The <i>Staphylococcus aureus</i> Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression

<div><p><i>Staphylococcus aureus</i> is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of <i>S</i>. <i>aureus</i> is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking <i>mgrA</i> fail to clump in the presence of fibrinogen, and clumping can be restored to an <i>arlRS</i> mutant by overexpressing either <i>arlRS</i> or <i>mgrA</i>, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses <i>ebh</i>, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of <i>ebh</i> and <i>sraP</i>. Clumping can be restored to an <i>mgrA</i> mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that <i>mgrA</i> mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins <i>ebh</i>, <i>sraP</i>, and <i>sasG</i>. While <i>mgrA</i> mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins.</p></div

<i>mgrA</i> mutants are less virulent in a combined rabbit model of sepsis and endocarditis.

<p>Prior to infection, aortic valve damage was induced by temporary placement of plastic catheters in the carotid artery. Catheters were removed and rabbits were infected intravenously with either strain 502a (A-C), or strain MW2 (D-F). WT strains were compared to the <i>mgrA</i> single mutant, and for MW2 the <i>mgrA ebh sraP sasG</i> quadruple mutant (Δquad) was also tested. Rabbits were monitored for up to four days, and survival (A, D), total heart valve vegetation weights (B, E), and CFUs per total vegetations (C, F) were monitored. Statistical significance was determined using a log-rank (Mantel-Cox) test (survival), or two-tailed t test (vegetation weights and CFU counts). ** p<0.005, * p<0.05, NS not significant.</p

Comparison of clumping phenotypes in strains with truncated <i>ebh</i> genes.

<p>(A) Schematic of predicted Ebh proteins from a collection of <i>S</i>. <i>aureus</i> strains, showing only the product of the first predicted <i>ebh</i> ORF for each strain. Key domains, as well as the secretion signal sequence and transmembrane (TM) domain are indicated. (B) Clumping of <i>arlRS</i> and <i>mgrA</i> mutants in each of the strains shown in (A) after 1 h of incubation with human plasma. (C) Clumping time course of LAC, <i>mgrA</i> mutant and <i>mgrA ebh</i> double mutants incubated with human plasma. All clumping results represent averages of three separate experiments.</p

ArlRS is required for <i>mgrA</i> expression.

<p>The <i>mgrA</i> gene has two promoters, depicted in (C). Lines labeled P2 and P1 show the fragments used to make transcriptional reporters. (A) Each <i>mgrA</i> promoter was independently cloned upstream of a promoterless copy of GFP in plasmid pCM11. Expression from the upstream promoter <i>mgrA</i> P2 (left) and downstream <i>mgrA</i> P1 (right) was assessed in the WT strain LAC (black) and the <i>arlRS</i> mutant (blue). (B) Western blot showing ArlR and MgrA protein levels at various time points (hours). (D) Quantification of MgrA protein levels in LAC (black, solid line) and the <i>arlRS</i> mutant (blue, solid line), and growth curves of the same strains (dotted lines). Quantification is representative of three separate experiments.</p

Epistasis analysis indicates MgrA acts downstream of ArlRS.

<p>LAC WT, <i>arlRS</i>, and <i>mgrA</i> mutants contained either the empty vector or plasmids for constitutive expression of <i>arlRS</i> or <i>mgrA</i>. (A) Clumping ability was measured after two hours of incubation with human plasma, and results from three separate experiments were averaged. (B) Dot blot showing Ebh protein levels in 2-fold dilutions of culture supernatants from overnight cultures. Strains were identical to those used in Fig 3A except that they lacked <i>spa</i>, to prevent non-specific antibody binding.</p

Proposed model for how the ArlRS-MgrA regulatory cascade controls clumping (A) and biofilm formation (B).

<p>In response to an unknown signal, the ArlRS two component system activates expression of MgrA, which in turn represses expression of the large surface proteins Ebh, SraP, and SasG. ClfA interacts with fibrinogen, allowing clumping to occur (inset). When ArlRS is inhibited or inactivated, MgrA production is diminished and expression of Ebh, SraP, and SasG is de-repressed (B). These surface proteins interfere with clumping, while up-regulation of SasG can also promote biofilm formation (inset). Fg, fibrinogen.</p

Surface proteins regulated by MgrA.

<p>Surface proteins regulated by MgrA.</p

MgrA directly represses <i>sraP</i>.

<p>(A) The <i>sraP</i> promoter region, showing the two putative transcription start sites (marked with black circles) determined using 5’ RACE. Potential -10 and -35 promoter elements are boxed, and possible MgrA binding sites are shown in red text. The <i>sraP</i> ATG start codon is indicated in bold font. The promoter probe used for EMSA experiments is underlined. (B) Schematic of the <i>sraP</i> gene cluster, including its dedicated secretory system (<i>secY2</i>, <i>asp1-3</i>, and <i>secA2</i>) as well as the putative glycosyltransferases <i>gtfA</i> and <i>gtfB</i>. Inset below shows the two promoters mapped in (A) and the regions used to make transcriptional reporter plasmids (labeled rep 1 and rep 3). (C) Expression of a GFP transcriptional fusion containing both P1 and P2 (labeled rep 1 in panel B) in LAC and the isogenic <i>mgrA</i> mutant. (D) Expression of transcriptional reporter 3, containing just <i>sraP</i> P2 driving production of GFP. (E) Expression of each gene in the <i>sraP</i> gene cluster in LAC and the <i>mgrA</i> mutant, measured by qRT-PCR. Values are averages and standard deviations of three biological replicates and are normalized to expression in LAC for each gene. (F) EMSA assessing MgrA binding to the <i>sraP</i> P2 promoter. Increasing concentrations of purified MgrA were incubated with the probe underlined in panel A. Unbound probe and shifted probe are indicated on the left. In the last three lanes competition with a 10-fold excess of unlabeled probes was assessed. SP, specific probe identical to the labeled probe; MP, specific probe in which the putative MgrA binding site has been mutated; NSP, non-specific probe.</p

Identification of other surface proteins involved in inhibiting clumping.

<p>(A) Genes encoding surface proteins predicted to be regulated by MgrA were inactivated in an <i>mgrA ebh</i> double mutant background, and clumping was measured after 2 h of incubation with human plasma. (B) Comparison of clumping in LAC strains lacking combinations of <i>mgrA</i>, <i>ebh</i>, and <i>sraP</i>, measured after incubation with human plasma for two hours. (C) Clumping of MW2 strains lacking combinations of <i>mgrA</i>, <i>ebh</i>, <i>sraP</i>, and <i>sasG</i>. Graph shows clumping after 1 h of incubation with human plasma. All clumping results represent averages of three separate experiments.</p

MgrA directly represses <i>ebh</i>.

<p>(A) Quantification of transcripts of genes related to clumping in LAC and the <i>mgrA</i> mutant using qRT-PCR. Values are averages and standard deviations of three biological replicates, normalized to expression in LAC for each gene. (B) Expression of <i>ebh</i> was measured in LAC and the <i>mgrA</i> mutant using a transcriptional fusion of the entire intergenic region upstream of <i>ebh</i> to GFP. (C) The putative <i>ebh</i> transcription start site, indicated by the black circle, was determined by 5’ RACE. Possible -10 and -35 promoter elements are boxed, and the <i>ebh</i> start codon (GTG) is shown in bold. Potential MgrA binding sites are shown in red text, and the sequence of the DNA probe used for EMSA experiments is underlined. (D) Ebh protein levels were measured by dot blot in 2-fold dilutions of supernatants from overnight cultures of the indicated strains. All strains also lacked the <i>spa</i> gene. (E) EMSA showing MgrA binding to the <i>ebh</i> promoter. Increasing concentrations of MgrA were incubated with an IRDye-labeled probe (P_<i>ebh</i>, sequence underlined in C) before separation by PAGE. Unbound probe (P_<i>ebh</i>) and MgrA-probe complex (shift) are indicated. The last three lanes show competition experiments, where the binding reaction included a 10-fold excess of unlabeled specific probe (SP), non-specific probe (NSP), or a version of the specific probe in which the proposed MgrA binding site was mutated (MP).</p