 647 (Invitrogen). The labeled cDNA probes from three different cultures of UA159 were hybridized to the S. mutans microarray slides obtained from PFGRC (http://pfgrc.tigr.org). Array hybridization was conducted using a protocol from the PFGRC with minor modification. After hybridization, washes and dried, the array slides were scanned by ScanArray 5000XL Reader (Perkin Elmer, Boston, MA). After the array slides were scanned, the resulting images were loaded into TIGR Spotfinder software (http://www.tigr.org/software/) and overlaid. A spot grid was created according to TIGR specifications and manually adjusted to fit all spots within the grid, and the intensity values of each spot were determined. Signal intensities of individual channels from an array slide were averaged and normalized using an array data analysis software (MIDAS) by using LOWESS and iterative log mean centering with default settings, followed by in-slide replicate analysis. A t-test was used to determine the consistency of ratios across replicate hybridizations. Only genes whose ratios were â¥ 2-fold changes (either increase or decrease) with 99% confidence interval (P â¤ 0.01) were considered statistically significant.

Project description:The two-component system (TCS) is a specific regulatory system in bacteria and plays an important role in sensing and adapting to the environment. In this study, we evaluated the roles of TCSs in the major cariogenic pathogen Streptococcus mutans in resistance to several types of bacteriocin. In a comprehensive analysis using individual TCS mutants, we found that two novel TCSs were associated with resistance against distinct lantibiotics nisin A (class I type A[I]) and nukacin ISK-1(class I type A[II]). One TCS, SMU.659-660 (designated as NsrRS), was related to resistance against nisin A produced by Lactococcus lactis ATCC 11454. The other TCS, SMU.1146-1145 (designated as LcrRS), was related to resistance against nukacin ISK-1 produced by Staphylococcus warneri ISK-1. NsrRS induced the expression of SMU.658 (designated as NsrX), which constitutes an operon with nsrRS, in response to nisin A. Inactivation of nsrX increased susceptibility to nisin A. Additionally, NsrX expression in S. mutans increased the binding affinity to nisin A compared to a no-expression strain. LcrRS induced the expression of SMU.1148-50 (lctFEG), which encodes an ABC transporter and is located upstream of lcrRS, in response to nukacin ISK-1. Inactivation of lctFEG significantly increased susceptibility to nukacin ISK-1. Electrophoretic mobility shift assay analysis revealed that NsrR and LcrR bound directly to regions upstream of nsrX and lctFEG, respectively. This is the first report that two distinct TCSs, NsrRS and LcrRS, are independently involved in resistance to nisin A and nukacin ISK-1 in S. mutans. Total of 14 samples were analyzed. Total RNA from each test strain and control described below were labeled with Alexa FluorÂ® 555 and Alexa FluorÂ® 647, respectively, and were cohybridized on a single array. Labeling and hybridization were performed once or twice independently. UA159 wild type as control vs. UA159 with nisinA or nukacin ISK-1, UA159 wild type with nisinA or nukacin ISK-1 vs. nsrRS or lcrRS deletion mutant in UA159 with nisinA or nukacin ISK-1.

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Expression analysis of Streptococcus mutans UA159 under Hyperosmotic stress environment

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