Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes

Leukocidin ED (LukED) is a pore-forming toxin produced by Staphylococcus aureus, which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which depends on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium, contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers: LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton–Valentine leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic. The interaction of LukE with DARC and the role this plays in hemolysis are incompletely characterized. To determine the domain(s) of LukE that are critical for DARC binding, we studied the hemolytic function of LukE–LukS-PV chimeras, in which areas of sequence divergence (divergence regions, or DRs) were swapped between the toxins. We found that two regions of LukE's rim domain contribute to hemolysis, namely residues 57–75 (DR1) and residues 182–196 (DR4). Interestingly, LukE DR1 is sufficient to render LukS-PV capable of DARC binding and hemolysis. Further, LukE, by binding DARC through DR1, promotes the recruitment of LukD to erythrocytes, likely by facilitating LukED oligomer formation. Finally, we show that LukE targets murine Darc through DR1 in vivo to cause host lethality. These findings expand our biochemical understanding of the LukE–DARC interaction and the role that this toxin-receptor pair plays in S. aureus pathophysiology.

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Ceftobiprole EfficacyIn Vitroagainst Panton-Valentine Leukocidin Production andIn Vivoagainst Community-Associated Methicillin-Resistant Staphylococcus aureus Osteomyelitis in Rabbits

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00926-12 ◽

2012 ◽

Vol 56 (12) ◽

pp. 6291-6297 ◽

Cited By ~ 9

Author(s):

Azzam Saleh-Mghir ◽

Oana Dumitrescu ◽

Aurélien Dinh ◽

Yassine Boutrad ◽

Laurent Massias ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant ◽

Content Type ◽

The Mean ◽

Mrsa Strains ◽

Time Kill ◽

Panton Valentine Leukocidin ◽

Valentine Leukocidin

ABSTRACTCommunity-associated methicillin-resistantStaphylococcus aureus(CA-MRSA) can cause osteomyelitis with severe sepsis and/or local complications in which a Panton-Valentine leukocidin (PVL) role is suspected.In vitrosub-MIC antibiotic effects on growth and PVL production by 11 PVL+MRSA strains, including the major CA-MRSA clones (USA300, including the LAC strain; USA400; and USA1000), and 11 PVL+methicillin-susceptibleS. aureus(MSSA) strains were tested in microplate culture. Time-kill analyses with ceftobiprole at its MIC were also run with LAC. Efficacies of ceftobiprole (40 mg/kg of body weight subcutaneously [s.c.] four times a day [q.i.d.]) or vancomycin (60 mg/kg intramuscularly [i.m.] twice a day [b.i.d.]) alone or combined with rifampin (10 mg/kg b.i.d.) against rabbit CA-MRSA osteomyelitis, induced by tibial injection of 3.4 × 107CFU of LAC, were compared. Treatment, started 14 days postinoculation, lasted 14 days.In vitro, 6/11 strains cultured with sub-MICs of ceftobiprole produced 1.6- to 4.8-fold more PVL than did the controls, with no link to specific clones. Rifampin decreased PVL production by all tested strains. In time-kill analyses at the LAC MIC (0.75 mg/liter), PVL production rose transiently at 6 and 8 h and then declined 2-fold at 16 h, concomitant with a 2-log10-CFU-count decrease.In vivo, the mean log10CFU/g of bone for ceftobiprole (1.44 ± 0.40) was significantly lower than that for vancomycin (2.37 ± 1.22) (P= 0.034), with 7/10 versus 5/11 bones sterilized, respectively. Combination with rifampin enhanced ceftobiprole (1.16 ± 0.04 CFU/g of bone [P= 0.056], 11/11 sterile bones) and vancomycin (1.23 ± 0.06 CFU/g [P= 0.011], 11/11 sterile bones) efficacies. Ceftobiprole bactericidal activity and the rifampin anti-PVL effect could play a role in these findings, which should be of interest for treating CA-MRSA osteomyelitis.

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Immune-Activating Properties of Panton-Valentine Leukocidin Improve the Outcome in a Model of Methicillin-Resistant Staphylococcus aureus Pneumonia

Infection and Immunity ◽

10.1128/iai.06360-11 ◽

2012 ◽

Vol 80 (8) ◽

pp. 2894-2904 ◽

Cited By ~ 31

Author(s):

Pauline Yoong ◽

Gerald B. Pier

Keyword(s):

Staphylococcus Aureus ◽

Severe Disease ◽

White Blood Cells ◽

Host Cells ◽

Mitogen Activated Protein Kinase ◽

Human Neutrophils ◽

Infection Model ◽

Methicillin Resistant ◽

Panton Valentine Leukocidin ◽

Valentine Leukocidin

ABSTRACTThe Panton-Valentine leukocidin (PVL) is a cytotoxin expressed by many methicillin-resistantStaphylococcus aureus(MRSA) strains that cause community-acquired infections (CA-MRSA). Its role in virulence however, is controversial, with clinical data suggesting that PVL-producing strains may cause less severe disease in humans. PVL is capable of lysing human white blood cells, but at sublytic amounts, PVL can activate protective host immunity in the absence of cell damage. The concentration-dependent reactions it elicits from host cells could be the reason for seemingly contradictory results about PVL's role in virulence. We hypothesized that a key to understanding PVL's action on host cells and, possibly, outcomes from infection is the amount of toxin present, a hypothesis previously supported in studies using a low-inoculum skin infection model, where low levels of PVL augmented innate immune resistance to infection. Here, we present additional data supporting this hypothesis using a mouse model of MRSA pneumonia, wherein we found increased virulence of isogenic Δpvlstrains and further confirmed PVL's capacity to activate proinflammatory responses from mouse and human neutrophils and pulmonary cells. Activation was measured as the production of phosphorylated p38 mitogen-activated protein kinase (MAPK) and proinflammatory cytokines interleukin-8 (IL-8) and KC (from human and mouse cells, respectively), as well as the release of antibacterial factors. Conversely, PVL lowered the levels of tumor necrosis factor alpha (TNF-α) produced in active pulmonary infection, while low doses induced apoptosis, suggesting that PVL also has the capacity to regulate inflammation. Our data indicate that, independent of its cytotoxic effects, PVL also plays an important and positive immunomodulatory role during MRSA infections.

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In VivoEfficacy of Ceftaroline Fosamil in a Methicillin-Resistant Panton-Valentine Leukocidin-Producing Staphylococcus aureus Rabbit Pneumonia Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01707-13 ◽

2014 ◽

Vol 58 (4) ◽

pp. 1855-1861 ◽

Cited By ~ 11

Author(s):

Delphine Croisier-Bertin ◽

Davy Hayez ◽

Sonia Da Silva ◽

Delphine Labrousse ◽

Donald Biek ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Enzyme Linked Immunosorbent Assay ◽

Pulmonary Tissue ◽

Ceftaroline Fosamil ◽

Methicillin Resistant ◽

Content Type ◽

Panton Valentine Leukocidin ◽

Valentine Leukocidin

ABSTRACTCeftaroline, the active metabolite of the prodrug ceftaroline fosamil, is a cephalosporin with broad-spectrumin vitroactivity against Gram-positive organisms, including methicillin-resistantStaphylococcus aureus(MRSA) and multidrug-resistantStreptococcus pneumoniae(MDRSP), and common Gram-negative pathogens. This study investigated thein vivoactivity of ceftaroline fosamil compared with clindamycin, linezolid, and vancomycin in a severe pneumonia model due to MRSA-producing Panton-Valentine leukocidin (PVL). A USA300 PVL-positive clone was used to induce pneumonia in rabbits. Infected rabbits were randomly assigned to no treatment or simulated human-equivalent dosing with ceftaroline fosamil, clindamycin, linezolid, or vancomycin. Residual bacterial concentrations in the lungs and spleen were assessed after 48 h of treatment. PVL expression was measured using a specific enzyme-linked immunosorbent assay (ELISA). Ceftaroline, clindamycin, and linezolid considerably reduced mortality rates compared with the control, whereas vancomycin did not. Pulmonary and splenic bacterial titers and PVL concentrations were greatly reduced by ceftaroline, clindamycin, and linezolid. Ceftaroline, clindamycin, and linezolid were associated with reduced pulmonary tissue damage based on significantly lower macroscopic scores. Ceftaroline fosamil, clindamycin, and, to a lesser extent, linezolid were efficient in reducing bacterial titers in both the lungs and spleen and decreasing macroscopic scores and PVL production compared with the control.

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