Haipeng Sun

Staphylococcus aureus AI-2 Quorum Sensing Associates with the KdpDE Two-Component System To Regulate Capsular Polysaccharide Synthesis and Virulence

ABSTRACT Autoinducer 2 (AI-2) is widely recognized as a signal molecule for intra- and interspecies communication in Gram-negative bacteria, but its signaling function in Gram-positive bacteria, especially in Staphylococcus aureus, remains obscure. Here we reveal the role of LuxS in the regulation of capsular polysaccharide synthesis in S. aureus NCTC8325 and show that AI-2 can regulate gene expression and is involved in some physiological activities in S. aureus as a signaling molecule. Inactivation of luxS in S. aureus NCTC8325 resulted in higher levels of transcription of capsular polysaccharide synthesis genes. The survival rate of the luxS mutant was higher than that of the wild type in both human blood and U937 macrophages. In comparison to the luxS mutant, a culture supplemented with chemically synthesized 4,5-dihydroxy-2,3-pentanedione (DPD), the AI-2 precursor molecule, restored all the parental phenotypes, suggesting that AI-2 has a signaling function in S. aureus. Furthermore, we demonstrated that the LuxS/AI-2 signaling system regulates capsular polysaccharide production via a two-component system, KdpDE, whose function has not yet been clarified in S. aureus. This regulation occurred via the phosphorylation of KdpE binding to the cap promoter.

The Staphylococcus aureus KdpDE Two-Component System Couples Extracellular K+ Sensing and Agr Signaling to Infection Programming

ABSTRACT The Kdp system is widely distributed among bacteria. In Escherichia coli, the Kdp-ATPase is a high-affinity K+ uptake system and its expression is activated by the KdpDE two-component system in response to K+ limitation or salt stress. However, information about the role of this system in many bacteria still remains obscure. Here we demonstrate that KdpFABC in Staphylococcus aureus is not a major K+ transporter and that the main function of KdpDE is not associated with K+ transport but that instead it regulates transcription for a series of virulence factors through sensing external K+ concentrations, indicating that this bacterium might modulate its infectious status through sensing specific external K+ stimuli in different environments. Our results further reveal that S. aureus KdpDE is upregulated by the Agr/RNAIII system, which suggests that KdpDE may be an important virulence regulator coordinating the external K+ sensing and Agr signaling during pathogenesis in this bacterium.

LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing

In quorum sensing (QS) process, bacteria regulate gene expression by utilizing small signaling molecules called autoinducers in response to a variety of environmental cues. Autoinducer 2 (AI-2), a QS signaling molecule proposed to be involved in interspecies communication, is produced by many species of gram-negative and gram-positive bacteria. In Escherichia coli and Salmonella typhimurium, the extracellular AI-2 is imported into the cell by a transporter encoded by the lsr operon. Upstream of the lsr operon, there is a divergently transcribed gene encoding LsrR, which was reported previously to repress the transcription of the lsr operon and itself. Here, we have demonstrated for the first time that LsrR represses the transcription of the lsr operon and itself by directly binding to their promoters using gel shift and DNase I footprinting assays. The β-galactosidase reporter assays further suggest that two motifs in both the lsrR and lsrA promoter regions are crucial for the LsrR binding. Furthermore, in agreement with the conclusion that phosphorylated AI-2 can relieve the repression of LsrR in previous studies, our data show that phospho-AI-2 renders LsrR unable to bind to its own promoter in vitro.

The Staphylococcus aureus GGDEF Domain-Containing Protein, GdpS, Influences Protein A Gene Expression in a Cyclic Diguanylic Acid-Independent Manner

ABSTRACT Staphylococcus aureus is an important human pathogen that is the principal cause of a variety of diseases, ranging from localized skin infections to life-threatening systemic infections. The success of the organism as a pathogen and its ability to cause such a wide range of infections are due to its extensive virulence factors. In this study, we identified the role of the only GGDEF domain protein (GdpS [GGDEF domain protein from Staphylococcus]) in the virulence of S. aureus NCTC8325. Inactivation of gdpS results in an alteration in the production of a range of virulence factors, such as serine and cysteine proteases, fibrinogen-binding proteins, and, specifically, protein A (Spa), a major surface protein of S. aureus. The transcript level of spa decreases eightfold in the gdpS mutant compared with the parental NCTC8325 strain. Furthermore, the transcript level of sarS, which encodes a direct positive regulator of spa, also decreases in the gdpS mutant compared with the wild type, while the transcript levels of agr, sarA, sarT, and rot display no apparent changes in the gdpS mutant, suggesting that GdpS affects the expression of spa through interaction with SarS by unknown mechanisms. Furthermore, the complementation assays show that the influences of GdpS on spa and sarS depend on its N-terminal domain, which is predicted to be the sensor of a two-component system, rather than its C-terminal GGDEF domain with conserved GGDEF, suggesting that GdpS functions in S. aureus by an unknown mechanism independent of 3′,5′-cyclic diguanylic acid signaling.

ArtR, a novel sRNA of Staphylococcus aureus, regulates α-toxin expression by targeting the 5' UTR of sarT mRNA.

Recent studies point to the importance of small-noncoding RNAs (sRNAs) in bacterial virulence control. In Staphylococcus aureus, functional dissections of sRNAs are limited to RNA III, SprD, RsaE, SprA1, and SSR42 only. Here, we report the identification and functional analyses of a novel sRNA, which we have designated ArtR. Our data show that the AgrA protein can bind to the artR promoter and repress artR transcription, suggesting that, after RNA III, ArtR is the second sRNA regulated by AgrA. Furthermore, ArtR is unique in S. aureus and involved in virulence regulation by activating α-toxin expression. ArtR promotes the degradation of sarT mRNA by RNase III and arrests the translation of SarT by direct binding to the 5′ untranslated region of the sarT mRNA, suggesting that the activation of ArtR on the α-toxin expression was through SarT. This study reveals another kind of staphylococcal regulatory small RNA that plays a role in virulence control. It also indicates the diversity of small RNA-target mRNA interactions and how these multiple interactions can mediate virulence regulation in this pathogen.

Staphylococcus aureus glucose-induced biofilm accessory proteins, GbaAB, influence biofilm formation in a PIA-dependent manner

The Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis are capable of attaching to a biomaterial surface and forming resistant biofilms. The identification of biomolecular and regulatory factors involved in staphylococcal adhesion and biofilm formation is needed to understand biofilm-associated infection in humans. Here, we have identified a new operon, gbaAB (glucose induced biofilm accessory gene), that affects biofilm formation in S. aureus NCTC8325. Real-time reverse transcription PCR (RT-PCR) and electrophoretic mobility shift assay showed that GbaA and GbaB are transcribed from the same transcript, and GbaA directly inhibits the transcription of the gbaAB operon through self-repression. Our results indicated that the gbaA mutant displayed enhanced biofilm formation compared with the wild type. However, the gbaB and the gbaAB double mutant displayed reduced biofilm formation, suggesting that the gbaAB operon is involved in biofilm formation and that gbaB might be the key gene in biofilm regulation. Phenotypic analysis suggested that the gbaAB operon mediated biofilm formation of S. aureus at the multicellular aggregation stage rather than during initial attachment. In addition, real-time RT-PCR analysis showed that icaA was upregulated in the gbaA mutant and downregulated in the gbaB and gbaAB mutants compared with the wild type. In addition, the gbaA and the gbaB mutants affected the induction of biofilm formation by glucose. Our results suggest that the gbaAB operon is involved in the regulation of the multicellular aggregation step of S. aureus biofilm formation in response to glucose and that this regulation may be mediated through the ica operon.

AI-2 quorum sensing negatively regulates rbf expression and biofilm formation in Staphylococcus aureus

Staphylococcus aureus is an important pathogen that is capable of forming biofilms on biomaterial surfaces to cause biofilm-associated infections. Autoinducer 2 (AI-2), a universal language for interspecies communication, is involved in a variety of physiological activities, although its exact role in Gram-positive bacteria, especially in S. aureus, is not yet thoroughly characterized. Herein we demonstrate that inactivation of luxS, which encodes AI-2 synthase, resulted in increased biofilm formation and higher polysaccharide intercellular adhesion (PIA) production compared with the wild-type strain in S. aureus NCTC8325. The transcript level of rbf, a positive regulator of biofilm formation, was significantly increased in the luxS mutant. All of the parental phenotypes could be restored by genetic complementation and chemically synthesized 4,5-dihydroxy-2,3-pentanedione, the AI-2 precursor molecule, suggesting that AI-2 has a signaling function to regulate rbf transcription and biofilm formation in S. aureus. Phenotypic analysis revealed that the luxS rbf double mutant produced approximately the same amount of biofilms and PIA as the rbf mutant. In addition, real-time quantitative reverse transcription-PCR analysis showed that the icaA transcript level of the rbf mutant was similar to that of the luxS rbf double mutant. These findings demonstrate that the LuxS/AI-2 system regulates PIA-dependent biofilm formation via repression of rbf expression in S. aureus. Furthermore, we demonstrated that Rbf could bind to the sarX and rbf promoters to upregulate their expression.

Modulation of cell wall synthesis and susceptibility to vancomycin by the two-component system AirSR in Staphylococcus aureus NCTC8325

BackgroundVancomycin has been the medication of last resort to cure infections caused by Staphylococcus aureus since the increase in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA). Some strains have developed vancomycin-intermediate resistance, which is generally associated with altered expression of or mutations in some part of the two-component system (TCS), such as GraSR, VraSR, and WalKR.ResultsWe deleted the AirSR TCS in S. aureus NCTC8325 and compared the resultant transcript levels with those of its parent strain using microarray analysis. The results indicated that more than 20 genes that are related to cell wall metabolism were down-regulated in the airSR mutant. The airSR mutant exhibited reduced autolysis rates and reduced viability in the presence of vancomycin. Real-time reverse transcription PCR and DNA mobility shift assays verified that AirR can directly bind to and regulate genes that function in cell wall metabolism (cap, pbp1, and ddl) and autolysis (lytM).ConclusionsAirSR acts as a positive regulator in cell wall biosynthesis and turnover in Staphylococcus aureus NCTC8325.

The Staphylococcus aureus Protein-Coding Gene gdpS Modulates sarS Expression via mRNA-mRNA Interaction

ABSTRACT Staphylococcus aureus is an important Gram-positive pathogen responsible for numerous diseases ranging from localized skin infections to life-threatening systemic infections. The virulence of S. aureus is essentially determined by a wide spectrum of factors, including cell wall-associated proteins and secreted toxins that are precisely controlled in response to environmental changes. GGDEF domain protein from Staphylococcus (GdpS) is the only conserved staphylococcal GGDEF domain protein that is involved not in c-di-GMP synthesis but in the virulence regulation of S. aureus NCTC8325. Our previous study showed that the inactivation of gdpS generates an extensive change of virulence factors together with, in particular, a major Spa (protein A) surface protein. As reported, sarS is a direct positive regulator of spa. The decreased transcript levels of sarS in the gdpS mutant compared with the parental NCTC8325 strain suggest that gdpS affects spa through interaction with sarS. In this study, site mutation and complementary experiments showed that the translation product of gdpS was not involved in the regulation of transcript levels of sarS. We found that gdpS functioned through direct RNA-RNA base pairing with the 5′ untranslated region (5′UTR) of sarS mRNA and that a putative 18-nucleotide region played a significant role in the regulatory process. Furthermore, the mRNA half-life analysis of sarS in the gdpS mutant showed that gdpS positively regulates the mRNA levels of sarS by contributing to the stabilization of sarS mRNA, suggesting that gdpS mRNA may regulate spa expression in an RNA-dependent pathway.

Regulatory mechanism of the three-component system HptRSA in glucose-6-phosphate uptake in Staphylococcus aureus.

Glucose-6-phosphate (G6P) is a common alternative carbon source for various bacteria, and its uptake usually relies on the hexose phosphate antiporter UhpT. In the human pathogenic bacterium Staphylococcus aureus, the ability to utilize different nutrients, particularly alternative carbon source uptake in glucose-limiting conditions, is essential for its fitness in the host environment during the infectious process. It has been reported that G6P uptake in S. aureus is regulated by the three-component system HptRSA. When G6P is provided as the only carbon source, HptRSA could sense extracellular G6P and activate uhpT expression to facilitate G6P utilization. However, the regulatory mechanism of HptRSA is still unclear. In this study, we further investigated the HptRSA system in S. aureus. First, we confirmed that HptRSA is necessary for the normal growth of this pathogen in chemically defined medium with G6P supplementation, and we discovered that HptRSA could exclusively sense extracellular G6P compared to the other organophosphates we tested. Next, using isothermal titration calorimetry, we found that HptA could bind to G6P, suggesting that it may be the G6P sensor. After that experiment, using an electrophoresis mobility shift assay, we verified that the response regulator HptR could directly bind to the uhpT promoter and identified a putative binding site from −67 to −96-bp. Subsequently, we created different point mutations in the putative binding site and revealed that the entire 30-bp sequence is essential for HptR regulation. In summary, we unveiled the regulatory mechanism of the HptRSA system in S. aureus, HptA most likely functions as the G6P sensor, and HptR could implement its regulatory function by directly binding to a conserved, approximately 30-bp sequence in the uhpT promoter.