Biology subjects

Nair, S.

Publications and source records attributed to Nair, S..

82 records · Page 5Linked to original sources

bioRxiv2019

Mode and tempo of microsatellite length change in a malaria parasite mutation accumulation experiment

Microsatellite sequences are widely assumed to evolve neutrally, but also play an important role in bacterial pathogenesis, human disease and transcript abundance. The malaria parasite Plasmodium falciparum genome is extraordinarily AT-rich, containing 132,449 microsatellites-stretches of perfect 1-9 bp repeats between 10-1000bp, which comprise 10.74% of the 23 Mb genome. This project was designed to determine the mode and tempo of microsatellite mutations in malaria parasites. We maintained 31 parasite lines derived from a single 3D7 parasite cell for 114-267 days, with frequent bottlenecking to a single cell to minimize effective population size, allowing us to measure mutations accumulated over ~13,207 mitotic divisions. We Illumina sequenced the genomes of both progenitor and end-point mutation accumulation (MA) parasite lines in duplicate to validate stringent calling parameters. Calls were 99.89% (GATK), 99.99% (freeBayes) and 99.96% (HipSTR) concordant in duplicate sequence runs from independent sequence libraries. We observed 98 microsatellite mutations, giving rates of 2.11 x 10-7 - 1.46 x 10-8 /cell division that were strongly influenced by repeat motif and array length. Mutation rate was low relative to other organisms. However, despite this, in a single infection (1011 parasites) there will be 1.46 x 103 - 2.11 x 104 independent mutations at any single microsatellite locus. Given that many microsatellites are found in promotors, introns, within or close to coding sequences, we suggest that they may be important regulators of transcriptional and phenotypic variation in this pathogen. Author summaryMutation is central to evolution: in pathogens, the rate of mutation may determine how rapidly drug resistance evolves or how effectively pathogens can escape immune attack. Malaria parasites have small extremely AT-rich genomes, and genetic variation in natural populations is dominated by repeat number changes in short tandem repeats (microsatellites) rather than point mutations. We therefore focused on quantifying microsatellite mutation. We established 31 parasite cultures in the laboratory all derived from a single parasite cell. These were maintained for 114-267 days with frequent reductions to a single cell, so parasites accumulated mutations during ~13,207 cell divisions. We sequenced the parasite genomes at the end of the experiment to count the mutations. We highlight several conclusions: like other organisms studied, microsatellite mutation rates are associated with both repeat number and repeat motif. However, 41% of changes resulted from loss or gain of more than one repeat: this was particularly true for long repeat arrays. Unlike other eukaryotes, we found no insertions or deletions that were not associated with repeats or homology regions. Overall, we found that microsatellite mutation rates in malaria were amongst the lowest recorded and comparable to those in another AT-rich protozoan (the slime mold Dictyostelium).

evolutionary biology↗

bioRxiv2019

A real-time PCR for the differentiation of typhoidal and non-typhoidal Salmonella

Rapid and accurate differentiation of Salmonella spp. causing enteric fever from non-typhoidal Salmonella is essential for clinical management of cases, laboratory risk management and implementation of public health measures. Current methods used for confirmation of identification including biochemistry and serotyping as well as whole genome sequencing analyses, takes several days. Here we report the development and evaluation of a real-time PCR assay that can be performed directly on crude DNA extracts from bacterial colonies, for the rapid identification of typhoidal and non-typhoidal Salmonella. This novel two-hour assay identifies the genus Salmonella by detecting the ttr gene, encoding tetrathionate reductase, and defines typhoidal Salmonella by the detection of S. Typhi and Paratyphi-specific gene combinations. PCR assay performance was determined using 211 clinical cultures of Salmonella (114 non-typhoidal and 97 Typhoidal strains) and 7 clinical non-Salmonella cultures. In addition, the specificity of the assay was evaluated in silico using a diverse in-house collection of 1882 Salmonella whole genome sequences. The real-time PCR results for 218 isolates and the genomic analysis of the 1882 isolates produced 100% sensitivity and 100% specificity (based on a 7 gene profile) for identifying typhoidal Salmonella compared to the Salmonella whole genome sequening identification methods currently used at Public Health England. This paper describes a robust real-time PCR assay for the rapid, accurate identification of typhoidal and non-typhoidal Salmonella which will be invaluable for the urgent screening of isolates from symptomatic individuals, the safe processing of isolates in laboratories and for assisting the management of public health risks.

microbiology↗

bioRxiv2019

Informal genomic surveillance of regional distribution of Salmonella Typhi genotypes and antimicrobial resistance via returning travellers

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a systemic human infection with a burden exceeding 20 million cases each year that occur disproportionately among children in low and middle income countries. Antimicrobial therapy is the mainstay for treatment, but resistance to multiple agents is common. Here we report genotypes and antimicrobial resistance (AMR) determinants detected from routine whole-genome sequencing (WGS) of 533 S. Typhi isolates referred to Public Health England between April 2014 and April 2017, 488 (92%) of which had accompanying patient travel information obtained via an enhanced surveillance questionnaire. The majority of cases involved S. Typhi 4.3.1 (H58) linked with travel to South Asia (58%). Travel to East and West Africa were associated with genotypes 4.3.1 and 3.3.1, respectively. Point mutations in the quinolone resistance determining region (QRDR), associated with reduced susceptibility to fluoroquinolones, were very common (85% of all cases) but the frequency varied significantly by region of travel: 95% in South Asia, 43% in East Africa, 27% in West Africa. QRDR triple mutants, resistant to ciprofloxacin, were restricted to 4.3.1 lineage II and associated with travel to India, accounting for 23% of cases reporting travel to the country. Overall 24% of isolates were MDR, however the frequency varied significantly by region and country of travel: 27% in West Africa, 52% in East Africa, 55% in Pakistan, 24% in Bangladesh, 3% in India. MDR determinants were plasmid-borne (IncHI1 PST2 plasmids) in S. Typhi 3.1.1 linked to West Africa, but in all other regions MDR was chromosomally integrated in 4.3.1 lineage I. We propose that routine WGS data from travel-associated cases in industrialised countries could serve as informal sentinel AMR genomic surveillance data for countries where WGS is not available or routinely performed.\n\nAuthor SummaryOur data demonstrate how routine WGS data produced by Public Health England can be further mined for informal passive surveillance of Salmonella Typhi circulating in different geographical regions where typhoid is endemic. We have shown the public health utility of a simplified approach to WGS reporting based on the GenoTyphi genotyping framework and nomenclature, which doesnt require the generation of a phylogenetic tree or other phylogenetic analysis. These approaches yielded results consistent with previously reported antimicrobial resistance (AMR) patterns of S. Typhi, including prevalence of multi-drug resistant (MDR) and fluoroquinolone resistance in different regions in association with different pathogen variants. These data provide a rationale and framework for the extraction and reporting of geographically stratified genotype and AMR data from public health labs in non-endemic countries. Prospective analysis and reporting of such data could potentially detect shifts in regional S. Typhi populations, such as replacement or spread of different subclades and the emergence and dissemination of MDR, fluoroquinolone resistant and/or extensively drug resistant S. Typhi, providing valuable data to inform typhoid control measures in low and middle income countries that are still building their genomics capacity.

microbiology↗

bioRxiv2018

Cell size heterogeneity early in development is required for collective cell migration during gastrulation in zebrafish

Current knowledge of the mechanisms of cell migration is based on differentiated cells in culture where it is known that the actomyosin machinery drives migration via dynamic interactions with the extracellular matrix and adhesion complexes. However, unlike differentiated cells, cells in early metazoan embryos must also dynamically change cell sizes as they migrate. The relevance of cell size to cell migration and embryonic development is not known. Here we investigate this phenomena in zebrafish embryos, a model system in which reductive cell divisions causes cell sizes to decrease naturally over time as cells migrate collectively to sculpt the embryonic body plan. We show that cell size reduction during early development follows power-law scaling. Because mutations that can perturb cell sizes so early in development do not exist, we generate haploid and tetraploid zebrafish embryos and show that cell sizes in such embryos are smaller and larger than the diploid norm, respectively. Cells in embryos made of smaller or larger than normal cells migrate sub-optimally, leading to gastrulation defects. Multiple lines of evidence suggest that the observed defects originate from altered cell size rather than from pleotropic effects of altered ploidy. This interpretation is strengthened by the result wherein restoring cell sizes to normal diploid-like values rescues gastrulation defects. Live imaging of chimeric embryos where haploid/tetraploid cells are introduced into diploid embryos reveal the cell-autonomous nature of the migration defects. Additionally, aberrant intracellular actin dynamics with respect to the vectorial direction of motion suggests a cellular mechanism behind the migration defects. Taken together, early reductive cell divisions potentially allow dynamic, stage-specific cell size norms to emerge, which enables efficient collective cell migration to correctly position cells in space and time to shape an amorphous ball of blastoderm into an embryo.

developmental biology↗

bioRxiv2018

Resolving within-host malaria parasite diversity using single-cell sequencing

Malaria patients can carry one or more clonal lineage of the parasite, Plasmodium falciparum, but the composition of these infections cannot be directly inferred from bulk sequence data. Well-defined, complete haplotypes at single-cell resolution are ideal for describing within-host population structure and unambiguously determining parasite diversity, transmission dynamics and recent ancestry but have not been analyzed on a large scale. We generated 485 near-complete single-cell genome sequences isolated from fifteen P. falciparum patients from Chikhwawa, Malawi, an area of intense malaria transmission. Matched single-cell and bulk genomic analyses revealed patients harbored up to seventeen unique lineages. Estimation of parasite relatedness within patients suggests superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Our single-cell analysis indicates strong barriers to establishment of new infections in malaria-infected patients and allows high resolution dissection of intra-host variation in malaria parasites.

genetics↗

bioRxiv2018

Substrate-assisted Enzymatic Formation of Lysinoalanine in Duramycin

Duramycin is a heavily post-translationally modified peptide that binds phosphatidylethanolamine. It has been investigated as an antibiotic, inhibitor of viral entry, therapeutic for cystic fibrosis, and tumor and vasculature imaging agent. Duramycin contains a {beta}-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) crosslink. The mechanism of Lal formation is not known. We here show that Lal is installed stereospecifically by DurN via addition of Lys19 to a dehydroalanine. The structure of DurN reveals an unusual dimer with a new fold. Surprisingly, in the structure of duramycin bound to DurN, no residues of the enzyme are near the Lal. Instead, Hya15 of the substrate makes interactions with Lal suggesting it acts as a base to deprotonate Lys19 during catalysis. Biochemical data suggest that DurN preorganizes the reactive conformation of the substrate, such that the Hya15 of the substrate can serve as the catalytic base for Lal formation.

biochemistry↗

bioRxiv2017

Salmonella enterica Serovar Typhimurium ST313 Responsible For Gastroenteritis In The UK Are Genetically Distinct From Isolates Causing Bloodstream Infections In Africa

The ST313 sequence type of Salmonella enterica serovar Typhimurium causes invasive non-typhoidal salmonellosis amongst immunocompromised people in sub-Saharan Africa (sSA). Previously, two distinct phylogenetic lineages of ST313 have been described which have rarely been found outside sSA. Following the introduction of routine whole genome sequencing of Salmonella enterica by Public Health England in 2014, we have discovered that 2.7% (79/2888) of S. Typhimurium from patients in England and Wales are ST313. Of these isolates, 59/72 originated from stool and 13/72 were from extra-intestinal sites. The isolation of ST313 from extra-intestinal sites was significantly associated with travel to Africa (OR 12 [95% CI: 3,53]). Phylogenetic analysis revealed previously unsampled diversity of ST313, and distinguished UK-linked isolates causing gastroenteritis from African-associated isolates causing invasive disease. Bayesian evolutionary investigation suggested that the two African lineages diverged from their most recent common ancestors independently, circa 1796 and 1903. The majority of genome degradation of African ST313 lineage 2 is conserved in the UK ST313 lineages and only 10/44 pseudogenes were lineage 2-specific. The African lineages carried a characteristic prophage and antibiotic resistance gene repertoire, suggesting a strong selection pressure for these horizontally-acquired genetic elements in the sSA setting. We identified an ST313 isolate associated with travel to Kenya that carried a chromosomally-located blaCTX-M-15, demonstrating the continual evolution of this sequence type in Africa in response to selection pressure exerted by antibiotic usage.\n\nThe S. Typhimurium ST313 sequence type has been primarily associated with invasive disease in Africa. Here, we highlight the power of routine whole-genome-sequencing by public health agencies to make epidemiologically-significant deductions that would be missed by conventional microbiological methods. The discovery of ST313 isolates responsible for gastroenteritis in the UK reveals new diversity in this important sequence type. We speculate that the niche specialization of sub-Saharan African ST313 lineages is driven in part by the acquisition of accessory genome elements.

microbiology↗

bioRxiv2017

High-resolution single-cell sequencing of malaria parasites

Single-cell genomics is a powerful tool for determining the genetic structure of complex communities of unicellular organisms. Patients infected with the malaria-causing parasite, Plasmodium falciparum, often carry multiple, genetically distinct parasites. Little is known about the diversity and relatedness of these lineages. We have developed an improved single-cell genomics protocol to reconstruct individual haplotypes from infections, a necessary step in uncovering parasite ecology within the host. This approach captures singly-infected red blood cells (iRBCs) by fluorescence-activated cell sorting (FACS) prior to whole genome amplification (WGA) and whole genome sequencing (WGS). Here, we demonstrate that parasites in late cell cycle stages, which contain increased DNA content, are far superior templates for generating high quality genomic data. Targeting of these cells routinely generates near-complete capture of the 23Mb P. falciparum genome (mean breadth of coverage 90.7%) at high efficiency. We used this approach to analyze the genomes of 48 individual cells from a polyclonal malaria infection sampled in Chikhwawa, Malawi. This comprehensive dataset enabled high-resolution estimation of the clonality and the relatedness of parasite haplotypes within the infection, long-standing problems in malaria biology.

genomics↗

bioRxiv2017

Millennia of genomic stability within the invasive Para C Lineage of Salmonella enterica

Salmonella enterica serovar Paratyphi C is the causative agent of enteric (paratyphoid) fever. While today a potentially lethal infection of humans that occurs in Africa and Asia, early 20th century observations in Eastern Europe suggest it may once have had a wider-ranging impact on human societies. We recovered a draft Paratyphi C genome from the 800-year-old skeleton of a young woman in Trondheim, Norway, who likely died of enteric fever. Analysis of this genome against a new, significantly expanded database of related modern genomes demonstrated that Paratyphi C is descended from the ancestors of swine pathogens, serovars Choleraesuis and Typhisuis, together forming the Para C Lineage. Our results indicate that Paratyphi C has been a pathogen of humans for at least 1,000 years, and may have evolved after zoonotic transfer from swine during the Neolithic period.\n\nOne Sentence SummaryThe combination of an 800-year-old Salmonella enterica Paratyphi C genome with genomes from extant bacteria reshapes our understanding of this pathogens origins and evolution.

microbiology↗

bioRxiv2016

Longitudinal genomic surveillance of Plasmodium falciparum malaria parasites reveals complex genomic architecture of emerging artemisinin resistance in western Thailand

BackgroundArtemisinin-based combination therapies are the first line of treatment for Plasmodium falciparum infections worldwide, but artemisinin resistance (ART-R) has risen rapidly in in Southeast Asia over the last decade. Mutations in kelch13 have been associated with artemisinin (ART) resistance in this region. To explore the power of longitudinal genomic surveillance to detect signals in kelch13 and other loci that contribute to ART or partner drug resistance, we retrospectively sequenced the genomes of 194 P. falciparum isolates from five sites in Northwest Thailand, bracketing the era in which there was a rapid increase in ART-R in this region (2001-2014).\n\nResultsWe evaluated statistical metrics for temporal change in the frequency of individual SNPs, assuming that SNPs associated with resistance should increase frequency over this period. After Kelch13-C580Y, the strongest temporal change was seen at a SNP in phosphatidylinositol 4-kinase (PI4K), situated in a pathway recently implicated in the ART-R mechanism. However, other loci exhibit temporal signatures nearly as strong, and warrant further investigation for involvement in ART-R evolution. Through genome-wide association analysis we also identified a variant in a kelch-domain-containing gene on chromosome 10 that may epistatically modulate ART-R.\n\nConclusionsThis analysis demonstrates the potential of a longitudinal genomic surveillance approach to detect resistance-associated loci and improve our mechanistic understanding of how resistance develops. Evidence for additional genomic regions outside of the kelch13 locus associated with ART-R parasites may yield new molecular markers for resistance surveillance and may retard the emergence or spread of ART-R in African parasite populations.

genomics↗