Ocular conjunctival microbiome profiling in dry eye disease: A case control pilot study.

Purpose: Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is a multifactorial disease that results in discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. A pilot study was undertaken to determine if there were any major substantial differences in the ocular microbiome in DED patients versus healthy controls. Methods: The bacterial communities residing in the conjunctiva of patients with DED (n = 4) and healthy controls (n = 4) were assessed by 16S ribosomal RNA (rRNA) gene sequencing of the V4-V5 region. Results: The phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were most dominant and accounted for 97% and 94.5% of all bacterial sequences in patients and controls, respectively. At the genus level, 27 bacterial genera were found with more than two-fold difference between patients and controls. Four of these - Acinetobacter, Corynebacterium, Lactobacillus, and Pseudomonas spp. - dominated the ocular microbiome of all subjects, but were proportionately lower in DED (16.5%) compared to controls (37.7%). Several bacterial genera were found to be unique in DED (34) and controls (24). Conclusion: This pilot study is an attempt to profile the ocular microbiome in patients with DED that demonstrated a higher concentration of microbial DNA compared to controls, with Firmicutes phyla dominating the bacterial population in patients with DED.

Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.

Structural characterization of glutamyl-tRNA synthetase (GluRS) from Plasmodium falciparum.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein translation machinery that provide the charged tRNAs needed for protein synthesis. Over the past decades, aaRSs have been studied as anti-parasitic, anti-bacterial, and anti-fungal drug targets. This study focused on the cytoplasmic glutamyl-tRNA synthetase (GluRS) from Plasmodium falciparum, which belongs to class Ib in aaRSs. GluRS unlike most other aaRSs requires tRNA to activate its cognate amino acid substrate L-Glutamate (L-Glu), and fails to form an intermediate adenylate complex in the absence of tRNA. The crystal structures of the Apo, ATP, and ADP-bound forms of Plasmodium falciparum glutamyl-tRNA synthetase (PfGluRS) were solved at 2.1 Å, 2.2 Å, and 2.8 Å respectively. The structural comparison of the Apo- and ATP-bound holo-forms of PfGluRS showed considerable conformational changes in the loop regions around the ATP-binding pocket of the enzyme. Biophysical characterization of the PfGluRS showed binding of the enzyme substrates L-Gluand ATP.. The sequence and structural conservation were evident across GluRS compared to other species. The structural dissection of the PfGluRS gives insight into the critical residues involved in the binding of ATP substrate, which can be harvested to develop new antimalarial drugs.

Insecticide resistance status of malaria vectors in the malaria endemic states of India: implications and way forward for malaria elimination.

Background: In 2012, the World Health Organization (WHO) released the Global Plan for Insecticide Resistance Management in malaria vectors to stress the need to address insecticide resistance. In a prospective multi-centric study commissioned by the Indian Council of Medical Research (ICMR), we assessed the insecticide susceptibility status of the primary malaria vectors in India from 2017 through 2019. Methods: The insecticide susceptibility status of the prevalent primary malaria vectors - An. culicifacies, An. fluviatilis, An. stephensi, An. minimus and An. baimaii and secondary malaria vectors - An. aconitus, An. annularis and An. philippinensis/nivepes from 328 villages in 79 districts of 15 states of India were assessed following the WHO method mainly to insecticides used in vector control, organochlorine (DDT), organophosphate (malathion), and other pyrethroids (alpha-cypermethrin, cyfluthrin, lambda-cyhalothrin and permethrin). The study sites were selected as suggested by the National Vector Borne Disease Control Programme. Results: The primary malaria vector An. culicifacies showed resistance to DDT (50/50 districts including two districts of Northeastern India), malathion (27/44 districts), and deltamethrin (17/44 districts). This species was resistant to DDT alone in 19 districts, double resistant to DDT-malathion in 16 districts, double resistant to DDT-deltamethrin in 6 districts, and triple resistant to DDT-malathion-deltamethrin in 9 districts. An. minimus and An. baimaii were susceptible in Northeastern India while An. fluviatilis and the secondary malaria vector An. annularis was resistant to DDT in Jharkhand. Conclusion: In this study we report that among the primary vectors An. culicifacies is predominantly resistant to multiple insecticides. Our data suggest that periodic monitoring of insecticide susceptibility is vital. The national malaria program can take proactive steps for insecticide resistance management to continue its push toward malaria elimination in India.

Profiling of metabolic alterations in mice infected with malaria parasites via high-resolution metabolomics.

BACKGROUND: Malaria infection can result in distinct clinical outcomes from asymptomatic to severe. The association between patho-physiological changes and molecular changes in the host, and their correlation with severity of malaria progression is not fully understood. METHODS: In this study, we addressed mass spectrometry-based temporal profiling of serum metabolite levels from mice infected with Plasmodium berhgei (strain ANKA). RESULTS: We show global perturbations and identify changes in specific metabolites in correlation with disease progression. While metabolome-wide changes were apparent in late-stage malaria, a subset of metabolites exhibited highly correlated changes with disease progression. These metabolites changed early on following infection and either continued or maintained the change as mice developed severe disease. Some of these have the potential to be sentinel metabolites for severe malaria. Moreover, glycolytic metabolites, purine nucleotide precursors, tryptophan and its bioactive derivatives were many fold decreased in late-stage disease. Interestingly, uric acid, a metabolic waste reported to be elevated in severe human malaria, increased with disease progression, and subsequently appears to be detoxified into allantoin. This detoxification mechanism is absent in humans as they lack the enzyme uricase. CONCLUSIONS: We have identified candidate marker metabolites that may be of relevance in the context of human malaria.

A single amino acid substitution alters activity and specificity in Plasmodium falciparum aspartyl & asparaginyl-tRNA synthetases.

The specificity of each aminoacyl-tRNA synthetase (aaRS) for its cognate amino acid ensures correct tRNA esterification and allows fidelity in protein synthesis. The aaRSs discriminate based on the chemical properties of their amino acid substrates and structural features of the binding pockets. In this study, we characterized aspartyl-(DRS) and asparaginyl-tRNA synthetase (NRS) from Plasmodium falciparum to determine the basis of their specificity towards L-asp and L-asn respectively. The negatively charged L-asp and its analogue L-asn differ only in their side-chain groups i.e., -OH and -NH2. Further, the amino acid binding sites are highly conserved within these two enzymes. Analysis of the substrate (L-asp/L-asn) binding sites across species revealed two highly conserved residues in PfDRS (D408 and K372) and PfNRS (E395 and L360) that are involved in recognition of the Oδ2/Nδ2 of L-asp/L-asn respectively. These residues were mutated and swapped between the D408→E in PfDRS and the corresponding E395→D in PfNRS. A similar approach was employed for residue number K372→L in PfDRS and L360→K in PfNRS. The mutated PfDRSD408E retained its enzymatic activity during step 1 of aminoacylation reaction towards L-asp and L-asn and esterified tRNAAsp with L-asp like wild type enzyme, while the PfDRSK372L was rendered enzymatically inactive. The correspondingly mutated PfNRSE395D was enzymatically inactive. The mutated PfNRSL360K had an altered specificity and esterified tRNAAsn with non-cognate amino acid L-asp and not L-asn. These data suggest that the residue K372 is crucial for the enzymatic activity of PfDRS while the residue L360 in PfNRS imparts specificity towards L-asn.

Drug targeting of aminoacyl-tRNA synthetases in Anopheles species and Aedes aegypti that cause malaria and dengue.

BACKGROUND: Mosquito-borne diseases have a devastating impact on human civilization. A few species of Anopheles mosquitoes are responsible for malaria transmission, and while there has been a reduction in malaria-related deaths worldwide, growing insecticide resistance is a cause for concern. Aedes mosquitoes are known vectors of viral infections, including dengue, yellow fever, chikungunya, and Zika. Aminoacyl-tRNA synthetases (aaRSs) are key players in protein synthesis and are potent anti-infective drug targets. The structure-function activity relationship of aaRSs in mosquitoes (in particular, Anopheles and Aedes spp.) remains unexplored. METHODS: We employed computational techniques to identify aaRSs from five different mosquito species (Anopheles culicifacies, Anopheles stephensi, Anopheles gambiae, Anopheles minimus, and Aedes aegypti). The VectorBase database ( https://vectorbase.org/vectorbase/app ) and web-based tools were utilized to predict the subcellular localizations (TargetP-2.0, UniProt, DeepLoc-1.0), physicochemical characteristics (ProtParam), and domain arrangements (PfAM, InterPro) of the aaRSs. Structural models for prolyl (PRS)-, and phenylalanyl (FRS)-tRNA synthetases-were generated using the I-TASSER and Phyre protein modeling servers. RESULTS: Among the vector species, a total of 37 (An. gambiae), 37 (An. culicifacies), 37 (An. stephensi), 37 (An. minimus), and 35 (Ae. aegypti) different aaRSs were characterized within their respective mosquito genomes. Sequence identity amongst the aaRSs from the four Anopheles spp. was > 80% and in Ae. aegypti was > 50%. CONCLUSIONS: Structural analysis of two important aminoacyl-tRNA synthetases [prolyl (PRS) and phenylanalyl (FRS)] of Anopheles spp. suggests structural and sequence similarity with potential antimalarial inhibitor [halofuginone (HF) and bicyclic azetidine (BRD1369)] binding sites. This suggests the potential for repurposing of these inhibitors against the studied Anopheles spp. and Ae. aegypti.

Validation of a Mobile Health Technology Platform (FeverTracker) for Malaria Surveillance in India: Development and Usability Study.

BACKGROUND: A surveillance system is the foundation for disease prevention and control. Malaria surveillance is crucial for tracking regional and temporal patterns in disease incidence, assisting in recorded details, timely reporting, and frequency of analysis. OBJECTIVE: In this study, we aim to develop an integrated surveillance graphical app called FeverTracker, which has been designed to assist the community and health care workers in digital surveillance and thereby contribute toward malaria control and elimination. METHODS: FeverTracker uses a geographic information system and is linked to a web app with automated data digitization, SMS text messaging, and advisory instructions, thereby allowing immediate notification of individual cases to district and state health authorities in real time. RESULTS: The use of FeverTracker for malaria surveillance is evident, given the archaic paper-based surveillance tools used currently. The use of the app in 19 tribal villages of the Dhalai district in Tripura, India, assisted in the surveillance of 1880 suspected malaria patients and confirmed malaria infection in 93.4% (114/122; Plasmodium falciparum), 4.9% (6/122; P vivax), and 1.6% (2/122; P falciparum/P vivax mixed infection) of cases. Digital tools such as FeverTracker will be critical in integrating disease surveillance, and they offer instant data digitization for downstream processing. CONCLUSIONS: The use of this technology in health care and research will strengthen the ongoing efforts to eliminate malaria. Moreover, FeverTracker provides a modifiable template for deployment in other disease systems.

Epidemiological profiles and associated risk factors of SARS-CoV-2 positive patients based on a high-throughput testing facility in India.

We describe the epidemiological characteristics and associated risk factors of those presenting at a large testing centre for SARS-CoV-2 infection. This is a retrospective record review of individuals who underwent SARS-CoV-2 testing by reverse transcription-polymerase chain reaction (RT-PCR) at a high-throughput national-level government facility located in the north of India. Samples collected from 6 April to 31 December 2020 are included in this work and represent four highly populous regions. Additionally, there was a prospective follow-up of 1729 cases through telephone interviews from 25 May 2020 to 20 June 2020. Descriptive analysis has been performed for profiling clinic-epidemiological aspects of suspect cases. Multivariable logistic regression analysis was undertaken to determine risk factors that are associated with SARS-CoV-2 test positivity and symptom status. A total of 125 600 participants' details have been included in this report. The mean (s.d.) age of the participants was 33.1 (±15.3) years and 66% were male. Among these tested, 9515 (7.6%) were positive for COVID-19. A large proportion of positive cases were asymptomatic. In symptomatic positive cases, the commonest symptoms were cough and fever. Increasing age (groups 20-59 and ≥60 years compared to age group less than 5 years), male sex, history of international travel, symptoms for SARS-CoV-2, and participants from Delhi and Madhya Pradesh were positively associated with SARS-CoV-2 test positivity. Having co-morbidity, risk behaviours and intra-familial positivity were associated with a positive odds ratio for exhibiting SARS-CoV-2 symptoms. Intensified testing and isolation of cases, identification of both asymptomatic and symptomatic individuals and additional care of those with co-morbidities and risk behaviours will all be collectively important for disease containment in India. Reasons for differentials in testing between men and women remain an important area for in-depth study. The increased deployment of vaccines is likely to impact the trajectory of COVID-19 in the coming time, and therefore our data will serve as a comparative resource as India experiences the second wave of infection in light of newer variants that are likely to accelerate disease spread.

Structural analyses of the malaria parasite aminoacyl-tRNA synthetases provide new avenues for antimalarial drug discovery.

Malaria is a parasitic illness caused by the genus Plasmodium from the apicomplexan phylum. Five plasmodial species of P. falciparum (Pf), P. knowlesi, P. malariae, P. ovale, and P. vivax (Pv) are responsible for causing malaria in humans. According to the World Malaria Report 2020, there were 229 million cases and ~ 0.04 million deaths of which 67% were in children below 5 years of age. While more than 3 billion people are at risk of malaria infection globally, antimalarial drugs are their only option for treatment. Antimalarial drug resistance keeps arising periodically and thus threatens the main line of malaria treatment, emphasizing the need to find new alternatives. The availability of whole genomes of P. falciparum and P. vivax has allowed targeting their unexplored plasmodial enzymes for inhibitor development with a focus on multistage targets that are crucial for parasite viability in both the blood and liver stages. Over the past decades, aminoacyl-tRNA synthetases (aaRSs) have been explored as anti-bacterial and anti-fungal drug targets, and more recently (since 2009) aaRSs are also the focus of antimalarial drug targeting. Here, we dissect the structure-based knowledge of the most advanced three aaRSs-lysyl- (KRS), prolyl- (PRS), and phenylalanyl- (FRS) synthetases in terms of development of antimalarial drugs. These examples showcase the promising potential of this family of enzymes to provide druggable targets that stall protein synthesis upon inhibition and thereby kill malaria parasites selectively.