Abstract
Staphylococcus pseudintermedius is responsible for severe and necrotizing infections in humans and dogs. Contrary to S. aureus, the pathophysiological mechanisms involved in this virulence are incompletely understood. We previously showed the intracellular cytotoxicity induced after internalization of S. pseudintermedius. Herein, we aimed to identify the virulence factors responsible for this cytotoxic activity. After addition of filtered S. pseudintermedius supernatants in culture cell media, MG63 cells, used as representative of non-professional phagocytic cells (NPPc), released a high level of LDH, indicating that the cytotoxicity was mainly mediated by secreted factors. Accordingly, we focused our attention on S. pseudintermedius toxins. In silico analysis found the presence of two PSMs (δ-toxin and PSMε) as well as Luk-I leukotoxin, the presence of which was confirmed by PCR in all clinical strains tested (n = 17). Recombinant Luk-I leukotoxin had no cytotoxic activity on NPPc but the ectopic expression of the CXCR2 receptor in U937 cells conferred cytotoxity to Luk-I. This is in agreement with the lack of Luk-I effect on NPPc and the previous report of Luk-I cytoxic activity on immune cells. Contrary to Luk-I, synthetic δ-toxin and PSMε had a strong cytotoxic activity on NPPc. The secretion of δ-toxin and PSMε at cytotoxic concentrations by S. pseudintermedius in culture supernatant was confirmed by HPLC-MS. In addition, the supplementation of such supernatants with human serum, known to inhibit PSM, induced a complete abolition of cytotoxicity which indicates that PSMs are the key players in the cytotoxic phenotype of NPPc. The results suggest that the severity of S. pseudintermedius infections is, at least in part, explained by a combined action of Luk-I that specifically targets immune cells expressing the CXCR2 receptor, and PSMs that disrupt cell membranes whatever the cell types. The present study strengthens the key role of PSMs in virulence of the different species belonging to Staphylococcus genus.
Highlights
Staphylococcus pseudintermedius is a skin and mucous commensal bacterium in dogs, with carriage reaching more than 80% in some populations of healthy dogs (Rubin and Chirino-Trejo, 2011; Bannoehr and Guardabassi, 2012)
Cytolytic activity expressed as the means ± standard deviations quantified by Lactate dehydrogenase (LDH) release from osteoblasts incubated with S. pseudintermedius LMG 20220 culture supernatant (773 ± 92%), was significantly higher than those observed in the control condition (100 ± 12%, p < 0.001; Figure 1A)
These results demonstrate that the cytotoxicity induced by S. pseudintermedius is mediated by secreted virulence factors, which prompted us to investigate the specific bacterial factors involved, especially among staphylococcal toxins with cellular tropism
Summary
Staphylococcus pseudintermedius is a skin and mucous commensal bacterium in dogs, with carriage reaching more than 80% in some populations of healthy dogs (Rubin and Chirino-Trejo, 2011; Bannoehr and Guardabassi, 2012). S. pseudintermedius, often described as the “dog’s golden staph,” shares several features with S. aureus, notably the capacity to express a variety of virulence factors such as: (i) proteolytic enzymes including coagulase and protease (Garbacz et al, 2013), (ii) microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), such as the staphylococcal protein A, that are host surface proteins and participate to the active tissue colonization and the evasion of the host immune system (Bannoehr et al, 2011, 2012; Balachandran et al, 2017), and (iii) toxins (Dziewanowska et al, 1996; Futagawa-Saito et al, 2004) Among the latter, pore-forming toxins (PFTs) have been extensively investigated for their ability to damage plasma membranes and lead to cell lysis (Prévost et al, 2001). Various types of PSMs (α, β, δ-toxin. . . ) have been described and are highly conserved among S. aureus or Staphylococcus non-aureus (SNA) strains (Cheung et al, 2014; Cameron et al, 2015; Da et al, 2017); whole genome sequencing analysis of S. pseudintermedius has revealed the presence of several PSMs: δ-toxin, PSMβ, and PSMε (Cheung et al, 2014)
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