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.     

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.     

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.     

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.     

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.     

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.     

Methicillin-resistant Staphylococcus aureus: a pervasive pathogen highlights the need for new antimicrobial development

Staphylococcus aureus (S. aureus) has entered the spotlight as a globally pervasive drug-resistant pathogen. While historically associated exclusively with hospital-acquired infections in immunocompromised hosts, the methicillin-resistant form of S. aureus has been spreading throughout communities since the 1990s. Indeed, it has now become a common household term: MRSA. S. aureus has developed numerous mechanisms of virulence and strategies to evade the human immune system, including a host of surface proteins, secreted enzymes, and toxins. In hospital intensive care units, the proportion of MRSA-related S. aureus infections has increased strikingly from just 2 percent in 1974 to 64 percent in 2004. Its presence in the community has been rising similarly, posing a significant public health burden. The growing incidence of MRSA unfortunately has been met with dwindling efforts to develop new, more effective antibiotics. The continued emergence of resistant strains of bacteria such as MRSA demands an urgent revival of the search for new antibiotics.     

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.     

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.     

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.     

Quadruplex real-time quantitative PCR assay for the detection of pathogens related to late-onset ventilator-associated pneumonia: A preliminary report

A quadruplex real-time (RT) qPCR assay for the detection and quantification in 4 h of Staphylococcusaureus, Pseudomonasaeruginosa, Acinetobacterbaumannii and Stenotrophomonasmaltophilia directly from bronchoalveolar lavage specimens was developed. The specificity of the assay was 100% for all four species.     

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.     

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.     

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).     

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.     

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.     

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.     

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.