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Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection.

Tseng CW, Biancotti JC, Berg BL, Gate D, Kolar SL, Müller S, Rodriguez MD, Rezai-Zadeh K, Fan X, Beenhouwer DO, Town T, Liu GY - PLoS Pathog. (2015)

Bottom Line: Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide.However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology.PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Infectious Diseases and the Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT
Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγ (NSG) mice engrafted with human CD34+ umbilical cord blood cells. These "humanized" NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL+ versus isogenic PVL- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL+ S. aureus but not PVL- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.

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PMX53 inhibits PVL-mediated pore formation and cytotoxicity in vitro.Human PMN were incubated with C5aR blockers (PMX53 or hC5aR antibody) and then exposed to rLukS-PV with or without rLukF-PV. (A) PVL binding to PMN was measured and % binding inhibition vs untreated cells was calculated. rLukS-PV:100 ng/mL. (B-C) PVL-induced pore formation was assayed by PI staining. rPVL:200 ng/mL in B. (D) PVL-induced PMN cytotoxicity in the presence of 10 μg/mL blockers. Representative images are shown. Scale: 10 μm. (n > 3 for A-D). *: p <0.05, **: p <0.01; ***: p < 0.005.
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ppat.1005292.g004: PMX53 inhibits PVL-mediated pore formation and cytotoxicity in vitro.Human PMN were incubated with C5aR blockers (PMX53 or hC5aR antibody) and then exposed to rLukS-PV with or without rLukF-PV. (A) PVL binding to PMN was measured and % binding inhibition vs untreated cells was calculated. rLukS-PV:100 ng/mL. (B-C) PVL-induced pore formation was assayed by PI staining. rPVL:200 ng/mL in B. (D) PVL-induced PMN cytotoxicity in the presence of 10 μg/mL blockers. Representative images are shown. Scale: 10 μm. (n > 3 for A-D). *: p <0.05, **: p <0.01; ***: p < 0.005.

Mentions: Because hC5aR serves as the primary receptor for PVL, the humanized NSG mouse represents a unique tool to address the question whether blockade of hC5aR could ameliorate PVL+CA-MRSA diseases. We first sought out inhibitors of hC5aR that also blocked PVL binding. Based on the study by Spaan and colleagues, two antibodies to hC5aR, both directed at the N-terminal domain of hC5aR, competed with LukS-PV for binding to PMN albeit a high concentration of PVL (313 nM) was required [15]. We tested one of the antibodies (S5/1) for inhibition of PVL activities, including binding to PMN, pore formation, and cytotoxicity, but found the antibody to be a relatively ineffective blocker (Fig 4A, 4B and 4D). Alternatively, we tested a well-characterized synthetic peptide blocker of hC5aR, PMX53, which has demonstrated C5aR inhibitory activity in vitro and in various models of C5a–related diseases [25,26]. PMX53 binds with high affinity to hC5aR and with relatively low affinity to mC5aR [27]. In the human PMN assays, PMX53 showed good inhibition of PVL binding, pore formation, and cellular toxicity compared to the S5/1 antibody to hC5aR (Fig 4A–4D), but the effect was modest when a higher concentration of PVL (400 ng/mL) was used (Fig 4C).


Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection.

Tseng CW, Biancotti JC, Berg BL, Gate D, Kolar SL, Müller S, Rodriguez MD, Rezai-Zadeh K, Fan X, Beenhouwer DO, Town T, Liu GY - PLoS Pathog. (2015)

PMX53 inhibits PVL-mediated pore formation and cytotoxicity in vitro.Human PMN were incubated with C5aR blockers (PMX53 or hC5aR antibody) and then exposed to rLukS-PV with or without rLukF-PV. (A) PVL binding to PMN was measured and % binding inhibition vs untreated cells was calculated. rLukS-PV:100 ng/mL. (B-C) PVL-induced pore formation was assayed by PI staining. rPVL:200 ng/mL in B. (D) PVL-induced PMN cytotoxicity in the presence of 10 μg/mL blockers. Representative images are shown. Scale: 10 μm. (n > 3 for A-D). *: p <0.05, **: p <0.01; ***: p < 0.005.
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Related In: Results  -  Collection

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ppat.1005292.g004: PMX53 inhibits PVL-mediated pore formation and cytotoxicity in vitro.Human PMN were incubated with C5aR blockers (PMX53 or hC5aR antibody) and then exposed to rLukS-PV with or without rLukF-PV. (A) PVL binding to PMN was measured and % binding inhibition vs untreated cells was calculated. rLukS-PV:100 ng/mL. (B-C) PVL-induced pore formation was assayed by PI staining. rPVL:200 ng/mL in B. (D) PVL-induced PMN cytotoxicity in the presence of 10 μg/mL blockers. Representative images are shown. Scale: 10 μm. (n > 3 for A-D). *: p <0.05, **: p <0.01; ***: p < 0.005.
Mentions: Because hC5aR serves as the primary receptor for PVL, the humanized NSG mouse represents a unique tool to address the question whether blockade of hC5aR could ameliorate PVL+CA-MRSA diseases. We first sought out inhibitors of hC5aR that also blocked PVL binding. Based on the study by Spaan and colleagues, two antibodies to hC5aR, both directed at the N-terminal domain of hC5aR, competed with LukS-PV for binding to PMN albeit a high concentration of PVL (313 nM) was required [15]. We tested one of the antibodies (S5/1) for inhibition of PVL activities, including binding to PMN, pore formation, and cytotoxicity, but found the antibody to be a relatively ineffective blocker (Fig 4A, 4B and 4D). Alternatively, we tested a well-characterized synthetic peptide blocker of hC5aR, PMX53, which has demonstrated C5aR inhibitory activity in vitro and in various models of C5a–related diseases [25,26]. PMX53 binds with high affinity to hC5aR and with relatively low affinity to mC5aR [27]. In the human PMN assays, PMX53 showed good inhibition of PVL binding, pore formation, and cellular toxicity compared to the S5/1 antibody to hC5aR (Fig 4A–4D), but the effect was modest when a higher concentration of PVL (400 ng/mL) was used (Fig 4C).

Bottom Line: Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide.However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology.PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Infectious Diseases and the Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT
Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγ (NSG) mice engrafted with human CD34+ umbilical cord blood cells. These "humanized" NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL+ versus isogenic PVL- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL+ S. aureus but not PVL- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.

Show MeSH
Related in: MedlinePlus