S. aureus haemolysin A‐induced IL‐8 and IL‐6 release from human airway epithelial cells is mediated by activation of p38‐ and Erk‐MAP kinases and additional,cell type‐specific signalling mechanisms

Soluble virulence‐associated factors of Staphylococcus aureus like haemolysin A (Hla) induce secretion of chemo/cytokines from airway epithelial cells. To elucidate the potential roles of specific signalling pathways in this response, we treated 16HBE14o‐, S9 or A549 cells with recombinant Hla (rHla). In a dose‐dependent manner, rHla induced secretion of IL‐8 in all three cell types, but IL‐6 release only in 16HBE14o‐ and S9 cells. rHla‐mediated secretion of IL‐8 and IL‐6 was suppressed by pre‐incubation of cells with inhibitors of Erk type or p38 MAP kinases, indicating that activation of these signalling pathways is essential for IL‐8 release in all three cell types and for IL‐6 release in 16HBE14o‐ and S9 cells. The rHla‐mediated phosphorylation and activation of p38 MAP kinase seem to depend on elevations in [Ca²⁺]_i, an early response in rHla‐treated cells. Inhibitors of calmodulin or calcium/calmodulin‐dependent kinase II attenuated rHla‐mediated release of IL‐8 in 16HBE14o‐ and A549 cells and of IL‐6 in 16HBE14o‐ cells. This indicates that rHla may mediate simultaneous activation of calmodulin‐dependent processes as additional prerequisites for chemo/cytokine secretion.However, the inhibitors of calmodulin‐dependent signalling did not affect rHla‐induced p38 MAP kinase phosphorylation, indicating that this pathway works in parallel with p38 MAP kinase.     

Staphylococcus aureus promotes autophagy by decreasing intracellular cAMP levels

Staphylococcus aureus is an intracellular bacterium responsible for serious infectious processes. This pathogen escapes from the phagolysosomal pathway into the cytoplasm, a strategy that allows intracellular bacterial replication and survival with the consequent killing of the eukaryotic host cell and spreading of the infection. S. aureus is able to secrete several virulence factors such as enzymes and toxins. Our recent findings indicate that the main virulence factor of S. aureus, the pore-forming toxin α-hemolysin (Hla), is the secreted factor responsible for the activation of an alternative autophagic pathway. We have demonstrated that this noncanonical autophagic response is inhibited by artificially elevating the intracellular levels of cAMP. This effect is mediated by RAPGEF3/EPAC (Rap guanine nucleotide exchange factor (GEF)3/exchange protein activated by cAMP), a cAMP downstream effector that functions as a GEF for the small GTPase Rap. We have presented evidence that RAPGEF3 and RAP2B, through calpain activation, are the proteins involved in the regulation of Hla and S. aureus-induced autophagy. In addition, we have found that both, RAPGEF3 and RAP2B, are recruited to the S. aureus–containing phagosome. Of note, adding purified α-toxin or infecting the cells with S. aureus leads to a decrease in intracellular cAMP levels, which promotes autophagy induction, a response that favors pathogen intracellular survival, as previously demonstrated. We have identified some key signaling molecules involved in the autophagic response upon infection with a bacterial pathogen, which have important implications in understanding innate immune defense mechanisms.     

cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla) is the S. aureus–secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus–containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.     

Staphylococcal α-hemolysin does not induce cell damage in murine mast cells but it augments the degranulation induced by FcεRI cross-linking and ionomycin

Staphylococcal α-hemolysin (Hla) is a principal small β-barrel pore forming toxin. It targets a variety of mammalian cells including immune cells; however little is known about its effects on mast cells. In this study, we examined whether Hla affects the degranulation of mast cells. Although Hla bound to the surface of bone marrow-derived mast cells (BMMCs) and formed SDS-stable oligomers on the cells, Hla alone induced neither cytotoxicity nor obvious release of a granule enzyme, β-hexosaminidase. However, Hla more than doubled the releases of β-hexosaminidase from BMMCs induced by FcεRI cross-linking or treatment with ionomycin. The augmentation of the enzyme release by rHla was impaired in the presence of 130?mM of extracellular KCl. The mutants of Hla that lacked pore-formation did not augment the release of the enzyme. These findings demonstrate that Hla is able to enhance the degranulation of mast cells induced by FcεRI cross-linking and ionomycin, although it alone does not induce the degranulation, and the pore-formation of Hla followed by potassium efflux is involved in the augmentation. These findings propose a previously unrecognized role for Hla in S. aureus-associated allergic and inflammatory processes via augmentation of mast cell responses.     

The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions

Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16⁻ and ATG9⁻/16⁻ cells and compare them to the previously reported ATG9⁻ mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9⁻ and ATG16⁻ cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9⁻/16⁻ double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9⁻/16⁻ cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9⁻/16⁻ cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.     

A protocol for the investigation of the intracellular Staphylococcus aureus metabolome

Systems biology studies assume the acquisition of reliable and reproducible data sets. Metabolomics, in particular, requires comprehensive evaluated workflows to enable the analysis of hundreds of different compounds. Therefore, a protocol to elucidate the metabolome of the gram-positive pathogen, Staphylococcus aureus COL strain, grown in a chemically defined medium is introduced here. Different standard operating procedures in the field of metabolome experiments were tested for common pitfalls. These included suitable and fast sampling processes, efficient metabolite extraction, quenching effectiveness (energy charge), and estimation of leakage and recovery of metabolites. Moreover, a cell disruption protocol for S. aureus was developed and optimized for metabolome analyses, for the express purpose of obtaining reproducible data. We used complementary methods (e.g., gas chromatography and/or liquid chromatography coupled with mass spectrometry) to detect the highly chemically diverse groups of metabolites for a global insight into the intracellular metabolism of S. aureus.     

Nasal Colonisation by Staphylococcus aureus Depends upon Clumping Factor B Binding to the Squamous Epithelial Cell Envelope Protein Loricrin

Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the “dock, lock and latch” mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB⁺ S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor^−/−) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB⁻ mutant colonised wild-type mice less efficiently than the parental ClfB⁺ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB⁻ mutant in the Lor^−/− mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor^−/− mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by S. aureus.     

Staphylococcus aureus promotes autophagy by decreasing intracellular cAMP levels

Staphylococcus aureus is an intracellular bacterium responsible for serious infectious processes. This pathogen escapes from the phagolysosomal pathway into the cytoplasm, a strategy that allows intracellular bacterial replication and survival with the consequent killing of the eukaryotic host cell and spreading of the infection. S. aureus is able to secrete several virulence factors such as enzymes and toxins. Our recent findings indicate that the main virulence factor of S. aureus, the pore-forming toxin α-hemolysin (Hla), is the secreted factor responsible for the activation of an alternative autophagic pathway. We have demonstrated that this noncanonical autophagic response is inhibited by artificially elevating the intracellular levels of cAMP. This effect is mediated by RAPGEF3/EPAC (Rap guanine nucleotide exchange factor (GEF)3/exchange protein activated by cAMP), a cAMP downstream effector that functions as a GEF for the small GTPase Rap. We have presented evidence that RAPGEF3 and RAP2B, through calpain activation, are the proteins involved in the regulation of Hla and S. aureus-induced autophagy. In addition, we have found that both, RAPGEF3 and RAP2B, are recruited to the S. aureus–containing phagosome. Of note, adding purified α-toxin or infecting the cells with S. aureus leads to a decrease in intracellular cAMP levels, which promotes autophagy induction, a response that favors pathogen intracellular survival, as previously demonstrated. We have identified some key signaling molecules involved in the autophagic response upon infection with a bacterial pathogen, which have important implications in understanding innate immune defense mechanisms.     

Size-Dependent Antimicrobial Effects of Novel Palladium Nanoparticles

Investigating the interactions between nanoscale materials and microorganisms is crucial to provide a comprehensive, proactive understanding of nanomaterial toxicity and explore the potential for novel applications. It is well known that nanomaterial behavior is governed by the size and composition of the particles, though the effects of small differences in size toward biological cells have not been well investigated. Palladium nanoparticles (Pd NPs) have gained significant interest as catalysts for important carbon-carbon and carbon-heteroatom reactions and are increasingly used in the chemical industry, however, few other applications of Pd NPs have been investigated. In the present study, we examined the antimicrobial capacity of Pd NPs, which provides both an indication of their usefulness as target antimicrobial compounds, as well as their potency as potential environmental pollutants. We synthesized Pd NPs of three different well-constrained sizes, 2.0±0.1 nm, 2.5±0.2 nm and 3.1±0.2 nm. We examined the inhibitory effects of the Pd NPs and Pd²⁺ ions toward gram negative Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) bacterial cultures throughout a 24 hour period. Inhibitory growth effects of six concentrations of Pd NPs and Pd²⁺ ions (2.5×10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, and 10⁻⁹ M) were examined. Our results indicate that Pd NPs are generally much more inhibitory toward S. aureus than toward E. coli, though all sizes are toxic at ≥10⁻⁵ M to both organisms. We observed a significant difference in size-dependence of antimicrobial activity, which differed based on the microorganism tested. Our work shows that Pd NPs are highly antimicrobial, and that fine-scale (<1 nm) differences in size can alter antimicrobial activity.     

Design,synthesis, biological evaluation and molecular docking studies of novel pleuromutilin derivatives containing nitrogen heterocycle and alkylamine groups

A series of pleuromutilin derivatives containing alkylamine and nitrogen heterocycle groups were designed and synthesised under mild conditions. The in vitro antibacterial activity of these semisynthetic derivatives against four strains of Staphylococcus aureus (MRSA ATCC 43300, S.aureus ATCC 29213, S.aureus AD3, and S.aureus 144) were evaluated by the broth dilution method. Compound 13 was found to have excellent antibacterial activity against MRSA (MIC = 0.0625 μg/mL). Furthermore, compound 13 was further studied by the time-killing kinetics and the post-antibiotic effect approach. In the mouse thigh infection model, compound 13 exhibited superior antibacterial efficacy than that of tiamulin. Meanwhile, compound 13 showed a lower inhibitory effect than that of tiamulin on RAW264.7 and 16HBE cells at the concentration of 10 μg/mL. Molecular docking study revealed that compound 13 can effectively bind to the active site of the 50S ribosome (the binding free energy = −9.66 kcal/mol).     

Staphylococcus aureus α-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles

Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla) to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.     

Evaluation of the Protective Immunity of a Novel Subunit Fusion Vaccine in a Murine Model of Systemic MRSA Infection

Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB) is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla) is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on IsdB and Hla. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla). Based on the results presented here, the chimeric bivalent vaccine affords higher levels of protection against S. aureus and has potential as a more effective candidate vaccine.     

Human anti-peptidoglycan-IgG-mediated opsonophagocytosis is controlled by calcium mobilization in phorbol myristate acetate-treated U937 cells

Recently, we demonstrated that human serum amyloid P component (SAP) specifically recognizes exposed bacterial peptidoglycan (PGN) of wall teichoic acid (WTA)-deficient Staphylococcus aureus ΔtagO mutant cells and then induces complement-independent phagocytosis. In our preliminary experiments, we found the existence of human serum immunoglobulins that recognize S. aureus PGN (anti-PGNIgGs), which may be involved in complement-dependent opsonophagocytosis against infected S. aureus cells. We assumed that purified serum anti-PGN-IgGs and S. aureus ΔtagO mutant cells are good tools to study the molecular mechanism of anti-PGN-IgG-mediated phagocytosis. Therefore, we tried to identify the intracellular molecule(s) that is involved in the anti-PGN-IgG-mediated phagocytosis using purified human serum anti-PGN-IgGs and different S. aureus mutant cells. Here, we show that anti-PGN-IgG-mediated phagocytosis in phorbol myristate acetate-treated U937 cells is mediated by Ca²⁺ release from intracellular Ca²⁺ stores and anti-PGN-IgGdependent Ca²⁺ mobilization is controlled via a phospholipase Cγ-2-mediated pathway. [BMB Reports 2015; 48(1): 36-41]     

Bioenergetic Mechanism for Nisin Resistance, Induced by the Acid Tolerance Response of Listeria monocytogenes

This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR⁺) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR⁺ cells had lower transmembrane pH (ΔpH) and electric potential (Δψ) than the control (ATR⁻) cells. The decreased proton motive force (PMF) of ATR⁺ cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR⁺ cells were ~7-fold higher than those in ATR⁻ cells. This suggested a role for the F_oF₁ ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F_oF₁ ATPase enzyme complex by N′-N′-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR⁻ but not in ATR⁺ cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR⁺ listeriae, the downregulation of the proton-translocating c subunit of the F_oF₁ ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F_oF₁ ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.     

Rotating Magnetic Field-Assisted Reactor Enhances Mechanisms of Phage Adsorption on Bacterial Cell Surface

Growing interest in bacteriophage research and use, especially as an alternative treatment option for multidrug-resistant bacterial infection, requires rapid development of production methods and strengthening of bacteriophage activities. Bacteriophage adsorption to host cells initiates the process of infection. The rotating magnetic field (RMF) is a promising biotechnological method for process intensification, especially for the intensification of micromixing and mass transfer. This study evaluates the use of RMF to enhance the infection process by influencing bacteriophage adsorption rate. The RMF exposition decreased the t₅₀ and t₇₅ of bacteriophages T4 on Escherichia coli cells and vb_SauM_A phages on Staphylococcus aureus cells. The T4 phage adsorption rate increased from 3.13 × 10⁻⁹ mL × min⁻¹ to 1.64 × 10⁻⁸ mL × min⁻¹. The adsorption rate of vb_SauM_A phages exposed to RMF increased from 4.94 × 10⁻⁹ mL × min⁻¹ to 7.34 × 10⁻⁹ mL × min⁻¹. Additionally, the phage T4 zeta potential changed under RMF from −11.1 ± 0.49 mV to −7.66 ± 0.29 for unexposed and RMF-exposed bacteriophages, respectively.     

Upregulation of ABC transporters contributes to chemoresistance of sphingosine 1-phosphate lyase-deficient fibroblasts

Sphingosine 1-phosphate (S1P) is an extra- and intracellular mediator that regulates cell growth, survival, migration, and adhesion in many cell types. S1P lyase is the enzyme that irreversibly cleaves S1P and thereby constitutes the ultimate step in sphingolipid catabolism. It has been reported previously that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1^−/−-MEFs) are resistant to chemotherapy-induced apoptosis through upregulation of B cell lymphoma 2 (Bcl-2) and Bcl-2-like 1 (Bcl-xL). Here, we demonstrate that the transporter proteins Abcc1/MRP1, Abcb1/MDR1, Abca1, and spinster-2 are upregulated in Sgpl1^−/−-MEFs. Furthermore, the cells efficiently sequestered the substrates of Abcc1 and Abcb1, fluo-4 and doxorubicin, in subcellular compartments. In line with this, Abcb1 was localized mainly at intracellular vesicular structures. After 16 h of incubation, wild-type MEFs had small apoptotic nuclei containing doxorubicin, whereas the nuclei of Sgpl1^−/−-MEFs appeared unchanged and free of doxorubicin. A combined treatment with the inhibitors of Abcb1 and Abcc1, zosuquidar and MK571, respectively, reversed the compartmentalization of doxorubicin and rendered the cells sensitive to doxorubicin-induced apoptosis. It is concluded that upregulation of multidrug resistance transporters contributes to the chemoresistance of S1P lyase-deficient MEFs.     

Phenotypic Variants of Staphylococci and Their Underlying Population Distributions Following Exposure to Stress

This study investigated whether alterations in environmental conditions would induce the formation of small colony variant phenotypes (SCV) with associated changes in cell morphology and ultra-structure in S. aureus, s. epidermidis, and S. lugdunensis. Wild-type clinical isolates were exposed to low temperature (4°C), antibiotic stress (penicillin G and vancomycin; 0-10,000 µg mL^-1), pH stress (pH 3-9) and osmotic challenge (NaCl concentrations of 0-20%). Changes in cell diameter, cell-wall thickness, and population distribution changes (n ≥ 300) were assessed via scanning and transmission electron microscopy (SEM and TEM), and compared to control populations. Our analyses found that prolonged exposure to all treatments resulted in the subsequent formation of SCV phenotypes. Observed SCVs manifested as minute colonies with reduced haemolysis and pigmentation (NaCl, pH and 4°C treatments), or complete lack thereof (antibiotic treatments). SEM comparison analyses revealed significantly smaller cell sizes for SCV populations except in S. aureus and S. epidermidis 10% NaCl, and S. epidermidis 4°C (p<0.05). Shifts in population distribution patterns were also observed with distinct sub-populations of smaller cells appearing for S. epidermidis, and S. lugdunensis. TEM analyses revealed significantly thicker cell-walls in all treatments and species except S. lugdunensis exposed to 4°C. These findings suggest that staphylococci adapted to environmental stresses by altering their cell size and wall thickness which could represent the formation of altered phenotypes which facilitate survival under harsh conditions. The phenotypic response was governed by the type of prevailing environmental stress regime leading to appropriate alterations in ultra-structure and size, suggesting downstream changes in gene expression, the proteome, and metabolome.