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Staphylococcus epidermidis expresses glycerol phosphate wall teichoic acid (WTA), but some health care-associated methicillin-resistant S. epidermidis (HA-MRSE) clones produce a second, ribitol phosphate (RboP) WTA, resembling that of the aggressive pathogen Staphylococcus aureus. RboP-WTA promotes HA-MRSE persistence and virulence in bloodstream infections. We report here that the TarM enzyme of HA-MRSE [TarM(Se)] glycosylates RboP-WTA with glucose, instead of N-acetylglucosamine (GlcNAc) by TarM(Sa) in S. aureus. Replacement of GlcNAc with glucose in RboP-WTA impairs HA-MRSE detection by human immunoglobulin G, which may contribute to the immune-evasion capacities of many invasive S. epidermidis. Crystal structures of complexes with uridine diphosphate glucose (UDP-glucose), and with UDP and glycosylated poly(RboP), reveal the binding mode and glycosylation mechanism of this enzyme and explain why TarM(Se) and TarM(Sa) link different sugars to poly(RboP). These structural data provide evidence that TarM(Se) is a processive WTA glycosyltransferase. Our study will support the targeted inhibition of TarM enzymes, and the development of RboP-WTA targeting vaccines and phage therapies.

Abstract Background Bacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation. Results In order to elucidate how global metabolic processes are affected by the absence of formylated proteins the exometabolome of an S. aureus fmt mutant was compared with that of the parental strain and the transcription of corresponding enzymes was analyzed to identify possible regulatory changes. The mutant consumed glucose and other carbon sources slower than the wild type. While the turnover of several metabolites remained unaltered fmt inactivation led to increases pyruvate release and, concomitantly, reduced pyruvate dehydrogenase activity. In parallel, the release of the pyruvate-derived metabolites lactate, acetoin, and alanine was reduced. The anaerobic degradation of arginine was also reduced in the fmt mutant compared to the wild-type strain. Moreover, the lack of formylated proteins caused increased susceptibility to the antibiotics trimethoprim and sulamethoxazole suggesting that folic acid-dependant pathways were perturbed in the mutant. Conclusions These data indicate that formylated proteins are crucial for specific bacterial metabolic processes and they may help to understand why it has remained important during bacterial evolution to initiate protein biosynthesis with a formylated tRNAMet.

Abstract Background Modification of teichoic acids with D-alanine by the products of the dlt operon protects Gram-positive bacteria against major antimicrobial host defense molecules such as defensins, cathelicidins, myeloperoxidase or phospholipase. The graRS regulatory genes have recently been implicated in the control of D-alanylation in Staphylococcus aureus. Results To determine the impact of the GraRS regulatory system on resistance to antimicrobial host defense mechanisms and virulence of S. aureus, we compared inactivation of S. aureus SA113 wild type and its isogenic graRS deletion mutant by the human cathelicidin LL-37 or human neutrophil granulocytes in vitro, and the ability to cause infection in vivo. We show here that graRS deletion considerably alters bacterial surface charge, increases susceptibility to killing by human neutrophils or the defense peptide LL-37, and attenuates virulence of S. aureus in a mouse infection model. Conclusion Our results indicate that S. aureus can regulate its surface properties in order to overcome innate host defenses.

Daptomycin, a calcium-dependent lipopeptide antibiotic whose full mode of action is still not entirely understood, has become a standard-of-care agent for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Daptomycin-resistant (DAP-R) S. aureus mutants emerge during therapy, featuring isolates which in most cases possess point mutations in the mprF gene. MprF is a bifunctional bacterial resistance protein that synthesizes the positively charged lipid lysyl-phosphatidylglycerol (LysPG) and translocates it subsequently from the inner membrane leaflet to the outer membrane leaflet. This process leads to increased positive S. aureus surface charge and reduces susceptibility to cationic antimicrobial peptides and cationic antibiotics. We characterized the most commonly reported MprF mutations in DAP-R S. aureus strains in a defined genetic background and found that only certain mutations, including the frequently reported T345A single nucleotide polymorphism (SNP), can reproducibly cause daptomycin resistance. Surprisingly, T345A did not alter LysPG synthesis, LysPG translocation, or the S. aureus cell surface charge. MprF-mediated DAP-R relied on a functional flippase domain and was restricted to daptomycin and a related cyclic lipopeptide antibiotic, friulimicin B, suggesting that the mutations modulate specific interactions with these two antibiotics. Notably, the T345A mutation led to weakened intramolecular domain interactions of MprF, suggesting that daptomycin and friulimicin resistance-conferring mutations may alter the substrate range of the MprF flippase to directly translocate these lipopeptide antibiotics or other membrane components with crucial roles in the activity of these antimicrobials. Our study points to a new mechanism used by S. aureus to resist calcium-dependent lipopeptide antibiotics and increases our understanding of the bacterial phospholipid flippase MprF.IMPORTANCE Ever since daptomycin was introduced to the clinic, daptomycin-resistant isolates have been reported. In most cases, the resistant isolates harbor point mutations in MprF, which produces and flips the positively charged phospholipid LysPG. This has led to the assumption that the resistance mechanism relies on the overproduction of LysPG, given that increased LysPG production may lead to increased electrostatic repulsion of positively charged antimicrobial compounds, including daptomycin. Here we show that the resistance mechanism is highly specific and relies on a different process that involves a functional MprF flippase, suggesting that the resistance-conferring mutations may enable the flippase to accommodate daptomycin or an unknown component that is crucial for its activity. Our report provides a new perspective on the mechanism of resistance to a major antibiotic.

Bicarbonate and CO2 are essential substrates for carboxylation reactions in bacterial central metabolism. In Staphylococcus aureus, the bicarbonate transporter, MpsABC (membrane potential-generating system) is the only carbon concentrating system. An mpsABC deletion mutant can hardly grow in ambient air. In this study, we investigated the changes that occur in S. aureus when it suffers from CO2/bicarbonate deficiency. Electron microscopy revealed that ΔmpsABC has a twofold thicker cell wall thickness compared to the parent strain. The mutant was also substantially inert to cell lysis induced by lysostaphin and the non-ionic surfactant Triton X-100. Mass spectrometry analysis of muropeptides revealed the incorporation of alanine into the pentaglycine interpeptide bridge, which explains the mutant's lysostaphin resistance. Flow cytometry analysis of wall teichoic acid (WTA) glycosylation patterns revealed a significantly lower α-glycosylated and higher ß-glycosylated WTA, explaining the mutant's increased resistance towards Triton X-100. Comparative transcriptome analysis showed altered gene expression profiles. Autolysin-encoding genes such as sceD, a lytic transglycosylase encoding gene, were upregulated, like in vancomycin-intermediate S. aureus mutants (VISA). Genes related to cell wall-anchored proteins, secreted proteins, transporters, and toxins were downregulated. Overall, we demonstrate that bicarbonate deficiency is a stress response that causes changes in cell wall composition and global gene expression resulting in increased resilience to cell wall lytic enzymes and detergents. [ABSTRACT FROM AUTHOR]

Polycyclic polyprenylated acylphloroglucinols (PPAPs) comprise a large group of compounds of mostly plant origin. The best-known compound is hyperforin from St. John's wort with its antidepressant, antitumor and antimicrobial properties. The chemical synthesis of PPAP variants allows the generation of compounds with improved activity and compatibility. Here, we studied the antimicrobial activity of two synthetic PPAP-derivatives, the water-insoluble PPAP23 and the water-soluble sodium salt PPAP53. In vitro, both compounds exhibited good activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium. Both compounds had no adverse effects on Galleria mellonella wax moth larvae. However, they were unable to protect the larvae from infection with S. aureus because components of the larval coelom neutralized the antimicrobial activity; a similar effect was also seen with serum albumin. In silico docking studies with PPAP53 revealed that it binds to the F1 pocket of human serum albumin with a binding energy of −7.5 kcal/mol. In an infection model of septic arthritis, PPAP23 decreased the formation of abscesses and S. aureus load in kidneys; in a mouse skin abscess model, topical treatment with PPAP53 reduced S. aureus counts. Both PPAPs were active against anaerobic Gram-positive gut bacteria such as neurotransmitter-producing Clostridium, Enterococcus or Ruminococcus species. Based on these results, we foresee possible applications in the decolonization of pathogens. [ABSTRACT FROM AUTHOR]

BackgroundPrevious studies of both clinically-derived and in vitro passage-derived daptomycin-resistant (DAP-R) Staphylococcus aureus strains demonstrated the coincident emergence of increased DAP MICs and resistance to host defense cationic peptides (HDP-R).MethodsIn the present investigation, we studied a parental DAP-susceptible (DAP-S) methicillin-resistant Staphylococcus aureus (MRSA) strain and three isogenic variants with increased DAP MICs which were isolated from both DAP-treated and DAP-untreated rabbits with prosthetic joint infections. These strains were compared for: in vitro susceptibility to distinct HDPs differing in size, structure, and origin; i.e.; thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]; cell membrane (CM) phospholipid and fatty acid content; CM order; envelope surface charge; cell wall thickness; and mprF single nucleotide polymorphisms (SNPs) and expression profiles.ResultsIn comparison with the parental strain, both DAP-exposed and DAP-naive strains exhibited: (i) significantly reduced susceptibility to each HDP (P