Faculty Bibliography

The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station's history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

Malaria is a mosquito-borne disease caused by the Plasmodium parasite. Of the four Plasmodium species that routinely cause human malaria, Plasmodium vivax is the most widespread species outside Africa, causing approximately 18.9 million cases in 2012. P. vivax cannot be cultured continuously in vitro, which severely hampers research in nonendemic and endemic countries alike. Consequently, whole-genome sequencing has become an effective means to interrogate the biology of the P. vivax parasite. Our comparative genomic analysis of five P. vivax reference genomes and several whole-genome sequences of the closely related monkey malaria species P. cynomolgi has revealed an extraordinary level of genetic diversity and enabled characterization of novel multigene families and important single-copy genes. The generation of whole-genome sequences from multiple clinical isolates is also driving forward knowledge concerning the biology and evolution of the species. Understanding the biology of P. vivax is crucial to develop potential antimalarial drugs and vaccines and to achieve the goal of eliminating malaria.

Parasites, defined as eukaryotic microbes and parasitic worms that cause global diseases of human and veterinary importance, span many lineages in the eukaryotic Tree of Life. Historically challenging to study due to their complicated life-cycles and association with impoverished settings, their inherent complexities are now being elucidated by genome sequencing. Over the course of the last decade, projects in large sequencing centers, and increasingly frequently in individual research labs, have sequenced dozens of parasite reference genomes and field isolates from patient populations. This 'tsunami' of genomic data is answering questions about parasite genetic diversity, signatures of evolution orchestrated through anti-parasitic drug and host immune pressure, and the characteristics of populations. This brief review focuses on the state of the art of parasitic protist genomics, how the peculiar genomes of parasites are driving creative methods for their sequencing, and the impact that next-generation sequencing is having on our understanding of parasite population genomics and control of the diseases they cause.

The genus Plasmodium is a diversified group of parasites with more than 200 known species that includes those causing malaria in humans. These parasites use numerous proteins in a complex process that allows them to invade the red blood cells of their vertebrate hosts. Many of those proteins are part of multi-gene families; one of which is the merozoite surface protein-3 (msp3) family. The msp3 multi-gene family is considered important in the two main human parasites, Plasmodium vivax and Plasmodium falciparum, as its paralogs are simultaneously expressed in the blood stage (merozoite) and are immunogenic. There are large differences among Plasmodium species in the number of paralogs in this family. Such differences have been previously explained, in part, as adaptations that allow the different Plasmodium species to invade their hosts. To investigate this, we characterized the array containing msp3 genes among several Plasmodium species, including P. falciparum and P. vivax. We first found no evidence indicating that the msp3 family of P. falciparum was homologous to that of P. vivax. Subsequently, by focusing on the diverse clade of nonhuman primate parasites to which P. vivax is closely related, where homology was evident, we found no evidence indicating that the interspecies variation in the number of paralogs was an adaptation related to changes in host range or host switches. Overall, we hypothesize that the evolution of the msp3 family in P. vivax is consistent with a model of multi-allelic diversifying selection where the paralogs may have functionally redundant roles in terms of increasing antigenic diversity. Thus, we suggest that the expressed MSP3 proteins could serve as "decoys", via antigenic diversity, during the critical process of invading the host red blood cells.

Trichomonads are common parasites of many vertebrate and invertebrate species, with four species classically recognized as human parasites: Dientamoeba fragilis, Pentatrichomonas hominis, Trichomonas vaginalis, and Trichomonas tenax. The latter two species are considered human-specific; by contrast, D. fragilis and P. hominis have been isolated from domestic and farm mammals, demonstrating a wide host range and potential zoonotic origin. Several new studies have highlighted the zoonotic dimension of trichomonads. First, species typically known to infect birds and domestic mammals have been identified in human clinical samples. Second, several phylogenetic analyses have identified animal-derived trichomonads as close sister taxa of the two human-specific species. It is our opinion, therefore, that these observations prompt further investigation into the importance of zoonotic trichomonads for human health.

Malaria continues to be a major health problem in more than 100 endemic countries located primarily in tropical and sub-tropical regions around the world. Malaria transmission is a dynamic process and involves many interlinked factors, from uncontrollable natural environmental conditions to man-made disturbances to nature. Almost half of the population at risk of malaria lives in forest areas. Forests are hot beds of malaria transmission as they provide conditions such as vegetation cover, temperature, rainfall and humidity conditions that are conducive to distribution and survival of malaria vectors. Forests often lack infrastructure and harbor tribes with distinct genetic traits, socio-cultural beliefs and practices that greatly influence malaria transmission dynamics. Here we summarize the various topographical, entomological, parasitological, human ecological and socio-economic factors, which are crucial and shape malaria transmission in forested areas. An in-depth understanding and synthesis of the intricate relationship of these parameters in achieving better malaria control in various types of forest ecosystems is emphasized.

BACKGROUND: Assessing the Plasmodium vivax burden in India is complicated by the potential threat of an emerging chloroquine (CQ) resistant parasite population from neighbouring countries in Southeast Asia. Chennai, the capital of Tamil Nadu and an urban setting for P. vivax in southern India, was selected as a sentinel site for investigating CQ efficacy and sensitivity in vivax malaria. METHODS: CQ efficacy was evaluated with a 28-day in vivo therapeutic study, while CQ sensitivity was measured with an in vitro drug susceptibility assay. In both studies, isolates also underwent molecular genotyping to investigate correlations between parasite diversity and drug susceptibility to CQ. Molecular genotyping included sequencing a 604 base pair (bp) fragment of the P. vivax multidrug resistant gene-1 (Pvmdr1) for single nucleotide polymorphisms (SNPs) and also the amplification of eight microsatellite (MS) loci located across the genome on eight different chromosomes. RESULTS: In the 28-day in vivo study (N=125), all subjects were aparasitaemic by Day 14. Passive case surveillance continuing beyond Day 28 in 22 subjects exposed 17 recurrent infections, which ranged from 44 to 148 days post-enrollment. Pvmdr1 sequencing of these recurrent infections revealed that 93.3% had identical mutant haplotypes (958M/Y976/1076L) to their baseline Day 0 infection. MS genotyping further revealed that nine infection pairs were related with >/=75% haplotype similarity (same allele at six or more loci). To test the impact of this mutation on CQ efficacy, an in vitro drug assay (N=68) was performed. No correlation between IC50 values and the percentage of ring-stage parasites prior to culture was observed (rsadj: -0.00063, p = 0.3307) and the distribution of alleles among the Pvmdr1 SNPs and MS haplotypes showed no significant associations with IC50 values. CONCLUSIONS: Plasmodium vivax was found to be susceptible to CQ drug treatment in both the in vivo therapeutic drug study and the in vitro drug assay. Though the mutant 1076L of Pvmdr1 was found in a majority of isolates tested, this single mutation did not associate with CQ resistance. MS haplotypes revealed strong heterogeneity in this population, indicating a low probability of reinfection with highly related haplotypes.

BACKGROUND: Trichomonas vaginalis is the most prevalent non-viral sexually transmitted parasite. Although the protist is presumed to reproduce asexually, 60% of its haploid genome contains transposable elements (TEs), known contributors to genome variability. The availability of a draft genome sequence and our collection of >200 global isolates of T. vaginalis facilitate the study and analysis of TE population dynamics and their contribution to genomic variability in this protist. RESULTS: We present here a pilot study of a subset of class II Tc1/mariner TEs that belong to the T. vaginalis Tvmar1 family. We report the genetic structure of 19 Tvmar1 loci, their ability to encode a full-length transposase protein, and their insertion frequencies in 94 global isolates from seven regions of the world. While most of the Tvmar1 elements studied exhibited low insertion frequencies, two of the 19 loci (locus 1 and locus 9) show high insertion frequencies of 1.00 and 0.96, respectively. The genetic structuring of the global populations identified by principal component analysis (PCA) of the Tvmar1 loci is in general agreement with published data based on genotyping, showing that Tvmar1 polymorphisms are a robust indicator of T. vaginalis genetic history. Analysis of expression of 22 genes flanking 13 Tvmar1 loci indicated significantly altered expression of six of the genes next to five Tvmar1 insertions, suggesting that the insertions have functional implications for T. vaginalis gene expression. CONCLUSIONS: Our study is the first in T. vaginalis to describe Tvmar1 population dynamics and its contribution to genetic variability of the parasite. We show that a majority of our studied Tvmar1 insertion loci exist at very low frequencies in the global population, and insertions are variable between geographical isolates. In addition, we observe that low frequency insertion is related to reduced or abolished expression of flanking genes. While low insertion frequencies might be expected, we identified two Tvmar1 insertion loci that are fixed across global populations. This observation indicates that Tvmar1 insertion may have differing impacts and fitness costs in the host genome and may play varying roles in the adaptive evolution of T. vaginalis.

Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite's acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST=0.253, P<0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r=0.49, P=0.003, N=83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both neutral and adaptive loci across India.