Molecular typing of methicillin-resistant Staphylococcus aureus strains by PCR-RFLP of SPA gene: a reference laboratory perspective.

PURPOSE: To characterize methicillin-resistant Staphylococcus aureus (MRSA) strains by molecular typing based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of spa gene and to assess the utility of spa genotyping over bacteriophage typing in the discrimination of the strains. MATERIALS AND METHODS: Studies were undertaken on 125 MRSA strains representing the most predominant phage types and the non phage typeable strains. Strains were typed by bacteriophage typing and PCR-RFLP of spa gene. DNA sequence analysis of the amplified spa gene fragment of the representative RFLP patterns was performed using standard protocols. RESULTS: All the strains resistant to oxacillin were found to contain mec A gene. Fifty-two per cent of these strains were typeable by the international basic set of 23 phages. Five different PCR-RFLP patterns were observed among 125 MRSA strains. Non phage typeable strains were differentiated into four PCR-RFLP patterns. Sequencing of the spa gene from the representative strains of each RFLP pattern confirmed the length of these restriction fragments due to variation in the 24 bp and the 174 bp tandem repeats. It also revealed the presence of three new spa repeat patterns. CONCLUSION: The study demonstrates the importance of spa genotyping in the discrimination of MRSA strains, which were otherwise indistinguishable by bacteriophage typing. spa genotyping allowed differentiation of strains within a particular phage type. Nucleotide sequencing of isolates of different PCR-RFLP patterns indicated a correlation between the RFLP patterns of a variable number of tandem repeats and the phage type. The study provides valuable information on the epidemiological characterization of MRSA strains.

Histone as future drug target for malaria.

Malaria continues to be a major cause of mortality and morbidity in tropical countries and affecting around 100 countries of the world. As per WHO estimates, 300-500 million are being infected and 1-3 million deaths annually due to malaria. With the emerging knowledge about genome sequence of all the three counterparts involved in the disease of malaria, the parasite Plasmodium, vector Anopheles and host Homo sapien have helped the scientists to understand interactions between them. Simultaneous advancement in technology further improves the prospects to discover new targets for vaccines and drugs. Though the malaria vaccine is still far away in this situation there is need to develop a potent and affordable drug(s). Histones are the key protein of chromatin and play an important role in DNA packaging, replication and gene expression. They also show frequent post-translation modifications. The specific combinations of these posttranslational modifications are thought to alter chromatin structure by forming epigenetic bar codes that specify either transient or heritable patterns of genome function. Chromatin regulators and upstream pathways are therefore seen as promising targets for development of therapeutic drugs.

Genetic alteration in drug resistance markers of Plasmodium falciparum.

Plasmodium falciparum shows plasticity in its genome. For its survival it can delete certain genes (or portions) if not needed for its growth and has the capability to regulate its genes under various stages of its life cycle as well as under unfavourable environmental conditions. Parasite shows enormous amount of antigenic variation under immune pressure leading to the emergence of vaccine resistant strains. Similarly, under drug pressure it allows mutations to settle in the target genes. It is becoming more and more clear that with the continuous exposure to a drug, the parasite accumulates more and more number of mutations in these genes. By measuring the number of these point mutations among field isolates one can predict the efficacy of a particular drug. Therefore, these markers are useful tools at epidemiological level. This molecular surveillance can also help in slowing down the drug resistance if supported by a careful drug usage policy. Further studies are required to develop molecular markers for rest of the antimalarial drugs as well as the improvement on the existing molecular tools for accurate and rapid detection of drug resistant malaria.

Malaria genome project and its impact on the disease.

Malaria remains uncontrolled to-date due to lack of effective parasite and vector control strategies. With the completion of the host, parasite and vector genome projects more suitable and effective disease control measures can be achieved. Here we have reviewed the Plasmodium falciparum genome project and its impact on malaria research in future. The parasite genome project has revealed certain metabolic pathways which can be targeted to develop antimalarial drugs. It has also identified large number of potential antigens for the future potential vaccines. Now the researchers in the malaria field can plan to take up the studies, which can yield more fruitful results within the limited financial resources using bioinformatics, proteomics, structural, functional and comparative genomics, etc.

Partial sequence analysis of a Plasmodium vivax cation transporting ATPase gene homologue & a putative pseudohomologue.

Molecular characterization of P. vivax is essential to develop suitable antimalarial drugs and vaccines. We describe here isolation and sequence analysis of a partial cDNA of a calcium ATPase as well as a putative pseudogene from this parasite. The immunoscreening of lambda gtll- P. vivax DNA library with patients serum has earlier resulted in the isolation of several seroreactive clones including Pv14. This clone contains a 299 bp insert having 18 amino acids (aa) reading frame fused with beta galactosidase. A larger fragment of approximately 15 kb was isolated from the EMBL3 library for Pv14 but it had only 2 extra aa in its reading frame. The far upstream region of Pv14 revealed a 101aa long putative open reading frame (ORF) showing homology to a variety of calcium ATPases in the M8 and M9 transmembrane region. But in the absence of a transcript in the parasite could indicate that it represents a pseudogene. However, the real gene for calcium ATPase in P. vivax was detected by RT-PCR using degenerate primers, designed from the conserved sequences of energy transduction and phosphorylation domains. The amplified cDNA-PCR product of 550 bp was cloned and sequenced which showed a significant aa homology to the calcium ATPase4 of P. falciparum. The present study, therefore, establishes the existence of calcium ion pumps in P. vivax which will be useful in drug development.

Knob proteins in falciparum malaria.

Knob proteins play a significant role in the pathophysiology of cerebral malaria caused by Plasmodium falciparum. Most of these proteins are of parasite origin and can be divided into two major classes: (i) the cytoadherent proteins present at the surface of the knobs; and (ii) the submembranous structural proteins which are placed towards the cytoplasmic side in the knobs. Several surface proteins [viz., P. falciparum-infected erythrocyte membrane protein-1 (PFEMP-1), sequestrin, pfalhesin] and submembranous structural proteins [viz., knob-associated histidine-rich protein (KAHRP), PFEMP-2, PFEMP-3] of the knobs have been identified and characterized to a certain extent. The structural proteins interact with several host (e.g., spectrin, actin, band 4.1 etc.) as well as parasite (e.g., PFEMP-1) molecules to produce functional knobs. The surface proteins on the other hand interact with several adhesion molecules of the endothelial cell through receptor-ligand type of binding. Knob proteins are important from the point of view of malaria control since immunotherapeutic agents can be developed to block as well as reverse the cytoadherence phenomenon. The surface proteins are also good vaccine candidates except that they show a high rate of antigenic variation. Nevertheless, the use of ribozyme or antisense oligonucleotides to inhibit the expression of knob proteins (e.g., KAHRP alone or with surface protein) can be used as a molecular therapeutic agent.