Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense, enhancing neutrophil killing of Staphylococcus aureus

By sequestering manganese and zinc, the neutrophil protein calprotectin plays a crucial role in host defense against bacterial and fungal pathogens. However, the essential processes disrupted by calprotectin remain unknown. We report that calprotectin enhances the sensitivity of Staphylococcus aureus to superoxide through inhibition of manganese-dependent bacterial superoxide defenses, thereby increasing superoxide levels within the bacterial cell. Superoxide dismutase activity is required for full virulence in a systemic model of S.?aureus infection, and disruption of staphylococcal superoxide defenses by calprotectin augments the antimicrobial activity of neutrophils promoting in?vivo clearance. Calprotectin mutated in two transition metal binding sites and therefore defective in binding manganese and zinc does not inhibit microbial growth, unequivocally linking the antimicrobial properties of calprotectin to metal chelation. These results suggest that calprotectin contributes to host defense by rendering bacterial pathogens more sensitive to host immune effectors and reducing bacterial growth.     

IFN-gamma regulated chemokine production determines the outcome of Staphylococcus aureus infection

Immunomodulatory therapy represents an attractive approach in treating multidrug-resistant infections. Developing this therapy necessitates a lucid understanding of host defense mechanisms. Neutrophils represent the first line of systemic defense during Staphylococcus aureus infections. However, recent research suggests that survival of S. aureus inside neutrophils may actually contribute to pathogenesis, indicating that neutrophil trafficking to the infection site must be tightly regulated to ensure efficient microbial clearance. We demonstrate that neutrophil-regulating T cells are activated during S. aureus infection and produce cytokines that control the local neutrophil response. S. aureus capsular polysaccharide activates T cell production of IFN-gamma in a novel MHC class II-dependent mechanism. During S. aureus surgical wound infection, the presence of IFN-gamma at the infection site depends upon alphabetaTCR+ cells and functions to regulate CXC chemokine production and neutrophil recruitment in vivo. We note that the reduced neutrophil response seen in IFN-gamma-/- mice during S. aureus infection is associated with reduced tissue bacterial burden. CXC chemokine administration to the infection site resulted in an increased survival of viable S. aureus inside neutrophils isolated from the wound. These data demonstrate that T cell-derived IFN-gamma generates a neutrophil-rich environment that can potentiate S. aureus pathogenesis by facilitating bacterial survival within the neutrophil. These findings suggest avenues for novel immunomodulatory approaches to control S. aureus infections.     

Specificity for human hemoglobin enhances Staphylococcus aureus infection

Iron is required for bacterial proliferation, and Staphylococcus aureus steals this metal from host hemoglobin during invasive infections. This process involves hemoglobin binding to the cell wall of S.?aureus, heme extraction, passage through the cell envelope, and degradation to release free iron.?Herein, we demonstrate an enhanced ability of S.?aureus to bind hemoglobin derived from humans as compared to other mammals. Increased specificity for human hemoglobin (hHb) translates into an improved ability to acquire iron and is entirely dependent on the staphylococcal hemoglobin receptor IsdB. This feature affects host-pathogen interaction?as demonstrated by the increased susceptibility of?hHb-expressing mice to systemic staphylococcal?infection. Interestingly, enhanced utilization of human hemoglobin is not a uniform property of all bacterial pathogens. These results suggest a step in the evolution of S. aureus to better colonize the human host and establish hHb-expressing mice as a model of S. aureus pathogenesis.     

High phenotypic diversity in infecting but not in colonizing Staphylococcus aureus populations

In hostile environments diversity within a bacterial population may be beneficial for the fitness of the microbial community as a whole. Here we analysed the population diversity of Staphylococcus aureus in infecting and colonizing situations. In the study, performed independently in two German centres, the heterogeneity of the S. aureus population was determined by quantifying the occurrence of phenotypic variants (differences in haemolysis, pigmentation, colony morphology) in primary cultures from nose, oropharyngeal and sputum specimens from cystic fibrosis (CF) patients and in nose swabs from healthy S. aureus carriers. The proportion of heterogeneous samples, the number of clearly distinguishable isolates per sample and the qualitative differences between phenotypes was significantly higher in CF sputum specimens than in the other samples. The heterogeneity of the S. aureus population could be correlated with high bacterial densities in the sputum samples. In patients co-infected with Pseudomonas aeruginosa lower S. aureus bacterial loads and less heterogeneity in the S. aureus population were observed. Typing of all S. aureus isolates from heterogeneous samples by pulsed-field gel electrophoresis or spa typing revealed that the bacteria were polyclonal in 30%, monoclonal with minor genetic alterations in 25% or not distinguishable in 69% of the specimens. Some specimens harboured monoclonal and polyclonal variants simultaneously. Importantly, differences in antibiotic susceptibility were detected in phenotypic S. aureus variants within a single specimen. Diversification of a S. aureus population is highly favoured during chronic CF lung infection, supporting the general hypothesis that maintenance of intrahost diversity can be of adaptive value, increasing the fitness of the bacterial community.     

Environment-mediated accumulation of diacyl lipoproteins over their triacyl counterparts in Staphylococcus aureus

Bacterial lipoproteins are believed to exist in only one specific lipid-modified structure, such as the diacyl form or the triacyl form, in each bacterium. In the case of Staphylococcus aureus, recent extensive matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis revealed that S. aureus lipoproteins exist in the α-aminoacylated triacyl form. Here, we discovered conditions that induce the accumulation of diacyl lipoproteins that lack α-aminoacylation in S. aureus. The accumulation of diacyl lipoproteins required a combination of conditions, including acidic pH and a post-logarithmic-growth phase. High temperatures and high salt concentrations additively accelerated the accumulation of the diacyl lipoprotein form. Following a post-logarithmic-growth phase where S. aureus MW2 cells were grown at pH 6, SitC lipoprotein was found almost exclusively in its diacyl structure rather than in its triacyl structure. This is the first report showing that the environment mediates lipid-modified structural alterations of bacterial lipoproteins.     

Immunomagnetic separation and MS/SPR end-detection combined procedure for rapid detection of Staphylococcus aureus and protein A

The aim of this study was to establish an IMS-MS/SPR technique for the detection of Staphylococcus aureus (S. aureus) and Staphylococcus protein A (SPA) at the same time, which consists of isolating S. aureus and trapping-enrichmenting its SPA by IMS, and the end point is determined by using either MS or SPR measurements. Magnetic bead (MB) containing aldehyde group was synthesized with latex-polymerization and immunomagnetic bead (IMB) was fabricated by modifying its surface with an oriented layer of human IgG in covalent linkage. As soon as sample of pulverator-treated bacterial cell lysate (10(8) cfu/mL) was incubated with IMB at 4 degrees C for 30 min, SPA was captured and separated from the mixed solution in a few minutes by the IMB and then detected with mass spectrometry after washing. SPR was used to detect S. aureus quantitatively in situ at the end-detection procedure. All in all, this technique can be employed to detect rapidly SPA and S. aureus within 2h and also be applied to detect other cells or their membrane proteins with changed modified antibodies.     

Whole-genome sequencing of staphylococcus haemolyticus uncovers the extreme plasticity of its genome and the evolution of human-colonizing staphylococcal species

Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of S.haemolyticus to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of S.haemolyticus, Staphylococcus aureus, and Staphylococcus epidermidis were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the "oriC environ," likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of S.haemolyticus, S.aureus, and S.epidermidis elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the S.haemolyticus chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.     

Antibiotic resistant Staphylococcus aureus: a paradigm of adaptive power

Nothing documents better the spectacular adaptive capacity of Staphylococcus aureus than the response of this important human and animal pathogen to the introduction of antimicrobial agents into the clinical environment. The effectiveness of penicillin introduced in the early 1940s was virtually annulled within a decade because of the plasmid epidemics that spread the ss-lactamase gene through the entire species of S. aureus. In 1960 within one to two years of the introduction of penicillinase resistant ss-lactams (methicillin), methicillin resistant S. aureus (MRSA) strains were identified in clinical specimens. By the 1980s, epidemic clones of MRSA acquired multidrug resistant traits and spread worldwide to become one of the most important causative agents of hospital acquired infections. In the early 2000s, MRSA strains carrying the Tn1546 transposon-based enterococcal vancomycin resistant mechanism were identified in clinical specimens, bringing the specter of a totally resistant bacterial pathogen closer to reality. Then, in the late 1990s, just as effective hygienic and antibiotic use policies managed to bring down the frequency of MRSA in hospitals of several countries, MRSA strains began to show up in the community.     

Efficacy of tetracycline encapsulated O-carboxymethyl chitosan nanoparticles against intracellular infections of Staphylococcus aureus

Intracellular bacterial infections are recurrent, persistent and are difficult to treat because of poor penetration and limited availability of antibiotics within macrophages and epithelial cells. We developed biocompatible, 200nm sized tetracycline encapsulated O-carboxymethyl chitosan nanoparticles (Tet-O-CMC Nps) via ionic gelation for its sustained delivery of Tet into cells. S. aureus binds and aggregates with Tet-O-CMC Nps increasing drug concentrations at the infection site. Tet-O-CMC Nps were sixfold more effective in killing intracellular S. aureus compared to Tet alone in HEK-293 and differentiated THP1 macrophage cells proving it to be an efficient nanomedicine to treat intracellular S. aureus infections.     

Host-pathogen interactions between the skin and Staphylococcus aureus

Staphylococcus aureus is responsible for the vast majority of bacterial skin infections in humans. The propensity for S. aureus to infect skin involves a balance between cutaneous immune defense mechanisms and virulence factors of the pathogen. The tissue architecture of the skin is different from other epithelia especially since it possesses a corneal layer, which is an important barrier that protects against the pathogenic microorganisms in the environment. The skin surface, epidermis, and dermis all contribute to host defense against S. aureus. Conversely, S. aureus utilizes various mechanisms to evade these host defenses to promote colonization and infection of the skin. This review will focus on host-pathogen interactions at the skin interface during the pathogenesis of S. aureus colonization and infection.     

Neutrophil-stimulating activity of staphylococci based on reactive chemiluminescence data

The neutrophil-stimulating properties of 38 S. aureus strains and 32 S. epidermidis strains were studied in the reaction of luminol-mediated chemiluminescence. All S. aureus strains and 29 S. epidermidis strains were found to possess neutrophil-stimulating activity, the mean activity index for S. aureus being significantly higher. The stimulating activity of the strains varied within a wide range (the variation coefficient was 120.0 +/- 21.9%) and did not correlate with the content of protein A in bacterial cells and the degree of their hydrophoby. The opsonization of staphylococci with normal human serum enhanced the neutrophil reaction 1.5- to 100-fold and simultaneously leveled out the chemiluminescence indices in experiments with different strains (the variation coefficient was 8.0 +/- 1.5%). The nature of the neutrophil-stimulating effect of staphylococci and its relationship to the exploratory reactions of phagocytes are discussed.     

Mapping of interactions between human macrophages and Staphylococcus aureus reveals an involvement of MAP kinase signaling in the host defense

Staphylococcus aureus is a dangerous opportunistic human pathogen that causes serious invasive diseases when it reaches the bloodstream. Recent studies have shown that S. aureus is highly resistant to killing by professional phagocytes and that such cells even provide a favorable environment for intracellular survival of S. aureus. Importantly, the reciprocal interactions between phagocytes and S. aureus have remained largely elusive. Here we have employed kinase profiling to define the nature and time resolution of the human THP-1 macrophage response toward S. aureus and proteomics to identify the response of S. aureus toward macrophages. The results of these studies reveal major macrophage signaling pathways triggered by S. aureus and proteomic signatures of the responses of S. aureus to macrophages. We also identify human proteins bound to S. aureus that have potential roles in bacterial killing and internalization. Most noticeably, our observations challenge the classical concept that macrophage responses are mainly mediated through Toll-like receptor 2 and NF-κB signaling and highlight the important role of the stress-activated MAP kinase signaling in orchestrating the host defense.     

Studies on the antibacterial activity of phanquone: chelating properties in relation to mode of action against Escherichia coli and Staphylococcus aureus

Phanquone is active against a wide range of Gram-positive and Gram-negative organisms. Its activity is affected by the nature of the suspending fluid, pH and anaerobic growth conditions. Its ability to chelate metal ions was examined and found to be related to its antibacterial activity, which was reduced by the presence of added metal ions, e.g. Co (II), Cu(II), Fe(II) and Fe(III) in nutrient media for both E. coli and S. aureus. When antibacterial activity was examined in dis-nutrient media for both E. coli and S. aureus. When antibacterial activity was examined in distilled water, then certain added metal ions, whilst antagonizing activity was examined in distilled water, then certain added metal ions, whilst antagonizing the activity of Phanquone against E. coli, exerted a co-operative effect in the case of S. aureus. The addition of EDTA and NTA lowered the activity of Phanquone against S. aureus, but not E. coli, while the addition of thiol-containing compounds lowered its activity against E. coli but not S. aureus. concentration quenching was observed for S. aureus but not for E. coli, while overnight pre-incubation at 4 degrees C resulted in the appearance of a growth zone inside the zone of inhibition in the case of S. aureus but not E. coli. Phanquone may have a different mode of action against the two organisms.     

Activation of syndecan-1 ectodomain shedding by Staphylococcus aureus alpha-toxin and beta-toxin

Exploitation of host components by microbes to promote their survival in the hostile host environment has been a recurring theme in recent years. Available data indicate that bacterial pathogens activate ectodomain shedding of host cell surface molecules to enhance their virulence. We reported previously that several major bacterial pathogens activate ectodomain shedding of syndecan-1, the major heparan sulfate proteoglycan of epithelial cells. Here we define the molecular basis of how Staphylococcus aureus activates syndecan-1 shedding. We screened mutant S. aureus strains devoid of various toxin and protease genes and found that only strains lacking both alpha-toxin and beta-toxin genes do not stimulate shedding. Mutations in the agr global regulatory locus, which positively regulates expression of alpha- and beta-toxins and other exoproteins, also abrogated the capacity to stimulate syndecan-1 shedding. Furthermore, purified S. aureus alpha- and beta-toxins, but not enterotoxin A and toxic shock syndrome toxin-1, rapidly potentiated shedding in a concentration-dependent manner. These results establish that S. aureus activates syndecan-1 ectodomain shedding via its two virulence factors, alpha- and beta-toxins. Toxin-activated shedding was also selectively inhibited by antagonists of the host cell shedding mechanism, indicating that alpha- and beta-toxins shed syndecan-1 ectodomains through stimulation of the host cell's shedding machinery. Interestingly, beta-toxin, but not alpha-toxin, also enhanced ectodomain shedding of syndecan-4 and heparin-binding epidermal growth factor. Because shedding of these ectodomains has been implicated in promoting bacterial pathogenesis, activation of ectodomain shedding by alpha-toxin and beta-toxin may be a previously unknown virulence mechanism of S. aureus.     

Attachment of Staphylococcus aureus to eukaryotic cells and experimental pitfalls in staphylococcal adherence assays: a critical appraisal.

Staphylococcus aureus is a bacterial species with pathogenic potential to both humans and animals. The primary natural niche is said to be the human vestibulum nasi from where bacterial cells may spread to the environment or additional anatomical sites such as the perineum or the hands, where residence is usually transient. Apparently, S. aureus is capable of a precise and balanced interaction with specific types of eukaryotic nasal cells. Although a wide variety of important bacterial ligands and possible eukaryote receptors have been described, the precise mechanisms leading to persistent bacterial colonization and, even more importantly, associated infection have not yet been elucidated in detail. This may be a consequence of the fact that most of the adherence factors have been studied individually in simplified in vitro systems, not taking the complexity of multi-factorial in vivo cell-cell interactions into account. An overall scheme of the initial and sequential interactions leading to S. aureus colonization of eukaryotic cell surfaces has not yet emerged. This review concisely describes the current state of affairs in the multi-disciplinary field of staphylococcal adherence research. Specific emphasis is placed upon the pros and cons of the various artificial, mostly in vitro models employed to study the interaction between bacterial and human or animal cells.     

CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses

We have developed a mouse brain abscess model by using Staphylococcus aureus, one of the main etiologic agents of brain abscesses in humans. Direct damage to the blood-brain barrier was observed from 24 h to 7 days after S. aureus exposure as demonstrated by the accumulation of serum IgG in the brain parenchyma. Evaluation of brain abscesses by immunohistochemistry and flow cytometry revealed a prominent neutrophil infiltrate. To address the importance of neutrophils in the early containment of S. aureus infection in the brain, mice were transiently depleted of neutrophils before implantation of bacteria-laden beads. Neutrophil-depleted animals consistently demonstrated more severe brain abscesses and higher CNS bacterial burdens compared with control animals. S. aureus led to the induction of numerous chemokines in the brain, including macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL1, monocyte chemoattractant protein-1/CCL2, and TCA-3/CCL1, within 6 h after bacterial exposure. These chemokines also were expressed by both primary cultures of neonatal mouse microglia and astrocytes exposed to heat-inactivated S. aureus in vitro. Because neutrophils constitute the majority of the cellular infiltrate in early brain abscess development, subsequent analysis focused on MIP-2 and KC/CXCL1, two neutrophil-attracting CXC chemokines. Both MIP-2 and KC protein levels were significantly elevated in the brain after S. aureus exposure. Neutrophil extravasation into the brain parenchyma was impaired in CXCR2 knockout mice and was associated with increased bacterial burdens. These studies demonstrate the importance of the CXCR2 ligands MIP-2 and KC and neutrophils in the acute host response to S. aureus in the brain.     

Molecular analysis of fusidic acid resistance in Staphylococcus aureus

Fusidic acid is a potent antibiotic against severe Gram-positive infections that interferes with the function of elongation factor G (EF-G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF-G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid-resistant clinical S. aureus strains as well as in 10 fusidic acid-resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF-G. To confirm the importance of observed amino acid exchanges in EF-G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF-G derivatives with the exchanges P406L, H457Y and L461K were constructed by site-directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid-susceptible S. aureus strain RN4220 caused a fusidic acid-resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid-resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF-G for the generation of fusidic acid resistance in S. aureus.     

An acute osteomyelitis model in traumatized rat tibiae involving sand as a foreign body, thermal injury, and bimicrobial contamination

The multfactorial nature of bone injuries in modern warfare and emergency trauma patients warrants enhancement of existing models. To develop a more appropriate model, rat tibiae (n = 195) were mechanically injured, divided into 2 groups (with or without thermal injury), and contaminated with a range of Staphylococcus aureus (Cowan 1) inocula. In some experiments, S. aureus inocula also contained Escherichia coli or foreign bodies (sand or soil). The primary outcome measure was the amount of S. aureus remaining in the tibia (tibial bacterial load) 24 h after contamination, reported as log10 cfu/g bone. S. aureus showed ID50 and ID95 values of 72 and 977 cfu, respectively. Values were lower than seen previously by using S. aureus strain SMH. S. aureus tibial bacterial loads were higher in tibiae with mechanical and thermal injury (log10 4.15 +/- 0.27 cfu/g) versus mechanical injury alone (log10 3.1 +/- 0.47 cfu/g, P = 0.028). The addition of E. coli to the S. aureus inoculum had no effect on tibial bacterial loads (S. aureus only, log10 4.24 +/- 0.92 cfu/g; S. aureus + E. coli, log10 4.1 +/- 1.0 cfu/g, P = 0.74). Sand, added as a foreign body, increased tibial bacterial load. Combined mechanical and thermal trauma of the tibia is associated with increased S. aureus tibial bacterial loads, increasing the risk of acute osteomyelitis. Understanding the interplay of mechanical and thermal injuries, bimicrobial contamination, and foreign bodies may improve our understanding of traumatic bone injuries and the pathogenesis of osteomyelitis.