Cell wall thickening is a common feature among daptomycin-resistant Staphylococcus aureus strains. However, the mechanism(s) leading to this phenotype is unknown. We examined a number of cell wall synthesis pathway parameters in an isogenic strain set of S. aureus bloodstream isolates obtained from a patient with recalcitrant endocarditis who failed daptomycin therapy, including the initial daptomycin-susceptible parental strain (strain 616) and two daptomycin-resistant strains (strains 701 and 703) isolated during daptomycin therapy. Transmission electron microscopy demonstrated significantly thicker cell walls in the daptomycinresistant strains than in the daptomycin-susceptible strain, a finding which was compatible with significant differences in dry cell weight of strain 616 versus strains 701 to 703 (P < 0.05). Results of detailed analysis of cell wall muropeptide composition, the degree of peptide side chain cross-linkage, and the amount of the peptidoglycan precursor, UDP-MurNAc-pentapeptide, were similar in the daptomycin-susceptible and daptomycin-resistant isolates. In contrast, the daptomycin-resistant strains contained less O-acetylated peptidoglycan. Importantly, both daptomycin-resistant strains synthesized significantly more wall teichoic acid (WTA) than the parental strain (P < 0.001). Moreover, the proportion of D-alanylated WTA species was substantially higher in the daptomycin-resistant strains than in the daptomycin-susceptible parental strain (P < 0.05 in comparing strain 616 versus strain 701). The latter phenotypic findings correlated with (i) enhanced tagA and dltA gene expression, respectively, and (ii) an increase in surface positive charge observed in the daptomycinresistant versus daptomycin-susceptible isolates. Collectively, these data suggest that increases in WTA synthesis and the degree of its D-alanylation may play a major role in the daptomycin-resistant phenotype in some S. aureus strains.Daptomycin has become a key agent for the management of serious Staphylococcus aureus infections, especially for drug-resistant strains, such as methicillin-resistant S. aureus (MRSA) (21, 35). However, a number of recent reports have documented the emergence of in vivo daptomycin-resistant S. aureus strains during unsuccessful therapy with this agent (3,11,27). There appear to be several potential mechanisms associated with the daptomycin-resistant phenotype at both the genotypic and phenotypic levels, including (i) increased expression of genes involved in maintenance of the bacterial surface positive charge (e.g., dltABCD or mprF [37,38,40]), (ii) perturbations in cell membrane fluidity (18), and (iii) altered cell membrane permeabilization (12). We have recently shown that a common (although not universal) accompaniment of the daptomycin-resistant phenotype is a notably thickened cell wall among such strains (18,37,38). Although some investigations have shown the presence of selected cell wall synthetic alterations in daptomycin-resistant S. aureus strains (e.g., changes in peptidoglycan...

Development of in vivo daptomycin resistance (DAP-R) among Staphylococcus aureus clinical isolates, in association with clinical treatment failures, has become a major therapeutic problem. This issue is especially relevant to methicillin-resistant S. aureus (MRSA) strains in the context of invasive endovascular infections. In the current study, we used three well-characterized and clinically-derived DAP-susceptible (DAP-S) vs. resistant (DAP-R) MRSA strain-pairs to elucidate potential genotypic mechanisms of the DAP-R phenotype. In comparison to the DAP-S parental strains, DAP-R isolates demonstrated (i) altered expression of two key determinants of net positive surface charge, either during exponential or stationary growth phases (i.e., dysregulation of dltA and mprF), (ii) a significant increase in the D-alanylated wall teichoic acid (WTA) content in DAP-R strains, reflecting DltA gain-in-function; (iii) heightened elaboration of lysinylated-phosphatidylglyderol (L-PG) in DAP-R strains, reflecting MprF gain-in-function; (iv) increased cell membrane (CM) fluidity, and (v) significantly reduced susceptibility to prototypic cationic host defense peptides of platelet and leukocyte origins. In the tested DAP-R strains, genes conferring positive surface charge were dysregulated, and their functionality altered. However, there were no correlations between relative surface positive charge or cell wall thickness and the observed DAP-R phenotype. Thus, charge repulsion mechanisms via altered surface charge may not be sufficient to explain the DAP-R outcome. Instead, changes in the compositional or biophysical order of the DAP CM target of such DAP-R strains (i.e., increased fluidity) may be essential to this phenotype. Taken together, DAP-R in S. aureus appears to involve multi-factorial and strain-specific adaptive mechanisms.

Multiple mechanisms have been correlated with daptomycin-resistance (DAP-R) in Staphylococcus aureus. However, one common phenotype observed in many DAP-R S. Aureus strains is a thickened cell wall (CW). The first evidence for an impact of CW-linked glycopolymers on this phenotype was recently demonstrated in a single, well-characterized DAP-R methicillin-susceptible S. aureus (MSSA) strain. In this isolate the thickened CW phenotype was linked to an increased production and D-alanylation of wall teichoic acids (WTA). In the current report, we extended these observations to methicillin-resistant daptomycin-sensitive/daptomyin-resistant (DAP-S/DAP-R) strain-pairs. These pairs included methicillin-resistant S. aureus (MRSA) isolates with and without single nucleotide polymorphisms (SNPs) in mprF (a genetic locus linked to DAP-R phenotype). We found increased CW dry mass in all DAP-R vs DAP-S isolates. This correlated with an increased expression of the WTA biosynthesis gene tagA, as well as an increased amount of WTA in the DAP-R vs DAP-S isolates. In addition, all DAP-R isolates showed a higher proportion of WTA D-alanylation vs their corresponding DAP-S isolate. We also detected an increased positive surface charge amongst the DAP-R strains (presumably related to the enhanced D-alanylation). In comparing the detailed CW composition of all isolate pairs, substantive differences were only detected in one DAP-S/DAP-R pair. The thickened CW phenotype, together with an increased surface charge most likely contributes to either: i) a charge-dependent repulsion of calcium complexed-DAP; and/or ii) steric-limited access of DAP to the bacterial cell envelope target. Taken together well-defined perturbations of CW structural and functional metrics contribute to the DAP-R phenotype and are common phenotypes in DAP-R S. Aureus isolates, both MSSA and MRSA. Note: Although “daptomycin-nonsusceptibility” is the generally accepted terminology, we have utilized the term “daptomycin resistance” for ease of presentation in this manuscript

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are the cause of a severe pandemic consisting primarily of skin and soft tissue infections. The underlying pathomechanisms have not been fully understood and we report here a mechanism that plays an important role for the elevated virulence of CA-MRSA. Surprisingly, skin abscess induction in an animal model was correlated with the amount of a major cell wall component of S. aureus, termed wall teichoic acid (WTA). CA-MRSA exhibited increased cell-wall-associated WTA content (WTA) and thus were more active in inducing abscess formation via a WTA-dependent and T-cell-mediated mechanism than S. aureus strains with a WTA phenotype. We show here that WTA is directly involved in S. aureus strain-specific virulence and provide insight into the underlying molecular mechanisms that could guide the development of novel anti-infective strategies.

We propose here a feedback model in which misfolded mutant K14 triggers enhanced expression of K14, which in turn amplifies the JNK-/AP1-mediated MAPK stress response. The exact mechanism cannot be deduced from these preliminary data. However, based on the observation that extrachromosomal overexpression of wild-type K14 in non-mutant cells also leads to the same effects, we suggest that an imbalance between K14 incorporated into the IF network and K14 existing as cytoplasmic keratin monomers triggers elevated MAPK signalling, potentially altering IF dynamics by phosphorylation. In this context, the MAPK family member p38 has previously been shown to modulate IF organization through phosphorylation of keratins (14).These data should help broaden the still narrow field of therapeutic approaches to EBS (17) and raise awareness to the possibility that as a secondary effect of the K14 mutation, imbalance of IF components and the associated amplification of MAPK signalling could play an important role in the pathophysiology of the disease. AcknowledgementsMW performed the research, analysed the data and wrote the manuscript, KO designed the research study, AT contributed essential tools and expertise in laser microscopy, and JWB and HH critically reviewed the manuscript. We gratefully thank Prof. Birgit Lane for kindly supplying the wild-type NEB1 and the patient KEB7 keratinocyte cell lines. This work was supported by the Dystrophic Epidermolysis Bullosa Research Association (DEBRA), Austria and by the Paracelsus Medical University, Salzburg, Austria (project number E-11/13/067-OEN). Conflict of interestsThe authors have declared no conflict of interest. Supporting InformationAdditional Supporting Information may be found in the online version of this article: Kiel, Schittenhelmstr. 7, 24105 Kiel, Germany, Tel.: 49-431-5971598, Fax: 49-431-5975243, e-mail: jharder@dermatology.uni-kiel.de Abstract: The Gram-positive bacterium Staphylococcus aureus is a frequent skin colonizer that often causes severe skin infections. It has been reported that neutralizing the negatively charged bacterial surface through the incorporation of D-alanine in its teichoic acids confers reduced susceptibility of S. aureus towards cationic antimicrobial peptides (AMPs). Using a S. aureus strain deficient in D-alanylated teichoic acids (dltA mutant), we demonstrate that D-alanylation of its surface reduces the susceptibility of S. aureus to skin-derived AMPs such as RNase 7 and human beta-defensins. This is accompanied by a higher killing activity of skin extracts towards the S. aureus dltA mutant as well as towards clinical isolates expressing lower levels of dltA. We conclude that modulation of cell envelope D-alanylation may help S. aureus to persist on human skin through evasion of cutaneous innate defense provided by cationic skin-derived AMPs.