1997. a green fluorescent protein (GFP)-P3 promoter reporter strain revealed enhanced activation of the staphylococcal quorum sensing system during polymicrobial versus monomicrobial growth. Analyses by quantitative real-time PCR (qPCR), Western blot, and toxin functional assays confirmed enhanced dependent, and genetic knockout and Tretinoin complementation of recognized alpha-toxin as the key staphylococcal virulence factor driving lethal synergism. Analysis of mono- and polymicrobial infections 8?h p.i. exhibited equivalent bacterial burdens in the peritoneal cavity but significantly elevated levels of alpha-toxin (3-fold) and the eicosanoid prostaglandin E2 (PGE2) (4-fold) during coinfection. Importantly, prophylactic passive immunization using the monoclonal anti-alpha-toxin antibody MEDI4893* led to significantly improved survival Tretinoin rates compared to those following treatment with isotype control antibody. Collectively, these results define alpha-toxin as an essential virulence determinant during IAI and describe a novel mechanism by which a human-pathogenic fungus can augment the virulence of a highly pathogenic bacterium and the ubiquitous bacterial pathogen methicillin-resistant (MRSA) remain serious clinical threats (1, 2). Together, these microorganisms rank among the most prevalent causes of nosocomial sepsis and catheter-related bloodstream infections, and recent reports have recognized their coisolation with increasing frequency (3, 4). While polymicrobial contamination STAT2 is usually often associated with poor patient prognosis, studies designed to mechanistically evaluate microbial community composition and fungal-bacterial interactions in the context of host immunity are still in their infancy (5). Comprehensive epidemiological data on has identified several unique clades, each characterized by unique disease pathology and virulence factors. Among these are USA200 and USA300 strains, which are commonly referred to as hospital-acquired (HA) and community-acquired (CA) MRSA, respectively. Generally, HA-MRSA strains are strong formers of staphylococcal biofilm and demonstrate wider antimicrobial resistance profiles, and some clades (e.g., CC30) exhibit lower levels of secreted bacterial toxins (6). These strains are often associated with orthopedic and medical device-related infections. Conversely, CA-MRSA strains secrete comparatively higher levels of bacterial toxin, including Panton-Valentine leukocidin Tretinoin (PVL) and the pore-forming cytolytic alpha-toxin (1). These strains have been associated with skin contamination outbreaks (e.g., in prisons, care facilities, locker rooms), and it is believed that high levels of toxin as well as others secreted factors allow for efficient skin-skin Tretinoin and skin-fomite transfer. Toxin expression in has been linked to multiple bacterial sensory regulators, but perhaps the best studied is the (accessory gene regulator) quorum sensing system (7). The system is the product of the RNAII transcript, consisting of an operon composed of four genes ((repressor of toxin), thereby increasing exotoxin secretion (9). Both alpha- and delta-toxin are well-characterized staphylococcal virulence factors, mediating a variety of pathological effects, including hemolytic activity, dermonecrosis, inflammasome activation, abscess formation, leukocyte oxidative burst, and reduced macrophage phagocytic killing (10,C14). Toxin production by is crucial for systemic disease, as high titers of antibody to staphylococcal exotoxins (including alpha-toxin) directly correlates with improved survival rates during clinical sepsis (15). A series of studies by Carlson exhibited that peritoneal coinoculation of mice with and resulted in synergistic increases in mortality, while mice inoculated with each of these microbes alone efficiently cleared the infection (16,C18). It was also observed that not all strains of resulted in polymicrobial infectious synergism equally, with some decreasing the staphylococcal 50% lethal dose (LD50) by as much as 70,000-fold while others exhibited only modest synergistic effects (2- to 3-fold) (16). It was proposed that this expression of specific staphylococcal toxins governed increased mortality, namely, alpha-toxin and delta-toxin. Unfortunately, lack of isogenic controls complicated data interpretation. Recently, our labs recapitulated these findings using a USA200 strain of (NRS383) and recognized that the host eicosanoid prostaglandin E2 (PGE2) is usually associated with disease severity and that pharmacologic blockade of PGE2 synthesis and PGE2 receptors Tretinoin 1 and 3 dramatically improves survival rate (19,C21). Interestingly, staphylococcal exotoxins have been implicated in the activation of phospholipase A2 and subsequent prostaglandin release (22, 23). Furthermore, synergistic effects on mortality and PGE2 generation during coinfection with are independent of the capacity to undergo fungal morphogenesis (the major virulence attribute of species (including and evidence that staphylococcal alpha-toxin is necessary for strong infectious synergism and that augments staphylococcal toxin production via engagement of the quorum sensing system. RESULTS Previous observations by Carlson revealed that strains capable of producing numerous exotoxins demonstrate.