Production of an Attenuated Phenol-Soluble Modulin Variant Unique to the MRSA Clonal Complex 30 Increases Severity of Bloodstream Infection

et al. (2014) Production of an Attenuated Phenol-Soluble Modulin Variant Unique to the MRSA Clonal Complex 30 Increases Severity of Bloodstream Infection. PLoS Pathog 10(8): e1004298. doi:10.1371/journal.ppat.1004298 Production of an Attenuated Phenol-Soluble Modulin Variant Unique to the MRSA Clonal Complex 30 Increases Severity of Bloodstream Infection Gordon Y. C. Cheung Dorothee Kretschmer Anthony C. Duong Anthony J. Yeh Trung V. Ho Yan Chen Hwang-Soo Joo Barry N. Kreiswirth Andreas Peschel Michael Otto Alice Prince, Columbia University, United States of America Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of morbidity and death. Phenol-soluble modulins (PSMs) are recently-discovered toxins with a key impact on the development of Staphylococcus aureus infections. Allelic variants of PSMs and their potential impact on pathogen success during infection have not yet been described. Here we show that the clonal complex (CC) 30 lineage, a major cause of hospital-associated sepsis and hematogenous complications, expresses an allelic variant of the PSMa3 peptide. We found that this variant, PSMa3N22Y, is characteristic of CC30 strains and has significantly reduced cytolytic and pro-inflammatory potential. Notably, CC30 strains showed reduced cytolytic and chemotactic potential toward human neutrophils, and increased hematogenous seeding in a bacteremia model, compared to strains in which the genome was altered to express non-CC30 PSMa3. Our findings describe a molecular mechanism contributing to attenuated pro-inflammatory potential in a main MRSA lineage. They suggest that reduced pathogen recognition via PSMs allows the bacteria to evade elimination by innate host defenses during bloodstream infections. Furthermore, they underscore the role of point mutations in key S. aureus toxin genes in that adaptation and the pivotal importance PSMs have in defining key S. aureus immune evasion and virulence mechanisms. - Funding: This study was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), The National Institutes of Health (NIH) to MO, and grants from the German Research Foundation (SFB685 to AP, TR34 to AP and DK), the German Ministry of Education and Research (Menage, to AP), and the Fortu ne program of the Medical Faculty, University of Tu bingen to DK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Staphylococcus aureus is a dangerous human pathogen that is responsible for thousands of deaths annually in the U. S. alone [1]. Virulence of S. aureus is due to a large repertoire of virulence factors, including immune evasion factors and aggressive cytolytic toxins [2]. S. aureus infections become particularly dangerous when they are caused by strains that are resistant to commonly used antibiotics. Methicillin-resistant S. aureus (MRSA) is of especially great concern, as identification of MRSA eliminates the therapeutic use of most beta-lactam antibiotics, which are antibiotics of first choice against pathogenic staphylococci. Many countries report high rates of methicillin resistance among hospital-associated infections caused by S. aureus [3]. In addition, community-associated strains of MRSA (CA-MRSA) have emerged over the last two decades that have the capacity to infect healthy individuals outside of hospital settings [4]. MRSA strains belonging to clonal complex (CC) 30 are a major cause of hospital-associated infections in the U. S., Europe, and elsewhere [57]. Infections with CC30 MRSA present predominantly as bloodstream infections with complications such as hematogenous seeding [8]. Historical methicillin-susceptible CC30 strains (phage type 80/81) caused serious, in part communityassociated infections of the skin and lungs in addition to blood infections. In contrast to contemporary CC30 isolates, many historical phage type 80/81 clones had genes encoding the Panton-Valentine leukocidin (PVL) [7]. Furthermore, contemporary CC30 clones contain mutations in the global virulence regulator Agr (agrC gene, non-synonymous mutation, G55R) and the gene encoding a-toxin (hla, STOP mutation) [9]. The resulting overall lower expression of cytolytic toxins in contemporary compared to historic CC30 clones has been linked to the fact that contemporary CC30 clones predominantly cause hospital-associated infections [9]. PSMs are short, amphipathic, a-helical peptides with a major impact on S. aureus virulence [10,11]. The PSMa peptides of S. aureus in particular cause lysis of a variety of cell types, including neutrophils (or polymorphonuclear leukocytes, PMNs), monocytes, erythrocytes, and osteoblasts [1113]. The rather low target specificity of PSM-mediated cytolysis is due to the fact that lysis is believed to be receptor-independent [14,15], which is reflected by the capacity of PSMs to lyse artificial vesicles [14]. In addition, Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality and a great concern for public health. The CC30 MRSA lineage is especially notorious for causing bloodstream infections with complications such as seeding into organs. In our study, we show that this lineage produces an attenuated form of a key S. aureus toxin with decreased proinflammatory features. Our results suggest that attenuation of this toxin allows the bacteria to evade recognition and subsequent elimination by host defenses, thereby increasing pathogen success during blood infection. PSMs have pro-inflammatory capacities that are receptor-dependent, leading for example to neutrophil chemotaxis and activation [11,14]. Similar to other S. aureus toxins, these immune-stimulatory activities are observed at sublytic concentrations [14,16]. PSMa3 is the by far most pro-inflammatory and cytolytic PSM of S. aureus [11]. Notably, the capacity of PSMa3 to elicit chemotaxis by neutrophils by far exceeds that of any other PSM of S. aureus [11]. Except for a variation in the PSM d-toxin sequence (serine substitution for glycine at position 10 in some strains), whose effect on peptide function has not yet been analyzed, naturally occurring variants of PSM peptides have not yet been reported; and in general, the consequences of non-synonymous variations in psm genes are not understood. Here, we report an allelic variation in the PSMa3-encoding gene that is characteristic of CC30 strains and leads to significantly lower cytolytic and chemotactic activity, and increased hematogenous seeding in a bacteremia model. For the first time, our study describes an allelic variant of a psm gene that has key biological consequences and whose appearance is strongly correlated with a specific MRSA lineage. Furthermore, our findings reveal a molecular mechanism supporting the notion that MRSA strains such as those of (...truncated)