Recombinant expression, purification and preliminary characterization of the mRNA export factor MEX67 of Saccharomyces cerevisiae.

The nuclear export of macromolecules is facilitated by the nuclear pore complexes (NPCs), embedded in the nuclear envelope and consists of multi-protein complexes. MEX67 is one of the nuclear export factor responsible for the transport of the majority of cellular mRNAs from the nucleus to the cytoplasm. The mechanism of mRNA transport through NPCs is unclear due to the unavailability of structures and the known interacting partners of MEX67. The mex67 gene was cloned in pQE30A and was expressed in Escherichia coli. A strategy has been developed to purify the insoluble MEX67 using a nickel affinity column with chelating Sepharose fast flow media, after solubilizing with sodium lauroyl sarcosinate (Sarkosyl). The IMAC purified recombinant MEX67 was further purified using SEC to apparent homogeneity (∼8 mg/L). Following SEC, MEX67 was stable and observed to be a 67 kDa monomeric protein as determined by PAGE and the size exclusion chromatography. The availability of large quantities of the protein will help in its biochemical and biophysical characterization, which may lead to the identification of new interaction partners of MEX67 or MEX67 complex.

Sulfur mobilization for Fe-S cluster assembly by the essential SUF pathway in the Plasmodium falciparum apicoplast and its inhibition.

The plastid of the malaria parasite, the apicoplast, is essential for parasite survival. It houses several pathways of bacterial origin that are considered attractive sites for drug intervention. Among these is the sulfur mobilization (SUF) pathway of Fe-S cluster biogenesis. Although the SUF pathway is essential for apicoplast maintenance and parasite survival, there has been limited biochemical investigation of its components and inhibitors of Plasmodium SUFs have not been identified. We report the characterization of two proteins, Plasmodium falciparum SufS (PfSufS) and PfSufE, that mobilize sulfur in the first step of Fe-S cluster assembly and confirm their exclusive localization to the apicoplast. The cysteine desulfurase activity of PfSufS is greatly enhanced by PfSufE, and the PfSufS-PfSufE complex is detected in vivo. Structural modeling of the complex reveals proximal positioning of conserved cysteine residues of the two proteins that would allow sulfide transfer from the PLP (pyridoxal phosphate) cofactor-bound active site of PfSufS. Sulfide release from the l-cysteine substrate catalyzed by PfSufS is inhibited by the PLP inhibitor d-cycloserine, which forms an adduct with PfSufS-bound PLP. d-Cycloserine is also inimical to parasite growth, with a 50% inhibitory concentration close to that reported for Mycobacterium tuberculosis, against which the drug is in clinical use. Our results establish the function of two proteins that mediate sulfur mobilization, the first step in the apicoplast SUF pathway, and provide a rationale for drug design based on inactivation of the PLP cofactor of PfSufS.

Nuclear-encoded DnaJ homologue of Plasmodium falciparum interacts with replication ori of the apicoplast genome.

The apicoplast of Plasmodium falciparum carries a 35 kb circular genome (plDNA) that replicates at the late trophozoite stage of the parasite intraerythocytic cycle. plDNA replication proceeds predominantly via a d-loop/bi-directional ori mechanism with replication ori localized within inverted repeat region. Although replication of the apicoplast genome is a validated drug target, the proteins involved in the replication process are only partially characterized. We analysed DNA-protein interactions at a plDNA replication ori region and report the identification of a nuclear-encoded DnaJ homologue that binds directly to ori elements of the plDNA molecule. PfDnaJ(A) interacted with the minor groove of the DNA double-helix and recognized a 13 bp sequence within the ori. Inhibition of binding with anti-PfDnaJ(A) antibodies confirmed identity of the protein in DNA-binding experiments with organellar protein fractions. The DNA-binding domain of the approximately 69 kDa PfDnaJ(A) lay within the N-terminal 38 kDa region that carries DnaJ signature motifs. In contrast to PfDnaJ(A) in parasite organellar fractions, the recombinant protein interacted with DNA in a sequence non-specific manner. Our results suggest a role for PfDnaJ(A) in replication/repair of the apicoplast genome.

Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system.

The maintenance of cellular redox homeostasis is a crucial adaptive problem faced by parasites, and its disruption can shift the biochemical balance toward the host. The thioredoxin (Trx) system plays a key role in redox metabolism and defense against oxidative stress. In this study, biochemical experiments were performed on Fasciola gigantica Thioredoxin1 (FgTrx1). The recombinant FgTrx1 exists as a monomer and catalyzes the reduction of insulin. FgTrx1 is preferentially regenerated by the glutathione (GSH) system using glutathione reductase (GR). The regeneration of FgTrx1 by the conventional Trx system is much less as compared to the GSH system, suggesting that FgTrx1 could be acting as glutaredoxin (Grx). DNA nicking and hydroperoxide assay suggests that it protects the DNA from radical-induced oxidative damage. Thus, FgTrx1 might play a role in parasite survival as it can regenerate itself even in the absence of the canonical Trx system and also protect the cells from ROS induced damage. Further, we propose that the GR activity of FgTrx1 is not restricted to -CXXC- motif but is regulated by residues present in close proximity to the -CXXC- motif, through manipulation of the redox potential or the pKa of the active site Cys residues.

Functional classification and biochemical characterization of a novel rho class glutathione S-transferase in Synechocystis PCC 6803.

We report a novel class of glutathione S-transferase (GST) from the model cyanobacterium Synechocystis PCC 6803 (sll1545) which catalyzes the detoxification of the water pollutant dichloroacetate and also shows strong glutathione-dependent peroxidase activity representing the classical activities of zeta and theta/alpha class respectively. Interestingly, sll1545 has very low sequence and structural similarity with these classes. This is the first report of dichloroacetate degradation activity by any bacterial GST. Based on these results we classify sll1545 to a novel GST class, rho. The present data also indicate potential biotechnological and industrial applications of cyanobacterial GST in dichloroacetate-polluted areas.

"Neonatal Sepsis": Bacteria & their Susceptibility Pattern towards Antibiotics in Neonatal Intensive Care Unit.

BACKGROUND: Neonatal sepsis is one of the most common causes of neonatal mortality and morbidity, particularly in the developing countries. Its causative bacteria and their respective sensitivity patterns are different in each hospital and region. The objective of this study was to determine the causative bacteria and pattern of susceptibility to antibiotics in NICU of a tertiary care centre, which in turn may help in implementation of empirical therapy. MATERIAL AND METHODS: This prospective study was carried out at a medical college during the period from 1st April 2011 to 31st March 2013. A total of 364 cases of suspected sepsis were admitted in our NICU during the mentioned period. Out of which, 137 cases were positive for culture. All the neonates of suspected sepsis were screened by using a panel consisting of CRP, ANC, I/T ratio, micro ESR and culture and sensitivity. RESULTS: A total of 137 cultures were found to be positive out of 364 cases. The most common organism isolated was Staphylococcus aureus (37.22%) followed by Klebsiella pneumoniae (27.01%) and Escherichia coli (19.70%). Other organisms were much less in number, which included pathogenic Streptococci, Coagulase negative Staphylococci (CoNS), Pseudomonas, Acinetobacter and Enterobacter species. The gram positive organisms except Streptococci displayed a high degree of resistance to most penicillins and ciprofloxacin but were sensitive to vancomycin, amikacin and cefepime. There was a high incidence of resistance noted with ampicillin, gentamicin and ciprofloxacin amongst most gram negative organisms' where-in cefepime, amikacin and meropenem were effective in most cases. CONCLUSION: There is an increasing trend of antibiotic resistance to the commonly used first line drugs. Continuous surveillance for antibiotic susceptibility is needed to ensure proper empirical therapy.

Interaction between sulphur mobilisation proteins SufB and SufC: evidence for an iron-sulphur cluster biogenesis pathway in the apicoplast of Plasmodium falciparum.

The plastid of Plasmodium falciparum, the apicoplast, performs metabolic functions essential to the parasite. Various reactions in the plastid require the assembly of [Fe-S] prosthetic groups on participating proteins as well as the reductant activity of ferredoxin that is converted from its apo-form by the assembly of [Fe-S] clusters inside the apicoplast. The [Fe-S] assembly pathway involving sulphur mobilising Suf proteins has been predicted to function in the apicoplast with one component (PfSufB) encoded by the plastid genome itself. We demonstrate the ATPase activity of recombinant P. falciparum nuclear-encoded SufC and its localisation in the apicoplast. Further, an internal region of apicoplast SufB was used to detect PfSufB-PfSufC interaction in vitro; co-elution of SufB from parasite lysate with recombinant PfSufC on an affinity column also indicated an interaction of the two proteins. As a departure from bacterial SufB and similar to reported plant plastid SufB, apicoplast SufB exhibited ATPase activity, suggesting the evolution of specialised functions in the plastid counterparts. Our results provide experimental evidence for an active Suf pathway in the Plasmodium apicoplast.