Resistance to leukocytes ties benefits of quorum sensing dysfunctionality to biofilm infection.

Social interactions play an increasingly recognized key role in bacterial physiology1. One of the best studied is quorum sensing (QS), a mechanism by which bacteria sense and respond to the status of cell density2. While QS is generally deemed crucial for bacterial survival, QS-dysfunctional mutants frequently arise in in vitro culture. This has been explained by the fitness cost an individual mutant, a 'quorum cheater', saves at the expense of the community3. QS mutants are also often isolated from biofilm-associated infections, including cystic fibrosis lung infection4, as well as medical device infection and associated bacteraemia5-7. However, despite the frequently proposed use of QS blockers to control virulence8, the mechanisms underlying QS dysfunctionality during infection have remained poorly understood. Here, we show that in the major human pathogen Staphylococcus aureus, quorum cheaters arise exclusively in biofilm infection, while in non-biofilm-associated infection there is a high selective pressure to maintain QS control. We demonstrate that this infection-type dependence is due to QS-dysfunctional bacteria having a significant survival advantage in biofilm infection because they form dense and enlarged biofilms that provide resistance to phagocyte attacks. Our results link the benefit of QS-dysfunctional mutants in vivo to biofilm-mediated immune evasion, thus to mechanisms that are specific to the in vivo setting. Our findings explain why QS mutants are frequently isolated from biofilm-associated infections and provide guidance for the therapeutic application of QS blockers.

Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 µg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 µg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 µg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors.

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.

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.

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.

Distribution and regulation of the mobile genetic element-encoded phenol-soluble modulin PSM-mec in methicillin-resistant Staphylococcus aureus.

The phenol-soluble modulin PSM-mec is the only known staphylococcal toxin that is encoded on a mobile antibiotic resistance determinant, namely the staphylococcal cassette chromosome (SCC) element mec encoding resistance to methicillin. Here we show that the psm-mec gene is found frequently among methicillin-resistant Staphylococcus aureus (MRSA) strains of SCCmec types II, III, and VIII, and is a conserved part of the class A mec gene complex. Controlled expression of AgrA versus RNAIII in agr mutants of all 3 psm-mec-positive SCCmec types demonstrated that expression of psm-mec, which is highly variable, is controlled by AgrA in an RNAIII-independent manner. Furthermore, psm-mec isogenic deletion mutants showed only minor changes in PSMα peptide production and unchanged (or, as previously described, diminished) virulence compared to the corresponding wild-type strains in a mouse model of skin infection. This indicates that the recently reported regulatory impact of the psm-mec locus on MRSA virulence, which is opposite to that of the PSM-mec peptide and likely mediated by a regulatory RNA, is minor when analyzed in the original strain background. Our study gives new insight in the distribution, regulation, and role in virulence of the PSM-mec peptide and the psm-mec gene locus.

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.

Understanding the significance of Staphylococcus epidermidis bacteremia in babies and children.

PURPOSE OF REVIEW: This article provides an overview of Staphylococcus epidermidis bacteremia/sepsis and coagulase-negative staphylococci (CoNS) infections in neonates and children. RECENT FINDINGS: The incidence of S. epidermidis and CoNS sepsis in neonates is still very high and preventing and treating disease remains difficult. There has been recent progress in understanding the pathogenesis of S. epidermidis infection, interaction of S. epidermidis with host defenses, and risk factors for the development of S. epidermidis disease. For example, we have gained more insight into the development of biofilm-associated catheter infections, which are responsible for recurrent CoNS infections in hospitalized premature neonates and are especially difficult to treat owing to intrinsic resistance of biofilms to antibiotics. SUMMARY: Biofilm-associated catheter infections by S. epidermidis occur frequently in neonates and adults. S. epidermidis bloodstream infections are particularly problematic in neonates. Prophylaxis in the form of eradicating colonizing S. epidermidis may be a double-edged sword, as S. epidermidis colonization may be beneficial to the host. New drugs may arise from a better understanding of S. epidermidis virulence and analysis of risk factors may help identify neonates susceptible to bacterial sepsis. However, reducing morbidity should always begin by increasing hygiene in hospital settings to reduce the introduction of potentially harmful opportunistic pathogens such as S. epidermidis on indwelling medical devices or during surgery.

Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis.

A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration>1 microg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations<0.5 microg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 microg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 microg/ml, whereas a approximately 500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high beta-sheet content or a beta-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations.

Transcriptional responses of murine macrophages to the adenylate cyclase toxin of Bordetella pertussis.

Three different recombinant forms of CyaA were used to investigate transcriptional responses of murine bone marrow-derived macrophages (BMMs) using Affymetrix Mouse Genome GeneChips. These forms were enzymically active, invasive CyaA, non-enzymically active, invasive CyaA (CyaA*) and non-enzymically active, non-invasive CyaA (proCyaA*). BMMs, treated with 20 ng/ml of CyaA for 24h, showed over 1000 significant changes in gene transcription compared with control cells. CyaA caused an increase in transcription of many inflammatory genes and genes associated with various signalling cascades such as those involved in cyclic AMP-dependent protein kinase A signalling. Most strikingly, CyaA caused down-regulation of numerous genes involved in cell proliferation. CyaA* at 20 ng/ml significantly up-regulated the transcription of only twelve genes after 24h whereas proCyaA* at this concentration significantly increased the transcription of only two genes.