Corey P. Parlet

Castanea sativa (European Chestnut) Leaf Extracts Rich in Ursene and Oleanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance

The Mediterranean is home to a rich history of medical traditions that have developed under the influence of diverse cultures over millennia. Today, many such traditions are still alive in the folk medical practices of local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections led us to study Castanea sativa (European Chestnut) for its potential antibacterial activity. Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes), against all Staphylococcus aureus accessory gene regulator (agr) alleles. We present layers of evidence of agr blocking activity (IC50 1.56–25 μg mL-1), as measured in toxin outputs, reporter assays hemolytic activity, cytotoxicity studies, and an in vivo abscess model. We demonstrate the extract’s lack of cytotoxicity to human keratinocytes and murine skin, as well as lack of growth inhibitory activity against S. aureus and a panel of skin commensals. Lastly, we demonstrate that serial passaging of the extract does not result in acquisition of resistance to the quorum quenching composition. In conclusion, through disruption of quorum sensing in the absence of growth inhibition, this study provides insight into the role that non-biocide inhibitors of virulence may play in future antibiotic therapies.

Virulence Inhibitors from Brazilian Peppertree Block Quorum Sensing and Abate Dermonecrosis in Skin Infection Models.

Widespread antibiotic resistance is on the rise and current therapies are becoming increasingly limited in both scope and efficacy. Methicillin-resistant Staphylococcus aureus (MRSA) represents a major contributor to this trend. Quorum sensing controlled virulence factors include secreted toxins responsible for extensive damage to host tissues and evasion of the immune system response; they are major contributors to morbidity and mortality. Investigation of botanical folk medicines for wounds and infections led us to study Schinus terebinthifolia (Brazilian Peppertree) as a potential source of virulence inhibitors. Here, we report the inhibitory activity of a flavone rich extract “430D-F5” against all S. aureus accessory gene regulator (agr) alleles in the absence of growth inhibition. Evidence for this activity is supported by its agr-quenching activity (IC50 2–32 μg mL−1) in transcriptional reporters, direct protein outputs (α-hemolysin and δ-toxin), and an in vivo skin challenge model. Importantly, 430D-F5 was well tolerated by human keratinocytes in cell culture and mouse skin in vivo; it also demonstrated significant reduction in dermonecrosis following skin challenge with a virulent strain of MRSA. This study provides an explanation for the anti-infective activity of peppertree remedies and yields insight into the potential utility of non-biocide virulence inhibitors in treating skin infections.

Characterization of a novel mouse model with genetic deletion of CD177

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.

Chronic Ethanol Feeding Induces Subset Loss and Hyporesponsiveness in Skin T Cells

BACKGROUND Chronic alcoholism is associated with increased incidence and severity of cutaneous infection. Skin-resident T cells orchestrate numerous immunological functions that are critically involved in both tissue homeostasis and cutaneous immunity. The impact of chronic ethanol (EtOH) exposure on skin T cells has not previously been examined; given their important role in maintaining the immune barrier function of the skin further study is warranted. METHODS Mice were administered EtOH in the drinking water for 12 to 16 weeks. Flow cytometry was used to evaluate impact of EtOH feeding on skin T cell numbers, rates of proliferation, and apoptosis as well as activation marker expression and cytokine production after ex vivo stimulation. RESULTS Chronic EtOH feeding caused a baseline reduction in dendritic epidermal T cell (DETC) numbers that corresponded with reduced expression of the activation marker JAML following phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation. Chronic EtOH feeding did not alter total numbers of dermal T cells, but specific subset loss was observed in Foxp3(+) regulatory T cells (Tregs) as well as CD3hi, Vγ3(+) and CD3int, Vγ3(-) dermal γδ T cells. EtOH-induced dysfunction in the latter population, which represents prototypical interleukin-17 (IL-17)-producing dermal γδT17s, was made evident by diminished IL-17 production following anti-CD3 stimulation. Additionally, the capacity of lymph node γδ T cells to produce IL-17 following anti-CD3 and PMA/ionomycin stimulation was impaired by chronic EtOH feeding. CONCLUSIONS Chronic EtOH feeding induced defects in both numbers and function of multiple skin T cell subsets. The decreased density and poor responsiveness of DETCs and γδT17 cells in particular would be expected to compromise immune effector mechanisms necessary to maintain a protective barrier and restrict pathogen invasion. These findings demonstrate the sensitivity of skin T cells to EtOH and provide new mechanisms to help explain the propensity of alcoholics to suffer skin infection.

Signal Biosynthesis Inhibition with Ambuic Acid as a Strategy To Target Antibiotic-Resistant Infections

ABSTRACT There has been major interest by the scientific community in antivirulence approaches against bacterial infections. However, partly due to a lack of viable lead compounds, antivirulence therapeutics have yet to reach the clinic. Here we investigate the development of an antivirulence lead targeting quorum sensing signal biosynthesis, a process that is conserved in Gram-positive bacterial pathogens. Some preliminary studies suggest that the small molecule ambuic acid is a signal biosynthesis inhibitor. To confirm this, we constructed a methicillin-resistant Staphylococcus aureus (MRSA) strain that decouples autoinducing peptide (AIP) production from regulation and demonstrate that AIP production is inhibited in this mutant. Quantitative mass spectrometric measurements show that ambuic acid inhibits signal biosynthesis (50% inhibitory concentration [IC50] of 2.5 ± 0.1 μM) against a clinically relevant USA300 MRSA strain. Quantitative real-time PCR confirms that this compound selectively targets the quorum sensing regulon. We show that a 5-μg dose of ambuic acid reduces MRSA-induced abscess formation in a mouse model and verify its quorum sensing inhibitory activity in vivo. Finally, we employed mass spectrometry to identify or confirm the structure of quorum sensing signaling peptides in three strains each of S. aureus and Staphylococcus epidermidis and single strains of Enterococcus faecalis, Listeria monocytogenes, Staphylococcus saprophyticus, and Staphylococcus lugdunensis. By measuring AIP production by these strains, we show that ambuic acid possesses broad-spectrum efficacy against multiple Gram-positive bacterial pathogens but does not inhibit quorum sensing in some commensal bacteria. Collectively, these findings demonstrate the promise of ambuic acid as a lead for the development of antivirulence therapeutics.

Hyaluronan modulation impacts Staphylococcus aureus biofilm infection

ABSTRACT Staphylococcus aureus is a leading cause of chronic biofilm infections. Hyaluronic acid (HA) is a large glycosaminoglycan abundant in mammalian tissues that has been shown to enhance biofilm formation in multiple Gram-positive pathogens. We observed that HA accumulated in an S. aureus biofilm infection using a murine implant-associated infection model and that HA levels increased in a mutant strain lacking hyaluronidase (HysA). S. aureus secretes HysA in order to cleave HA during infection. Through in vitro biofilm studies with HA, the hysA mutant was found to accumulate increased biofilm biomass compared to the wild type, and confocal microscopy showed that HA is incorporated into the biofilm matrix. Exogenous addition of purified HysA enzyme dispersed HA-containing biofilms, while catalytically inactive enzyme had no impact. Additionally, induction of hysA expression prevented biofilm formation and also dispersed an established biofilm in the presence of HA. These observations were corroborated in the implant model, where there was decreased dissemination from an hysA mutant biofilm infection compared to the S. aureus wild type. Histopathology demonstrated that infection with an hysA mutant caused significantly reduced distribution of tissue inflammation compared to wild-type infection. To extend these studies, the impact of HA and S. aureus HysA on biofilm-like aggregates found in joint infections was examined. We found that HA contributes to the formation of synovial fluid aggregates, and HysA can disrupt aggregate formation. Taken together, these studies demonstrate that HA is a relevant component of the S. aureus biofilm matrix and HysA is important for dissemination from a biofilm infection.

Chronic ethanol feeding increases the severity of Staphylococcus aureus skin infections by altering local host defenses

Alcoholics are at increased risk of Staphylococcus aureus skin infection and serious sequelae, such as bacteremia and death. Despite the association between alcoholism and severe S. aureus skin infection, the impact of EtOH on anti‐S. aureus cutaneous immunity has not been investigated in a model of chronic EtOH exposure. To test the hypothesis that EtOH enhances the severity of S. aureus skin infection, mice were fed EtOH for ≥12 weeks via the Meadows‐Cook model of alcoholism and inoculated with S. aureus following epidermal abrasion. Evidence of exacerbated staphylococcal disease in EtOH‐fed mice included: skin lesions that were larger and contained more organisms, greater weight loss, and increased bacterial dissemination. Infected EtOH‐fed mice demonstrated poor maintenance and induction of PMN responses in skin and draining LNs, respectively. Additionally, altered PMN dynamics in the skin of these mice corresponded with reduced production of IL‐23 and IL‐1β by CD11b+ myeloid cells and IL‐17 production by γδ T cells, with the latter defect occurring in the draining LNs as well. In addition, IL‐17 restoration attenuated S. aureus‐induced dermatopathology and improved bacterial clearance defects in EtOH‐fed mice. Taken together, the findings show, in a novel model system, that the EtOH‐induced increase in S. aureus‐related injury/illness corresponds with defects in the IL‐23/IL‐17 inflammatory axis and poor PMN accumulation at the site of infection and draining LNs. These findings offer new information about the impact of EtOH on cutaneous host‐defense pathways and provide a potential mechanism explaining why alcoholics are predisposed to S. aureus skin infection.