Poly- N -Acetylglucosamine Production by Staphylococcus epidermidis Cells Increases Their In Vivo Proinflammatory Effect

Abstract

Staphylococcus epidermidis is a commensal bacterium of human skin and mucous membranes (1). The ability to establish biofilms on the surfaces of indwelling medical devices is a major virulence mechanism of this bacterium, which is the most commonly isolated etiological agent of nosocomial infections (2). S. epidermidis biofilm formation involves initial cellular adherence to a surface, followed by intercellular aggregation and accumulation in multilayer cell clusters (1). This process is dependent on adhesive extracellular molecules (3) such as poly-N-acetylglucosamine (PNAG). PNAG is a major factor mediating cell-to-cell adhesion in staphylococci (4, 5), and its production depends on genes encoded within the intercellular adhesion (ica) locus (6). The final stage of the biofilm life cycle comprises cell disassembly and subsequent growth in a planktonic mode, a process contributing to S. epidermidis biofilm pathogenesis by disseminating infection (7).S. epidermidis strains with impaired PNAG production display attenuated virulence in foreign body infection models (8, 9), while the production of this polysaccharide conferred a survival advantage on this bacterium infecting the nematode host Caenorhabditis elegans (10). Moreover, ica mutants display growth attenuation when competing in vivo with the corresponding wild-type (WT) strains (11) and commensal ica-negative strains became invasive when transformed with a plasmid containing the ica locus (12). In the clinical setting, the presence of icaA/D genes was associated with therapeutic failure in device-related infections with coagulase-negative staphylococci (13). Despite the observed association of PNAG production with S. epidermidis virulence, it is not entirely clear how this polysaccharide may affect the host immune response. Previous reports have shown that PNAG stimulates the production of proinflammatory mediators (14, 15) but also that it impairs phagocytosis and phagocyte-mediated bacterial killing (16-18). Additionally, little is known about the effects of this polysaccharide on acquired immunity. Here, by using the PNAG-producing S. epidermidis 9142 strain and a PNAG-negative isogenic M10 mutant, we have addressed in the murine model how this polysaccharide affected the host inflammatory response. The results obtained show that mice infected with a PNAG-producing S. epidermidis strain exhibited a more intense inflammatory response than mice infected with an isogenic PNAG-negative mutant. MATERIALS AND METHODSBacteria and growth conditions. Biofilm-forming S. epidermidis strain 9142 (19) and the isogenic PNAG-negative M10 mutant (5) were used. Absence of growth defects in the M10 strain was confirmed, as the two strains had similar generation times, as determined by growing 9142 and M10 bacteria in tryptic soy broth (TSB; Merck, Darmstadt, Germany).

Pathogenic Mechanisms and Host Interactions in Staphylococcus epidermidis Device-Related Infection

“…Similar observations have been made from in vivo studies: IL-6, TNF-α, and IL-1β are typically observed in serum in the first hours post-challenge with live or inactivated S. epidermidis (Wakabayashi et al, 1991 ; Simojoki et al, 2011 ; Bi et al, 2015 ; Ferreirinha et al, 2016 ; Qin et al, 2017 ), or in tissue exudates/homogenates from experimental DRI models (Boelens et al, 2000b ; Svensson et al, 2015 ). The regulatory cytokine IL-10 is also present in vivo (Ferreirinha et al, 2016 ) and it has been shown that S. epidermidis inoculation result in higher IL-10 levels compared to P. aeruginosa in an intradermal infection model (Bialecka et al, 2005 ). In a S. epidermidis DRI mouse model it was shown that IL-10 was involved in reducing infection-associated morbidity, with higher levels of pro-inflammatory cytokines and greater weight loss in IL-10 KO animals.…”

“…PIA may contribute to innate immune system evasion by promoting generation of complement C5a fragment (Satorius et al, 2013 ; Al-Ishaq et al, 2015 ), inhibiting phagocytes and neutrophil killing (Vuong et al, 2004b , c ), and reducing the activity of AMPs (Vuong et al, 2004b ; Otto, 2006 ). Recently, other studies have reported slightly opposite findings, with PIA-producing bacteria inducing greater inflammatory responses and enhanced phagocytosis (Spiliopoulou et al, 2012 ; Ferreirinha et al, 2016 ), although Spiliopoulou et al did observe reduced killing in PIA-producing strains as discussed elsewhere recently (Nguyen et al, 2017 ). S. epidermidis also produces a second exopolymer, the poly-γ-glutamic acid (PGA), although at comparatively lower levels.…”

“…Secretion of IL-8, important for neutrophil recruitment, has been described in vitro and in the first hours post-infection in in vivo studies (Wakabayashi et al, 1991 ; Boelens et al, 2000b ; Simojoki et al, 2011 ; Svensson et al, 2015 ). CXCL-1 and CXCL-2, mostly produced by macrophages (via TLR-2 recognition but also by other mechanisms), have also been observed in bacteraemia and peritonitis models (Strunk et al, 2010 ; Bi et al, 2015 ; Ferreirinha et al, 2016 ; Qin et al, 2017 ). Additionally, a murine peritonitis model revealed increasing levels of numerous chemokines upon challenge with S. epidermidis supernatants (Perks et al, 2016 ).…”

“…Based on cytokines induced by S. epidermidis in the different studies (e.g., IL-6, IFN-γ, or IL-12), a Th1/Th17 polarization may be expected in such infections. This goes in line with the findings of Ferreirinha et al ., who observed that injection of PNAG-producing S. epidermidis in mice lead to IFN-γ and IL-17A producing T cells (Ferreirinha et al, 2016 ). Also, as mentioned above, immunization of mice with Atl-AM led to an increase in Th1 and Th2 cells (Th17 cells were not evaluated on that study).…”

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes

“…PNAG is a surface polysaccharide that is a key virulence factor in highly divergent bacterial species 37,38 . A. baumannii PNAG plays a crucial role in biofilm formation 35 .…”