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.     

Homology Modeling of Coagulase in Staphylococcus aureus

The close correlation between the ability of coagulase to clot blood plasma and their capacity to produce disease, and the corresponding absence of this property in nonpathogenic strains, have led to the assumption that the coagulase, plays important role in the pathogenesis of disease. Currently, crystal structure of coagulase in Staphylococcus aureus remains indefinable. Thus, the objectives of this research is to generate the three dimensional model of coagulase in S. aureus by using homology modeling approach. In this study, we used bioinformatics tools and databases such as BLAST (Basic Local Alignment Search Tool), GenBank, PDB (Protein Databank), and Discovery Studio to gain specific functional insights into coagulase. The model was validated using protein structure checking tools such as PROCHECK, Verify 3D and CE (Combinatorial Extension) for reliability. Therefore, structure prediction of coagulase in S. aureus can provide preliminary knowledge for understanding the function of the protein. The information from this finding will provide important information into the action and regulation mechanism of the coagulase protein in S. aureus.     

Identification of potential targets in Staphylococcus aureus N315 using computer aided protein data analysis

Staphylococcus aureus is a gram positive bacterium, responsible for both community-acquired and hospital-acquired infection, resulting in a mortality rate of 39%. 43.2% resistance to methicilin and emerging resistance to Fluroquinolone and Oxazolidinone, have evoked the necessity of the establishment of alternative and effective therapeutic approach to treat this bacteria. In this computational study, various database and online software are used to determine some specific targets of Staphylococcus aureus N315 other than those used by Penicillin, Quinolone and Oxazolidinone. For this purpose, among 302 essential proteins, 101 nonhomologous proteins were accrued and 64 proteins which are unique in several metabolic pathways of S. aureus were isolated by using metabolic pathway analysis tools. Furthermore, 7 essentially unique enzymes involved in exclusive metabolic pathways were revealed by this research, which can be potential drug target. Along with these important enzymes, 15 non-homologous proteins located on membrane were identified, which can play a vital role as potential therapeutic targets for the future researchers.     

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.     

Crystal structure and substrate specificity of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

beta-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 A resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- > acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.     

Computational structural and functional analysis of hypothetical proteins of Staphylococcus aureus

Genome sequencing projects has led to an explosion of large amount of gene products in which many are of hypothetical proteins with unknown function. Analyzing and annotating the functions of hypothetical proteins is important in Staphylococcus aureus which is a pathogenic bacterium that cause multiple types of diseases by infecting various sites in humans and animals. In this study, ten hypothetical proteins of Staphylococcus aureus were retrieved from NCBI and analyzed for their structural and functional characteristics by using various bioinformatics tools and databases. The analysis revealed that some of them possessed functionally important domains and families and protein-protein interacting partners which were ABC transporter ATP-binding protein, Multiple Antibiotic Resistance (MAR) family, export proteins, Helix-Turn-helix domains, arsenate reductase, elongation factor, ribosomal proteins, Cysteine protease precursor, Type-I restriction endonuclease enzyme and plasmid recombination enzyme which might have the same functions in hypothetical proteins. The structural prediction of those proteins and binding sites prediction have been done which would be useful in docking studies for aiding in the drug discovery.     

Determinants of murein hydrolase targeting to cross-wall of Staphylococcus aureus peptidoglycan

Cells of eukaryotic or prokaryotic origin express proteins with LysM domains that associate with the cell wall envelope of bacteria. The molecular properties that enable LysM domains to interact with microbial cell walls are not yet established. Staphylococcus aureus, a spherical microbe, secretes two murein hydrolases with LysM domains, Sle1 and LytN. We show here that the LysM domains of Sle1 and LytN direct murein hydrolases to the staphylococcal envelope in the vicinity of the cross-wall, the mid-cell compartment for peptidoglycan synthesis. LysM domains associate with the repeating disaccharide β-N-acetylmuramic acid, (1→4)-β-N-acetylglucosamine of staphylococcal peptidoglycan. Modification of N-acetylmuramic acid with wall teichoic acid, a ribitol-phosphate polymer tethered to murein linkage units, prevents the LysM domain from binding to peptidoglycan. The localization of LytN and Sle1 to the cross-wall is abolished in staphylococcal tagO mutants, which are defective for wall teichoic acid synthesis. We propose a model whereby the LysM domain ensures septal localization of LytN and Sle1 followed by processive cleavage of peptidoglycan, thereby exposing new LysM binding sites in the cross-wall and separating bacterial cells.     

The Genome Sequence of a Type ST239 Methicillin-Resistant Staphylococcus aureus Isolate from a Malaysian Hospital

We report the genome sequence of a healthcare-associated MRSA type ST239 clone isolated from a patient with septicemia in Malaysia. This clone typifies the characteristics of ST239 lineage, including resistance to multiple antibiotics and antiseptics.     

Characterization of cell membrane parameters of clinical isolates of Staphylococcus aureus with varied susceptibility to alpha-melanocyte stimulating hormone

Staphylococcus aureus (S. aureus), a major human pathogen of hospital and community acquired infections, is becoming resistant to almost all commercially available antibiotics. This has prompted development of antimicrobial peptides as therapeutic options. Alpha melanocyte stimulating hormone (α-MSH) is one such peptide known to possess antimicrobial properties. In the present study, we analyzed the antimicrobial activity of α-MSH against 75 clinical strains of S. aureus including both methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) strains. Results of our previous study showed that membrane damage is the major mechanism of staphylocidal activity of α-MSH. In this context, we compared the various bacterial membrane parameters, viz., membrane fluidity, lipid composition, and surface charge of a few selected MSSA and MRSA strains that showed variable susceptibility to the melanocortin peptide. Our results showed that α-MSH killed both type of strains efficiently (≥70% killing in 84% clinical strains after exposure with 6μM of α-MSH for 1h). It was observed that compared to the α-MSH-susceptible strains, the α-MSH-non-susceptible strains had a different membrane order and phospholipid pattern. There was no consistent pattern of cell surface charge to distinguish α-MSH-susceptible strain from a non-susceptible strain. In conclusion, α-MSH possessed potential staphylocidal activity for both against MSSA and MRSA strains. S. aureus strains not susceptible to the peptide exhibited a rigid membrane and a higher amount of the cationic phospholipid as compared to the α-MSH-susceptible strains.     

A new platform for ultra-high density Staphylococcus aureus transposon libraries

Background

Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed.

Results

Here we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how null mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.

Conclusions

The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1361-3) contains supplementary material, which is available to authorized users.     

A molecular modeling based screening for potential inhibitors to alpha hemolysin from Staphylococcus aureus

Staphylococcus aureus, a Gram-positive bacterium is pathogenic in nature. It is known that secreted toxins remain active after antibiotic treatment. The alpha hemolysin or alpha toxin damages cell membrane and induces apoptosis and degradation of DNA. The titer of alphahemolysin increases and causes hemostasis disturbances, thrombocytopenia, and pulmonary lesions during staphylococcal infection. Therefore, it is of interest to inhibit alpha hemolysin using novel compounds. We used the structure of alpha hemolysin(PDB: 7AHL) to screen structures for 100,000 compounds from the ZINC database using molecular docking with AutoDock VINA. Nine (9) successive hits were then subjected for pharmacokinetic and toxicity properties by PROTOX (a webserver for the prediction of oral toxicities of small molecules) and FAFDrugs (a tool for prediction of ADME and Toxicity). This exercise further identified hit #1 ({[3a-(Dihydroxymethyl)-6-hydroxy-2,2-dimethyl-1,3,4-trioxatetrahydro-2H-pentalen-5- yl]methyl}amino(9H-fluoren-9-yl)acetate with binding affinity: -10.3 kcal/mol) and hit #2 (6-(Dihydroxymethyl)-2-{2-[3- (methylamino)propyl]-2-azatricyclo[9.4.0.03,8]pentadeca-1(11),3,5,7,12,14-hexaen-6-yloxy}tetrahydro-2H-pyran-3,4,5-triol with binding affinity: -9.6 kcal/mol) with acceptable toxicity and ADME properties for potential predicted hemolysin inhibition. These compounds should then be evaluated in vitro using inhibitory studies.     

The role of IL-27 in susceptibility to post-influenza Staphylococcus aureus pneumonia

Background

Influenza is a common respiratory virus and Staphylococcus aureus frequently causes secondary pneumonia during influenza infection, leading to increased morbidity and mortality. Influenza has been found to attenuate subsequent Type 17 immunity, enhancing susceptibility to secondary bacterial infections. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection.

Methods

A murine model of influenza and Staphylococcus aureus infection was used to mimic human viral, bacterial co-infection. C57BL/6 wild-type, IL-27 receptor α knock-out, and IL-10 knock-out mice were infected with Influenza H1N1 (A/PR/8/34) or vehicle for 6 days followed by challenge with Staphylococcus aureus or vehicle for 24 hours. Lung inflammation, bacterial burden, gene expression, and cytokine production were determined.

Results

IL-27 receptor α knock-out mice challenged with influenza A had increased morbidity compared to controls, but no change in viral burden. IL-27 receptor α knock-out mice infected with influenza displayed significantly decreased IL-10 production compared to wild-type. IL-27 receptor α knock-out mice co-infected with influenza and S. aureus had improved bacterial clearance compared to wild-type controls. Importantly, there were significantly increased Type 17 responses and decreased IL-10 production in IL-27 receptor α knock-out mice. Dual infected IL-10−/− mice had significantly less bacterial burden compared to dual infected WT mice.

Conclusions

These data reveal that IL-27 regulates enhanced susceptibility to S. aureus pneumonia following influenza infection, potentially through the induction of IL-10 and suppression of IL-17.     

Mapping ultra-weak protein-protein interactions between heme transporters of Staphylococcus aureus

Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.     

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.     

Characterisation of the Virulence Factors and Genetic Types of Methicillin Susceptible Staphylococcus aureus from Patients and Healthy Individuals

Methicillin sensitive Staphylococcus aureus is an important bacterial pathogen associated with hospital- and community-acquired infections leading to endocarditis, skin tissue infection and pneumonia. The objective of this study was to determine both the genetic characteristics of methicillin-sensitive S. aureus (MSSA) strains, and the occurrence of virulence factors produced by S. aureus strains isolated from UMMC and healthy students in the University from year 2009. Out of 429 nasal swab samples, 67 were MSSA. The prevalence of 21 different virulence genes among 67 Malaysian clinical and community MSSA strains was determined by PCR, and their genetic features were assessed by PCR-RFLP of coa gene, agr types, spa typing and PFGE. The five predominant virulence genes were ica (79 %), efb and fnbA (61 % each), sdrE (57 %) and hlg (45 %). Toxin genes (enterotoxin, etd and pvl) were significantly more common (P < 0.05) in clinical strains compared to community strains. Three agr genotypes were observed: agr type I (45 %), agr type III (25 %) and agr type II (19 %). All 67 MSSA strains were distinguished into 26 profiles by PCR-RFLP of coa, 55 pulsotypes and 21 spa types. Four novel spa types (t7312, t7581, t7582 and t7583) were observed. In conclusion, different virulence profiles were observed in MSSA strains in Malaysia where toxin genes were more prevalent among clinical strains. No correlation between DNA profiles (coa-RFLP, PFGE and spa) and virulotypes was observed. The Malaysian MSSA strains from clinical and community sources were genetically diverse and heterogeneous.     

N-Acetylglucosaminylation of Serine-Aspartate Repeat Proteins Promotes Staphylococcus aureus Bloodstream Infection

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.     

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.     

Signal peptides direct surface proteins to two distinct envelope locations of Staphylococcus aureus

Surface proteins of Gram-positive bacteria are covalently linked to the cell wall envelope by a mechanism requiring an N-terminal signal peptide and a C-terminal LPXTG motif sorting signal. We show here that surface proteins of Staphylococcus aureus arrive at two distinct destinations in the bacterial envelope, either distributed as a ring surrounding each cell or as discrete assembly sites. Proteins with ring-like distribution (clumping factor A (ClfA), Spa, fibronectin-binding protein B (FnbpB), serine-aspartate repeat protein C (SdrC) and SdrD) harbour signal peptides with a YSIRK/GS motif, whereas proteins directed to discrete assembly sites (S. aureus surface protein A (SasA), SasD, SasF and SasK) do not. Reciprocal exchange of signal peptides between surface proteins with (ClfA) or without the YSIRK/GS motif (SasF) directed recombinant products to the alternate destination, whereas mutations that altered only the YSIRK sequence had no effect. Our observations suggest that S. aureus distinguishes between signal peptides to address proteins to either the cell pole (signal peptides without YSIRK/GS) or the cross wall, the peptidoglycan layer that forms during cell division to separate new daughter cells (signal peptides with YISRK/GS motif).