The Virulence Regulator Sae of Staphylococcus aureus: Promoter Activities and Response to Phagocytosis-Related Signals (original) (raw)
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Journal of Bacteriology, 2003
We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.
Journal of Bacteriology, 2006
The two-component system SaeRS consisting of the histidin kinase SaeS and the response regulator SaeR is known to act on virulence gene expression in Staphylococcus aureus. In order to get a more comprehensive picture on SaeR-regulated genes, we studied the contribution of the two-component system on global gene expression by using both the proteomic and transcriptomic approach. Altogether, a loss of SaeRS resulted in a decreased amount of at least 17 extracellular proteins and two cell surface-associated proteins, among them several important virulence factors such as HlgA, HlgB, HlgC, LukF, and LukM. SaeRS activates the expression of these genes at the transcriptional level. The amount of the five proteins Aur, SspA, SsaA, Plc, and GlpQ was negatively influenced by SaeRS. However, the transcription of the corresponding genes was not affected by the two-component system. SaeRS had also no measurable influence on the transcription of the regulatory genes agr, sarA, arlRS, and sigB that contribute to the regulation of SaeRS-dependent virulence factors identified in this investigation. Our results clearly show that SaeRS is strongly involved in the tight temporal control of virulence factor expression in S. aureus. Its precise role within the regulatory network remains to be determined.
Studies on the Expression of Regulatory Locus sae in Staphylococcus aureus
Current Microbiology, 2003
Global regulatory locus sae consists of a two-component signal transduction system coded by saeR and saeS genes that upregulates the transcription of several exoproteins. Northern analysis carried out in this study reveals the synthesis at late and post-exponential phases of a cotranscript of saeR and saeS structural genes of about 2.4 kb. This transcript is diminished in the isogenic agr::tetM mutant. Likewise, transcriptional fusion experiments show that sae expression is downregulated in the agr null mutant. Complementation analyses with plasmids carrying fragments of about 1.2 or 0.2 kbp upstream of saeR-saeS genes, which restore fully or only partially, respectively, the wild-type phenotype to the sae mutant, are in agreement with two initiation start points of transcription revealed by primer extension experiments. This work, as well as previous studies, reveals a complex hierarchical regulatory network involving several loci that control the expression of virulence determinants in S. aureus. Staphylococcus aureus is a major pathogen of man and animals; it synthesizes a large number of extracellular and cell wall-associated proteins that contribute to its virulence. Several global regulatory loci, such as agr, sar and sae, have been found to regulate the production of these virulence factors [2, 7, 13]. The agr locus consists of two divergent transcripts, RNAIII transcribed from the P3 promoter, which encodes ␦-hemolysin and acts in the regulation of secretory and cell wall-associated proteins, and RNAII, transcribed from the P2 promoter, which encodes the products of agrB, D, C, and A. Genes agrD and agrB encode and process an autoinducing peptide that acts as a signal that activates the expression of RNAII and RNAIII through a two-component signal system coded by agrC and agrA. Activation of RNAIII leads to increased production of ␣and -hemolysins, serine protease, DNase, and other exotoxins and to repression of the production of protein A, coagulase, and other cell wall-associated proteins [12, 14, 18]. The main product of the sar locus is a DNA-binding protein, SarA, which is transcribed from three different promoters. SarA acts on the expression of virulent factors indirectly by upregulating RNAIII transcription and also stimulating or repressing the transcription of several virulence genes such as hla, fnbA, cna, spa, and ssp (which code for ␣-hemolysin, fibronectin, and collagenbinding proteins, proteinA and serin-protease, respectively) in an agr-independent way [1, 4, 15, 25]. The regulatory locus designated sae (for S. aureus exoprotein expression) encodes a two-component regulatory system involving saeR, a response regulator, and saeS, a histidine protein kinase, of 687 and 1062 bp, respectively, and upregulates the production of ␣and -hemolysins, DNase, and coagulase at the transcriptional level [9, 10]. More recently, other virulence regulators have been described. The sarH1 or sarS locus, which belongs to a family of sar homologs, represses the transcription of ␣-hemolysin and activates spa transcription while its own transcription is repressed by sarA and agr [3, 23]. Other characterized genes of this family are sarT, which like sarH1 is negatively controlled by sarA and agr and † Died October 11, 2000.
PLoS ONE, 2008
S. aureus is a highly successful pathogen that is speculated to be the most common cause of human disease. The progression of disease in S. aureus is subject to multi-factorial regulation, in response to the environments encountered during growth. This adaptive nature is thought to be central to pathogenesis, and is the result of multiple regulatory mechanisms employed in gene regulation. In this work we describe the existence of a novel S. aureus regulator, an as yet uncharacterized ECF-sigma factor (s S ), that appears to be an important component of the stress and pathogenic responses of this organism. Using biochemical approaches we have shown that s S is able to associates with core-RNAP, and initiate transcription from its own coding region. Using a mutant strain we determined that s S is important for S. aureus survival during starvation, extended exposure to elevated growth temperatures, and Triton X-100 induced lysis. Coculture studies reveal that a s S mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth (7 days), or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS. Assays performed hourly for 72h revealed expression in typically background ranges. Analysis of a potential anti-sigma factor, encoded downstream of sigS, revealed it to have no obvious role in the upregulation of sigS expression. Using a murine model of septic arthritis, sigS-mutant infected animals lost significantly less weight, developed septic arthritis at significantly lower levels, and had increased survival rates. Studies of mounted immune responses reveal that sigS-mutant infected animals had significantly lower levels of IL-6, indicating only a weak immunological response. Finally, strains of S. aureus lacking sigS were far less able to undergo systemic dissemination, as determined by bacterial loads in the kidneys of infected animals. These results establish that s S is an important component in S. aureus fitness, and in its adaptation to stress. Additionally it appears to have a significant role in its pathogenic nature, and likely represents a key component in the S. aureus regulatory network. Citation: Shaw LN, Lindholm C, Prajsnar TK, Miller HK, Brown MC, et al. (2008) Identification and Characterization of s S , a Novel Component of the Staphylococcus aureus Stress and Virulence Responses. PLoS ONE 3(12): e3844.
The SaeRS Two-Component System of Staphylococcus aureus
In the Gram-positive pathogenic bacterium Staphylococcus aureus, the SaeRS two-component system (TCS) plays a major role in controlling the production of over 20 virulence factors including hemolysins, leukocidins, superantigens, surface proteins, and proteases. The SaeRS TCS is composed of the sensor histidine kinase SaeS, response regulator SaeR, and two auxiliary proteins SaeP and SaeQ. Since its discovery in 1994, the sae locus has been studied extensively, and its contributions to staphylococcal virulence and pathogenesis have been well documented and understood; however, the molecular mechanism by which the SaeRS TCS receives and processes cognate signals is not. In this article, therefore, we review the literature focusing on the signaling mechanism and its interaction with other global regulators.
The Role of Innate Immunity in Promoting SaeR/S-Mediated Virulence in Staphylococcus aureus
Journal of Innate Immunity, 2013
The ability of Staphylococcus aureus to infect tissues is dependent on precise control of virulence through gene-regulatory systems. While the SaeR/S two-component system has been shown to be a major regulator of S. aureus virulence, the influence of the host environment on SaeR/S-regulated genes (saeR/S targets) remains incompletely defined. Using QuantiGene 2.0 transcriptional assays, we examined expression of genes with the SaeR binding site in USA300 exposed to human and mouse neutrophils and host-derived peptides and during subcutaneous skin infection. We found that only some of the saeR/S targets, as opposed to the entire SaeR/S virulon, were activated within 5 and 10 min of interacting with human neutrophils as well as α-defensin. Furthermore, mouse neutrophils promoted transcription of saeR/S targets despite lacking α-defensin, and the murine skin environment elicited a distinctive expression profile of saeR/S targets. These findings indicate that saeR/S-mediated transcripti...
Journal of Bacteriology, 2010
Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length SaeR could be restored by sensor kinase SaeS-induced phosphorylation. Phosphorylated SaeR is more resistant to digestion by trypsin, suggesting conformational changes. DNase I footprinting assays revealed that the SaeR protection region in the P1 promoter contains a direct repeat sequence (GTTAAN 6 GTTAA [where N is any nucleotide]). This sequence is critical to the binding of phosphorylated SaeR. Mutational changes in the repeat sequence greatly reduced both the in vitro binding of SaeR and the in vivo function of the P1 promoter. From these results, we concluded that SaeR recognizes the direct repeat sequence as a binding site and that binding requires phosphorylation by SaeS.
SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ
PLoS ONE, 2013
The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeS L but causing activation in strains containing SaeS P. Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/ phosphatase activity.
Proceedings of the National Academy of Sciences, 2014
Significance Staphylococcus aureus is a ubiquitous pathogen that causes disease in a variety of tissues. Our studies provide insight into how this pathogen uses the SaeS sensor kinase to recognize innate immunity signals and induce a transcriptional response tailored for its environment. Our results demonstrate that the specificity of these responses is determined by individual amino acids predicted to be in an extracellular domain. These amino acids include aromatic anchors and a methionine residue essential for activation of target gene transcription and virulence. Our findings provide a putative mechanism for the ability of bacterial sensory systems to integrate and diversify the responses to host stimuli. They underscore the exquisite nature of bacterial signaling and the complexity of host-pathogen interactions. Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Sta...