SUMMARY Development of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but despite its importance, its molecular basis has been unknown. We now report a parasite gene family responsible for PSAC activity. We performed high-throughput screening to find transport inhibitors specific for distinct lines of the human pathogen P. falciparum. One inhibitor, 800-fold more active against PSAC from the Dd2 line than from HB3 parasites, was used with a genetic cross to map a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are determined by two clag genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein establishes novel ion channel activity on the erythrocyte surface.

Background: Malaria parasites acquire antimalarial resistance through incompletely understood mechanisms. Results: Resistance to blasticidin S results from reversible silencing of parasite clag genes through histone modifications without DNA level changes. Conclusion: Sophisticated epigenetic control of clag genes permits regulated control of nutrient and antimalarial transport at the host membrane. Significance: This resistance mechanism allows rapid parasite adaptation to environmental pressures and is worrisome for drug discovery efforts.

Summary Malaria parasites grow within erythrocytes, but are also free in host plasma between cycles of asexual replication. As a result, the parasite is exposed to fluctuating levels of Na+ and K+, ions assumed to serve important roles for the human pathogen, Plasmodium falciparum. We examined these assumptions and the parasite's ionic requirements by establishing continuous culture in novel sucrose-based media. With sucrose as the primary osmoticant and K+ and Cl− as the main extracellular ions, we obtained parasite growth and propagation at rates indistinguishable from those in physiological media. These conditions abolish long-known increases in intracellular Na+ via parasite-induced channels, excluding a requirement for erythrocyte cation remodeling. We also dissected Na+, K+, and Cl− requirements and found that unexpectedly low concentrations of each ion meet the parasite's demands. Surprisingly, growth was not adversely affected by up to 148 mM K+, suggesting that low extracellular K+ is not an essential trigger for erythrocyte invasion. At the same time, merozoite egress and invasion required a threshold ionic strength, suggesting critical electrostatic interactions between macromolecules at these stages. These findings provide insights into transmembrane signaling in malaria and reveal fundamental differences between host and parasite ionic requirements.

Bovine mastitis caused by multidrug resistant Staphylococcus aureus is a huge problem reported worldwide, resulting in prolonged antibiotic treatment and death of livestock. The current study is focused on surveillance of antibiotic susceptibility along with genotypic and phenotypic characterization of the pathogenic S. aureus strains causing mastitis in India. One hundred and sixty seven milk samples were collected from mastitis-affected cows from different farms in India resulting in thirty nine isolated S. aureus strains. Antibiotic sensitivity profiling revealed the majority of the strains (n = 24) to be multidrug resistant and eleven strains showed reduced susceptibility to vancomycin (MICs = 2μg/ml). All strains were oxacillin sensitive, but 19 strains were positive for the mecA gene, which revealed the occurrence of oxacillin susceptible mecA positive strains (OS-MRSA) for the first time from India. Additionally, 32 strains were positive for the pvl gene, a virulence determinant; of these 17 were also OS-MRSA strains. Molecular characterization based on multilocus sequence typing (MLST), spa typing, agr typing and SCCmec classification revealed strains belonging to different groups. Moreover, strains showed spa types (t2526, t9602) and MLST sequence types, ST-72, ST-88 and ST-239 which have been earlier reported in human infections. The prevalence of OS-MRSA strains indicates the importance of including both the genetic and phenotypic tests in characterizing S. aureus strains. Increased genotypic variability with strain related to human infections and pvl positive isolates indicates a worrisome situation with the possibility of bilateral transfer.

Methicillin resistant Staphylococcus aureus causing bovine mastitis has been very well investigated worldwide. However, there are only limited reports on the characterization of methicillin resistant and sensitive coagulase negative staphylococci (CoNS) across the globe. Hence, in the present study, we aim to determine the phenotypic traits based on antimicrobial susceptibility profile and genotypic characterization by verifying the presence of resistance determinants, virulence and toxin genes present in the CoNS causing clinical mastitis. We obtained 62 CoNS isolates from 167 mastitic milk samples collected from three different states of India. The 62 isolates comprises of 10 different CoNS species S. sciuri, S. haemolyticus, S. chromogenes, S. saprophyticus, S. xylosus, S. simulans, S. agnetis, S. epidermidis, S. gallinarum, and S. cohinii. Susceptibility screening against 11 antibiotics determined 45.16% isolates as multidrug resistant (resistant to more than two class of antibiotic), 46.74% resistant (one or two antibiotic class) and 8.06% isolates were pan-sensitive (sensitive to all drugs). High resistance was observed against oxacillin and cefoxitin, whereas all isolates were susceptible toward vancomycin and linezolid. Fifty three isolates were methicillin resistant and 9 isolates were sensitive as determined by oxacillin susceptibility assay. The methicillin resistance gene, mecA was found in 95.16% isolates and staphylococcal cassette chromosome mec (SCCmec) typing predominantly revealed Type III (n = 34) and Type V (n = 18). Interestingly, 11.9% of mecA positive isolates were oxacillin susceptible and referred as oxacillin susceptible mecA positive staphylococci (OS-MRS). Additionally, genes encoding for enterotoxin, (sea, seb, seh, see) toxic shock syndrome (tsst), exfoliatin (eta, etb, etd) and virulence (pvl, Y-hlg) were also screened. Of all the genes examined, 67.74% of isolate were positive for the Y-hlg gene, followed by the sea gene in 25.8% whereas in none of the isolates the eta and the etb gene was amplified. The study also highlights the incidence of clinical isolates of CoNS, which are harboring the toxin and the virulence genes rendering them as a more potential threat. This is the first report of animal origin OS-MRS from India, which emphasizes on the inclusion of both the genetic and phenotypic test for proper characterization of CoNS and preventing resistant strain misidentification.

Plasmodium, Toxoplasma, Cryptosporidium, Babesia, and Theileria are the major apicomplexan parasites affecting humans or animals worldwide. These pathogens represent an excellent example of host manipulators who can overturn host signaling pathways for their survival. They infect different types of host cells and take charge of the host machinery to gain nutrients and prevent itself from host attack. The mechanisms by which these pathogens modulate the host signaling pathways are well studied for Plasmodium, Toxoplasma, Cryptosporidium, and Theileria, except for limited studies on Babesia. Theileria is a unique pathogen taking into account the way it modulates host cell transformation, resulting in its clonal expansion. These parasites majorly modulate similar host signaling pathways, however, the disease outcome and effect is different among them. In this review, we discuss the approaches of these apicomplexan to manipulate the host–parasite clearance pathways during infection, invasion, survival, and egress.

Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted ␣-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC.T he human malaria parasite Plasmodium falciparum remodels its host erythrocyte by exporting many proteins, generating membranous structures in the host cytosol, and increasing erythrocyte permeability to many solutes. Studies by multiple groups have determined that anions, sugars, purines, organic cations, and some vitamins have increased permeability after infection (1-4). The increase in permeability is primarily mediated by a parasitederived ion and nutrient channel known as the plasmodial surface anion channel (PSAC) (5). Importantly, both PSAC single-channel properties and the relative increases in solute permeabilities are conserved in divergent malaria parasites (6). Because Babesia parasites do not induce PSAC-like activity in erythrocytes that they invade (7), this channel is thought to be restricted to the genus Plasmodium.The clag multigene family, also conserved in and restricted to malaria parasites (8), has recently been linked to PSAC activity (9-11). Two paralogs on parasite chromosome 3, known as clag3.1 and clag3.2, appear to play a critical role, as identified by genetic mapping experiments with ISPA-28, an isolate-specific PSAC antagonist that blocks channels on the Dd2 laboratory clone but is ineffective against channels from other lines. These genes undergo epigenetic switching to encode a single protein that is initially packaged in specialized organelles known as rh...

BackgroundTick borne diseases impinge cattle worldwide causing mortality and resulting in huge economic losses. Theileriosis is one of the important tick borne diseases mainly caused by Theileria annulata and one of the commonly occurring infections among the livestock. T. annulata causes immense loss to the livestock industry and therefore, efficacious eradication and control strategies are needed for the control of the disease. Genetic diversity among T. annulata parasites is another important aspect which is overlooked in India. Thus, the present study aims to evaluate the prevalence along with genetic diversity and phylogeny of the prevailing T. annulata population of India.MethodsGenomic DNA was extracted from cattle blood samples (n = 862) from different regions of Andhra Pradesh. Molecular diagnosis using T. annulata 18S rRNA based PCR was performed to detect parasites in cattle. Further, 18S rRNA gene was cloned and sequenced to determine similarity and diversity from the known T. annulata sequences.ResultsWe observed an overall prevalence rate of 32.40 % T. annulata infection in Andhra Pradesh based on PCR assay. The sequence analysis revealed novel genotypes among the T. annulata strains from India. Thirteen strains showed closed proximity with a strain from China whereas one Indian strain showed similarity with a South African strain [Theileria sp (buffalo)] based on phylogenetic analysis. Nucleotide heterogeneity of the 18S rRNA sequence among the strains examined varied from 0.1 to 8.6 % when compared with the published strains.ConclusionThe present study provides us with the molecular prevalence of theileriosis, and will support the accomplishment of actions or in design of strategy to control theileriosis transmission to cattle. Additionally, it highlights the emergence of strains with novel genotypes from India.