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.

Bacterial virulence plays a crucial role in MRSA sepsis.

Bacterial sepsis is a major global cause of death. However, the pathophysiology of sepsis has remained poorly understood. In industrialized nations, Staphylococcus aureus represents the pathogen most commonly associated with mortality due to sepsis. Because of the alarming spread of antibiotic resistance, anti-virulence strategies are often proposed to treat staphylococcal sepsis. However, we do not yet completely understand if and how bacterial virulence contributes to sepsis, which is vital for a thorough assessment of such strategies. We here examined the role of virulence and quorum-sensing regulation in mouse and rabbit models of sepsis caused by methicillin-resistant S. aureus (MRSA). We determined that leukopenia was a predictor of disease outcome during an early critical stage of sepsis. Furthermore, in device-associated infection as the most frequent type of staphylococcal blood infection, quorum-sensing deficiency resulted in significantly higher mortality. Our findings give important guidance regarding anti-virulence drug development strategies for the treatment of staphylococcal sepsis. Moreover, they considerably add to our understanding of how bacterial sepsis develops by revealing a critical early stage of infection during which the battle between bacteria and leukocytes determines sepsis outcome. While sepsis has traditionally been attributed mainly to host factors, our study highlights a key role of the invading pathogen and its virulence mechanisms.

Virulence Mechanisms of Staphylococcal Animal Pathogens.

Staphylococci are major causes of infections in mammals. Mammals are colonized by diverse staphylococcal species, often with moderate to strong host specificity, and colonization is a common source of infection. Staphylococcal infections of animals not only are of major importance for animal well-being but have considerable economic consequences, such as in the case of staphylococcal mastitis, which costs billions of dollars annually. Furthermore, pet animals can be temporary carriers of strains infectious to humans. Moreover, antimicrobial resistance is a great concern in livestock infections, as there is considerable antibiotic overuse, and resistant strains can be transferred to humans. With the number of working antibiotics continuously becoming smaller due to the concomitant spread of resistant strains, alternative approaches, such as anti-virulence, are increasingly being investigated to treat staphylococcal infections. For this, understanding the virulence mechanisms of animal staphylococcal pathogens is crucial. While many virulence factors have similar functions in humans as animals, there are increasingly frequent reports of host-specific virulence factors and mechanisms. Furthermore, we are only beginning to understand virulence mechanisms in animal-specific staphylococcal pathogens. This review gives an overview of animal infections caused by staphylococci and our knowledge about the virulence mechanisms involved.

Contribution of Staphylococcal Enterotoxin B to Staphylococcus aureus Systemic Infection.

Staphylococcal enterotoxin B (SEB), which is produced by the major human pathogen, Staphylococcus aureus, represents a powerful superantigenic toxin and is considered a bioweapon. However, the contribution of SEB to S. aureus pathogenesis has never been directly demonstrated with genetically defined mutants in clinically relevant strains. Many isolates of the predominant Asian community-associated methicillin-resistant S. aureus lineage sequence type (ST) 59 harbor seb, implying a significant role of SEB in the observed hypervirulence of this lineage. We created an isogenic seb mutant in a representative ST59 isolate and assessed its virulence potential in mouse infection models. We detected a significant contribution of seb to systemic ST59 infection that was associated with a cytokine storm. Our results directly demonstrate that seb contributes to S. aureus pathogenesis, suggesting the value of including SEB as a target in multipronged antistaphylococcal drug development strategies. Furthermore, they indicate that seb contributes to fatal exacerbation of community-associated methicillin-resistant S. aureus infection.

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.

Staphylococcus δ-toxin induces allergic skin disease by activating mast cells.

Atopic dermatitis is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca(2+)) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in Kit(W-sh/W-sh) mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

Antimicrobial Peptide Resistance Mechanism Contributes to Staphylococcus aureus Infection.

Antimicrobial peptides (AMPs) constitute an important part of innate host defense. Possibly limiting the therapeutic potential of AMPs is the fact that bacteria have developed AMP resistance mechanisms during their co-evolution with humans. However, there is no direct evidence that AMP resistance per se is important during an infection. Here we show that the Staphylococcus aureus Pmt ABC transporter defends the bacteria from killing by important human AMPs and elimination by human neutrophils. By showing that Pmt contributes to virulence during skin infection in an AMP-dependent manner, we provide evidence that AMP resistance plays a key role in bacterial infection.

Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus.

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-ß1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

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.

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.

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.

Methicillin Resistance Elements in the Canine Pathogen Staphylococcus pseudintermedius and Their Association with the Peptide Toxin PSM-mec.

Staphylococcus pseudintermedius is a frequent cause of infections in dogs. Infectious isolates of this coagulase-positive staphylococcal species are often methicillin- and multidrug-resistant, which complicates therapy. In staphylococci, methicillin resistance is encoded by determinants found on mobile genetic elements called Staphylococcal Chromosome Cassette mec (SCCmec), which, in addition to methicillin resistance factors, sometimes encode additional genes, such as further resistance factors and, rarely, virulence determinants. In this study, we analyzed SCCmec in a collection of infectious methicillin-resistant S. pseudintermedius (MRSP) isolates from predominant lineages in the United States. We found that several lineages characteristically have specific types of SCCmec elements and Agr types and harbor additional factors in their SCCmec elements that may promote virulence or affect DNA uptake. All isolates had SCCmec-encoded restriction-modification (R-M) systems of types I or II, and sequence types (STs) ST84 and ST64 had one type II and one type I R-M system, although the latter lacked a complete methylation enzyme gene. ST68 isolates also had an SCCmec-encoded CRISPR system. ST71 isolates had a psm-mec gene, which, in all but apparently Agr-dysfunctional isolates, produced a PSM-mec peptide toxin, albeit at relatively small amounts. This study gives detailed insight into the composition of SCCmec elements in infectious isolates of S. pseudintermedius and lays the genetic foundation for further efforts directed at elucidating the contribution of identified accessory SCCmec factors in impacting SCCmec-encoded and thus methicillin resistance-associated virulence and resistance to DNA uptake in this leading canine pathogen.

Exacerbation of allergic rhinitis by the commensal bacterium Streptococcus salivarius.

Allergic rhinitis (AR)-commonly called hay fever-is a widespread condition that affects the quality of life of millions of people. The pathophysiology of AR remains incompletely understood. In particular, it is unclear whether members of the colonizing nasal microbiota contribute to AR. Here, using 16S ribosomal RNA sequencing, we show that the nasal microbiome of patients with AR (n = 55) shows distinct differences compared with that from healthy individuals (n = 105), including decreased heterogeneity and the increased abundance of one species, Streptococcus salivarius. Using ex vivo and in vivo models of AR, we demonstrate that this commensal bacterium contributes to AR development, promoting inflammatory cytokine release and morphological changes in the nasal epithelium that are characteristic of AR. Our data indicate that this is due to the ability of S. salivarius to adhere to the nasal epithelium under AR conditions. Our study indicates the potential of targeted antibacterial approaches for AR therapy.

Extensive re-modelling of the cell wall during the development of Staphylococcus aureus bacteraemia.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Extensive remodelling of the cell wall during the development of Staphylococcus aureus bacteraemia.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.