Pathogen elimination by probiotic Bacillus via signalling interference.

Probiotic nutrition is frequently claimed to improve human health. In particular, live probiotic bacteria obtained with food are thought to reduce intestinal colonization by pathogens, and thus to reduce susceptibility to infection. However, the mechanisms that underlie these effects remain poorly understood. Here we report that the consumption of probiotic Bacillus bacteria comprehensively abolished colonization by the dangerous pathogen Staphylococcus aureus in a rural Thai population. We show that a widespread class of Bacillus lipopeptides, the fengycins, eliminates S. aureus by inhibiting S. aureus quorum sensing-a process through which bacteria respond to their population density by altering gene regulation. Our study presents a detailed molecular mechanism that underlines the importance of probiotic nutrition in reducing infectious disease. We also provide evidence that supports the biological significance of probiotic bacterial interference in humans, and show that such interference can be achieved by blocking a pathogen's signalling system. Furthermore, our findings suggest a probiotic-based method for S. aureus decolonization and new ways to fight S. aureus infections.

Acidonemycins A-C, Glycosylated Angucyclines with Antivirulence Activity Produced by the Acidic Culture of Streptomyces indonesiensis.

The genome of Streptomyces indonesiensis is highly enriched with cryptic biosynthetic gene clusters (BGCs). The majority of these cryptic BGCs are transcriptionally silent in normal laboratory culture conditions as determined by transcriptome analysis. When cultured in acidic pH (pH 5.4), this strain has been shown to produce a set of new metabolites that were not observed in cultures of neutral pH (pH 7.4). The organic extract of the acidic culture displayed an antivirulence activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report the structures of new glycosylated aromatic polyketides, named acidonemycins A-C (1-3), belonging to the family of angucyclines. Type II polyketide synthase BGC responsible for the production of 1-3 was identified by a transcriptome comparison between acidic (pH 5.4) and neutral (pH 7.4) cultures and further confirmed by heterologous expression in Streptomyces albus J1074. Of the three new compounds, acidonemycins A and B (1 and 2) displayed antivirulence activity against MRSA. The simultaneous identification of both antivirulent compounds and their BGC provides a starting point for the future effort of combinatorial biosynthesis.

Simultaneous monitoring of each component on degradation of blended bioplastic using gas chromatography-mass spectrometry.

The increasing interest in bioplastics, with regard to future environmental issues, has rendered research on bioplastic biodegradation highly important. However, only a few tools directly monitor the degradation of bioplastics without measuring the levels of gaseous products, such as carbon dioxide. Classical nonquantitative methods, such as clear zone tests on solid plates, and less-sensitive weight-loss experiments in liquid media measured using a precision scale, are still employed to screen the microbial players associated with bioplastic degradation and monitor the biodegradation rates. However, the simultaneous monitoring of the degradation of each component of blended bioplastics has not been previously reported. In the present study, to provide information regarding the degradation rates and compositional changes of different bioplastics in a blend in a time-dependent manner, we simultaneously monitored and quantified the degradation of four bioplastics, polyhydroxybutyrate (PHB), polybutylene succinate (PBS), polycaprolactone (PCL), and poly(butylene adipate-co-terephthalate) (PBAT), by Bacillus sp. JY36 using gas chromatography-mass spectrometry (GC-MS) analysis after fatty acid methyl ester (FAME) derivatization. Our results demonstrate the feasibility of using the GC-MS-based method described here to obtain comprehensive data regarding blended bioplastics and their degradation. Moreover, our findings indicate that this method may support classical analytic tools for assessing bioplastic biodegradation.

Unusual bridged angucyclinones and potent anticancer compounds from Streptomyces bulli GJA1.

Two new unique angucyclinones (1 and 2) with unprecedented ether bridges connecting carbons 5 and 7 were isolated from the cultures of Streptomyces bulli GJA1, an endophyte of Gardenia jasminoides, together with two known ones (3 and 4). The MS2-based molecular networking system facilitated the isolation of compounds with target functionalities. The stereochemistry of 1 was completely established by ROESY and ECD experiments. Compound 3 showed antivirulence activities by inhibiting the peptide toxin (PSMs) production and the biofilm formation of MRSA. Compounds 3 and 4 showed very potent anti-proliferative effects against OV1 and ES2 ovarian cancer cells.

Toxin Mediates Sepsis Caused by Methicillin-Resistant Staphylococcus epidermidis.

Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the psm-mec locus that have been reported in S. aureus strains, inasmuch as our findings suggest that the psm-mec locus may exert effects in the background of S. aureus strains that differ from its original role in the CNS environment due to originally "unintended" interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.

Do amyloid structures formed by Staphylococcus aureus phenol-soluble modulins have a biological function?

Phenol-soluble modulins (PSMs) are alpha-helical, amphipathic peptides that have multiple functions in staphylococcal physiology and virulence. Recent research has suggested that PSMs form amyloid fibrils and amyloids are involved in PSM-mediated phenotypes such as cytolysis and biofilm stability. While we observed PSM amyloid formation using electron microscopy and dye assays, there were no apparent differences in the production of extracellular fibrous material between a PSM-deficient strain and the isogenic wild-type strain. Furthermore, we detected no correlation between cytolytic or pro-inflammatory activities with the propensity of PSM derivatives to form amyloids. In addition, we propose a model based on our finding of non-specific attachment of PSMs to DNA, which we here report results in resistance to DNase digestion, explaining previous findings on PSM-mediated biofilm stability without the necessity to assume amyloid involvement. Collectively, our results indicate that PSM amyloid formation may not be of major relevance for known key biological functions of PSMs. Intriguingly, however, we found that amyloid-forming capacity of PSMalpha3 allows almost no amino acid exchanges, suggesting importance of amyloid formation in possibly yet unknown functions of PSMs.

Mechanisms of resistance to antimicrobial peptides in staphylococci.

Staphylococci are commensal bacteria living on the epithelial surfaces of humans and other mammals. Many staphylococci, including the dangerous pathogen Staphylococcus aureus, can cause severe disease when they breach the epithelial barrier. Both during their commensal life and during infection, staphylococci need to evade mechanisms of innate host defense, of which antimicrobial peptides (AMPs) play a key role in particular on the skin. Mechanisms that staphylococci have developed to evade the bactericidal activity of AMPs are manifold, comprising repulsion of AMPs via alteration of cell wall and membrane surface charges, proteolytic inactivation, sequestration, and secretion. Furthermore, many staphylococci form biofilms, which represents an additional way of protection from antimicrobial agents, including AMPs. Finally, staphylococci can sense the presence of AMPs by sensor/regulator systems that control many of those resistance mechanisms. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

Production of an attenuated phenol-soluble modulin variant unique to the MRSA clonal complex 30 increases severity of bloodstream infection.

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 PSMα3 peptide. We found that this variant, PSMα3N22Y, 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 PSMα3. 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.

Basis of virulence in a Panton-Valentine leukocidin-negative community-associated methicillin-resistant Staphylococcus aureus strain.

Community-associated (CA) infections with methicillin-resistant Staphylococcus aureus (MRSA) are on a global rise. However, analysis of virulence characteristics has been limited almost exclusively to the US endemic strain USA300. CA-MRSA strains that do not produce Panton-Valentine leukocidin (PVL) have not been investigated on a molecular level. Therefore, we analyzed virulence determinants in a PVL-negative CA-MRSA strain, ST72, from Korea. Genome-wide analysis identified 3 loci that are unique to that strain, but did not affect virulence. In contrast, phenol-soluble modulins (PSMs) and the global virulence regulator Agr strongly affected lysis of neutrophils and erythrocytes, while α-toxin and Agr had a major impact on in vivo virulence. Our findings substantiate the general key roles these factors play in CA-MRSA virulence. However, our analyses also showed noticeable differences to strain USA300, inasmuch as α-toxin emerged as a much more important factor than PSMs in experimental skin infection caused by ST72.

AraC-Type Regulator Rsp Adapts Staphylococcus aureus Gene Expression to Acute Infection.

Staphylococcus aureus is an important human pathogen that can cause two categories of severe infections. Acute infections are characterized by pronounced toxin production, while chronic infections often involve biofilm formation. However, it is poorly understood how S. aureus controls the expression of genes associated with acute versus biofilm-associated virulence. We here identified an AraC-type transcriptional regulator, Rsp, that promotes the production of key toxins while repressing major biofilm-associated genes and biofilm formation. Genome-wide transcriptional analysis and modeling of regulatory networks indicated that upregulation of the accessory gene regulator (Agr) and downregulation of the ica operon coding for the biofilm exopolysaccharide polysaccharide intercellular adhesin (PIA) were central to the regulatory impact of Rsp on virulence. Notably, the Rsp protein directly bound to the agrP2 and icaADBC promoters, resulting in strongly increased levels of the Agr-controlled toxins phenol-soluble modulins (PSMs) and alpha-toxin and reduced production of PIA. Accordingly, Rsp was essential for the development of bacteremia and skin infection, representing major types of acute S. aureus infection. Our findings give important insight into how S. aureus adapts the expression of its broad arsenal of virulence genes to promote different types of disease manifestations and identify the Rsp regulator as a potential target for strategies to control acute S. aureus infection.

Role of Phenol-Soluble Modulins in Formation of Staphylococcus aureus Biofilms in Synovial Fluid.

Staphylococcus aureus is a leading cause of prosthetic joint infections, which, as we recently showed, proceed with the involvement of biofilm-like clusters that cause recalcitrance to antibiotic treatment. Here we analyzed why these clusters grow extraordinarily large, reaching macroscopically visible extensions (>1 mm). We found that while specific S. aureus surface proteins are a prerequisite for agglomeration in synovial fluid, low activity of the Agr regulatory system and subsequent low production of the phenol-soluble modulin (PSM) surfactant peptides cause agglomerates to grow to exceptional dimensions. Our results indicate that PSMs function by disrupting interactions of biofilm matrix molecules, such as the polysaccharide intercellular adhesin (PIA), with the bacterial cell surface. Together, our findings support a two-step model of staphylococcal prosthetic joint infection: As we previously reported, interaction of S. aureus surface proteins with host matrix proteins such as fibrin initiates agglomeration; our present results show that, thereafter, the bacterial agglomerates grow to extremely large sizes owing to the lack of PSM expression under the specific conditions present in joints. Our findings provide a mechanistic explanation for the reported extreme resistance of joint infection to antibiotic treatment, lend support to the notions that Agr functionality and PSM production play a major role in defining different forms of S. aureus infection, and have important implications for antistaphylococcal therapeutic strategies.

Insight into structure-function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (PSM) α3 peptide.

Phenol-soluble modulins (PSMs) are a family of peptides with multiple functions in staphylococcal pathogenesis. To gain insight into the structural features affecting PSM functions, we analyzed an alanine substitution library of PSMα3, a strongly cytolytic and proinflammatory PSM of Staphylococcus aureus with a significant contribution to S. aureus virulence. Lysine residues were essential for both receptor-dependent proinflammatory and receptor-independent cytolytic activities. Both phenotypes also required additional structural features, with the C terminus being crucial for receptor activation. Biofilm formation was affected mostly by hydrophobic amino acid positions, suggesting that the capacity to disrupt hydrophobic interactions is responsible for the effect of PSMs on biofilm structure. Antimicrobial activity, absent from natural PSMα3, could be created by the exchange of large hydrophobic side chains, indicating that PSMα3 has evolved to exhibit cytolytic rather than antimicrobial activity. In addition to gaining insight into the structure-function relationship in PSMs, our study identifies nontoxic PSMα3 derivatives for active vaccination strategies and lays the foundation for future efforts aimed to understand the biological role of PSM recognition by innate host defense.

How Staphylococcus aureus biofilms develop their characteristic structure.

Biofilms cause significant problems in the environment and during the treatment of infections. However, the molecular mechanisms underlying biofilm formation are poorly understood. There is a particular lack of knowledge about biofilm maturation processes, such as biofilm structuring and detachment, which are deemed crucial for the maintenance of biofilm viability and the dissemination of cells from a biofilm. Here, we identify the phenol-soluble modulin (PSM) surfactant peptides as key biofilm structuring factors in the premier biofilm-forming pathogen Staphylococcus aureus. We provide evidence that all known PSM classes participate in structuring and detachment processes. Specifically, absence of PSMs in isogenic S. aureus psm deletion mutants led to strongly impaired formation of biofilm channels, abolishment of the characteristic waves of biofilm detachment and regrowth, and loss of control of biofilm expansion. In contrast, induced expression of psm loci in preformed biofilms promoted those processes. Furthermore, PSMs facilitated dissemination from an infected catheter in a mouse model of biofilm-associated infection. Moreover, formation of the biofilm structure was linked to strongly variable, quorum sensing-controlled PSM expression in biofilm microenvironments, whereas overall PSM production remained constant to ascertain biofilm homeostasis. Our study describes a mechanism of biofilm structuring in molecular detail, and the general principle (i.e., quorum-sensing controlled expression of surfactants) seems to be conserved in several bacteria, despite the divergence of the respective biofilm-structuring surfactants. These findings provide a deeper understanding of biofilm development processes, which represents an important basis for strategies to interfere with biofilm formation in the environment and human disease.

Oxacillin alters the toxin expression profile of community-associated methicillin-resistant Staphylococcus aureus.

The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a growing cause for concern. These strains are more virulent than health care-associated MRSA (HA-MRSA) due to higher levels of toxin expression. In a previous study, we showed that the high-level expression of PBP2a, the alternative penicillin binding protein encoded by the mecA gene on type II staphylococcal cassette chromosome mec (SCCmec) elements, reduced toxicity by interfering with the Agr quorum sensing system. This was not seen in strains carrying the CA-MRSA-associated type IV SCCmec element. These strains express significantly lower levels of PBP2a than the other MRSA type, which may explain their relatively high toxicity. We hypothesized that as oxacillin is known to increase mecA expression levels, it may be possible to attenuate the toxicity of CA-MRSA by using this antibiotic. Subinhibitory oxacillin concentrations induced PBP2a expression, repressed Agr activity, and, as a consequence, decreased phenol-soluble modulin (PSM) secretion by CA-MRSA strains. However, consistent with other studies, oxacillin also increased the expression levels of alpha-toxin and Panton-Valentine leucocidin (PVL). The net effect of these changes on the ability to lyse diverse cell types was tested, and we found that where the PSMs and alpha-toxin are important, oxacillin reduced overall lytic activity, but where PVL is important, it increased lytic activity, demonstrating the pleiotropic effect of oxacillin on toxin expression by CA-MRSA.

Genome-wide analysis of the regulatory function mediated by the small regulatory psm-mec RNA of methicillin-resistant Staphylococcus aureus.

Several methicillin resistance (SCCmec) clusters characteristic of hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains harbor the psm-mec locus. In addition to encoding the cytolysin, phenol-soluble modulin (PSM)-mec, this locus has been attributed gene regulatory functions. Here we employed genome-wide transcriptional profiling to define the regulatory function of the psm-mec locus. The immune evasion factor protein A emerged as the primary conserved and strongly regulated target of psm-mec, an effect we show is mediated by the psm-mec RNA. Furthermore, the psm-mec locus exerted regulatory effects that were more moderate in extent. For example, expression of PSM-mec limited expression of mecA, thereby decreasing methicillin resistance. Our study shows that the psm-mec locus has a rare dual regulatory RNA and encoded cytolysin function. Furthermore, our findings reveal a specific mechanism underscoring the recently emerging concept that S. aureus strains balance pronounced virulence and high expression of antibiotic resistance.

Genome-based cryptic gene discovery and functional identification of NRPS siderophore peptide in Streptomyces peucetius.

Identification of secondary metabolites produced by cryptic gene in bacteria may be difficult, but in the case of nonribosomal peptide (NRP)-type secondary metabolites, this study can be facilitated by bioinformatic analysis of the biosynthetic gene cluster and tandem mass spectrometry analysis. To illustrate this concept, we used mass spectrometry-guided bioinformatic analysis of genomic sequences to identify an NRP-type secondary metabolite from Streptomyces peucetius ATCC 27952. Five putative NRPS biosynthetic gene clusters were identified in the S. peucetius genome by DNA sequence analysis. Of these, the sp970 gene cluster encoded a complete NRPS domain structure, viz., C-A-T-C-A-T-E-C-A-T-C-A-T-C domains. Tandem mass spectrometry revealed that the functional siderophore peptide produced by this cluster had a molecular weight of 644.4 Da. Further analysis demonstrated that the siderophore peptide has a cyclic structure and an amino acid composition of AchfOrn-Arg-hOrn-hfOrn. The discovery of functional cryptic genes by analysis of the secretome, especially of NRP-type secondary metabolites, using mass spectrometry together with genome mining may contribute significantly to the development of pharmaceuticals such as hybrid antibiotics.

New meroterpenoids from a soil-derived fungus Penicillium sp. SSW03M2 GY and their anti-virulence activity.

Two new berkeley meroterpenoids (1 and 2), along with seven known compounds (3‒9) were isolated from a fungus, Penicillium sp. SSW03M2 GY derived from a sediment at Seosan bay, South Korea. Chemical structures of the isolated compounds were elucidated on the basis of 1D, 2D NMR, HRESIMS, and optical rotation. All the isolated compounds, 1 showed anti-virulence activity by significantly inhibiting α-toxin (Hla) secreted by methicillin-resistant Staphylococcus aureus without its growth inhibition.

A Study on the Epidemiological-Molecular Role of Staphylococcus aureus Strains in the Development of Ventilator-Associated Pneumonia in a Tertiary Hospital in Brazil.

This study aimed to explore the molecular epidemiology of Staphylococcus aureus isolated from patients on mechanical ventilation and the participation of virulence factors in the development of ventilator-associated pneumonia (VAP). A prospective cohort study was conducted on patients under mechanical ventilation, with periodic visits for the collection of tracheal aspirates and clinical data. The S. aureus isolates were analyzed regarding resistance profile, virulence, expression of protein A and alpha-toxin using Western blot, clonal profile using PFGE, sequence type using MLST, and characterization and quantification of phenol-soluble modulins. Among the 270 patients in the study, 51 S. aureus strains were isolated from 47 patients. The incidence density of S. aureus and MRSA VAP was 2.35/1000 and 1.96/1000 ventilator days, respectively; of these, 45% (n = 5) were resistant to oxacillin, with 100% (n = 5) harboring SCCmec types II and IV. The most frequent among the tested virulence factors were icaA, hla, and hld. The clonal profile showed a predominance of sequence types originating from the community. Risk factors for VAP were the presence of solid tumors and the sea gene. In conclusion, patient-related risk factors, together with microbiological factors, are involved in the development of S. aureus VAP, which is caused by the patient's own strains.

Antimicrobial activity of community-associated methicillin-resistant Staphylococcus aureus is caused by phenol-soluble modulin derivatives.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are causing an ongoing pandemic of mostly skin and soft tissue infections. The success of CA-MRSA as pathogens is due to a combination of antibiotic resistance with high virulence. In addition, it has been speculated that CA-MRSA strains such as the epidemic U.S. clone USA300 have increased capacity to colonize human epithelia, owing to bacteriocin-based bacterial interference. We here analyzed the molecular basis of antimicrobial activity detected in S. aureus strains, including those of the USA300 lineage. In contrast to a previous hypothesis, we found that this activity is not due to expression of a lantibiotic-type bacteriocin, but proteolytically processed derivatives of the phenol-soluble modulin (PSM) peptides PSMα1 and PSMα2. Notably, processed PSMα1 and PSMα2 exhibited considerable activity against Streptococcus pyogenes, indicating a role of PSMs in the interference of S. aureus strains with the competing colonizing pathogen. Furthermore, by offering a competitive advantage during colonization of the human body, the characteristically high production of PSMs in USA300 and other CA-MRSA strains may thus contribute not only to virulence but also the exceptional capacity of those strains to sustainably spread in the population, which so far has remained poorly understood.

Neutrophil responses to staphylococcal pathogens and commensals via the formyl peptide receptor 2 relates to phenol-soluble modulin release and virulence.

The mechanisms used by the immune system to discriminate between pathogenic and commensal bacteria have remained largely unclear. Recently, we have shown that virulence of Staphylococcus aureus depends on secretion of phenol-soluble modulin (PSM) peptides that disrupt neutrophils at micromolar concentrations. Moreover, all S. aureus PSMs stimulate and attract neutrophils at nanomolar concentrations via interaction with the formyl-peptide receptor 2 (FPR2). Here, we demonstrate that FPR2 allows neutrophils to adjust their responses in relation to the aggressiveness of staphylococcal species, which differ largely in their capacity to infect or colonize humans and animals. PSM-related peptides were detected in all human and animal pathogenic staphylococci, but were absent from most commensal species. Three PSMß-like peptides produced by the serious human pathogen Staphylococcus lugdunensis were identified as the previously described S. lugdunensis-synergistic hemolysins (SLUSHs). SLUSHs attracted and stimulated human leukocytes in a FPR2-dependent manner, indicating that FPR2 is a general receptor for all PSM-like peptide toxins. Remarkably, the release of PSMs correlated closely with the apparent capacity of staphylococcal species to cause invasive infections and with their ability to activate FPR2. These findings suggest that the innate immune system may be able to respond in different ways to pathogenic or innocuous staphylococci by monitoring the presence of PSMs via FPR2.