Leukocidins: staphylococcal bi-component pore-forming toxins find their receptors.

Staphylococcus aureus is a major bacterial pathogen that causes disease worldwide. The emergence of strains that are resistant to commonly used antibiotics and the failure of vaccine development have resulted in a renewed interest in the pathophysiology of this bacterium. Staphylococcal leukocidins are a family of bi-component pore-forming toxins that are important virulence factors. During the past five years, cellular receptors have been identified for all of the bi-component leukocidins. The identification of the leukocidin receptors explains the cellular tropism and species specificity that is exhibited by these toxins, which has important biological consequences. In this Review, we summarize the recent discoveries that have reignited interest in these toxins and provide an outlook for future research.

Sublytic concentrations of Staphylococcus aureus Panton-Valentine leukocidin alter human PMN gene expression and enhance bactericidal capacity.

CA-MRSA infections are often caused by strains encoding PVL, which can cause lysis of PMNs and other myeloid cells in vitro, a function considered widely as the primary means by which PVL might contribute to disease. However, at sublytic concentrations, PVL can function as a PMN agonist. To better understand this phenomenon, we investigated the ability of PVL to alter human PMN function. PMNs exposed to PVL had enhanced capacity to produce O(2)(-) in response to fMLF, but unlike priming by LPS, this response did not require TLR signal transduction. On the other hand, there was subcellular redistribution of NADPH oxidase components in PMNs following exposure of these cells to PVL--a finding consistent with priming. Importantly, PMNs primed with PVL had an enhanced ability to bind/ingest and kill Staphylococcus aureus. Priming of PMNs with other agonists, such as IL-8 or GM-CSF, altered the ability of PVL to cause formation of pores in the plasma membranes of these cells. Microarray analysis revealed significant changes in the human PMN transcriptome following exposure to PVL, including up-regulation of molecules that regulate the inflammatory response. Consistent with the microarray data, mediators of the inflammatory response were released from PMNs after stimulation with PVL. We conclude that exposure of human PMNs to sublytic concentrations of PVL elicits a proinflammatory response that is regulated in part at the level of gene expression. We propose that PVL-mediated priming of PMNs enhances the host innate immune response.

Spread of epidemic MRSA-ST5-IV clone encoding PVL as a major cause of community onset staphylococcal infections in Argentinean children.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus-(CA-MRSA) strains have emerged in Argentina. We investigated the clinical and molecular evolution of community-onset MRSA infections (CO-MRSA) in children of Córdoba, Argentina, 2005-2008. Additionally, data from 2007 were compared with the epidemiology of these infections in other regions of the country. METHODOLOGY/PRINCIPAL FINDINGS: Two datasets were used: i) lab-based prospective surveillance of CA-MRSA isolates from 3 Córdoba pediatric hospitals-(CBAH1-H3) in 2007-2008 (compared to previously published data of 2005) and ii) a sampling of CO-MRSA from a study involving both, healthcare-associated community-onset-(HACO) infections in children with risk-factors for healthcare-associated infections-(HRFs), and CA-MRSA infections in patients without HRFs detected in multiple centers of Argentina in 2007. Molecular typing was performed on the CA-MRSA-(n: 99) isolates from the CBAH1-H3-dataset and on the HACO-MRSA-(n: 51) and CA-MRSA-(n: 213) isolates from other regions. Between 2005-2008, the annual proportion of CA-MRSA/CA-S. aureus in Córdoba hospitals increased from 25% to 49%, P<0.01. Total CA-MRSA infections increased 3.6 fold-(5.1 to 18.6 cases/100,000 annual-visits, P<0.0001), associated with an important increase of invasive CA-MRSA infections-(8.5 fold). In all regions analyzed, a single genotype prevailed in both CA-MRSA (82%) and HACO-MRSA(57%), which showed pulsed-field-gel electrophoresis-(PFGE)-type-"I", sequence-type-5-(ST5), SCCmec-type-IVa, spa-t311, and was positive for PVL. The second clone, pulsotype-N/ST30/CC30/SCCmecIVc/t019/PVL(+), accounted for 11.5% of total CA-MRSA infections. Importantly, the first 4 isolates of Argentina belonging to South American-USA300 clone-(USA300/ST8/CC8/SCCmecIVc/t008/PVL(+)/ACME(-)) were detected. We also demonstrated that a HA-MRSA clone-(pulsotype-C/ST100/CC5) caused 2% and 10% of CA-MRSA and HACO-MRSA infections respectively and was associated with a SCCmec type closely related to SCCmecIV(2B&5). CONCLUSIONS/SIGNIFICANCE: The dissemination of epidemic MRSA clone, ST5-IV-PVL(+) was the main cause of increasing staphylococcal community-onset infections in Argentinean children (2003-2008), conversely to other countries. The predominance of this clone, which has capacity to express the h-VISA phenotype, in healthcare-associated community-onset cases suggests that it has infiltrated into hospital-settings.

Ovarian hyperstimulation syndrome complicated by severe community-acquired pneumonia due to methicillin-resistant Staphylococcus aureus positive for Panton-Valentine leukocidin.

We report a case of severe ovarian hyperstimulation syndrome (OHSS) complicated by community-acquired methicillin-resistant Staphylococcus aureus-Panton-Valentine leukocidin positive (CAMRSA-PVL[+]) necrotizing pneumonia, sepsis and multiple organ failure (MOF) in a previously immunocompetent female. The patient required prolonged ventilatory support and intensive care unit (ICU) hospitalization. Multiple cavities and severely affected lung function persist 1 year after discharge.

Panton-Valentine leukocidin in the pathogenesis of community-associated methicillin-resistant Staphylococcus aureus infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that causes serious infectious diseases and was endemic in hospitals by the late 1960s. Beginning with its first report in the late 1990s, the rapid emergence of community-associated MRSA (CA-MRSA) worldwide responsible for a wide spectrum of diseases ranging from minor skin infections to fatal necrotizing pneumonia has been found in previously healthy individuals without established risk factors for MRSA acquisition. Recently, various virulence determinants unique to CA-MRSA have been uncovered, which explain how the pathogen spreads easily and causes severe CA-MRSA infections among humans. However, the role of Panton-Valentine leukocidin (PVL) in the pathogenesis of CA-MRSA infection is currently a matter of much debate because of conflicting data from epidemiologic studies of CA-MRSA infections and various murine disease models. Identifying specialized pathogenic traits of CA-MRSA and the concerted regulation of these factors remains a challenge that will foster development of vaccines and therapies designed to control CA-MRSA infections. This review focuses on the current status of molecular epidemiology associated with CA-MRSA in Taiwan and progresses toward understanding the enhanced virulence properties of CA-MRSA, with an emphasis on the role of Panton-Valentine leukocidin.

[Secreted proteins of Staphylococcus aureus].

Modern views on secreted proteins of Staphylococcus aureus are analyzed in the review. Spread of this pathogen is partially explained by wide spectrum of proteins secreted by this microorganism. Four groups of proteins secreted by staphylococci could be assigned. These are peptidoglycanhydrolases, intracellular enzymes (proreases, lipases etc.), membrane toxins (hemolyzins and leukocidins), and superantigens. Role of some proteins, especially belonging to latter groups, for the staphylococcal virulence and pathogenesis of staphylococcal infectious is known at this time. Genome sequencing of several strains of S. aureus also allowed to predict main characteristics of many secreted proteins such as molecular mass and spatial structure. Further studies of these proteins could be useful for development of staphylococcal vaccines and other immunity-modifying drugs.

Short communication: methicillin-resistant Staphylococcus aureus infections in children and young adults infected with HIV.

The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) infections, in particular with Panton-Valentine leukocidin (PVL)-positive strains, has not been well characterized in children and young adults with HIV infection. It is not known if PVL-positive strains of MRSA cause an increased morbidity in this population compared to PVL-negative strains. The purpose of this study was to retrospectively analyze the epidemiology of PVL-positive and PVL-negative MRSA infections in children and young adults with HIV from 2000 to 2007. Molecular typing was performed by polymerase chain reaction (PCR) for detection of the PVL genes. Staphylococcus Cassette Chromosome (SCC) mec and spa typing were performed on all PVL-positive isolates. The number of HIV patients with MRSA infection increased significantly between 2000 and 2007 ( p=0.0015). Twenty seven (87%) of the 31 MRSA isolates were from skin and soft tissue infections (SSTI). Clindamycin resistance was observed in 19% of the MRSA isolates. PVL-positive isolates bearing the type IV SCC mec element comprised 16 of 31 (52%) MRSA isolates. All the PVL-positive isolates belonged to the USA300 pulsed-field type. There was no difference in the mean CD4 count and HIV viral load between patients with PVL-positive and PVL-negative MRSA infections. PVL-positive MRSA infections were associated with more SSTI ( p=0.043) but not with increased morbidity or a higher risk of complications compared to PVL-negative MRSA infections in children and young adults with HIV.

Panton-valentine leukocidin enhances the severity of community-associated methicillin-resistant Staphylococcus aureus rabbit osteomyelitis.

BACKGROUND: Extensive spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the United States, and the concomitant increase in severe invasive staphylococcal infections, including osteomyelitis, in healthy children, has led to renewed interest in Panton-Valentine leukocidin (PVL). However, the pathogenetic role of PVL in staphylococcal infections remains controversial, possibly because it depends on the site of infection. METHODOLOGY/PRINCIPAL FINDINGS: We compared the course of experimental rabbit osteomyelitis due to the PVL-positive CA-MRSA strain USA 300 (LAC) and its PVL-negative isogenic derivative (LACDeltapvl), using a low and a high inoculum (8x10(5) and 4x10(8) CFU). With the low inoculum, bone infection was less frequent on day 7 (D7) and day 28 (D28) with LACDeltapvl than with LAC (respectively 12/19 and 18/19 animals, p = 0.042). With the high inoculum of both strains, all the animals were infected on D7 and the infection persisted on D28 in almost every case. However, tibial bacterial counts and the serum CRP concentration fell significantly between D7 and D28 with LACDeltapvl but not with LAC. Respectively 67% and 60% of LAC-infected rabbits had bone deformation and muscle/joint involvement on D7, compared to 0% and 7% of LACDeltapvl-infected rabbits (p = 0.001 and p = 0.005 respectively). Between D0 and D28, the anti-PVL antibody titer increased significantly only with the high inoculum of LAC. CONCLUSIONS/SIGNIFICANCE: PVL appears to play a role in the persistence and rapid local extension of rabbit osteomyelitis, in keeping with the greater severity of human bone infections due to PVL-positive S. aureus. The possible therapeutic implications of these findings are discussed.

Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) threatens public health worldwide, and epidemiologic data suggest that the Panton-Valentine Leukocidin (PVL) expressed by most CA-MRSA strains could contribute to severe human infections, particularly in young and immunocompetent hosts. PVL is proposed to induce cytolysis or apoptosis of phagocytes. However, recent comparisons of isogenic CA-MRSA strains with or without PVL have revealed no differences in human PMN cytolytic activity. Furthermore, many of the mouse studies performed to date have failed to demonstrate a virulence role for PVL, thereby provoking the question: does PVL have a mechanistic role in human infection? In this report, we evaluated the contribution of PVL to severe skin and soft tissue infection. We generated PVL mutants in CA-MRSA strains isolated from patients with necrotizing fasciitis and used these tools to evaluate the pathogenic role of PVL in vivo. In a model of necrotizing soft tissue infection, we found PVL caused significant damage of muscle but not the skin. Muscle injury was linked to induction of pro-inflammatory chemokines KC, MIP-2, and RANTES, and recruitment of neutrophils. Tissue damage was most prominent in young mice and in those strains of mice that more effectively cleared S. aureus, and was not significant in older mice and mouse strains that had a more limited immune response to the pathogen. PVL mediated injury could be blocked by pretreatment with anti-PVL antibodies. Our data provide new insights into CA-MRSA pathogenesis, epidemiology and therapeutics. PVL could contribute to the increased incidence of myositis in CA-MRSA infection, and the toxin could mediate tissue injury by mechanisms other than direct killing of phagocytes.

The Panton-Valentine leukocidin vaccine protects mice against lung and skin infections caused by Staphylococcus aureus USA300.

Methicillin-resistant Staphylococcus aureus is increasingly responsible for staphylococcal infections in the community. A large percentage of the community-acquired methicillin-resistant (CA-MRSA) strains in the USA produce Panton-Valentine leukocidin (PVL), which is associated with severe infections. The virulence of the clinical CA-MRSA strain USA300 was compared to that of its isogenic pvl-deleted mutant, and it was shown that PVL contributes to lung and muscle tissue destruction, respectively, in murine necrotizing pneumonia and skin infection models. Mice infected with the USA300 strain developed a dominant anti-PVL response. The PVL subunits were therefore tested as vaccinogens against this isolate, and their vaccine efficacy correlated with both the route of vaccination and infection. These data suggest that PVL is a virulence factor in murine CA-MRSA infections.

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity.

We have previously shown that ExoU, a type III secreted cytotoxin of Pseudomonas aeruginosa, causes acute cytotoxicity towards corneal epithelial cells in vitro, and contributes to corneal disease pathology and ocular colonization in vivo. Subsequently, we reported that ExoU represses phagocyte infiltration of infected corneas in vivo. ExoU has patatin-like phospholipase activity that is required for cytotoxic activity in vitro (mammalian cell injury and death) and for disease in a murine model of pneumonia. We hypothesized that the phospholipase activity was required for ExoU-mediated corneal disease and ocular colonization. Using the murine scarification model, corneal disease pathology was examined after inoculation with approximately 10(6)cfu of a P. aeruginosa effector mutant (PA103DeltaexoUexoT::Tc) complemented with either exoU (pUCPexoU), phospholipase-inactive exoU (pUCPexoUD344A) or a plasmid control (pUCP18). Eyes were photographed and disease severity scored at 24 and 48h post-infection. Viable bacteria colonizing infected eyes were quantified at 6 and 48h. Complementation with exoU caused significantly more pathology (increased disease severity scores) and enabled bacteria to better colonize (by approximately 1000-fold) at 48h as compared to phospholipase-inactive exoU which did not differ from plasmid control. Surprisingly, exoU did not contribute to early (6h) colonization. In-vitro assays confirmed that the phospholipase domain of exoU was required for cytotoxicity towards human corneal epithelial cells. Taken together these data show that the phospholipase activity of the P. aeruginosa cytotoxin, ExoU, plays a role in the pathogenesis of corneal infection via mechanism(s) occurring after initial colonization of a susceptible cornea.

Intracellular trafficking of Pseudomonas ExoS, a type III cytotoxin.

Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin that disrupts Ras- and Rho-signaling pathways in mammalian cells. A hydrophobic region (residues 51-77, termed the membrane localization domain) targets ExoS to the plasma membrane (PM) and late endosomes of host cells. In the current study, metabolic inhibitors and dominant-negative proteins that disrupt known vesicle-trafficking pathways were used to define the intracellular trafficking of ExoS. Release of ExoS from PM was independent of dynamin and ADP ribosylation factor 6 but inhibited by methyl-beta-cyclodextrin, a cholesterol-depleting reagent, and perinuclear localization of ExoS was disrupted by nocodazole. p50 dynamitin, a dynein inhibitor partially disrupted perinuclear localization of ExoS. Methyl-beta-cyclodextrin and nocodazole inhibited the ability of type-III-delivered ExoS to ADP-ribosylated Golgi/endoplasmic reticulum-resident Ras. Methyl-beta-cyclodextrin also relocated ExoS from the perinuclear region to the PM, indicating that ExoS can cycle through anterograde as well as through retrograde trafficking pathways. These findings show that ExoS endocytosis is cholesterol dependent, and it utilizes host microtubules, for intracellular trafficking. Understanding how type III cytotoxins enter and traffic within mammalian cells may identify new targets for therapeutic intervention of gram-negative bacterial pathogens.

Community-acquired methicillin-resistant Staphylococcus aureus (MRSA): new issues for infection control.

The emergence and clonal expansion of strains of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) have created new challenges with their entrance to hospitals. The increased virulence of CA-MRSA in concert with the depressed immunity of inpatients may cause added morbidity and mortality expected from healthcare-associated infections. Questions about changing prophylactic and empirical therapy as well as the use of intravenous immunoglobulin for life-threatening infections are addressed.

Staphylococcus aureus Panton-Valentine leukocidin causes necrotizing pneumonia.

The Staphylococcus aureus Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by strains epidemiologically associated with the current outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and with the often-lethal necrotizing pneumonia. To investigate the role of PVL in pulmonary disease, we tested the pathogenicity of clinical isolates, isogenic PVL-negative and PVL-positive S. aureus strains, as well as purified PVL, in a mouse acute pneumonia model. Here we show that PVL is sufficient to cause pneumonia and that the expression of this leukotoxin induces global changes in transcriptional levels of genes encoding secreted and cell wall-anchored staphylococcal proteins, including the lung inflammatory factor staphylococcal protein A (Spa).

In vitro production of panton-valentine leukocidin among strains of methicillin-resistant Staphylococcus aureus causing diverse infections.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus strains have recently been associated with severe necrotizing infections. Greater than 75% of these strains carry the genes for Panton-Valentine leukocidin (PVL), suggesting that this toxin may mediate these severe infections. However, to date, studies have not provided evidence of toxin production. METHODS: Twenty-nine community-acquired methicillin-resistant Staphylococcus aureus and 2 community-acquired methicillin-susceptible S. aureus strains were collected from patients with infections of varying severity. Strains were analyzed for the presence of lukF-PV and SCCmecA type. PVL production in lukF-PV gene-positive strains was measured by ELISA, and the amount produced was analyzed relative to severity of infection. RESULTS: Only 2 of the 31 strains tested, 1 methicillin-resistant Staphylococcus aureus abscess isolate and 1 nasal carriage methicillin-susceptible S. aureus isolate, were lukF-PV negative. All methicillin-resistant Staphylococcus aureus strains were SCCmec type IV. PVL was produced by all strains harboring lukF-PV, although a marked strain-to-strain variation was observed. Twenty-six (90%) of 29 strains produced 50-350 ng/mL of PVL; the remaining strains produced PVL in excess of 500 ng/mL. The quantity of PVL produced in vitro did not correlate with severity of infection. CONCLUSIONS: Although PVL likely plays an important role in the pathogenesis of these infections, its mere presence is not solely responsible for the increased severity. Factors that up-regulate toxin synthesis in vivo could contribute to more-severe disease and worse outcomes in patients with community-acquired methicillin-resistant Staphylococcus aureus infection.