<i>E. faecalis</i> MN1 inhibits the IL-8 response of HVECs to the staphylococcal superantigen TSST-1.

<p><i>E. faecalis</i> MN1 (1×10⁷ CFU) was incubated with HVECs in the absence and presence of TSST-1 (100 µg/ml) for 6 h. Cell culture supernates were collected and IL-8 was measured by ELISA. *Significantly higher than all other conditions and #significantly lower than medium only control by one-way ANOVA [<i>F</i>(3,29) = 251.2, <i>p</i><0.0001] and Tukey's post-hoc test. N = 3–6 replicates.</p

<i>E. faecalis</i> MN1 low molecular weight fraction inhibits TSST-1-induced T cell proliferation.

<p><i>E. faecalis</i> MN1 supernate was treated with ethanol (80% final concentration), insoluble material was removed by centrifugation, soluble material (referred to as the low molecular weight fraction) was concentrated 480 times relative to the original culture fluid, and dilutions incubated with human PBMCs and TSST-1 (1 µg/well) in a superantigenicity assay to assess the ability of secreted factors to inhibit TSST-1-induced T cell proliferation. The PBMC control shows the average CPMs for PBMCs alone and TSST-1 shows the average CPMs for TSST-1 alone. Treatments 48× to 0.0048× were significantly different from TSST-1 alone by individual Student's <i>t</i> tests (*p<0.001). Three independent wells for each treatment were assayed for cell viability, and viability in all cases was >95%.</p

<i>E. faecalis</i> MN1 secretes a factor responsible for inhibition of IL-8 production by HVECs in response to TSST-1.

<p>Transwell permeable supports (0.4 µm pore size) were used to determine the ability of <i>E. faecalis</i> MN1 secreted factors to inhibit TSST-1-induced IL-8 production by HVECs. HVECs were grown to confluency in the bottom wells and transwell permeable supports were added on the day of experimentation. <i>E. faecalis</i> MN1 (2×10⁷ CFU) was added to either the transwell (to assess secreted factors) or the bottom well (as a positive control) in the presence or absence of TSST-1 (100 µg/ml) and incubated for 6 h. In one condition, catalase enzyme (100 µg/ml) was added to break down hydrogen peroxide produced by the lactobacilli. Cell culture supernates were collected and analyzed by ELISA for IL-8 production. These results contain data averaged from three separate experiments, with 6 total replicates in each experimental condition. *All conditions with <i>E. faecalis</i> MN1 present demonstrated significantly lower IL-8 levels compared to the TSST-1 only control by individual Student's <i>t</i> tests (p<0.01).</p

<i>E. faecalis</i> MN1 supernate inhibits TSST-1-induced T cell proliferation.

<p><i>E. faecalis</i> MN1 supernate was incubated with human PBMCs and TSST-1 (1 µg/well) in a superantigenicity assay to assess the ability of secreted factors to inhibit TSST-1-induced T cell proliferation. The two highest doses of supernate (20 and 2 µl per well) significantly inhibited T cell proliferation due to TSST-1 by individual Student's <i>t</i> tests (*p<0.001). The 0 µl control shows the average CPMs for PBMCs with and without TSST-1.</p

<i>E. faecalis</i> MN1 makes low levels of hydrogen peroxide.

<p><i>E. faecalis</i> MN1 and <i>L. crispatus</i> ATCC 33197 were grown overnight in KSFM tissue culture medium. Hydrogen peroxide production was measured using a H₂O₂ colorimetric detection assay kit.</p

<i>E. faecalis</i> MN1 secretes a small molecule responsible for inhibition of TSST-1-induced IL-8 production by HVECs.

<p>A) Eighty percent ethanol was used to precipitate high molecular weight factors from filter-sterilized <i>E. faecalis</i> MN1 supernate. Isolated factors were added to HVECs ± TSST-1 (100 µg/ml) for 6 h, and IL-8 was measured by ELISA. Only the 20 µl condition showed inhibition of TSST-1-induced IL-8 by Student's <i>t</i> test (p<0.001). B) Low molecular weight (unprecipitated) factors were concentrated by lyophilization, resuspended in PBS to the same concentration as the precipitated material, and incubated with cells as described above. Lower doses of unprecipitated material were used to minimize cytotoxicity to the HVECs. The 10 µl condition showed absolute inhibition of IL-8 production (*p<0.001), whereas the 1 µl condition actually enhanced IL-8 levels in response to TSST-1 (**p<0.001) by individual Student's <i>t</i> tests. These results are based on the average of two separate experiments, each containing three replicates per condition. C) Filter-sterilized <i>E. faecalis</i> MN1 supernate was separated into different molecular weight fractions using Microcon centrifugal filters and incubated with HVECs +/− TSST-1 (100 µg/ml) for 6 h. Inhibition of TSST-1-induced IL-8 production is maintained in all fractions (<3 kDa, <10 kDa, <30 kDa) when the cells were incubated with 20 µl of material (*p<0.005); a higher level of IL-8 was detected when cells were incubated with smaller amounts of the <30 kDa fraction (**p<0.05) by individual Student's <i>t</i> tests. These data are from a single experiment carried out in triplicate. D) Filter-sterilized <i>E. faecalis</i> MN1 supernate was treated with DNase, RNase, or protease prior to incubation with TSST-1 (100 µg/ml) on HVECs for 6 h. None of the treatments affected the ability of the secreted factor to inhibit TSST-1-induced IL-8 production from the cells by individual Student's <i>t</i> tests (*p<0.001). These data are the average of two experiments, each done in triplicate.</p

<i>E. faecalis</i> MN1 inhibits the IL-8 response of HVECs to vaginal pathogens.

<p>HVECs were incubated with <i>C. albicans</i> (2×10⁵ CFU/well), <i>G. vaginalis</i> (2×10⁶ CFU/well), or <i>N. gonorrhoeae</i> (2×10⁶ CFU/well) in the absence or presence of <i>E. faecalis</i> MN1 (8×10⁶ CFU/well) for 6 h and IL-8 production was measured by ELISA. *In each case, <i>E. faecalis</i> MN1 significantly inhibited the IL-8 response of the cells to the pathogen by individual Student's <i>t</i> tests (p<0.01). N = 3 replicates.</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

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

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