Shubha Gopal

A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis.

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, gamma-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate gamma-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a gamma-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.

Maltose and Maltodextrin Utilization by Listeria monocytogenes Depend on an Inducible ABC Transporter which Is Repressed by Glucose

Background In the environment as well as in the vertebrate intestine, Listeriae have access to complex carbohydrates like maltodextrins. Bacterial exploitation of such compounds requires specific uptake and utilization systems. Methodology/Principal Findings We could show that Listeria monocytogenes and other Listeria species contain genes/gene products with high homology to the maltodextrin ABC transporter and utilization system of B. subtilis. Mutant construction and growth tests revealed that the L. monocytogenes gene cluster was required for the efficient utilization of maltodextrins as well as maltose. The gene for the ATP binding protein of the transporter was located distant from the cluster. Transcription analyses demonstrated that the system was induced by maltose/maltodextrins and repressed by glucose. Its induction was dependent on a LacI type transcriptional regulator. Repression by glucose was independent of the catabolite control protein CcpA, but was relieved in a mutant defective for Hpr kinase/phosphorylase. Conclusions/Significance The data obtained show that in L. monocytogenes the uptake of maltodextrin and, in contrast to B. subtilis, also maltose is exclusively mediated by an ABC transporter. Furthermore, the results suggest that glucose repression of the uptake system possibly is by inducer exclusion, a mechanism not described so far in this organism.

Synthesis and xanthine oxidase inhibitory activity of 7-methyl-2-(phenoxymethyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one derivatives

An elevated level of blood uric acid (hyperuricemia) is the underlying cause of gout. Xanthine oxidase is the key enzyme that catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. Allopurinol, a widely used xanthine oxidase inhibitor is the most commonly used drug to treat gout. However, a small but significant portion of the population suffers from adverse effects of allopurinol that includes gastrointestinal upset, skin rashes and hypersensitivity reactions. Moreover, an elevated level of uric acid is considered as an independent risk factor for cardiovascular diseases. Therefore use of allopurinol-like drugs with minimum side effects is the ideal drug of choice against gout. In this study, we report the synthesis of a series of pyrimidin-5-one analogues as effective and a new class of xanthine oxidase inhibitors. All the synthesized pyrimidin-5-one analogues are characterized by spectroscopic techniques and elemental analysis. Four (6a, 6b, 6d and 6f) out of 20 synthesized molecules in this class showed good inhibition against three different sources of xanthine oxidase, which were more potent than allopurinol based on their respective IC(50) values. Molecular modeling and docking studies revealed that the molecule 6a has very good interactions with the Molybdenum-Oxygen-Sulfur (MOS) complex a key component in xanthine oxidase. These results highlight the identification of a new class of xanthine oxidase inhibitors that have potential to be more efficacious, than allopurinol, to treat gout and possibly against cardiovascular diseases.

Genotyping of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) in a tertiary care centre in Mysore, South India: ST2371-SCCmec IV emerges as the major clone.

The burden of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) is on the rise in population and clinical settings on account of the adaptability and virulence traits of this pathogen. We characterized 45 non-duplicate CA-MRSA strains implicated mainly in skin and soft tissue infections (SSTIs) in a tertiary care hospital in Mysore, South India. All the isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing, accessory gene regulator (agr) typing, and multi-locus sequence typing (MLST). Four sequence types (STs) belonging to three major clonal complexes (CCs) were identified among the isolates: CC22 (ST2371 and ST22), CC1 (ST772) and CC8 (ST8). The majority (53.3%) of the isolates was of the genotype ST2371-t852-SCCmec IV [sequence type-spa type-SCCmec type], followed by ST22-t852-SCCmec IV (22.2%), ST772-t657-SCCmec V (13.3%) and ST8-t008-SCCmec IV (11.1%). ST237I, a single locus variant of ST22 (EMRSA-15 clone), has not been reported previously from any of the Asian countries. Our study also documents for the first time, the appearance of ST8-SCCmec IV (USA300) strains in India. Representative strains of the STs were further analyzed by pulsed field gel electrophoresis (PFGE). agr typing detected type I or II alleles in the majority of the isolates. All the isolates were positive for the leukotoxin gene, pvl (Panton-Valentine leukocidin) and the staphylococcal enterotoxin gene cluster, egc. Interestingly, multidrug resistance (resistance to ⩾3 classes of non-beta-lactam antibiotics) was observed in 77.8% (n=35) of the isolates. The highest (75.5%) resistance was recorded for ciprofloxacin, followed by erythromycin (53.3%), and quinupristin-dalfopristin (51.1%). Inducible clindamycin-resistance was identified in 37.7% of the isolates and it was attributed to the presence of erm(A), erm(C) and a combination of erm(A) and erm(C) genes. Isolates which showed a phenotypic pattern of M(R)/L(S) (macrolide-resistance/lincosamide-sensitivity) harbored the msr(A) gene. In conclusion, we report a high rate of multidrug resistance among Indian strains of CA-MRSA and the emergence of the lineages ST2371 and ST8 in India.

Chemical and microbial dynamics during composting of herbal pharmaceutical industrial waste.

A study was performed to analyze the dynamics of chemical, biochemical and microbial parameters during composting of herbal pharmaceutical waste. All the parameters were analyzed at three different intervals of composting (1st, 15th and 60th days). Temperature of the compost pile was initially high (46.2 °C) and on 60th day it dropped to 33.3 °C. The pH of the sample was initially acidic (2.39) and with the progress of decomposition gradually changed to neutrality (7.55). Electrical conductivity (EC) value was high (3.8 mS) during last day of composting compared to other stages. The activity of degradative enzymes namely amylase, invertase and urease were initially high (4.1, 4.79 mg of glucose/g/h and 0.19 mg of ammonia/g/h respectively) while it decreased with composting. The beneficial microbial load was initially low and very high at the last stages of decomposition. The bioassay studies using compost extracts revealed that the 60th day old sample was not phytotoxic in nature.

Bacteriological properties and health-related biochemical components of fermented fish sauce: An overview

ABSTRACT Fish sauce is an amber-colored salty liquid in Southeast Asian cuisine used as an important condiment for improving the taste of foods. It is produced by fermenting the fish with salt in the ratios of 1:1 or 3:1 (fish:salt, wet wt) in underground concrete tanks or earthenware for 6–12 months at ambient conditions. Proteins of the raw materials are hydrolyzed into peptides and amino acids by microbial proteolytic enzymes during fermentation. This paper reviews fish sauce diversity and its bacteriological, biochemical, and biofunctional properties. Additionally, efforts to accelerate fish sauce production by employing halotolerant bacterial cultures and proteinases to reduce the fermentation period and also to improve the acceptability of the product are also reviewed. Further, the review provides an overview of bacterial proteinases that have been employed to enhance the sensory and microbiological quality of fish sauce. The review also outlines the effect of fermented fish sauces on health, especially in reference to several bioactive peptides and bacterial metabolites apart from discussing desalination technique to recover the metabolites from fish sauce.

Revamping the role of biofilm regulating operons in device-associated Staphylococci and Pseudomonas aeruginosa

Extensive use of indwelling devices in modern medicine has revoked higher incidence of device associated infections and most of these devices provide an ideal surface for microbial attachment to form strong biofilms. These obnoxious biofilms are responsible for persistent infections, longer hospitalization and high mortality rate. Gene regulations in bacteria play a significant role in survival, colonization and pathogenesis. Operons being a part of gene regulatory network favour cell colonization and biofilm formation in various pathogens. This review explains the functional role of various operons in biofilm expression and regulation observed in device-associated pathogens such as Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa.

Antihyperuricemic effects of thiadiazolopyrimidin-5-one analogues in oxonate treated rats

Hyperuricemia is a risk factor for not only gout, but also to a variety of disorders that affect the vital organ systems of the human body. The xanthine oxidase (XO) is the key enzyme in the production of uric acid and its inhibition can inhibit hyperuricemia. Although, XO inhibitor allopurinol is widely prescribed antigout agent but its use is not without any side effects. Previously, we described the synthesis of four novel thiadiazolopyrimidin-5-one analogues as effective XO inhibitors and molecular docking studies also confirmed this. When these analogues were tested in potassium oxonate treated rats, their serum uric acid and creatinine levels were dropped significantly from 4.85±0.03 mg/dl to 1.21±0.01 mg/dl and 0.92±0.02 mg/dl to 0.40±0.02 mg/dl respectively. Among the pyrimidine analogues tested, 6a was most potent. Histological examinations of both liver and kidney tissues exhibited severe necrosis in oxonate treated rats and pyrimidine analogues could significantly attenuate this with a correlative inhibitory profile of hepatic XO from the same rats. Our results demonstrate antihyperuricemic effect of novel thiadiazolopyrimidin-5-one analogues in oxonate treated rats, which can be further explored not only as antigout therapeutics but also in other systems where hyperuricemia is the driving cause of the disease.

Prediction of Proteins Putatively Involved in the Thiol: Disulfide Redox Metabolism of a Bacterium (Listeria): The CXXC Motif as Query Sequence

Thiol:disulfide redox metabolism (TDRM) is a central metabolic network in all living cells. However, numerous proteins with different biochemical functions and several structural domains are involved, making it not trivial to identify and annotate its constituents in sequenced genomes. We developed an uncomplicated approach to solve the problem using existing web-based tools and public databases with the gram-positive bacterium Listeria monocytogenes EGD-e as a model organism. A pattern search for the Cys-Xaa-Xaa-Cys (CXXC) motif--a hallmark of TDRM proteins--in the genome sequence of the bacterium yielded 156 proteins. After initial refinement by protein and domain analysis, 14 candidate proteins remained. Subsequent detailed analyses, supported by modeling of 3D structures and data integration yielded 6 thioredoxin-like proteins plus thioredoxin reductase, glutaredoxin, one redox-sensitive regulator, one peptide methionine reductase - all typical TDRM constituents - and three putative novel components of the TDRM. For all 14 proteins orthologues were found in other Listeria species. Homology searches and phylogenetic analyses showed that related proteins are present mainly in other Firmicutes. This fast approach required minimal resources. It is immediately applicable to any genome with appropriate modifications and should be practicable also for other conserved, functionally important amino acid motifs.

Fermentative properties of proteolytic pediococcus strains isolated from salt fermented fish hydrolysate prepared using freshwater Fish Rohu (Labeo Rohita)

ABSTRACT Salt fermented fish hydrolysate (SFFH) was prepared from freshwater fish Rohu (Labeo rohita), an Indian major carp, using salt at room temperature for 50 days. Total lactic acid bacteria (LAB) counts of SFFH prepared using 20% and 30% NaCl (w/w) were found to be in the range of 5.83–7.25 log cfu/ml and 3.93–5.46 log cfu/ml, respectively, whereas the log cfu/ml values for the halotolerant LAB counts varied from 2.09 to 7.17 and 2.02 to 4.77, respectively. Out of 96 isolates from SFFH, 21 isolates were screened based on their pH reducing ability, total titratable acidity, and proteolytic properties. Morphological, biochemical, and molecular characterization of four selected LAB strains were carried out. The isolates FSAP3-3 (Pediococcus pentosaceus), FSBP4-40 (Pediococcus pentosaceus), FSBP16-40 (Pediococcus pentosaceus), and FSBP28-50 (Pediococcus acidilactici) showed both halotolerant and proteolytic properties. The identified LAB isolates showed antagonistic properties against several human pathogens and exhibited sensitivity toward a broad spectrum of antibiotics. All the identified LAB isolates showed excellent proteolytic activity and phosphohydrolase activity. These halotolerant LAB isolates that had both proteolytic properties and acidifying ability have the potential for application in an acceleration of fermentation to produce salted fish products (i.e., fish sauce, fish paste, fish mince, etc.).