The Staphylococcus aureus GGDEF domain-containing protein, GdpS, influences protein A gene expression in a cyclic diguanylic acid-independent manner - PubMed (original) (raw)

The Staphylococcus aureus GGDEF domain-containing protein, GdpS, influences protein A gene expression in a cyclic diguanylic acid-independent manner

Fei Shang et al. Infect Immun. 2009 Jul.

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.

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Figures

FIG. 1.

FIG. 1.

Bioinformatics identification of gdpS (SAOUHSC_00760) in S. aureus NCTC8325. (A) Chromosomal organization of the gdpS gene and its surrounding region. Upstream of gdpS, there exists an operon predicted to be involved in glutathione metabolism. Downstream of gdpS, there exists a gene divergently transcribed from gdpS, encoding a lipophilic protein that affects the lysis rate and methicillin resistance level. (B) Predicted domain structure of GdpS. TM, transmembrane domain. 5TMR-LYT is a transmembrane domain proposed to be the sensor of the LytS-YhcK-type histidine protein kinase.

FIG. 2.

FIG. 2.

Correlation of microarray and real-time RT-PCR results (microarray versus real-time RT-PCR). The differences in the transcription of seven genes were log2 transformed and plotted against each other. The diagonal line indicates that the ratio from the microarray data is similar as that from the real-time RT-PCR data. The abscissa represents the log2 ratio of mutant to wild type according to the real-time RT-PCR data, and the ordinate represents the log2 ratio of mutant to wild type according to the microarray data. The two methods are better correlated if the points are nearer the diagonal line.

FIG. 3.

FIG. 3.

Comparative measurement of spa and its regulator gene transcripts by real-time RT-PCR in S. aureus NCTC8325 (wild type [WT]), SX3 (gdpS mutant), and SX4 (gdpS mutant with a plasmid encoding full-length GdpS). All the strains were grown in TSB medium to an OD600 of 1.7. The relative transcription of each gene compared to the constitutively expressed 16S rRNA gene in SX3 and SX4 was compared with that in the wild type, to which we assigned a value of 1. Error bars indicate standard deviations.

FIG. 4.

FIG. 4.

(A) The transcriptional regulation of spa and sarS expression by GdpS was compared using real-time RT-PCR in S. aureus NCTC8325 (wild type [WT]), SX3 (gdpS mutant), SX4 (gdpS mutant with a plasmid encoding full-length GdpS), SX5 (gdpS mutant with a plasmid encoding the N terminus of GdpS), SX6 (gdpS mutant with a plasmid encoding GdpS with a mutated GGDEF domain), and SX7 (gdpS mutant with a plasmid encoding GdpS with a mutated N terminus). All the strains were grown in TSB medium to an OD600 of 1.7. The relative transcription of each gene compared to the constitutively expressed 16S rRNA gene in strains SX3, SX4, SX5, SX6, and SX7 was compared with that in the wild type, to which we assigned a value of 1. (B) Western blot analysis of Spa in S. aureus NCTC8325, SX3, SX4, SX5, SX6, and SX7. All the strains were grown in TSB medium to an OD600 of 1.5.

FIG. 5.

FIG. 5.

Proposed regulation of Spa by GdpS, SarS, RNAIII, SarA, SarT, and Rot. Previous work has shown that expression of spa is negatively controlled by Agr/RNAIII and SarA through both direct regulation and interaction with SarS. SarT and Rot upregulate the expression of spa only though positive regulation of sarS. Our work indicates that GdpS influences expression of spa via SarS in an RNAIII-, SarA-, SarT-, and Rot-independent manner.

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