hcv protease inhibitors Livestock associated methicillin resistant Staphylococcus aureus LA MRSA

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex 398 (CC398) are widespread in livestock in Europe, and increasingly found in clinical settings (van Cleef et al., 2011; Schaumburg et al., 2012; Köck et al., 2013). A recent publication highlighted the importance of the exchange of mobile genetic elements (MGE) for the host-adaptation of the S. aureus CC398 clonal lineage when overcoming the species barrier (McCarthy et al., 2011). Of note, one of the major MGEs found in human methicillin sensitive S. aureus (MSSA) and MRSA isolates, the immune evasion cluster (IEC) encoded by β-hemolysin converting hcv protease inhibitors of the integrase group 3 (φSaint3, aka φSa3, βC-φs), is only occasionally found in LA-MRSA CC398 strains obtained from horses and cattle, and, if at all, very rarely found in porcine isolates (Kumagai et al., 2007; Sung et al., 2008; McCarthy et al., 2012; Nemeghaire et al., 2014; Cuny et al., 2015). The high prevalence of this bacteriophage among S. aureus isolates derived from humans (van Wamel et al., 2006; Verkaik et al., 2011; Cuny et al., 2015) and its uncommonness among animal-associated isolates on the one hand, and the repeated (re-) acquisition of φSaint3 in LA-MRSA CC398 isolates that were (re-) transmitted from animals to humans (McCarthy et al., 2011; Price et al., 2012) on the other hand, suggest an evolutionary driving force that favors carriage of this bacteriophage in human isolates. However, φSaint3 carriage does not seem to be a prerequisite for CC398 isolates to colonize or cause infections in humans (Verkaik et al., 2011; Cuny et al., 2015). φSaint3 represents a large phage group that may differ in their serogroup, holin genes, and IEC components (Goerke et al., 2009). The major virulence determinant of this phage group is thought to be the IEC, a combination of immune modulating factors including the chemotaxis inhibitory protein (CHIPS), the staphylococcal complement inhibitor (SCIN), staphylokinase (SAK), and some staphylococcal enterotoxins such as SEA or SEP (van Wamel et al., 2006). SCIN is the most prominent IEC encoded component found on nearly all φSaint3 (van Wamel et al., 2006). The small excreted protein inhibits all three complement pathways by interacting with bacterium-bound C3 convertases, thereby interfering with additional C3b deposition on the bacterial surface (Rooijakkers et al., 2005a). SAK is present in about ¾ of the IECs found on φSaint3 (van Wamel et al., 2006), and was shown to (i) inhibit the bactericidal activity of α-defensins (Jin et al., 2004), and (ii) possess anti-opsonic properties by activating human plasminogen into plasmin at the bacterial surface (Rooijakkers et al., 2005b). CHIPS is found in about half of the φSaint3 IECs (van Wamel et al., 2006), and interferes with neutrophil chemotaxis by binding the formylated peptide receptor and the C5a receptor on neutrophils (de Haas et al., 2004; Postma et al., 2004). Staphylococcal enterotoxins are only present in about 1/3 of the IEC found on φSaint3 (van Wamel et al., 2006), and are known to interfere with the host innate immune system by acting as superantigens [reviewed in (Grumann et al., 2014)]. Several studies highlighted the human specificity of selected IEC factors (Dohlsten et al., 1993; Gladysheva et al., 2003; de Haas et al., 2004; Rooijakkers et al., 2005a), which is in good accordance with the rareness of IEC positive S. aureus isolates found in animals. However, comparatively little is known about how the entire set of φSaint3 encoded factors modulate the host immune response, and if there are larger differences in host specificity. Notably, the removal of φSaint3 in S. aureus strain Newman was shown to result in a decreased potential of the bacterium to replicate in the liver in a murine abscess model (Bae et al., 2006), albeit the fact that individual components of the IEC were only slightly active when tested in this species (Dohlsten et al., 1993; Gladysheva et al., 2003; de Haas et al., 2004; Rooijakkers et al., 2005a). Here, we investigated the impact of φSaint3 on phagocytosis of LA-MRSA and MSSA CC398 isolates by human, equine, and porcine polymorphonuclear neutrophils (PMN) in whole blood. Additionally, we determined whether and how the disruption of the β-hemolysin encoding gene hlb, which is the preferred chromosomal insertion site of φSaint3 in S. aureus, affected the hemolytic potentials of φSaint3 positive and negative S. aureus CC398 isolates to lyse human-, horse-, and pig-derived erythrocytes, respectively.