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

Tumor Necrosis Factor Antagonists: Different Kinetics and/or Mechanisms of Action May Explain Differences in the Risk for Developing Granulomatous Infection ARTICLE IN PRESS

Objective Tumor necrosis factor (TNF) antagonists fall into 2 classes:etanercept (ETA) is a soluble TNF receptor, while infliximab (INF) and adalimumab (ADA) are monoclonal antibodies against TNF. All 3 drugs are effective in treating rheumatoid arthritis. However, these agents have been associated with an increased risk of granulomatous infections, such as tuberculosis and histoplasmosis. Several reports indicate that the incidence of granulomatous infections may potentially be higher in individuals treated with INF than ETA. Methods We conducted a comprehensive literature search (1966 to 2004) to review the role of TNF in normal and disease states, and the mechanisms of action of the TNF inhibitors. Specifically, we searched for possible mechanisms for the apparent increase in granulomatous infections associated with TNF inhibitors and for reasons that there may be differences between them. Results Infection may result from a number of differences between ETA and INF or ADA. First, binding avidities are different, with ETA binding in a 1:1 ratio and INF/ADA binding in 2 to 3:1 ratios. Second, the clearances of ADA, ETA, and INF are different, being about 13 times higher for ETA than INF or ADA, thus resulting in higher steady-state drug levels for ADA and INF. Also, the methods of administration are different, intravenously (for INF) versus subcutaneously (for ETA and ADA), which results in lower peak concentrations for ETA and ADA, potentially explaining some of the differences in effects on granuloma formation. Third, INF and ADA have somewhat different mechanisms of action from ETA: INF and ADA are associated with antibodymediated cell lysis, while ETA is not; INF may induce apoptosis in some tissues (eg, gastrointestinal [GI] mucosa) while ETA does not-although this is controversial and may not be true at steady state in synovium, where both drugs seem to cause apoptosis; ETA binds lymphotoxin-␣ while INF does not (ETA may thus be more efficient at preventing granuloma formation by this mechanism than INF); finally, ADA and INF seem to inhibit IFN-␥ expression (probably indirectly), while ETA does not. Conclusions There are significant differences between the 2 classes of TNF antagonists in terms of both their kinetics and mechanisms of action. These differences may help explain the apparent differences in the incidence of granuloma-dependent infections among them

Role of Complement in Protection against Cryptococcus gattii Infection▿

Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3- and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complement-deficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B- and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B- and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection

Separately or combined, LukG/LukH is functionally unique compared to other staphylococcal bicomponent leukotoxins.

Staphylococcus aureus is a major human pathogen that elaborates several exotoxins. Among these are the bicomponent leukotoxins (BCLs), which include γ-hemolysin, Panton-Valentine leukocidin (PVL), and LukDE. The toxin components are classified as either F or S proteins, which are secreted individually and assemble on cell surfaces to form hetero-oligomeric pores resulting in lysis of PMNs and/or erythrocytes. F and S proteins of γ-hemolysin, PVL and LukDE have ∼ 70% sequence homology within the same class and several heterologous combinations of F and S members from these three bicomponent toxin groups are functional. Recently, an additional BCL pair, LukGH (also called LukAB) that has only 30% homology to γ-hemolysin, PVL and LukDE, has been characterized from S. aureus. Our results showed that LukGH was more cytotoxic to human PMNs than PVL. However, LukGH-induced calcium ion influx in PMNs was markedly attenuated and slower than that induced by PVL and other staphylococcal BCLs. In contrast to other heterologous BCL combinations, LukG in combination with heterologous S components, and LukH in combination with heterologous F components did not induce calcium ion entry or cell lysis in human PMNs or rabbit erythrocytes. Like PVL, LukGH induced IL-8 production by PMNs. While individual components LukG and LukH had no cytolytic or calcium influx activity, they each induced high levels of IL-8 transcription and secretion. IL-8 production induced by LukG or LukH was dependent on NF-κB. Therefore, our results indicate LukGH differs functionally from other staphylococcal BCLs

Widespread severe acute respiratory coronavirus virus 2 (SARS-CoV-2) laboratory surveillance program to minimize asymptomatic transmission in high-risk inpatient and congregate living settings.

We describe a widespread laboratory surveillance program for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) at an integrated medical campus that includes a tertiary-care center, a skilled nursing facility, a rehabilitation treatment center, and temporary shelter units. We identified 22 asymptomatic cases of SARS-CoV-2 and implemented infection control measures to prevent SARS-CoV-2 transmission in congregate settings

512. Kinetics of SARS-CoV-2 IgG responses among hospitalized patients with COVID-19

Abstract Background The kinetics of antibody responses to SARS-CoV-2 infection are not fully understood. We analyzed IgG responses to the SARS-CoV-2 Spike protein receptor binding domain (RBD) in COVID-19 patients admitted to VA Greater Los Angeles (VAGLA) and correlated with clinical outcomes. Methods Serially admitted patients from March 20-May 10, 2020 with at least one available residual serum specimen were included in this analysis. Serum samples selected for analysis included first, last, and intermediaries spaced ≥ 5 days apart, as available. Anti-RBD IgG was detected with an enzyme immunoassay (EIA) using recombinant RBD protein. Serum from an uninfected individual collected April 2019 was used as control. The average optical density of the control in triplicate plus 3 standard deviations was considered the threshold positive/negative value. The highest dilution above the threshold value was considered the IgG titer. Clinical groups were defined as asymptomatic, moderate/severe (no ICU) or critical (mechanical ventilation, cytokine storm and/or death). Results Of the 43 consecutive patients admitted to VAGLA with COVID-19 in this analysis, 40 developed detectable RBD IgG responses with maximum inverse titers (MIT) ranging 100-819,200, geometric mean 12,152. Five patients remained asymptomatic but had positive EIAs with median MIT 3200 (IQR 800–3200). Twenty-five had moderate-severe illness with median MIT 25600 (IQR 6400–102400). Ten patients with critical disease had median MIT 38400 (IQR 8800–51200). The median time to positive IgG was 10 days for asymptomatic (IQR 10,10), 4 days for moderate-severe (IQR 3,15), and 7 days for critical (IQR 3.5,14.5). The figure depicts RBD IgG titers over time after onset of symptoms. Asymptomatic patients had a more gradual rate of increase and lower peak titers, while critical patients had the fastest rate of rise and the highest peak titers. Of the 21 patients with samples > 30 days after symptom onset (range 31–67 days), there was no evidence for decrease in anti-RBD IgG. Kinetics of IgG to SARS-CoV-2 receptor binding domain by clinical severity Conclusion Following infection with SARS-CoV-2, disease severity correlates with both the rate of increase and peak in antibody titers. Anti-RBD IgG titers did not decrease over the observation period. Disclosures All Authors: No reported disclosure

Calcium ion entry into human PMNs in presence of PVL (A) and LukGH (B) at the indicated concentrations.

<p>Leukotoxins were mixed with 2×10⁶/mL PMNs loaded with 4 µM Fluo-4 in presence of 1.1 mM CaCl₂. Fluorescence intensity was recorded immediately and then every minute. Percent Fluo-4 fluorescence was calculated according to the formula described in the methods section by using 1% Triton X-100 to estimate maximal fluorescence and 1 mM EGTA to determine minimal fluorescence. The results represent the mean of four independent experiments.</p