Michael E. Powers

A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.

Effect of strength and proprioception training on eversion to inversion strength ratios in subjects with unilateral functional ankle instability

Objectives: To examine the effect of six weeks of strength and proprioception training on eversion to inversion isokinetic strength ratios (E/I ratios) in subjects with unilateral functional ankle instability. Methods: Thirty eight subjects were randomly assigned to one of four treatment groups: strength training (S); proprioception training (P); strength + proprioception training (B); control (C). Isokinetic strength was tested before and after training using a Kin Com 125 automatic positioning isokinetic dynamometer. Subtalar joint eversion and inversion motions were tested both concentrically and eccentrically through a range of motion involving 40°. All peak torque and average torque values were normalised for body mass. E/I ratios were calculated from average torque and peak torque measures by taking the concentric eversion value and combining it with the eccentric inversion value. Data were analysed using a mixed model analysis of variance with repeated measures on the test factor. Average torque and peak torque E/I ratios at 30 and 120°/s were analysed separately. Results: There were no significant differences in average torque and peak torque E/I ratios of the functionally unstable ankle for any of the groups after training compared with before. Conclusions: Six weeks of strength and proprioception training (either alone or combined) had no effect on isokinetic measures of strength in subjects with self reported unilateral functional instability. Further studies examining this agonist (concentric) to antagonist (eccentric) muscle group strength ratio are needed.

ADAM10 Mediates Vascular Injury Induced by Staphylococcus aureus α-Hemolysin

Staphylococcus aureus is a leading cause of bacteremia and sepsis. The interaction of S. aureus with the endothelium is central to bloodstream infection pathophysiology yet remains ill-understood. We show herein that staphylococcal α-hemolysin, a pore-forming cytotoxin, is required for full virulence in a murine sepsis model. The α-hemolysin binding to its receptor A-disintegrin and metalloprotease 10 (ADAM10) upregulates the receptors metalloprotease activity on endothelial cells, causing vascular endothelial-cadherin cleavage and concomitant loss of endothelial barrier function. These cellular injuries and sepsis severity can be mitigated by ADAM10 inhibition. This study therefore provides mechanistic insight into toxin-mediated endothelial injury and suggests new therapeutic approaches for staphylococcal sepsis.

Assessment of Shoulder Proprioception in the Female Softball Athlete

Background There have been reports of overhand throwing athletes having decreased joint position sense in their dominant shoulder as compared with the nondominant shoulder. Very little research, however, exists concerning joint position sense in the female athlete. Hypothesis Female softball athletes have decreased joint position sense in their dominant shoulder as compared with their nondominant shoulder. Study Design Factorial design with investigation of multiple independent variables. Methods Joint position sense was assessed in 50 female softball players and 50 nonthrowing female athletes by using an inclinometer during four glenohumeral joint motions. Both the dominant and nondominant shoulders were assessed and error scores were calculated to describe joint position sense. Data were collected during the course of a fall semester and analyzed by using a mixed model analysis of variance with repeated measures on the dependent variable (error scores). Results A significant group by movement interaction was observed, with the softball athletes demonstrating significantly greater external rotation error scores than the nonthrowing athletes. Conclusion We failed to reject the null hypothesis. Increased error scores (less joint position sense) were observed in both arms of subjects in the softball group. Clinical Relevance This study suggests that there is decreased shoulder proprioception in asymptomatic female athletes involved in overyhand throwing sports, which may predispose them to injury.

Igniting the Fire: Staphylococcus aureus Virulence Factors in the Pathogenesis of Sepsis

Sepsis is a devastating disease process characterized by a systemic inflammatory response in the host, evoked by a known or suspected pathogen. Staphylococcus aureus has emerged as a leading etiologic agent of sepsis, owing to its propensity to cause deep-seated tissue infection and bacteremia [1]. S. aureus harbors an arsenal of virulence factors to facilitate tissue adhesion, immune evasion, and host cell injury. In the bloodstream, these factors cause inflammation, impair immune cell function, alter coagulation, and compromise vascular integrity. This review will discuss key secreted and surface-anchored proteins required for S. aureus infection in the hostile host environment of the bloodstream, emphasizing mechanistic insights on virulence factor function that illustrate the complex nature of the host–pathogen interaction. While we currently lack a clear understanding of the temporal and spatial integration of these virulence factors in the bloodstream, it is apparent that S. aureus triggers pathophysiologic disturbances that are further amplified by the host inflammatory response, culminating in the severe clinical manifestations of sepsis and septic shock.

Spine-board Transfer Techniques and the Unstable Cervical Spine

Study Design. A repeated-measures design using a cadaveric model was used in this preliminary investigation on the effectiveness of spine-board transfer techniques. Objectives. To compare the amount of angulation (flexion–extension) motion that results at the cervical spine during the execution of the log-roll maneuver and the lift-and-slide technique; and to examine how changes to the integrity of the cervical spine impacts the amount of motion generated during the transfer process. Summary of Background Data. Very little research has been performed to establish the efficacy of spine-board transfer techniques. Early studies have indicated that the log-roll maneuver may not be appropriate for transferring victims with thoracolumbar injuries. Also, there has not been a single study that has reported the impact of transfer techniques on the unstable cervical spine. This lack of data necessitated the present study. Methods. Four groups (with six participants each) were asked to execute the log-roll maneuver and the lift-and-slide technique on five cadavers. An electromagnetic motion analysis device was used to assess the amount of angulation motion generated at the C5–C6 segment during the execution of these transfer techniques. To examine how changes to the integrity of the cervical spine impacts the amount of motion that is produced during the transfer process, flexion–extension motion was assessed under various conditions: across a stable C5–C6 segment, after the creation of a posterior ligamentous injury, and after a complete segmental injury. Results. No significant differences in angulation motion were noted between transfer techniques. However, significant differences were noted between all three injury conditions. That is, as the severity of the injury increased, the average amount of angulation motion produced at the site of the lesion also increased, regardless of technique. Conclusion. The participants of this study were able to restrict flexion–extension motion equally well with thelog-roll maneuver as with the lift-and-slide technique. However, more research is needed to fully ascertain the effectiveness of spine-board transfer techniques.

Synergistic Action of Staphylococcus aureus α-Toxin on Platelets and Myeloid Lineage Cells Contributes to Lethal Sepsis.

Multi-organ failure contributes to mortality in bacterial sepsis. Platelet and immune cell activation contribute to organ injury during sepsis, but the mechanisms by which bacterial virulence factors initiate these responses remain poorly defined. We demonstrate that during lethal sepsis, Staphylococcus aureus α-toxin simultaneously alters platelet activation and promotes neutrophil inflammatory signaling through interactions with its cellular receptor ADAM10. Platelet intoxication prevents endothelial barrier repair and facilitates formation of injurious platelet-neutrophil aggregates, contributing to lung and liver injury that is mitigated by ADAM10 deletion on platelets and myeloid lineage cells. While platelet- or myeloid-specific ADAM10 knockout does not alter sepsis mortality, double-knockout animals are highly protected. These results define a pathway by which a single bacterial toxin utilizes a widely expressed receptor to coordinate progressive, multi-organ disease in lethal sepsis. As an expression-enhancing ADAM10 polymorphism confers susceptibility to severe human sepsis, these studies highlight the importance of understanding molecular host-microbe interactions.

In Vitro Activities of Arylomycin Natural-Product Antibiotics against Staphylococcus epidermidis and Other Coagulase-Negative Staphylococci

ABSTRACT The arylomycins are a class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), and we have found in diverse bacteria that their activity is limited by the presence of a resistance-conferring Pro residue in SPase that reduces inhibitor binding. We have also demonstrated that Staphylococcus epidermidis, which lacks this Pro residue, is extremely susceptible to the arylomycins. Here, to further explore the potential utility of the arylomycins, we report an analysis of the activity of a synthetic arylomycin derivative, arylomycin C16, against clinical isolates of S. epidermidis and other coagulase-negative staphylococci (CoNS) from distinct geographical locations. Against many important species of CoNS, including S. epidermidis, S. haemolyticus, S. lugdunensis, and S. hominis, we find that arylomycin C16 exhibits activity equal to or greater than that of vancomycin, the antibiotic most commonly used to treat CoNS infections. While the susceptibility was generally correlated with the absence of the previously identified Pro residue, several cases were identified where additional factors also appear to contribute.

Type I Signal Peptidase and Protein Secretion in Staphylococcus epidermidis

Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the β-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationary-phase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.

Synthesis and biological evaluation of penem inhibitors of bacterial signal peptidase.

We report the first synthesis of a 5S penem, known to bind bacterial type I signal peptidase, from the commercially available and inexpensive 6-aminopenicillanic acid. We report the first in vivo activity of the compound and use structure-activity relationship studies to begin to define the determinants of signal peptidase binding and also to begin to optimize the penem as an antibiotic.