Staphylococcus aureus protein A induced inflammatory response in human corneal epithelial cells

In the present study, we examined the role of Staphylococcus aureus protein A (SpA) in inducing inflammatory response in human corneal epithelial cells (HCECs). Exposure of HCECs to SpA induces rapid NF-kappaB activation and secretion of proinflammatory cytokine/chemokines (TNF-alpha and IL-8) in both concentration and time-dependent manner. Challenge of HCECs with live SpA(-/-) mutant S. aureus strains resulted in significantly reduced production of the cytokines when compared to the wild-type S. aureus strain. SpA also elicited the activation of MAP Kinases P38, ERK, but not JNK, in HCECs. SpA-induced production of proinflammatory cytokine were completely blocked by the NF-kappaB and p38 inhibitors and partially inhibited by the Jnk inhibitor. Pretreatment with anti-TLR2 neutralizing antibody had no effect on SpA-induced inflammatory response in HCECs, suggesting that this response is independent of TLR2 signaling. Moreover, unlike TLR2 ligands, SpA failed to induce the expression of antimicrobial peptides (hBD2 and LL-37) in HCECs. These studies indicate that SpA is a S. aureus virulence factor that stimulates HCEC inflammatory response through a pathway distinct from TLR2 in HCECs.     

Iron Acquisition and Transport in Staphylococcus aureus

Pathogenic Gram-positive bacteria encounter many obstacles in route to successful invasion and subversion of a mammalian host. As such, bacterial species have evolved clever ways to prevent the host from clearing an infection, including the production of specialized virulence systems aimed at counteracting host defenses or providing protection from host immune mechanisms. Positioned at the interface of bacteria/host interactions is the bacterial cell wall, a dynamic surface organelle that serves a multitude of functions, ranging from physiologic processes such as structural scaffold and barrier to osmotic lysis to pathogenic properties, for example the deposition of surface molecules and the secretion of cytotoxins. In order to succeed in a battle with host defenses, invading bacteria need to acquire the nutrient iron, which is sequestered within host tissues. A cell-wall based iron acquisition and import pathway was uncovered in Staphylococcus aureus. This pathway, termed the isd or iron-responsive surface determinant locus, consists of a membrane transporter, cell wall anchored heme-binding proteins, heme/haptoglobin receptors, two heme oxygenases, and sortase B, a transpeptidase that anchors substrate proteins to the cell wall. Identification of the isd pathway provides an additional function to the already bountiful roles the cell wall plays in bacterial pathogenesis and provides new avenues for therapeutics to combat the rise of antimicrobial resistance in S. aureus. This review focuses on the molecular attributes of this locus, with emphasis placed on the mechanism of iron transport and the role of such a system during infection.     

Identification of a heat-labile cellular nuclease in Staphylococcus aureus with properties similar to the extracellular nuclease (EC 3.1.4.7)

Besides the well-known heat-stable extracellular staphylococcal nuclease (EC 3.1.4.7) and cell surface bound nuclease, one more nuclease, which is heat-labile, has been identified and purified on phosphorylated cellulose column and characterized. Analyses by Sephadex G-75 gel chromatography indicates that the heat-labile cellular nuclease has molecular weight of about 16,000 similar to those of extracellular and cell-surface bound nucleases. Like the heat-stable nucleases, the heat-labile enzyme acts on both DNA and RNA, is more active on heat-denatured DNA, requires Ca²⁺ ions for activity and maximum catalytic activity is observed at pH 9.8–10 and at 45°C. The results suggest that the three enzymes have properties strikingly similar to one another and therefore may be related structurally.     

Antibacterial activities of essential oils from eight Greek aromatic plants against clinical isolates of Staphylococcus aureus

Aromatic plants have been used widely to extend the shelf life of foods but at the same time research is undergoes for their properties as antibacterial agents in clinical use. Although there are promising results for the antimicrobial properties of various essential oils against environmental or food-isolated strains of Staphylococcus aureus, limited work has been done concerning these properties against clinical isolates of this pathogen. S. aureus is responsible for an increase number of nosocomial infections and at the same time exhibits increased resistance to synthetic agents.In this study, essential oils from eight aromatic plants common in Greece were isolated by hydrodistillation, analyzed by gas chromatography (GC) and GC/mass spectrometry (GC/MS) for their chemical components and tested for their antimicrobial activities against 24 clinical isolates of S. aureus. The methods used were disk diffusion and broth dilution in order to determine the Minimum Inhibitory Concentration (MIC).Our results showed that essential oils from Origanum vulgare and Origanum dictamnus were active against S. aureus when tested by disk diffusion, but exhibited increased MIC values (>256 mg/L) with the dilution method. In contrast, the reference strain NCTC 6571 showed to be extremely sensitive in most of the oils tested (MICs 0.25−32.0 mg/L) and resistant only to the essential oil from Ocimum basilicum. Therefore, there is no evidence of a potential clinical use for those essential oils and further research is needed in order to determine if they could substitute efficiently synthetic antibiotics or, perhaps be used in combination.     

Functional and structural characterization of Spl proteases from Staphylococcus aureus

Staphylococcus aureus is the major cause of nosocomial infections world-wide, with increasing prevalence of community-acquired diseases. The recent dramatic increase in multi-antibiotic resistance, including resistance to the last-resort drug, vancomycin, together with the lack of an effective vaccine highlight the need for better understanding of S.aureus pathogenicity. Comparative analysis of available bacterial genomes allows for the identification of previously uncharacterized S.aureus genes with potential roles in pathogenicity. A good example is a cluster of six serine protease-like (spl) genes encompassed in one operon, which encode for putative proteases with similarity to staphylococcal glutamylendopeptidase (V8 protease). Here, we describe an efficient expression system for the production of recombinant SplB and SplC proteases in Escherichia coli, together with structural and functional characterization of the purified enzymes. A unique mechanism of cytoplasm protection against activity of misdirected SplB was uncovered. Apparently, the co-translated signal peptide maintains protease latency until it is cleaved by the signal peptidase during protein secretion. Furthermore, the crystal structure of the SplC protease revealed a fold resembling that of the V8 protease and epidermolytic toxins. Arrangement of the active site cleft and substrate-binding pocket of SplC explains the mechanism of enzyme latency and suggests that some Spl proteases possess restricted substrate specificity similar to that of the V8 protease and epidermolytic toxins.     

NADPH-oxidase but not inducible nitric oxide synthase contributes to resistance in a murine Staphylococcus aureus Newman pneumonia model

Staphylococcus aureus is a pathogen that often causes severe nosocomial infections including pneumonia. The present study was designed to examine innate phagocyte mediated immune mechanisms using a previously described murine S. aureus Newman pneumonia model. We found that BALB/c mice represent a more susceptible mouse strain compared to C57BL/6 mice after intranasal S. aureus Newman challenge. Depletion experiments revealed that neutrophils are a crucial determinant for resistance whereas depletion of alveolar macrophages protected mice to some degree from acute pulmonary S. aureus challenge. C57BL/6 mice lacking the subunit gp91phox of the NADPH-oxidase (gp91phox−/− mice) proved to be highly susceptible against the pathogen. In contrast, C57BL/6 inducible nitric oxidase synthase deficient (iNOS−/−) mice did not differ in their clinical outcome after infection. Neither bone marrow macrophages from iNOS−/− nor from gp91phox−/− mice were impaired in controlling intracellular persistence of S. aureus. Our data suggest that neutrophil and NADPH-oxidase mediated mechanisms are essential components in protecting the host against pulmonary S. aureus Newman challenge. On contrary, macrophages as well as NO mediated mechanisms do not seem to play a critical role for resistance in this model.     

Staphylococcus aureus giant protein Ebh is involved in tolerance to transient hyperosmotic pressure

Staphylococcus aureus is well known to colonize on human skin where the physiological condition is characterized by hypervariable water activity, i.e., repeated dehydration or rehydration. To determine the facilitating factors for the colonization under hypervariable water activity, we studied the giant protein Ebh (extracellular matrix (ECM)-binding protein homologue). The ebh mutant RAM8 showed invaginated vacuoles along the septum, similar to that found in partial plasmolysis, and the cells burst under osmotic upshift. RAM8 was also relatively susceptible to abrupt hyperosmotic upshift, teicoplanin, and Triton X-100. By using the green fluorescent protein (GFP) as a reporter, Ebh was localized over the entire cell surface. This suggests that Ebh might contribute to structural homeostasis by forming a bridge between the cell-wall and cytoplasmic membrane to avoid plasmolysis under hyperosmotic condition.     

Induced resistance to the antimicrobial peptide lactoferricin B in Staphylococcus aureus

This study was designed to investigate inducible intrinsic resistance against lactoferricin B in Staphylococcus aureus. Serial passage of seven S. aureus strains in medium with increasing concentrations of peptide resulted in an induced resistance at various levels in all strains. The induced resistance was unstable and decreased relatively rapidly during passages in peptide free medium but the minimum inhibitory concentration remained elevated after thirty passages. Cross-resistance to penicillin G and low-level cross-resistance to the antimicrobial peptides indolicidin and Ala(8,13,18)-magainin-II amide [corrected] was observed. No cross-resistance was observed to the human cathelicidin LL-37. In conclusion, this study shows that S. aureus has intrinsic resistance mechanisms against antimicrobial peptides that can be induced upon exposure, and that this may confer low-level cross-resistance to other antimicrobial peptides.     

Effect of specific growth limitations on cell wall composition of Staphylococcus aureus H

Conditions are described for the continuous culture of a derivative of Staphylococcus aureus H in a fully defined minimal medium in which cysteine is the sole amino acid. The effects of growth under various nutrient limitations on the composition and properties of the cell wall have been studied. The proportion of ribitol teichoic acid present in the wall, and the extent to which it is substituted with N-acetylglucosamine, varies in bacteria grown under different conditions as does the composition and extent of cross-linking of the peptidoglycan. Neither the derivative nor the original strain H produced teichuronic acid when grown under phosphate limitation.Non-Standard Abbreviation SDS Sodium dodecyl sulphate     

In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins

We report the first characterization of the in vivo porphyrin scavenging abilities of two components of a newly discovered heme scavenging system involving iron-regulated surface determinant (Isd) proteins. These proteins are present within the cell envelope of the Gram-positive human pathogen Staphylococcus aureus. IsdC and IsdE, when expressed heterologously in Escherichia coli, efficiently scavenged intracellular heme and resulted in de novo heme synthesis in excess of 100-fold above background. Magnetic circular dichroism analyses showed that the heme-binding properties of the two proteins differ significantly from one another. IsdC bound almost exclusively free-base protoporphyrin IX, whereas the IsdE protein was associated with low spin Fe(III) and Fe(II) heme. These properties provide important insight into the possible mechanisms of iron scavenging from bound heme by Isd proteins.     

Evidence for a new post-translational modification in Staphylococcus aureus: hydroxymethylation of asparagine and glutamine

Staphylococcus aureus is an opportunistic pathogen whose infectious capacity depends on surface proteins, which enable bacteria to colonize and invade host tissues and cells. We analyzed “trypsin-shaved” surface proteins of S. aureus cultures by high resolution LC-MS/MS at different growth stages and culture conditions. Some modified peptides were identified, with a mass shift corresponding to the addition of a CH₂O group (+ 30.0106 u). We present evidence that this shift corresponds to a hyxdroxymethylation of asparagine and glutamine residues. This known but poorly documented post-translational modification was only found in a few proteins of S. aureus grown under specific conditions. This specificity seemed to exclude the hypothesis of an artifact due to sample preparation. Altogether hydroxymethylation was observed in 35 peptides from 15 proteins in our dataset, which corresponded to 41 modified sites, 35 of them being univocally localized. While no function can currently be assigned to this post-translational modification, we hypothesize that it could be linked to modulation of virulence factors, since it was mostly found on some surface proteins of S. aureus.     

Lethal and mutational effects of solar and UV radiation on Staphylococcus aureus

Strains of Staphylococcus aureus, an opportunistic pathogen commonly found on human skin, were exposed to sunlight and UV C radiation, and the lethal and mutational effects measured. Sunlight killed cells with an inactivation constant of 3×10^-5 per joule per square metre; UV C was much more lethal, giving an inactivation constant of approximately 0.1 per joule per square metre. Some strains tested showed a sensitivity to sunlight that was dependent on the growth phase of the cells, exponentially growing cells showing a greater sensitivity. Mutational effects of irradiation were measured by the appearance of mutants sensitive to methicillin following irradiation of a multiresistant strain. Mutants appeared at a frequency of 10^-3; this high frequency of mutation in the region of the mec gene has also been observed when multiresistant strains are subjected to nutritional or thermal stress. Mutants showed the same chromosomal alteration (seen in pulse-field gel electrophoresis of Smal-digested DNA) whether induced by solar or UV C irradiation.     

A simple method of markerless gene deletion in Staphylococcus aureus

Staphylococcus aureus is a Gram-positive pathogen that causes opportunistic infections and a wide variety of diseases. Methicillin-resistant S. aureus (MRSA) is frequently isolated as multidrug-resistant in nosocomial and community infections. Molecular genetic manipulation is an important tool for understanding the molecular mechanism of S. aureus infection. However the number of available antibiotic markers is limited due to multidrug resistance. In this study, we constructed two Escherichia coli-S. aureus shuttle vectors, pKFT and pKFC, that carry a temperature-sensitive origin of replication in S. aureus, lacZ(a) enabling a simple blue-white screening in E. coli, an ampicillin resistant gene, and either a tetracycline resistance gene or a chloramphenicol resistance gene. We report a simple technique using pKFT to construct a markerless gene deletion mutant in S. aureus by allelic replacement without the use of a counter-selection marker. Subculture twice at 25 °C was critical to promote an allelic exchange rate in S. aureus. This technique is very simple and useful to facilitate genetic research on S. aureus.     

Immunization with glutathione S-transferase and mutant toxic shock syndrome toxin 1 fusion protein protects against Staphylococcus aureus infection

To investigate whether immunization with glutathione S-transferase (GST) and mutant toxic shock syndrome toxin 1 (mTSST-1) fusion protein can protect against Staphylococcus aureus infection, we purified a non-toxic mutant GST-mTSST-1 fusion protein. Mice were immunized with the GST-mTSST-1 plus alum adjuvant and then challenged with viable S. aureus. The results showed that the survival rate of GST-mTSST-1-immunized group was higher and the bacteria counts in the organs were significantly lower than those of the non-immunized mice. Immunization with GST-mTSST-1 induced strongly the production of TSST-1 specific antibodies, especially immunoglobulin G1 and immunoglobulin G2b. Furthermore, the serum samples from GST-mTSST-1-immunized mice also significantly inhibited interferon-gamma and tumor necrosis factor-alpha production from murine spleen cells by TSST-1. These results suggest that vaccination with GST-mTSST-1 provides protection against S. aureus infection and that the protection might be mediated by TSST-1-neutralizing antibody.     

Accuracy of tracheal aspirate gram stain in predicting Staphylococcus aureus infection in ventilator-associated pneumonia

Background

The Gram stain can be used to direct initial empiric antimicrobial therapy when complete culture is not available. This rapid test could prevent the initiation of inappropriate therapy and adverse outcomes. However, several studies have attempted to determine the value of the Gram stain in the diagnosis and therapy of bacterial infection in different populations of patients with ventilator-associated pneumonia (VAP) with conflicting results. The objective of this study is to evaluate the accuracy of the Gram stain in predicting the existence of Staphylococcus aureus infections from cultures of patients suspected of having VAP.

Methods

This prospective single-center open cohort study enrolled 399 patients from December 2005 to December 2010. Patients suspected of having VAP by ATS IDSA criteria were included. Respiratory secretion samples were collected by tracheal aspirate (TA) for standard bacterioscopic analysis by Gram stain and culture.

Results

Respiratory secretion samples collected by tracheal aspirates of 392 patients were analyzed by Gram stain and culture. When Gram-positive cocci were arranged in clusters, the sensitivity was 68.4%, specificity 97.8%, positive predictive value 88.1% and negative predictive value 92.8% for predicting the presence of Staphylococcus aureus in culture (p < 0.001).

Conclusions

A tracheal aspirate Gram stain can be used to rule out the presence of Staphylococcus aureus in patients with a clinical diagnosis of VAP with a 92.8% Negative Predictive Value. Therefore, 7.2% of patients with Staphylococcus aureus would not be protected by an empiric treatment that limits antimicrobial coverage to Staphylococcus aureus only when Gram positive cocci in clusters are identified.     

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd²⁺⁺⁺ influx via the Mn²⁺ porter down membrane potential () was fast due to involvement of energy generated by two proton pumps—the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd²⁺ influx is suggested to be due to a series of toxic events elicited by Cd²⁺ accumulation down generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient ( _H ⁺) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of _H ⁺-dependent ATP synthesis, (iii) acceleration of the initial Cd²⁺ influx down generated the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd²⁺ of _H ⁺-dependent ATP synthesis and of pH gradient (pH)-linked [¹⁴C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of _H ⁺ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd²⁺ accumulation was prevented due to activity of the plasmid-coded Cd²⁺ efflux system. Consequently, _H ⁺-producing and -requiring processes were not affected by Cd²⁺.     

Oxidative stress induced by ciprofloxacin in Staphylococcus aureus

Staphylococcus aureus with multiple sensitivity to ciprofloxacin, was investigated to detect alterations in the production of superoxide anion (O(2)(-)), other reactive oxidant species (ROS), and superoxide dismutase (SOD), and to relate them with ciprofloxacin accumulation and sensitivity. Oxidative stress was studied by means of Nitroblue Tetrazolium reaction (NBT) and chemiluminescence (CL); lucigenin was employed to detect O(2)(-), and luminol was used to measure other ROS. Sensitive strains exhibited higher intracellular O(2)(-) increase than resistant ones when incubated with ciprofloxacin. SOD was determined in normal conditions and induction was investigated in the presence of ciprofloxacin. These assays demonstrated that resistant and sensitive strains exported a great amount of SOD and that the induction of SOD intracellular was insufficient to counteract the augment of O(2)(-) in the cytoplasm of sensitive strains. Accumulation of ciprofloxacin, researched by spectrofluorometry, showed high levels of antibiotic in sensitive strains which increased the O(2)(-) causing more oxidative stress than in resistant S. aureus.     

Lipids and DNA oxidation in Staphylococcus aureus as a consequence of oxidative stress generated by ciprofloxacin

Ciprofloxacin induced an increment of reactive oxygen species in sensitive strains of Staphylococcus aureus leading to oxidative stress detected by chemiluminescence while resistant strains did not suffer such stress. Oxidation of lipids was performed by employing thiobarbituric acid reaction to detect the formation of the amplified intermediate between reactive species oxygen and cytoplasmic macromolecules, namely malondialdehyde (MDA). The sensitive strain presented higher peroxidation of lipids than the resistant strain. The oxidative consequence for DNA was investigated by means of bacteria incubation with ciprofloxacin and posterior extraction of DNA, which was studied by high performance liquid chromatography (HPLC). Sensitive S. aureus ATCC 29213 showed an increase of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) respect controls without antibiotic; there was evident increase of the ratio between 8-oxodG and deoxyguanosine (dG) as a consequence of oxidation of dG to 8-oxodG considered the major DNA marker of oxidative stress. The resistant strain showed low oxidation of DNA and the analysis of 8-oxodG/dG ratio indicated lesser formation of 8-oxodG than S. aureus ATCC 29213.