Glycerol Monolaurate and Dodecylglycerol Effects on Staphylococcus aureus and Toxic Shock Syndrome Toxin-1 In Vitro and In Vivo

BACKGROUND:Glycerol monolaurate (GML), a 12 carbon fatty acid monoester, inhibits Staphylococcus aureus growth and exotoxin production, but is degraded by S. aureus lipase. Therefore, dodecylglycerol (DDG), a 12 carbon fatty acid monoether, was compared in vitro and in vivo to GML for its effects on S. aureus growth, exotoxin production, and stability. METHODOLOGY/PRINCIPAL FINDINGS:Antimicrobial effects of GML and DDG (0 to 500 microg/ml) on 54 clinical isolates of S. aureus, including pulsed-field gel electrophoresis (PFGE) types USA200, USA300, and USA400, were determined in vitro. A rabbit Wiffle ball infection model assessed GML and DDG (1 mg/ml instilled into the Wiffle ball every other day) effects on S. aureus (MN8) growth (inoculum 3x10(8) CFU/ml), toxic shock syndrome toxin-1 (TSST-1) production, tumor necrosis factor-alpha (TNF-alpha) concentrations and mortality over 7 days. DDG (50 and 100 microg/ml) inhibited S. aureus growth in vitro more effectively than GML (p<0.01) and was stable to lipase degradation. Unlike GML, DDG inhibition of TSST-1 was dependent on S. aureus growth. GML-treated (4 of 5; 80%) and DDG-treated rabbits (2 of 5; 40%) survived after 7 days. Control rabbits (5 of 5; 100%) succumbed by day 4. GML suppressed TNF-alpha at the infection site on day 7; however, DDG did not (<10 ng/ml versus 80 ng/ml, respectively). CONCLUSIONS/SIGNIFICANCE:These data suggest that DDG was stable to S. aureus lipase and inhibited S. aureus growth at lower concentrations than GML in vitro. However, in vivo GML was more effective than DDG by reducing mortality, and suppressing TNF-alpha, S. aureus growth and exotoxin production, which may reduce toxic shock syndrome. GML is proposed as a more effective anti-staphylococcal topical anti-infective candidate than DDG, despite its potential degradation by S. aureus lipase

Epithelial Proinflammatory Response and Curcumin-Mediated Protection from Staphylococcal Toxic Shock Syndrome Toxin-1

Staphylococcus aureus initiates infections and produces virulence factors, including superantigens (SAgs), at mucosal surfaces. The SAg, Toxic Shock Syndrome Toxin-1 (TSST-1) induces cytokine secretion from epithelial cells, antigen presenting cells (APCs) and T lymphocytes, and causes toxic shock syndrome (TSS). This study investigated the mechanism of TSST-1-induced secretion of proinflammatory cytokines from human vaginal epithelial cells (HVECs) and determined if curcumin, an anti-inflammatory agent, could reduce TSST-1-mediated pathology in a rabbit vaginal model of TSS. TSST-1 caused a significant increase in NF-κB-dependent transcription in HVECs that was associated with increased expression of TNF- α, MIP-3α, IL-6 and IL-8. Curcumin, an antagonist of NF-κB-dependent transcription, inhibited IL-8 production from ex vivo porcine vaginal explants at nontoxic doses. In a rabbit model of TSS, co-administration of curcumin with TSST-1 intravaginally reduced lethality by 60% relative to 100% lethality in rabbits receiving TSST-1 alone. In addition, TNF-α was undetectable from serum or vaginal tissue of curcumin treated rabbits that survived. These data suggest that the inflammatory response induced at the mucosal surface by TSST-1 is NF-κB dependent. In addition, the ability of curcumin to prevent TSS in vivo by co-administration with TSST-1 intravaginally suggests that the vaginal mucosal proinflammatory response to TSST-1 is important in the progression of mTSS

An Oxygen-Sensing Two-Component System in the Burkholderia cepacia Complex Regulates Biofilm, Intracellular Invasion, and Pathogenicity

Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence

<i>Burkholderia</i> FixLJ functions as an oxygen sensor.

<p><i>B</i>. <i>dolosa</i> (strain AU0158 or its <i>fixLJ</i> deletion mutant), <i>B</i>. <i>cenocepacia</i> (strain J2315), and <i>B</i>. <i>multivorans</i> (strain ATCC23344) carrying a <i>pfixK-lacZ</i> reporter plasmid. Negative Control denotes reporter plasmid carrying <i>S</i>. <i>meliloti fixK</i> promoter sequence. EV denotes empty complementation vector. LacZ activity was quantified by Miller Units. Bars represent the means of triplicate biological replicates and error bars represent one standard deviation (representative of three independent experiments). *P<0.001 by 1-way ANOVA with Tukey’s multiple comparison test.</p

The <i>B</i>. <i>dolosa fixLJ</i> deletion mutant is cleared faster in a murine pneumonia model.

<p>(A-D) C57BL/6 mice were intranasally challenged with ~4x10⁸ CFU/mouse of <i>B</i>. <i>dolosa</i> strain AU0158 or its <i>fixLJ</i> deletion mutant. Bacterial loads were measured at the following sites and time points: (A) Lungs, 1 day after infection; (B) Lungs, 7 days after infection; (C) Spleen, 1 day after infection; (D) Spleen, 7 days after infection. Two <i>fixLJ</i> deletion-infected mice had undetectable bacterial levels in the spleen at day 7 (panel D, shown as 10² CFU/gm). Data is representative from 2 separate experiments with 7–8 mice per group. (E-F) C57BL/6 mice were intranasally challenged with <i>B</i>. <i>dolosa</i> AU0158 <i>ΔfixLJ</i> + <i>fixLJ</i> (6.7x10⁸ CFU/mouse) or <i>B</i>. <i>dolosa</i> AU0158 <i>ΔfixLJ</i> + empty vector (EV) (7.4x10⁸ CFU/mouse). Bacterial loads were measured 7 days after infection in the lungs (E) and spleen (F). Data are derived from one experiment done with 7–8 mice per group. Each point represents one mouse, and bars represent medians. *P<0.05 by Mann Whitney U test.</p

Strains used in this study.

<p>Strains used in this study.</p

<i>B</i>. <i>dolosa</i> virulence is independent of the presence of flagella.

<p>(A) <i>B</i>. <i>dolosa</i> strain AU0158 and its <i>fliC</i> deletion mutant were plated on low-density (0.3%) LB agar and swimming distance was measured after incubation for 48 hours. *P<0.05 by 1-way NOVA with Tukey’s multiple comparison test. Bars represent mean measurements of 3–4 replicates and error bars represent one standard deviation (representative of three independent experiments). (B) Overnight cultures of <i>B</i>. <i>dolosa</i> strain AU0158, the <i>fixLJ</i> deletion mutant, or the <i>fliC</i> deletion mutant were diluted 1:100 in TSB with 1% glucose, incubated for 48 hours, and then assessed for biofilm formation using crystal violet. Bars represent mean measurements of 6 replicates and error bars represent one standard deviation of the data (representative of three independent experiments). (C-D) C57BL/6 mice were intranasally challenged with 4.8 x10⁸ CFU/mouse of strain AU0158 or its <i>fliC</i> deletion mutant. Bacterial loads were measured 7 days after infection in the lungs (C) and spleen (D). Data are derived from one experiment done with 7–8 mice per group. Each point represents one mouse and bars represent medians.</p

Predicted domains of <i>B</i>. <i>dolosa</i> strain AU0158 FixL and FixJ.

<p>Domains predicted by SMART [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006116#ppat.1006116.ref025" target="_blank">25</a>]. PAS domains (named after 3 proteins in which they occur, namely, Per, Arnt, and Sim) are seen commonly in signaling proteins where they function as signal sensor and often include a cofactor such as heme. Domain abbreviations: TM- transmembrane, PAC- Motif C-terminal to PAS motif, HisKA-histidine kinase, HATPase- histidine kinase-associated ATPase, REC-CheY homologous receiver domain, and HTH LuxR-helix-turn-helix-Lux regulon (DNA binding domain).</p

The <i>B</i>. <i>dolosa fixLJ</i> deletion mutant is less invasive of epithelial and macrophage-like cells.

<p>A549 cells (A) or THP-1 cells treated with 200 nM PMA for 3 days (B) in 24-well plates were infected with ~2x10⁶ CFU/well (MOI of ~10:1) of strain AU0158, its <i>fixLJ</i> deletion mutant, or its <i>fliC</i> deletion mutant for 2 hours, after which the percent of internalized bacterial relative to the total bacterial growth was determined by killing extracellular bacteria with kanamycin (1 mg/mL). Means from 2–3 separate experiments with three replicates per experiment are plotted with error bars representing one standard deviation. *<i>P</i><0.05 by 1-way ANOVA with Tukey’s multiple comparison test. (C) THP-1-derived macrophages were infected with ~2x10⁶ CFU/well of <i>B</i>. <i>dolosa</i> for 2 hours, after which the extracellular bacteria were killed by treatment with kanamycin (1 mg/mL) and number of intracellular bacteria were determined after a 24-hour incubation. *<i>P</i><0.05 by t test. (D) THP-1-derived macrophages were infected with ~2x10⁶ CFU/well of <i>B</i>. <i>dolosa</i> for varying amounts of time (15 min-2 hours) after which the number of internalized bacteria was determined by killing extracellular bacteria with kanamycin (1 mg/mL). *<i>P</i><0.05 by t test compared to the <i>fixLJ</i> deletion mutant at that time point. (E) THP-1-derived macrophages were infected with ~2x10⁶ CFU/well of <i>B</i>. <i>dolosa</i> for 2 hours, after which the extracellular bacteria were treated with kanamycin (1 mg/mL) for varying amounts of time, after which the percent of internalized bacterial relative to the total bacterial growth within the initial 2-hour infection was determined. *<i>P</i><0.05 by t test compared to strain AU0158 at that time point. # <i>P</i><0.05 by 1-way ANOVA with Tukey’s multiple comparison test compared to hour-1 measurement. (C-E) Means from representative experiment repeated twice. Separate experiments with three replicates per experiment are plotted with error bars representing one standard deviation.</p