Paresh Sharma

Mitochondrial Associated Ubiquitin Fold Modifier-1 Mediated Protein Conjugation in Leishmania donovani

In this report, we demonstrate the existence of the ubiquitin fold modifier-1 (Ufm1) and its conjugation pathway in trypanosomatid parasite Leishmania donovani. LdUfm1 is activated by E1-like enzyme LdUba5. LdUfc1 (E2) specifically interacted with LdUfm1 and LdUba5 to conjugate LdUfm1 to proteinaceous targets. Mass spectrometry analysis revealed that LdUfm1 is conjugated to Leishmania protein targets that are associated with mitochondria. Immunofluorescence experiments showed that Leishmania Ufm1, Uba5 and Ufc1 are associated with the mitochondria. The demonstration that all the components of this system as well as the substrates are associated with mitochondrion suggests it may have physiological roles not yet described in any other organism. Overexpression of a non-conjugatable form of LdUfm1 and an active site mutant of LdUba5 resulted in reduced survival of Leishmania in the macrophage. Since mitochondrial activities are developmentally regulated in the life cycle of trypanosomatids, Ufm1 mediated modifications of mitochondrial proteins may be important in such regulation. Thus, Ufm1 conjugation pathway in Leishmania could be explored as a potential drug target in the control of Leishmaniasis.

Comparative in vivo expression of amastigote up regulated Leishmania genes in three different forms of Leishmaniasis.

In Old World Leishmania infections in India, Leishmania donovani is responsible for visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) while L. tropica is responsible for cutaneous leishmaniasis (CL) in humans. The molecular differences between the two species of Leishmania and within the same species causing distinct pathologies that govern the outcome of infection and pathogenesis in the human host are unknown. Quantitative expression of selected genes was evaluated directly in lesion tissues of VL, PKDL and CL patients. Assessment of in vivo mRNA level highlighted substantial differences in gene expression patterns, providing an indication of the genes involved in pathogenesis in the three different forms of Leishmaniasis.

Prevalence and Characterization of Oxacillin Susceptible mecA-Positive Clinical Isolates of Staphylococcus aureus Causing Bovine Mastitis in India.

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.

Identification of Variable Traits among the Methicillin Resistant and Sensitive Coagulase Negative Staphylococci in Milk Samples from Mastitic Cows in India

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.

Potential Sabotage of Host Cell Physiology by Apicomplexan Parasites for Their Survival Benefits

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.

Phylogenetic relationship and genotypic variability in Anaplasma marginale strains causing anaplasmosis in India

Anaplasma marginale is a tick borne rickesttsial parasite known to cause bovine anaplasmosis. There are prevalence reports from different parts of India, however, information regarding genetic diversity and phylogenetic association of the Indian strains are unknown. In the current study, 965 cattle blood samples from two states of India, Seemandhra and Telangana, were investigated for the presence of A. marginale by PCR using major surface protein 4 gene (msp4). We found an overall infection of 16.4%, with 3.4% prevalence in Seemandhra and 22.2% in Telangana. Sequence analysis of the 24 cloned msp4 gene indicated genetic diversity among Indian clinical strains of A. marginale which may be due to evolutionary pressure or migration of strains. Phylogenetic association analysis revealed that most of the strains showed close proximity with strains from Mexico and other strains showed closeness to strains reported from countries like Brazil, Zimbabwe, Prico and Hungary. This is the first report from India, identifying heterogeneous population of A. marginale strains causing anaplasmosis, and such data can play an important role in designing new control policies.

Leishmania donovani-specific Ub-related modifier-1: an early endosome-associated ubiquitin-like conjugation in Leishmania donovani.

Protein modification by ubiquitin (Ub) and Ub‐like molecules (Ubls) is a diverse biological process that regulates the activity of the target proteins. Systematic studies of Ubls in trypanosomatids like Leishmania, the causative organism of potentially fatal visceral leishmaniasis, would yield a better understanding of the disease pathogenesis and identify novel therapeutic targets. The present study is the first to characterize Leishmania donovani‐specific Ub‐related modifier‐1 (LdUrm1) and the associated conjugation pathway. Based on homology modeling, LdUrm1 was found to possess a β‐grasp fold and a C‐terminal di‐glycine motif unique to Ub/Ubls, essential for its conjugation to the target proteins. We identified LdUba4 as the E1 enzyme for LdUrm1 and demonstrated its energy‐dependent enzymatic activity. LdUrm1 was immunolocalized anteriorly near the flagellar reservoir, while LdUba4 was cytoplasmic, both in promastigotes and axenic amastigotes. Expression of nonconjugatable LdUrm1 in L. donovani resulted in depleted parasite growth suggesting its role in the pathogenesis. By mass spectrometry, we identified Rab5, a known mediator of early endosome regulated hemoglobin endocytosis in Leishmania, as a target of LdUrm1. Our data suggest that LdUrm1 conjugation pathway may have a role in early endosome‐mediated heme uptake in Leishmania that may be explored as a drug target.

Ubiquitin related modifier-1 (LdUrm1): an early endosome associated ubiquitin like conjugation in Leishmania donovani

Protein modification by ubiquitin (Ub) and Ub‐like molecules (Ubls) is a diverse biological process that regulates the activity of the target proteins. Systematic studies of Ubls in trypanosomatids like Leishmania, the causative organism of potentially fatal visceral leishmaniasis, would yield a better understanding of the disease pathogenesis and identify novel therapeutic targets. The present study is the first to characterize Leishmania donovani‐specific Ub‐related modifier‐1 (LdUrm1) and the associated conjugation pathway. Based on homology modeling, LdUrm1 was found to possess a β‐grasp fold and a C‐terminal di‐glycine motif unique to Ub/Ubls, essential for its conjugation to the target proteins. We identified LdUba4 as the E1 enzyme for LdUrm1 and demonstrated its energy‐dependent enzymatic activity. LdUrm1 was immunolocalized anteriorly near the flagellar reservoir, while LdUba4 was cytoplasmic, both in promastigotes and axenic amastigotes. Expression of nonconjugatable LdUrm1 in L. donovani resulted in depleted parasite growth suggesting its role in the pathogenesis. By mass spectrometry, we identified Rab5, a known mediator of early endosome regulated hemoglobin endocytosis in Leishmania, as a target of LdUrm1. Our data suggest that LdUrm1 conjugation pathway may have a role in early endosome‐mediated heme uptake in Leishmania that may be explored as a drug target.

A Real-Time PCR based assay for determining parasite to host ratio and parasitaemia in the clinical samples of Bovine Theileriosis

Theileria annulata is an intracellular parasite that causes active and latent forms of bovine theileriosis. Diagnosis of the disease is primarily based on traditional methods such as microscopy, however, PCR based methods have proven to be superior in the absence of clear disease symptoms. However, diagnosis is difficult in cases of lower parasitaemia by conventional PCR. Hence, a rapid and sensitive method which can detect early infection and low parasite load is required. Therefore, we have developed an absolute quantification based real-time PCR (qPCR) assay. Reference standard curve using recombinant plasmids of a host (hprt) and a parasite gene (tasp) was constructed, and the assay was initially standardised using in vitro T. annulata cell lines. Further, 414 blood samples from suspected theileriosis cases were also evaluated using qPCR. The assay can estimate host to parasite ratios, calculate parasitaemia and treatment effectiveness in the clinical cases of theileriosis. In comparison with the conventional PCR results, 44 additional positive cases were found. Therefore, the assay holds importance in a clinical setting due to its ability to quantify the parasite load in clinical samples. It may be further used in distinguishing active and latent theileriosis infections and detection of drug resistance in the field.

Antimicrobial and anti-biofilm activity of hexadentated macrocyclic complex of copper (II) derived from thiosemicarbazide against Staphylococcus aureus

Multidrug-resistant pathogens causing nosocomial and community acquired infections delineate a significant threat to public health. It had urged to identify new antimicrobials and thus, generated interest in studying macrocyclic metal complex, which has been studied in the past for their antimicrobial activity. Hence, in the present study, we have evaluated the antimicrobial activity of the hexadentated macrocyclic complex of copper (II) (Cu Complex) derived from thiosemicarbazide against Gram-positive and Gram-negative bacteria. We observed increased susceptibility against standard isolates of Staphylococcus aureus with a minimum inhibitory concentration (MIC) range of 6.25 to 12.5 μg/mL. Similar activity was also observed towards methicillin resistant and sensitive clinical isolates of S. aureus from human (n = 20) and animal (n = 20) infections. The compound has rapid bactericidal activity, and we did not observe any resistant mutant of S. aureus. The compound also exhibited antibiofilm activity and was able to disrupt pre-formed biofilms. Cu complex showed increased susceptibility towards intracellular S. aureus and was able to reduce more than 95% of the bacterial load at 10 μg/mL. Overall, our results suggest that Cu complex with its potent anti-microbial and anti-biofilm activity can be used to treat MRSA infections and evaluated further clinically.