Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are examples of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA–DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K_m = 12 μM) as a cofactor more effectively than NADH (K_m = 26 μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA–DHDPR has ∼20-fold greater binding affinity for NADPH (K_d = 1.5 μM) relative to NADH (K_d = 29 μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism; with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP⁺. This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA–DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.     

The prevalence of Staphylococcus aureus and methicillin resistant Staphylococcus aureus in milk and dairy products in Riyadh,Saudi Arabia

In terms of life- menaced contagion, methicillin resistant Staphylococcus aureus (MRSA) is known to be one of which and it is truly notable in the contaminated food causing a community health anxiety. However, the occurrence of S. aureus and MRSA in diverse kinds of dairy products have been tested in this study. Samples from: raw milk (unpasteurized) from horse, goat, camel, and cow origins and unpacked cheese were checked for the recovered strains of such bacterium and MRSA. Wholly, MRSA isolates were verified for antimicrobial susceptibility and further characterized by mecA and staphylococcal cassette chromosome mec (SCCmec) typing. Also, Panton-Valentine Leukocidin (PVL), Staphylococcus aureus protein A (spa), and Staphylococcal enterotoxins (SEs) were also tested between all positive MRSA isolates in order to discover the virulence factors. Consequently, 70% of the 100 collected dairy products samples were contaminated by S. aureus bacteria and 72.9% of them were defined as MRSA. 9.8% of MRSA isolates contained mecA genes with SCCmec type II (80%) as the most common SCCmec type. Moreover, large number of MRSA isolates were identified as multidrug resistance and 28.6% of MRSA-mecA positive isolates were also carried vancomycin resistance genes (i.e., vanB). Too, spa gene was detected between 9.8% of MRSA isolates but PVL gene was not spotted at all. Additionally, the existing of SEs was variable between MRSA isolates and the most common type was SEH (51%). In general, our results confirmed that raw milk and unpacked cheese in the Kingdom of Saudi Arabia (Riyadh) is a potential vehicle for multidrug resistant MRSA transmission. It is a critical civic health menace and stresses, thus; the need of applying well cleanliness practices is essential.     

Human neuroglobin functions as a redox-regulated nitrite reductase

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ～2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.     

The role of hypothiocyanous acid (HOSCN) in biological systems

Abstract

Hypohalous acids (HOX), produced by peroxidase-catalysed reactions of halide and pseudohalide ions with H₂O₂, play an important role in the human immune system. However, there is compelling evidence that these oxidants also mediate host tissue damage and contribute to the progression of a number of inflammatory diseases. Although it is well established that significant amounts of hypothiocyanous acid (HOSCN) are formed under physiological conditions, the reactions of this oxidant with host biological systems are relatively poorly characterized. It is generally accepted that HOSCN is a mild oxidant that reacts selectively with thiols. However, it is becoming increasingly recognized that this selectivity can result in the induction of significant cellular damage, which may contribute to disease. This review will outline the formation and reactivity of HOSCN and the role of this oxidant in biological systems.     

Cell wall thickness and the molecular mechanism of heterogeneous vancomycin-intermediate Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) with reduced sensitivity to vancomycin (VAN) has caused many clinical cases of VAN treatment failure, but the molecular mechanism underlying the reduced sensitivity to VAN is still unclear. We isolated a heterogeneous VAN-intermediate Staphylococcus aureus (hVISA), which was also a MRSA strain with reduced sensitivity to VAN. To investigate the molecular mechanism underlying the reduced sensitivity to VAN exhibited by the hVISA strain, we compared the hVISA strain with a VAN-sensitive MRSA strain, known as the N315 strain. The images captured by transmission electron microscopy showed that the cell wall of the hVISA strain was significantly thicker than that of the N315 strain (36·72 ± 1·04 nm vs 28·15 ± 1·25 nm, P < 0·05), and the results of real-time quantitative PCR analysis suggested that the expression levels of the cell wall thickness related genes (glmS, vraR/S, sgtB, murZ and PBP4) of the hVISA strain were significantly higher than those of the N315 strain (P < 0·05). In conclusion, this study indicated that the upregulation of the expression of the genes related to cell wall synthesis might be the molecular mechanism underlying the cell wall thickening of the hVISA strain and might be related to its resistance to VAN.     

Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress

Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p < 0.001) and diminished lipid peroxidation in a dose-dependent manner (p < 0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p = 0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p < 0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p = 0.01) associated with enhanced protection against lipid peroxidation (p = 0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.     

Role of interleukin-17 in a murine community-associated methicillin-resistant Staphylococcus aureus pneumonia model

Interleukin (IL)-17 is a key member of the Th17 cytokines and has been reported to be involved in the pathomechanisms underlying various diseases, including infectious diseases. Infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have garnered worldwide attention, and the representative USA300 strain is known to cause pneumonia in healthy people, which can be lethal. However, little is known about the role of IL-17 in CA-MRSA pneumonia. In this study, we investigated the role of IL-17 in a CA-MRSA pneumonia animal model. Mortality was higher and occurred at an earlier stage of infection in the IL-17A-knockout mice than in the wild-type (P < 0.01) and IL-17A/F-knockout mice (P < 0.05); however, no significant difference in the intrapulmonary bacterial counts was observed among the three groups of mice. Moreover, the IL-17A-knockout group showed significantly higher levels of IL-17F and granulocyte-colony stimulating factor (G-CSF) and a significantly higher neutrophil count in the bronchoalveolar lavage fluid than the other groups. These results confirmed that G-CSF expression significantly increased, and significant neutrophilic inflammation occurred under conditions of IL-17A deficiency in the murine CA-MRSA pneumonia model.     

耐甲氧西林金黄色葡萄球菌分型及流行现状

【目的】研究杭州地区耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)的基因型别,探讨MRSA菌株流行变化趋势及进化特点,为该地区MRSA的进一步防治提供科学依据。【方法】对86株MRSA进行葡萄球菌盒式染色体SCCmec基因、spa基因分型,并开展多位点序列分型(Multi-locus sequence typing,MLST),与国际上MRSA的流行型别进行比较,分析进化关系。【结果】86株MRSA共发现13个spa型(以t311型为主,占48.8%;其次为t6418型,占11.6%);MLST分型共发现9个ST型(以ST5为主,占59.3%;其次为ST239,占16.3%),经e BURST软件分析它们属于4个群(Group 1、Group 6、Group 8、Group 12)和8种克隆复合体(CC1、CC5、CC630、CC20、CC59、CC88、CC239、CC573);SCCmec基因分型以SCCmecⅡ型为主,占61.6%;其次为SCCmec III型,占22%;5株社区相关性MRSA(SCCmec-Ⅳ型)。其中第一流行克隆型为SCCmec-Ⅱ-ST5-t311-CC5(占47.7%)、其次为SCCmec-III-ST239-t030/t037-CC239(占12.8%)。【结论】SCCmec-Ⅱ-ST5-t311为杭州地区当前流行菌株;CA-MRSA菌株的出现,提示MRSA菌株有由医院向社区播散的趋势;此外,对新发展了单位点变体的菌株(SCCmec-Ⅰ-ST1921-t164-CC20和SCCmec-Ⅳ-ST965-t062-CC5),应加强重视。     

Detection of methicillin-resistant Staphylococcus aureus in Iberian pigs

Aims: Iberian pigs are bred in Spain for the production of high‐value dry‐cured products, whose export volumes are increasing. Animals are typically reared outdoors, although indoor farming is becoming popular. We compared carriage of methicillin‐resistant Staphylococcus aureus (MRSA) in Iberian pigs, raised indoors and outdoors, with intensively farmed Standard White pigs. Methods and Results: From June 2007 to February 2008, 106 skin swabs were taken from Iberian pigs and 157 samples from SWP at slaughterhouses in Spain. We found that Iberian pigs carried MRSA, although with a significantly lower prevalence (30/106; 28%) than SWP (130/157; 83%). A higher prevalence of indoor Iberian pigs compared with animals reared under outdoor conditions was not significant; however, all but one positive indoor Iberian pig samples were detected from one slaughterhouse. Overall, 16 different spa types were identified, with t011 predominating in all three animal populations. A subset of isolates was characterized by MLST. Most of these belonged to ST398. MRSA isolates from Iberian pigs presented a higher susceptibility to antibiotics than those isolated from SWP. Conclusions: Despite limited contact with humans, pigs raised outdoors are colonized by an MRSA population that genetically overlaps with that of intensively farmed pigs, although antimicrobial resistance is lower. Significance and Impact of the Study: To our knowledge, this is the first detection of MRSA in food animals raised in free‐range conditions.     

Geographic distribution of livestock-associated Staphylococcus aureus in the United States

Sampling for livestock-associated Staphylococcus aureus (LA-SA) in the United States is haphazard. The diversity of LA-S. aureus in the U.S. appears to be greater than in other countries. We review the epidemiology of LA-S. aureus in U.S. pigs, occupationally-exposed individuals, and environmental samples to assess the diversity and abundance of U.S. LA-S. aureus.     

Discovery and optimization of a new class of pyruvate kinase inhibitors as potential therapeutics for the treatment of methicillin-resistant Staphylococcus aureus infections

A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which would make it a target for development of novel antibiotics, but it is reported to be one of the most highly connected ‘hub proteins’ in MRSA, and thus should be very sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharmacophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that have similar anti-MRSA activity as compound 4. Structure–activity relationship (SAR) studies were carried out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10m with enzyme inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5 μg/ml. Some potent PK inhibitors, such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC Δpyk::Erm^R) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.     

Surfaceome and exoproteome of a clinical sequence type 398 methicillin resistant Staphylococcus aureus strain

For many years Staphylococcus aureus has been recognized as an important human pathogen. In this study, the surfacome and exoproteome of a clinical sample of MRSA was analyzed. The C2355 strain, previously typed as ST398 and spa-t011 and showing a phenotype of multiresistance to antibiotics, has several resistance genes. Using shotgun proteomics and bioinformatics tools, 236 proteins were identified in the surfaceome and 99 proteins in the exoproteome. Although many of these proteins are related to basic cell functions, some are related to virulence and pathogenicity like catalase and isdA, main actors in S. aureus infection, and others are related to antibiotic action or eventually resistance like penicillin binding protein, a cell-wall protein. Studying the proteomes of different subcellular compartments should improve our understanding of this pathogen, a microorganism with several mechanisms of resistance and pathogenicity, and provide valuable data for bioinformatics databases.     

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a K_m of 15 ± 4 μm, a k_cat of 5.2 ± 0.7 min⁻¹, and a k_cat/K_m of 5.8 × 10³ m⁻¹ s⁻¹. From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H₂O₂, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus.     

Discovery and initial structure–activity relationships of N-benzyl tricyclic indolines as antibacterials for methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent drug resistant bacteria. In 2012, over 11,000 fatalities in the United States were directly attributable to MRSA. In an effort to develop novel structural and mechanistic classes of antibacterial agents to fight against MRSA, we have optimized a hit compound, Of4, previously discovered in a screening campaign of a bio-inspired polycyclic indoline library previously developed in our lab. We took advantage of our concise and versatile synthetic strategy to conduct initial structure–activity relationship studies of Of4, and we now report the discovery of compound 4k as a more potent antibacterial agent against S. aureus. Compound 4k also displayed equivalent activity in four MRSA and a methicillin-susceptible strains while demonstrating an improved mammalian cytotoxicity profile compared to Of4. Interestingly, 4k shares the same tricyclic indoline core as Of1, a β-lactam-selective resistance-modifying agent, but harbors a distinct modification pattern conferring unique bioactivity. This phenomenon is reminiscent of many bioactive natural products.     

Presence of cna, emp and pls genes and pathogenicity of methicillin-resistant Staphylococcus aureus strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are important etiological factors responsible for hospital-acquired infections. The aim of this study was to analyze the influence of the presence of emp, pls and cna genes on the pathogenicity of MRSA strains. The presence of these genes was tested by PCR in 302 MRSA strains isolated from hospitalized patients and from carriers. For each tested gene, proportions of positive and negative strains were similar among the infected patients and carriers. We did not find any obvious correlation between the presence of the three tested genes and the infectivity of strains. Our results may also suggest that a lack of emp and presence of pls may correlate with reduced virulence of these strains.     

Molecular analysis of methicillin-resistant Staphylococcus aureus reveals an absence of plasmid DNA in multidrug-resistant isolates

The number, diversity and restriction enzyme fragmentation patterns of plasmids harboured by 44 multidrug-resistant hospital-acquired methicillin-resistant Staphylococcus aureus (MR-HA-MRSA) isolates, two multidrug-resistant community-acquired MRSA (MR-CA-MRSA), 50 hospital-acquired MRSA (HA-MRSA) isolates (from the University Hospital Birmingham, NHS Trust, UK) and 34 community-acquired MRSA (CA-MRSA) isolates (from general practitioners in Birmingham, UK) were compared. In addition, pulsed-field gel electrophoresis (PFGE) type following SmaI chromosomal digest and SCCmec element type assignment were ascertained for each isolate. All MR-HA-MRSA and MR-CA-MRSA isolates possessed the type II SCCmec, harboured no plasmid DNA and belonged to one of five PFGE types. Forty-three out of 50 HA-MRSA isolates and all 34 CA-MRSA isolates possessed the type IV SCCmec and all but 10 of the type IV HA-MRSA isolates and nine CA-MRSA isolates carried one or two plasmids. The 19 non-multidrug-resistant isolates (NMR) that did not harbour plasmids were only resistant to methicillin whereas all the NMR isolates harbouring at least one plasmid were resistant to at least one additional antibiotic. We conclude that although plasmid carriage plays an important role in antibiotic resistance, especially in NMR-HA-MRSA and CA-MRSA, the multidrug resistance phenotype from HA-MRSA is not associated with increased plasmid carriage and indeed is characterised by an absence of plasmid DNA.