Phenol-soluble modulin α and ß display divergent roles in mice with staphylococcal septic arthritis.

Phenol-soluble modulin α (PSMα) is identified as potent virulence factors in Staphylococcus aureus (S. aureus) infections. Very little is known about the role of PSMß which belongs to the same toxin family. Here we compared the role of PSMs in S. aureus-induced septic arthritis in a murine model using three isogenic S. aureus strains differing in the expression of PSMs (Newman, Δpsmα, and Δpsmß). The effects of PSMs on neutrophil NADPH-oxidase activity were determined in vitro. We show that the PSMα activates neutrophils via the formyl peptide receptor (FPR) 2 and reduces their NADPH-oxidase activity in response to the phorbol ester PMA. Despite being a poor neutrophil activator, PSMß has the ability to reduce the neutrophil activating effect of PSMα and to partly reverse the effect of PSMα on the neutrophil response to PMA. Mice infected with S. aureus lacking PSMα had better weight development and lower bacterial burden in the kidneys compared to mice infected with the parental strain, whereas mice infected with bacteria lacking PSMß strain developed more severe septic arthritis accompanied with higher IL-6 and KC. We conclude that PSMα and PSMß play distinct roles in septic arthritis: PSMα aggravates systemic infection, whereas PSMß protects arthritis development.

Trace amines produced by skin bacteria accelerate wound healing in mice.

Certain skin bacteria are able to convert aromatic amino acids (AAA) into trace amines (TA) that act as neuromodulators. Since the human skin and sweat contain a comparatively high content of AAA one can expect that such bacteria are able to produce TA on our skin. Here we show that TA-producing Staphylococcus epidermidis strains expressing SadA are predominant on human skin and that TA accelerate wound healing. In wounded skin, keratinocytes produce epinephrine (EPI) that leads to cell motility inhibition by ß2-adrenergic receptor (ß2-AR) activation thus delay wound healing. As ß2-AR antagonists, TA and dopamine (DOP) abrogate the effect of EPI thus accelerating wound healing both in vitro and in a mouse model. In the mouse model, the S. epidermidis wild type strain accelerates wound healing compared to its ΔsadA mutant. Our study demonstrates that TA-producing S. epidermidis strains present on our skin might be beneficial for wound healing.

Oxidative stress drives the selection of quorum sensing mutants in the Staphylococcus aureus population.

Quorum sensing (QS) is the central mechanism by which social interactions within the bacterial community control bacterial behavior. QS-negative cells benefit by exploiting public goods produced by the QS-proficient population. Mechanisms to keep the balance between producers and nonproducers within the population are expected but have not been elucidated for peptide-based QS systems in gram-positive pathogens. The Agr system of Staphylococcus aureus comprises the secretion and sensing of an autoinducing peptide to activate its own expression via the response regulator AgrA as well as the expression of a regulatory RNAIII and psmα/psmß coding for phenol-soluble modulins (PSMs). Agr mutants can be monitored on blood agar due to their nonhemolytic phenotype. In vitro evolution and competition experiments show that they readily accumulate in a process that is accelerated by ciprofloxacin, while the wild type (WT) is retained in the population at low numbers. However, agr mutants possess a fitness advantage only under aerobic conditions. Under hypoxia, Agr activity is increased but without the expected fitness cost. The Agr-imposed oxygen-dependent fitness cost is not due to a metabolic burden but due to the reactive oxygen species (ROS)-inducing capacity of the PSMs and RNAIII-regulated factors. Thus, selection of mutants is dictated by the QS system itself. Under aerobic conditions, emergence of agr-negative mutants may provide the population with a fitness advantage while hypoxia favors QS maintenance and even affords increased toxin production. The oxygen-driven tuning of the Agr system might be of importance to provide the pathogen with capabilities crucial for disease progression.

MpsAB is important for Staphylococcus aureus virulence and growth at atmospheric CO2 levels.

The mechanisms behind carbon dioxide (CO2) dependency in non-autotrophic bacterial isolates are unclear. Here we show that the Staphylococcus aureus mpsAB operon, known to play a role in membrane potential generation, is crucial for growth at atmospheric CO2 levels. The genes mpsAB can complement an Escherichia coli carbonic anhydrase (CA) mutant, and CA from E. coli can complement the S. aureus delta-mpsABC mutant. In comparison with the wild type, S. aureus mps mutants produce less hemolytic toxin and are less virulent in animal models of infection. Homologs of mpsA and mpsB are widespread among bacteria and are often found adjacent to each other on the genome. We propose that MpsAB represents a dissolved inorganic carbon transporter, or bicarbonate concentrating system, possibly acting as a sodium bicarbonate cotransporter.

Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus.

Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (RomR) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the RomR clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the RomR clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the RomR clone compared to its parental strain HG001. If farE is deleted in the RomR clone, or, if native farR is expressed in the RomR strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the RomR clone, that FarR is an important regulator, and that the point mutation in farR (RomR clone) makes the clone hyper-virulent.

A new host cell internalisation pathway for SadA-expressing staphylococci triggered by excreted neurochemicals.

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface-bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal-pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface-bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT-29. We found that TAs and DOP are agonists of the α2-adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F-actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F-actin formation without decreasing the cytoplasmic cAMP level. The neurochemical-induced internalisation in host cells is independent of the fibronectin-binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.

Bacterial Excretion of Cytoplasmic Proteins (ECP): Occurrence, Mechanism, and Function.

The excretion of cytoplasmic and signal-peptide-less proteins (ECP) by microorganisms and eukaryotes remains a fascinating topic. In principle, it appears to be a waste of energy. However, it turns out that - extracellularly - some cytoplasmic proteins (CPs) exert a completely different function such as contributing to pathogenicity or evasion of the immune system. Such CPs have been referred to as 'moonlighting' proteins. ECP is boosted by many endogenous or external factors that impair the membrane or cell wall structure. There are also differences regarding their mode of release. In some microorganisms they appear to be released directly, while in others they are embedded in membrane vesicles, or bound to the cell envelope. Some CPs might be promising candidates for vaccine developments against major bacterial pathogens.

Recovery of the Peptidoglycan Turnover Product Released by the Autolysin Atl in Staphylococcus aureus Involves the Phosphotransferase System Transporter MurP and the Novel 6-phospho-N-acetylmuramidase MupG.

The peptidoglycan of the bacterial cell wall undergoes a permanent turnover during cell growth and differentiation. In the Gram-positive pathogen Staphylococcus aureus, the major peptidoglycan hydrolase Atl is required for accurate cell division, daughter cell separation and autolysis. Atl is a bifunctional N-acetylmuramoyl-L-alanine amidase/endo-ß-N-acetylglucosaminidase that releases peptides and the disaccharide N-acetylmuramic acid-ß-1,4-N-acetylglucosamine (MurNAc-GlcNAc) from the peptido-glycan. Here we revealed the recycling pathway of the cell wall turnover product MurNAc-GlcNAc in S. aureus. The latter disaccharide is internalized and concomitantly phosphorylated by the phosphotransferase system (PTS) transporter MurP, which had been implicated previously in the uptake and phosphorylation of MurNAc. Since MurP mutant cells accumulate MurNAc-GlcNAc and not MurNAc in the culture medium during growth, the disaccharide represents the physiological substrate of the PTS transporter. We further identified and characterized a novel 6-phospho-N-acetylmuramidase, named MupG, which intracellularly hydrolyses MurNAc 6-phosphate-GlcNAc, the product of MurP-uptake and phosphorylation, yielding MurNAc 6-phosphate and GlcNAc. MupG is the first characterized representative of a novel family of glycosidases containing domain of unknown function 871 (DUF871). The corresponding gene mupG (SAUSA300_0192) of S. aureus strain USA300 is the first gene within a putative operon that also includes genes encoding the MurNAc 6-phosphate etherase MurQ, MurP, and the putative transcriptional regulator MurR. Using mass spectrometry, we observed cytoplasmic accumulation of MurNAc 6-phosphate-GlcNAc in ΔmupG and ΔmupGmurQ markerless non-polar deletion mutants, but not in the wild type or in the complemented ΔmupG strain. MurNAc 6-phosphate-GlcNAc levels in the mutants increased during stationary phase, in accordance with previous observations regarding peptidoglycan recycling in S. aureus.

The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus.

The innate immune system uses Toll-like receptor (TLR) 2 to detect conserved bacterial lipoproteins of invading pathogens. The lipid anchor attaches lipoproteins to the cytoplasmic membrane and prevents their release from the bacterial cell envelope. How bacteria release lipoproteins and how these molecules reach TLR2 remain unknown. Staphylococcus aureus has been described to liberate membrane vesicles. The composition, mode of release, and relevance for microbe-host interaction of such membrane vesicles have remained ambiguous. We recently reported that S. aureus can release lipoproteins only when surfactant-like small peptides, the phenol-soluble modulins (PSMs), are expressed. Here we demonstrate that PSM peptides promote the release of membrane vesicles from the cytoplasmic membrane of S. aureus via an increase in membrane fluidity, and we provide evidence that the bacterial turgor is the driving force for vesicle budding under hypotonic osmotic conditions. Intriguingly, the majority of lipoproteins are released by S. aureus as components of membrane vesicles, and this process depends on surfactant-like molecules such as PSMs. Vesicle disruption at high detergent concentrations promotes the capacity of lipoproteins to activate TLR2. These results reveal that vesicle release by bacterium-derived surfactants is required for TLR2-mediated inflammation.IMPORTANCE Our study highlights the roles of surfactant-like molecules in bacterial inflammation with important implications for the prevention and therapy of inflammatory disorders. It describes a potential pathway for the transfer of hydrophobic bacterial lipoproteins, the major TLR2 agonists, from the cytoplasmic membrane of Gram-positive bacteria to the TLR2 receptor at the surface of host cells. Moreover, our study reveals a molecular mechanism that explains how cytoplasmic and membrane-embedded bacterial proteins can be released by bacterial cells without using any of the typical protein secretion routes, thereby contributing to our understanding of the processes used by bacteria to communicate with host organisms and the environment.

Genetic Adaptation of a Mevalonate Pathway Deficient Mutant in Staphylococcus aureus.

In this study we addressed the question how a mevalonate (MVA)-auxotrophic Staphylococcus aureusΔmvaS mutant can revert to prototrophy. This mutant couldn't grow in the absence of MVA. However, after a long lag-phase of 4-6 days the mutant adapted from auxotrophic to prototrophic phenotype. During that time, it acquired two point mutations: One mutation in the coding region of the regulator gene spx, which resulted in an amino acid exchange that decreased Spx function. The other mutation in the upstream-element within the core-promoter of the mevalonolactone lactonase gene drp35. This mutation led to an increased expression of drp35. In repeated experiments the mutations always occurred in spx and drp35 and in the same order. The first detectable mutation appeared in spx and allowed slight growth; the second mutation, in drp35, increased growth further. Phenotypical characterizations of the mutant showed that small amounts of the lipid-carrier undecaprenol are synthesized, despite the lack of mvaS. The growth of the adapted clone, ΔmvaSad, indicates that the mutations reawake a rescue bypass. We think that this bypass enters the MVA pathway at the stage of MVA, because blocking the pathway downstream of MVA led to growth arrest of the mutant. In addition, the lactonase Drp35 is able to convert mevalonolactone to MVA. Summarized, we describe here a mutation-based two-step adaptation process that allows resuscitation of growth of the ΔmvaS mutant.

Lantibiotic production is a burden for the producing staphylococci.

Lantibiotics are antimicrobial peptides that contain non-proteinogenic amino acids lanthionine and 3-methyllanthionine and are produced by Gram-positive bacteria. Here we addressed the pros and cons of lantibiotic production for its producing strains. Two staphylococcal strains, S. gallinarum Tü3928 and S. epidermidis Tü3298 producing gallidermin and epidermin respectively were selected. In each of these parental strains, the structural genes gdmA and epiA were deleted; all the other biosynthetic genes including the immunity genes were left intact. Comparative analysis of the lantibiotic-producing strains with their non-producing mutants revealed that lantibiotic production is a burden for the cells. The production affected growth, caused release of ATP, lipids and increased the excretion of cytoplasmic proteins (ECP). The epidermin and gallidermin immunity genes were insufficient to protect the cells from their own product. Co-cultivation studies showed that the ΔgdmA mutant has an advantage over the parental strain; the latter was outcompeted. On the one hand, the production of staphylococcal lantibiotics is beneficial by suppressing competitors, but on the other hand they impose a burden on the producing-strains when they accumulate in higher amounts. Our observations explain why antibiotic-producing strains occur as a minority on our skin and other ecological niches, but retain corresponding antibiotic resistance.

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection.

SadA-Expressing Staphylococci in the Human Gut Show Increased Cell Adherence and Internalization.

A subgroup of biogenic amines, the so-called trace amines (TAs), are produced by mammals and bacteria and can act as neuromodulators. In the genus Staphylococcus, certain species are capable of producing TAs through the activity of staphylococcal aromatic amino acid decarboxylase (SadA). SadA decarboxylates aromatic amino acids to produce TAs, as well as dihydroxy phenylalanine and 5-hydroxytryptophan to thus produce the neurotransmitters dopamine and serotonin. SadA-expressing staphylococci were prevalent in the gut of most probands, where they are part of the human intestinal microflora. Furthermore, sadA-expressing staphylococci showed increased adherence to HT-29 cells and 2- to 3-fold increased internalization. Internalization and adherence was also increased in a sadA mutant in the presence of tryptamine. The α2-adrenergic receptor is required for enhanced adherence and internalization. Thus, staphylococci in the gut might contribute to gut activity and intestinal colonization.

Rhodomyrtone (Rom) is a membrane-active compound.

Particularly in Asia medicinal plants with antimicrobial activity are used for therapeutic purpose. One such plant-derived antibiotic is rhodomyrtone (Rom) isolated from Rhodomyrtus tomentosa leaves. Rom shows high antibacterial activity against a wide range of Gram-positive bacteria, however, its mode of action is still unclear. Reporter gene assays and proteomic profiling experiments in Bacillus subtilis indicate that Rom does not address classical antibiotic targets like translation, transcription or DNA replication, but acts at the cytoplasmic membrane. In Staphylococcus aureus, Rom decreases the membrane potential within seconds and at low doses, causes release of ATP and even the excretion of cytoplasmic proteins (ECP), but does not induce pore-formation as for example nisin. Lipid staining revealed that Rom induces local membrane damage. Rom's antimicrobial activity can be antagonized in the presence of a very narrow spectrum of saturated fatty acids (C15:0, C16:0, or C18:0) that most likely contribute to counteract the membrane damage. Gram-negative bacteria are resistant to Rom, presumably due to reduced penetration through the outer membrane and its neutralization by LPS. Rom is cytotoxic for many eukaryotic cells and studies with human erythrocytes showed that Rom induces eryptosis accompanied by erythrocyte shrinkage, cell membrane blebbing, and membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Rom's distinctive interaction with the cytoplasmic membrane reminds on the amphipathic, alpha-helical peptides, the phenol-soluble modulins (PSMs), and renders Rom an important tool for the investigation of membrane physiology.

Non-classical Protein Excretion Is Boosted by PSMα-Induced Cell Leakage.

Release of cytoplasmic proteins into the supernatant occurs both in bacteria and eukaryotes. Because the underlying mechanism remains unclear, the excretion of cytoplasmic proteins (ECP) has been referred to as "non-classical protein secretion." We show that none of the known specific protein transport systems of Gram-positive bacteria are involved in ECP. However, the expression of the cationic and amphipathic α-type phenol-soluble modulins (PSMs), particularly of PSMα2, significantly increase ECP, while PSMß peptides or δ-toxin have no effect on ECP. Because psm expression is strictly controlled by the accessory gene regulator (agr), ECP is also reduced in agr-negative mutants. PSMα peptides damage the cytoplasmic membrane, as indicated by the release of not only CPs but also lipids, nucleic acids, and ATP. Thus, our results show that in Staphylococcus aureus, PSMα peptides non-specifically boost the translocation of CPs by their membrane-damaging activity.

Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell Lysis.

Teixobactin represents the first member of a newly discovered class of antibiotics that act through inhibition of cell wall synthesis. Teixobactin binds multiple bactoprenol-coupled cell wall precursors, inhibiting both peptidoglycan and teichoic acid synthesis. Here, we show that the impressive bactericidal activity of teixobactin is due to the synergistic inhibition of both targets, resulting in cell wall damage, delocalization of autolysins, and subsequent cell lysis. We also find that teixobactin does not bind mature peptidoglycan, further increasing its activity at high cell densities and against vancomycin-intermediate Staphylococcus aureus (VISA) isolates with thickened peptidoglycan layers. These findings add to the attractiveness of teixobactin as a potential therapeutic agent for the treatment of infection caused by antibiotic-resistant Gram-positive pathogens.

Excreted Cytoplasmic Proteins Contribute to Pathogenicity in Staphylococcus aureus.

Excretion of cytoplasmic proteins in pro- and eukaryotes, also referred to as "nonclassical protein export," is a well-known phenomenon. However, comparatively little is known about the role of the excreted proteins in relation to pathogenicity. Here, the impact of two excreted glycolytic enzymes, aldolase (FbaA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), on pathogenicity was investigated in Staphylococcus aureus Both enzymes bound to certain host matrix proteins and enhanced adherence of the bacterial cells to host cells but caused a decrease in host cell invasion. FbaA and GAPDH also bound to the cell surfaces of staphylococcal cells by interaction with the major autolysin, Atl, that is involved in host cell internalization. Surprisingly, FbaA showed high cytotoxicity to both MonoMac 6 (MM6) and HaCaT cells, while GAPDH was cytotoxic only for MM6 cells. Finally, the contribution of external FbaA and GAPDH to S. aureus pathogenicity was confirmed in an insect infection model.

VraH Is the Third Component of the Staphylococcus aureus VraDEH System Involved in Gallidermin and Daptomycin Resistance and Pathogenicity.

In bacteria, extracellular signals are transduced into the cell predominantly by two-component systems (TCSs) comprising a regulatory unit triggered by a specific signal. Some of the TCSs control executing units such as ABC transporters involved in antibiotic resistance. For instance, inStaphylococcus aureus, activation of BraSR leads to the upregulation ofvraDEexpression that encodes an ABC transporter playing a role in bacitracin and nisin resistance. In this study, we show that the small staphylococcal transmembrane protein VraH forms, together with VraDE, a three-component system. Although the expression ofvraHin the absence ofvraDEwas sufficient to mediate low-level resistance, only this VraDEH entity conferred high-level resistance against daptomycin and gallidermin. In most staphylococcal genomes,vraHis located immediately downstream ofvraDE, forming an operon, whereas in some species it is localized differently. In an invertebrate infection model, VraDEH significantly enhancedS. aureuspathogenicity. In analogy to the TCS connectors, VraH can be regarded as an ABC connector that modulates the activity of ABC transporters involved in antibiotic resistance.

Excretion of cytoplasmic proteins in Staphylococcus is most likely not due to cell lysis.

The excretion of cytoplasmic proteins (ECP) is a long-known phenomenon in bacteria and eukaryotes. So far, it was not possible to associate either a signal peptide-dependent or a signal peptide-independent pathway to ECP. Nevertheless 25% of the proteins found in Staphylococcus aureus supernatants were cytoplasmic proteins. Because the excreted proteins do not possess a common motive, the most widespread opinion is that ECP is due to cell lysis. This explanation seems to be too easy since several indications imply that there exists a yet unknown mechanism for ECP. Certainly, the up-regulation of autolysins as well as decreased peptidoglycan cross-linking increased ECP. However, in recent years, several evidences arose that cell lysis is not the only reason for ECP. It seems that ECP is a part of the normal cell cycle of S. aureus as it turned out that ECP with several model proteins occurs mainly during cell growth. It has common features as proteins secreted via the Sec translocon and finally the excretion site is the cross wall of dividing cells.