Bacteriophage therapy for Staphylococcus aureus bacteremia in streptozotocin-induced diabetic mice

The protective effect of bacteriophage was assessed against experimental Staphylococcus aureus lethal bacteremia in streptozotocin (STZ) induced-diabetic and non-diabetic mice. Intraperitoneal administrations of S. aureus (RCS21) of 2 x 10(8) CFU caused lethal bacteremia in both diabetic and non-diabetic mice. A single administration of a newly isolated lytic phage strain (GRCS) significantly protected diabetic and nondiabetic mice from lethal bacteremia (survival rate 90% and 100% for diabetic and non-diabetic bacteremic groups versus 0% for saline-treated groups). Comparison of phage therapy to oxacillin treatment showed a significant decrease in RCS21 of 5 and 3 log units in diabetic and nondiabetic bacteremic mice, respectively. The same protection efficiency of phage GRCS was attained even when the treatment was delayed up to 4 h in both diabetic and non-diabetic bacteremic mice. Inoculation of mice with a high dose (10(10) PFU) of phage GRCS alone produced no adverse effects attributable to the phage per se. These results suggest that phages could constitute valuable prophylaxis against S. aureus infections, especially in immunocompromised patients. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of Staphylococcus aureus homoserine dehydrogenase

Staphylococcus aureus is a Gram-positive nosocomial pathogen. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in cell-wall maintenance and essential amino-acid biosynthesis are significant drug targets. Homoserine dehydrogenase (HSD) is an oxidoreductase that is involved in the reversible conversion of l-aspartate semialdehyde to l-homoserine in a dinucleotide cofactor-dependent reduction reaction. HSD is thus a crucial intermediate enzyme linked to the biosynthesis of several essential amino acids such as lysine, methionine, isoleucine and threonine.

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrC(Cyto)) and the response regulator domain of AgrA (AgrA(RR)) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrC(Cyto) provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections.

Genome sequencing unveils a novel sea enterotoxin-carrying PVL phage in staphylococcus aureus ST772 from India

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772. suggests that widespread mobility of the sea enterotoxin might be a selective force behind its `transfer' to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside he genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.

Novel Rearrangements in the Staphylococcal Cassette Chromosome Mec Type V Elements of Indian ST772 and ST672 Methicillin Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a commensal gram positive bacteria which causes severe and non severe infections in humans and livestock. In India, ST772 is a dominant and ST672 is an emerging clone of Staphylococcus aureus. Both cause serious human diseases, and carry type V SCCmec elements. The objective of this study was to characterize SCCmec type V elements of ST772 and ST672 because the usual PCR methods did not amplify all primers specific to the type. Whole genome sequencing analysis of seven ST772 and one ST672 S. aureus isolates revealed that the SCCmec elements of six of the ST772 isolates were the smallest of the extant type V elements and in addition have several other novel features. Only one ST772 isolate and the ST672 isolate carried bigger SCCmec cassettes which were composites carrying multiple ccrC genes. These cassettes had some similarities to type V SCCmec element from M013 isolate (ST59) from Taiwan in certain aspects. SCCmec elements of all Indian isolates had an inversion of the mec complex, similar to the bovine SCCmec type X. This study reveals that six out of seven ST772 S. aureus isolates have a novel type V (5C2) SCCmec element while one each of ST772 and ST672 isolates have a composite SCCmec type V element (5C2&5) formed by the integration of type V SCCmec into a MSSA carrying a SCC element, in addition to the mec gene complex inversions and extensive recombinations.

Allosteric regulation of serine hydroxymethyltransferase from mung bean (Phaseolus aureus)

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 104 times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with nH values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme. H4folate; (±)-L-tetrahydrofolate

Studies on Plant Aspartate Transcarbamylase Purification and Properties of the Enzyme from Mung-Bean (Phaseolus aureus) Seedlings

Aspartate transcarbamylase is purified from mung bean seedlings by a series of steps involving manganous sulphate treatment, ammonium sulphate fractionation, DEAE-cellulose chromatography, followed by a second ammonium sulphate fractionation and finally gel filtration on Sephadex-G 100. The enzyme is homogeneous on ultracentrifugation and on polyacrylamide gel electrophoresis. It functions optimally at 55°C. It has two pH optima, one at 8.0 and the other at 10.2. The enzyme follows Michaelis-Menten kinetics with l-aspartate as the variable substrate. However, it exhibits sigmoid saturation curves at both the pH optima when the concentration of carbamyl phosphate is varied. The enzyme is allosterically inhibited by UMP at both the pH optima. Increasing phosphorylation of the uridine nucleotide decreases the inhibitory effect. The enzyme is desensitized to inhibition by UMP on treatment with p-hydroxymercuribenzoate, gel electrophoresis indicating that the enzyme is dissociated by this treatment; the dissociated enzyme can be reassociated by treatment with 2-mercaptoethanol. The properties of the mung bean enzyme are compared with the enzyme from other sources.

Studies on Plant Aspartate Transcarbamylase Purification and Properties of the Enzyme from Mung-Bean (Phaseolus aureus) Seedlings

Aspartate transcarbamylase is purified from mung bean seedlings by a series of steps involving manganous sulphate treatment, ammonium sulphate fractionation, DEAE-cellulose chromatography, followed by a second ammonium sulphate fractionation and finally gel filtration on Sephadex-G 100. The enzyme is homogeneous on ultracentrifugation and on polyacrylamide gel electrophoresis. It functions optimally at 55°C. It has two pH optima, one at 8.0 and the other at 10.2. The enzyme follows Michaelis-Menten kinetics with l-aspartate as the variable substrate. However, it exhibits sigmoid saturation curves at both the pH optima when the concentration of carbamyl phosphate is varied. The enzyme is allosterically inhibited by UMP at both the pH optima. Increasing phosphorylation of the uridine nucleotide decreases the inhibitory effect. The enzyme is desensitized to inhibition by UMP on treatment with p-hydroxymercuribenzoate, gel electrophoresis indicating that the enzyme is dissociated by this treatment; the dissociated enzyme can be reassociated by treatment with 2-mercaptoethanol. The properties of the mung bean enzyme are compared with the enzyme from other sources.

Location of Pathogenic Bacteria during Persistent Infections: Insights from an Analysis Using Game Theory

Bacterial persistent infections are responsible for a significant amount of the human morbidity and mortality. Unlike acute bacterial infections, it is very difficult to treat persistent bacterial infections (e.g. tuberculosis). Knowledge about the location of pathogenic bacteria during persistent infection will help to treat such conditions by designing novel drugs which can reach such locations. In this study, events of bacterial persistent infections were analyzed using game theory. A game was defined where the pathogen and the host are the two players with a conflict of interest. Criteria for the establishment of Nash equilibrium were calculated for this game. This theoretical model, which is very simple and heuristic, predicts that during persistent infections pathogenic bacteria stay in both intracellular and extracellular compartments of the host. The result of this study implies that a bacterium should be able to survive in both intracellular and extracellular compartments of the host in order to cause persistent infections. This explains why persistent infections are more often caused by intracellular pathogens like Mycobacterium and Salmonella. Moreover, this prediction is in consistence with the results of previous experimental studies.

Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples

Antibiotic resistance in 40 Staphylococcus aureus clinical isolates from 110 diabetic patients (36%) was evaluated. Of these, 32 (80%) of the isolates showed multidrug-resistance to more than eight antibiotics and 35% isolates were found to be methicillin resistant S. aureus (MRSA). All 40 S. aureus strains (100%) screened from diabetic clinical specimens were resistant to penicillin, 63% to ampicillin, 55% to streptomycin, 50% to tetracycline and 50% to gentamicin. Where as low resistance rate was observed to ciprofloxacin (20%) and rifampicin (8%). In contrast, all (100%) S. aureus strains recorded susceptibility to teicoplanin, which was followed by vancomycin (95%). Genotypical examination revealed that 80% of the aminoglycoside resistant S. aureus (ARSA) have aminoglycoside modifying enzyme (AME) coding genes; however, 20% of ARSA which showed non-AME mediated (adaptive) aminoglycoside resistance lacked these genes in their genome. In contrast all MRSA isolates possessed mecA, femA genetic determinants in their genome.

Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant Staphylococcus aureus Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetyl-glucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. beta-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.

Crystal Structure of Staphylococcus aureus Metallopeptidase (Sapep) Reveals Large Domain Motions between the Manganese-bound and Apo-states

Proteases belonging to the M20 family are characterized by diverse substrate specificity and participate in several metabolic pathways. The Staphylococcus aureus metallopeptidase, Sapep, is a member of the aminoacylase-I/M20 protein family. This protein is a Mn2+-dependent dipeptidase. The crystal structure of this protein in the Mn2+-bound form and in the open, metal-free state suggests that large interdomain movements could potentially regulate the activity of this enzyme. We note that the extended inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys(155) and Cys(178), are not active site residues, the reduced form of this enzyme is substantially more active as a dipeptidase. These findings acquire further relevance given a recent observation that this enzyme is only active in methicillin-resistant S. aureus. The structural and biochemical features of this enzyme provide a template for the design of novel methicillin-resistant S. aureus-specific therapeutics.