Staphylococcus epidermidis strategies to avoid killing by human neutrophils

Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus.     

Oxygen-dependent killing of Staphylococcus aureus by human neutrophils

Luminol-dependent chemiluminescence was used as a monitor of reactive oxidant generation during phagocytosis of Staphylococcus aureus by human neutrophils. Reactive oxidants play a crucial role in the killing of this organism because: (a) S. aureus was killed most rapidly when the rate of increase of chemiluminescence was greatest; (b) neutrophils which had been activated to generate reactive oxidants by re-aeration of anaerobic suspensions killed this bacterium more efficiently than control suspensions; and (c) neutrophils from a patient with chronic granulomatous disease could neither generate reactive oxidants nor kill S. aureus.     

Structural Insights into SraP-Mediated Staphylococcus aureus Adhesion to Host Cells

Staphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelets. It remains unclear how SraP interacts with human host. Here we report the 2.05 Å crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a β-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differ in calcium coordination pattern. Under the conditions tested, small-angle X-ray scattering and molecular dynamic simulation indicated that the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outwards. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated host-pathogen interaction of S. aureus.     

Gamma interferon enhances the killing of Staphylococcus aureus by human neutrophils

The effect of purified human interferon-gamma on the responsiveness of human neutrophils was investigated. Pre-incubation of neutrophils with 100 U interferon ml-1 for 10 min at 37 degrees C resulted in a 2.5-fold increase in N-formylmethionyl-leucyl-phenylalanine-stimulated reactive oxygen metabolite generation (as assayed by luminol-dependent chemiluminescence). Pre-treatment of neutrophils with interferon also potentiated their ability to kill Staphylococcus aureus, and thus it is proposed that this lymphokine may also enhance neutrophil function in vivo under certain pathological conditions.     

Structural insight into binding of Staphylococcus aureus to human fibronectin

Staphylococcus aureus possesses cell-wall attached proteins that bind the human protein fibronectin (Fn). An intermodule interface between the 4F1 and 5F1 modules in the N-terminal domain of Fn is maintained on bacterial peptide binding but there is a small change in the intermodule orientation and alignment of beta-strands that are predicted to bind the peptide. The module pair is elongated, as in the unbound state. Combined with evidence that residues in both 4F1 and 5F1 are directly involved in peptide binding, this observation supports the hypothesis that, when bound to intact Fn, the bacterial protein adopts an unusual, highly extended conformation.     

Survival of Staphylococcus aureus inside neutrophils contributes to infection

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar-) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.     

Structural Insights into the Anti-methicillin-resistant Staphylococcus aureus (MRSA) Activity of Ceftobiprole

Methicillin-resistant Staphylococcus aureus (MRSA) is an antibiotic-resistant strain of S. aureus afflicting hospitals and communities worldwide. Of greatest concern is its development of resistance to current last-line-of-defense antibiotics; new therapeutics are urgently needed to combat this pathogen. Ceftobiprole is a recently developed, latest generation cephalosporin and has been the first to show activity against MRSA by inhibiting essential peptidoglycan transpeptidases, including the β-lactam resistance determinant PBP2a, from MRSA. Here we present the structure of the complex of ceftobiprole bound to PBP2a. This structure provides the first look at the molecular details of an effective β-lactam-resistant PBP interaction, leading to new insights into the mechanism of ceftobiprole efficacy against MRSA.     

Evaluation of electron spin resonance for studies of superoxide anion production by human neutrophils interacting with Staphylococcus aureus and Staphylococcus epidermidis

The present study evaluates electron spin resonance (ESR) and the spin trapper 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) for analysis of superoxide radical production by human neutrophils interacting with viable Staphylococcus aureus and Staphylococcus epidermidis bacteria. To avoid auto-activation due to interaction with glass surfaces, neutrophils were preincubated in plastic tubes until the peak response was reached, and then transferred to a quartz flat cell to record the ESR spectra. The time point for peak response was identified by parallel analysis of the bacteria–neutrophil interaction using luminol amplified chemiluminescence. We found detectable ESR spectra from neutrophils interacting with as few as five bacteria of the weak activating S. epidermidis per neutrophil. Addition of the NADPH oxidase inhibitor diphenylene iodonium totally abolished spectra. Catalase, DMSO or an iron chelator had no impact on the produced spectra and ionomycin, a selective activator of intracellular NADPH oxidase, gave significant ESR spectra. Taken together, our results indicate that DEPMPO is cell permeable and detects NADPH oxidase derived superoxide anions formed in phagosomes or released by human neutrophils phagocytosing viable S. aureus and S. epidermidis. The technique may be used as a sensitive tool to evaluate superoxide anion production in human neutrophils.     

Conditioning adhesive contacts between the neutrophils and the endotheliocytes by Staphylococcus aureus

We have developed a model for evaluating the integral intercellular interactions in the “endotheliocyte‐neutrophil” system and have shown the high variability of adhesion contacts in different donors associated with different expression profiles of neutrophils. Two methods (forсe spectroscopy‐spectroscopy and scanning ion‐conductance microscopy) showed a decrease in the rigidity of the membrane‐cytoskeletal complex of neutrophils under the influence of Staphylococcus aureus 2879 M. Adding this strain to the “endotheliocyte‐neutrophil” system caused a statistically significant decrease in the adhesion force and adhesion work, which indicates a change in the expression profile and physicochemical properties of membranes of both types of interacting cells (neutrophils and endotheliocytes).     

Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils

Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils were shown to upregulate hypoxia-inducible factor (HIF)-1α-dependent gene expression under hypoxic incubation conditions (3 kPa), with a consequent substantial delay in the onset of apoptosis. Despite this, polarization and chemotactic responsiveness to IL-8 and fMLP were entirely unaffected by hypoxia. Similarly, hypoxia did not diminish the ability of neutrophils to phagocytose serum-opsonized heat-killed streptococci. Of the secretory functions examined, IL-8 generation was preserved and elastase release was enhanced by hypoxia. Hypoxia did, however, cause a major reduction in respiratory burst activity induced both by the soluble agonist fMLP and by ingestion of opsonized zymosan, without affecting expression of the NADPH oxidase subunits. Critically, this reduction in respiratory burst activity under hypoxia was associated with a significant defect in the killing of Staphylococcus aureus. In contrast, killing of Escherichia coli, which is predominantly oxidase independent, was fully preserved under hypoxia. In conclusion, these studies suggest that although the NADPH oxidase-dependent bacterial killing mechanism may be compromised by hypoxia, neutrophils overall appear extremely well adapted to operate successfully under severely hypoxic conditions.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

Insights into Nasal Carriage of Staphylococcus aureus in an Urban and a Rural Community in Ghana

The epidemiology of Staphylococcus aureus in the community in Ghana was never investigated prior to this study. The aims of the study were: i) to assess prevalence of nasal S. aureus carriage in Ghanaian people living in an urban and a rural area, and ii) to identify phenotypic and genotypic traits of strains isolated from the two communities. Nasal swabs were collected from healthy individuals living in an urban community situated in the suburb of the capital city, Accra (n = 353) and in a rural community situated in the Dangme-West district (n = 234). The overall prevalence of nasal carriage was 21% with a significantly higher prevalence in the urban (28%) than in the rural community (11%) (p<0.0001). The levels of antimicrobial resistance were generally low (<5%) except for penicillin (91%) and tetracycline (25%). The only two (0.3%) MRSA carriers were individuals living in the urban area and had been exposed to hospitals within the last 12 months prior to sampling. Resistance to tetracycline (p = 0.0009) and presence of Panton-Valentine leukocidin (PVL) gene (p = 0.02) were significantly higher among isolates from the rural community compared to isolates from the urban community. Eleven MLST clonal complexes (CC) were detected based on spa typing of the 124 S. aureus isolates from the two communities: CC8 (n = 36), CC152 (n = 21), CC45 (n = 21), CC15 (n = 18), CC121 (n = 6), CC97 (n = 6), CC30 (n = 5), CC5 (n = 5), CC508 (n = 4), CC9 (n = 1), and CC707 (n = 1). CC8 and CC45 were less frequent in the rural area than in the urban area (p = 0.02). These results reveal remarkable differences regarding carriage prevalence, tetracycline resistance, PVL content and clonal distribution of S. aureus in the two study populations. Future research may be required to establish whether such differences in nasal S. aureus carriage are linked to socio-economic differences between urban and rural communities in this African country.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system.

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo

Bloodstream infection with Staphylococcus aureus is common and can be fatal. However, virulence factors that contribute to lethality in S. aureus bloodstream infection are poorly defined. We discovered that LukED, a commonly overlooked leucotoxin, is critical for S. aureus bloodstream infection in mice. We also determined that LukED promotes S. aureus replication in vivo by directly killing phagocytes recruited to sites of haematogenously seeded tissue. Furthermore, we established that murine neutrophils are the primary target of LukED, as the greater virulence of wild-type S. aureus compared with a lukED mutant was abrogated by depleting neutrophils. The in vivo toxicity of LukED towards murine phagocytes is unique among S. aureus leucotoxins, implying its crucial role in pathogenesis. Moreover, the tropism of LukED for murine phagocytes highlights the utility of murine models to study LukED pathobiology, including development and testing of strategies to inhibit toxin activity and control bacterial infection.