Identification of the amino acids essential for <scp>LytSR</scp>‐mediated signal transduction in <scp><i>S</i></scp><i>taphylococcus aureus</i> and their roles in biofilm‐specific gene expression

Lehman, McKenzie K.; Bose, Jeffrey L.; Sharma-Kuinkel, Batu K.; Moormeier, Derek E.; Endres, Jennifer L.; Sadykov, Marat R.; Biswas, Indranil; Bayles, Kenneth W.

doi:10.1111/mmi.12902

Cited by 37 publications

(23 citation statements)

References 57 publications

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“…␤-Galactosidase activity was determined as described previously, with minor modifications (53). Briefly, 1-ml samples were harvested from 12.5-ml late-exponentialgrowth-phase (5 h) or stationary-growth-phase (8 h) cultures, pelleted, and resuspended in 1.2 ml Z-buffer.…”

Section: Methodsmentioning

confidence: 99%

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System

2017

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In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of ␣-hemolysin and other virulence factors in Staphylococcus aureus. In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB, saeR, and saeS mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I (coa) and class II (hla) target genes were downregulated during the exponential growth phase in the vfrB mutant, compared to the wild-type strain. In addition, saePQRS expression was decreased in the vfrB mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, hla expression was decreased in the vfrB mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated ␣-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in S. aureus.IMPORTANCE The SaeRS two-component system is a key regulator of virulence determinant production in Staphylococcus aureus. Although the regulon of this twocomponent system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase VfrB as a positive regulatory modulator of SaeRS-mediated transcription of virulence factors in S. aureus. In addition to describing a new regulatory aspect of SaeRS, this study establishes a link between fatty acid kinase activity and virulence factor regulation.

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Section: Methodsmentioning

confidence: 99%

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System

2017

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“…TCSs are predominant signal transduction components used by prokaryotic microorganisms to convert rapid environmental changes into specific adaptive responses (Bourret and Silversmith, 2010; Capra and Laub, 2012; Lehman et al, 2015). They typically consist of a membrane-bound sensor histidine kinase (HK), which senses a specific environmental stimulus and undergoes autophosphorylation, and a cognate response regulator (RR), which receives the phosphoryl group via various phosphotransfer pathways and modulates gene transcription by binding to cis regulatory elements in the promoter region (Forst et al, 1989; Huang et al, 1997; Bilwes et al, 1999; Stock et al, 2000; Bourret and Silversmith, 2010).…”

Section: Introductionmentioning

confidence: 99%

The Two-Component System RsrS-RsrR Regulates the Tetrathionate Intermediate Pathway for Thiosulfate Oxidation in Acidithiobacillus caldus

Wang

Lin

et al. 2016

Front. Microbiol.

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Acidithiobacillus caldus (A. caldus) is a common bioleaching bacterium that possesses a sophisticated and highly efficient inorganic sulfur compound metabolism network. Thiosulfate, a central intermediate in the sulfur metabolism network of A. caldus and other sulfur-oxidizing microorganisms, can be metabolized via the tetrathionate intermediate (S4I) pathway catalyzed by thiosulfate:quinol oxidoreductase (Tqo or DoxDA) and tetrathionate hydrolase (TetH). In A. caldus, there is an additional two-component system called RsrS-RsrR. Since rsrS and rsrR are arranged as an operon with doxDA and tetH in the genome, we suggest that the regulation of the S4I pathway may occur via the RsrS-RsrR system. To examine the regulatory role of the two-component system RsrS-RsrR on the S4I pathway, ΔrsrR and ΔrsrS strains were constructed in A. caldus using a newly developed markerless gene knockout method. Transcriptional analysis of the tetH cluster in the wild type and mutant strains revealed positive regulation of the S4I pathway by the RsrS-RsrR system. A 19 bp inverted repeat sequence (IRS, AACACCTGTTACACCTGTT) located upstream of the tetH promoter was identified as the binding site for RsrR by using electrophoretic mobility shift assays (EMSAs) in vitro and promoter-probe vectors in vivo. In addition, ΔrsrR, and ΔrsrS strains cultivated in K2S4O6-medium exhibited significant growth differences when compared with the wild type. Transcriptional analysis indicated that the absence of rsrS or rsrR had different effects on the expression of genes involved in sulfur metabolism and signaling systems. Finally, a model of tetrathionate sensing by RsrS, signal transduction via RsrR, and transcriptional activation of tetH-doxDA was proposed to provide insights toward the understanding of sulfur metabolism in A. caldus. This study also provided a powerful genetic tool for studies in A. caldus.

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“…␤-Galactosidase assays were performed based on protocols previously described (26). Briefly, 1.0 ml of cell cultures was centrifuged, and the cell pellet was resuspended in 1 ml Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , 50 mM ␤-mercaptoethanol, pH 7.0) and was disrupted using a FastPrep FP120 (MP Biomedicals, Santa Ana, CA).…”

Section: Methodsmentioning

confidence: 99%

SrrAB Modulates Staphylococcus aureus Cell Death through Regulation of cidABC Transcription

et al. 2016

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The death and lysis of a subpopulation in Staphylococcus aureus biofilm cells are thought to benefit the surviving population by releasing extracellular DNA, a critical component of the biofilm extracellular matrix. Although the means by which S. aureus controls cell death and lysis is not understood, studies implicate the role of the cidABC and lrgAB operons in this process. Recently, disruption of the srrAB regulatory locus was found to cause increased cell death during biofilm development, likely as a result of the sensitivity of this mutant to hypoxic growth. In the current study, we extended these findings by demonstrating that cell death in the ⌬srrAB mutant is dependent on expression of the cidABC operon. The effect of cidABC expression resulted in the generation of increased reactive oxygen species (ROS) accumulation and was independent of acetate production. Interestingly, consistently with previous studies, cidC-encoded pyruvate oxidase was found to be important for the generation of acetic acid, which initiates the cell death process. However, these studies also revealed for the first time an important role of the cidB gene in cell death, as disruption of cidB in the ⌬srrAB mutant background decreased ROS generation and cell death in a cidCindependent manner. The cidB mutation also caused decreased sensitivity to hydrogen peroxide, which suggests a complex role for this system in ROS metabolism. Overall, the results of this study provide further insight into the function of the cidABC operon in cell death and reveal its contribution to the oxidative stress response. IMPORTANCEThe manuscript focuses on cell death mechanisms in Staphylococcus aureus and provides important new insights into the genes involved in this ill-defined process. By exploring the cause of increased stationary-phase death in an S. aureus ⌬srrAB regulatory mutant, we found that the decreased viability of this mutant was a consequence of the overexpression of the cidABC operon, previously shown to be a key mediator of cell death. These investigations highlight the role of the cidB gene in the death process and the accumulation of reactive oxygen species. Overall, the results of this study are the first to demonstrate a positive role for CidB in cell death and to provide an important paradigm for understanding this process in all bacteria.

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Identification of the amino acids essential for LytSR‐mediated signal transduction in Staphylococcus aureus and their roles in biofilm‐specific gene expression

Cited by 37 publications

References 57 publications

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System

The Two-Component System RsrS-RsrR Regulates the Tetrathionate Intermediate Pathway for Thiosulfate Oxidation in Acidithiobacillus caldus

SrrAB Modulates Staphylococcus aureus Cell Death through Regulation of cidABC Transcription

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