Systems analysis of host-parasite interactions.

Parasitic diseases caused by protozoan pathogens lead to hundreds of thousands of deaths per year in addition to substantial suffering and socioeconomic decline for millions of people worldwide. The lack of effective vaccines coupled with the widespread emergence of drug-resistant parasites necessitates that the research community take an active role in understanding host-parasite infection biology in order to develop improved therapeutics. Recent advances in next-generation sequencing and the rapid development of publicly accessible genomic databases for many human pathogens have facilitated the application of systems biology to the study of host-parasite interactions. Over the past decade, these technologies have led to the discovery of many important biological processes governing parasitic disease. The integration and interpretation of high-throughput -omic data will undoubtedly generate extraordinary insight into host-parasite interaction networks essential to navigate the intricacies of these complex systems. As systems analysis continues to build the foundation for our understanding of host-parasite biology, this will provide the framework necessary to drive drug discovery research forward and accelerate the development of new antiparasitic therapies

Infection of laboratory-colonized Anopheles darlingi mosquitoes by Plasmodium vivax.

Anopheles darlingi Root is the most important malaria vector in the Amazonia region of South America. However, continuous propagation of An. darlingi in the laboratory has been elusive, limiting entomological, genetic/genomic, and vector-pathogen interaction studies of this mosquito species. Here, we report the establishment of an An. darlingi colony derived from wild-caught mosquitoes obtained in the northeastern Peruvian Amazon region of Iquitos in the Loreto Department. We show that the numbers of eggs, larvae, pupae, and adults continue to rise at least to the F6 generation. Comparison of feeding Plasmodium vivax ex vivo of F4 and F5 to F1 generation mosquitoes showed the comparable presence of oocysts and sporozoites, with numbers that corresponded to blood-stage asexual parasitemia and gametocytemia, confirming P. vivax vectorial capacity in the colonized mosquitoes. These results provide new avenues for research on An. darlingi biology and study of An. darlingi-Plasmodium interactions

In silico discovery of transcription regulatory elements in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>With the sequence of the <it>Plasmodium falciparum </it>genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the <it>P. falciparum </it>genome sequence having revealed few <it>cis</it>-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of <it>P. falciparum </it>intergenic regions (~90% AT) presents significant challenges to <it>in silico cis</it>-regulatory element discovery.</p> <p>Results</p> <p>We have developed an algorithm called Gene Enrichment Motif Searching (GEMS) that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich <it>P. falciparum </it>genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from <it>P. falciparum </it>life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative <it>cis</it>-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the <it>Plasmodium </it>literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays were conducted.</p> <p>Conclusion</p> <p>This GEMS analysis demonstrates that <it>in silico </it>regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of <it>P. falciparum</it>. The fact that regulatory elements were predicted from a diverse range of functional gene clusters supports the hypothesis that <it>cis</it>-regulatory elements play a role in the transcriptional control of many <it>P. falciparum </it>biological processes. The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.</p

Esterase mutation is a mechanism of resistance to antimalarial compounds

Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance

Identification of pathogen genomic variants through an integrated pipeline

Background: Whole-genome sequencing represents a powerful experimental tool for pathogen research. We present methods for the analysis of small eukaryotic genomes, including a streamlined system (called Platypus) for finding single nucleotide and copy number variants as well as recombination events. Results: We have validated our pipeline using four sets of Plasmodium falciparum drug resistant data containing 26 clones from 3D7 and Dd2 background strains, identifying an average of 11 single nucleotide variants per clone. We also identify 8 copy number variants with contributions to resistance, and report for the first time that all analyzed amplification events are in tandem. Conclusions: The Platypus pipeline provides malaria researchers with a powerful tool to analyze short read sequencing data. It provides an accurate way to detect SNVs using known software packages, and a novel methodology for detection of CNVs, though it does not currently support detection of small indels. We have validated that the pipeline detects known SNVs in a variety of samples while filtering out spurious data. We bundle the methods into a freely available package

In vivo transcriptional profiling of Plasmodium falciparum

BACKGROUND: Both host and pathogen factors contribute to disease outcome in Plasmodium falciparum infection. The feasibility of studying the P. falciparum in vivo transcriptome to understand parasite transcriptional response while it resides in the human host is presented. METHODS: A custom made oligonucleotide array with probes based on the P. falciparum 3D7 laboratory strain chromosome 2 sequence was used to detect in vivo P. falciparum transcripts. This study analyzed transcripts from total RNA derived from small blood samples of P. falciparum infected patients and compared the in vivo expression profile to the in vitro cultivated 3D7 strain transcriptome. RESULTS: The data demonstrated that in vivo transcription can be studied from a small blood sample, despite the abundance of human RNA. The in vivo transcriptome is similar to the 3D7 ring stage transcriptome, but there are significant differences in genes encoding a sexual stage antigen and surface proteins. CONCLUSIONS: Whole genome transcription analysis of P. falciparum can be carried out successfully and further studies in selected patient cohorts may provide insight into parasite in vivo biology and defense against host immunity

Combining stage specificity and metabolomic profiling to advance antimalarial drug discovery

We report detailed susceptibility profiling of asexual blood stages of the malaria parasite Plasmodium falciparum to clinical and experimental antimalarials, combined with metabolomic fingerprinting. Results revealed a variety of stage-specific and metabolic profiles that differentiated the modes of action of clinical antimalarials including chloroquine, piperaquine, lumefantrine, and mefloquine, and identified late trophozoite-specific peak activity and stage-specific biphasic dose-responses for the mitochondrial inhibitors DSM265 and atovaquone. We also identified experimental antimalarials hitting previously unexplored druggable pathways as reflected by their unique stage specificity and/or metabolic profiles. These included several ring-active compounds, ones affecting hemoglobin catabolism through distinct pathways, and mitochondrial inhibitors with lower propensities for resistance than either DSM265 or atovaquone. This approach, also applicable to other microbes that undergo multiple differentiation steps, provides an effective tool to prioritize compounds for further development within the context of combination therapies

Covalent Plasmodium falciparum-selective proteasome inhibitors exhibit a low propensity for generating resistance in vitro and synergize with multiple antimalarial agents

Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the β2, β5 or β6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only &lt;five-fold decreases in parasite susceptibility. This result compares favorably against previously published non-covalent inhibitors of the Plasmodium proteasome that can select for resistant parasites with &gt;hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria

Noncoding RNA, antigenic variation, and the virulence genes of Plasmodium falciparum

Long non-coding RNAs (lncRNA) are being increasingly recognized as important regulators of gene expression. A recent paper in Genome Biology reports the identification of a lncRNA family in Plasmodium falciparum, the cause of the most deadly form of malaria, that may help to explain the mechanism of antigenic variation in virulence genes of this important pathogen