A drug repurposing approach reveals targetable epigenetic pathways in Plasmodium vivax hypnozoites

Radical cure of Plasmodium vivax malaria must include elimination of quiescent 'hypnozoite' forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials

<i>Staphylococcus aureus</i>in the House Fly: Temporospatial Fate of Bacteria and Expression of the Antimicrobial Peptide<i>defensin</i>

House flies disseminate numerous species of bacteria acquired during feeding and breeding activities in microbe-rich habitats. Previous house fly surveys have detected the pathogen Staphylococcus aureus Rosenbach 1884, which causes cutaneous and septic infections in mammals, and enterotoxic food poisoning. We assessed the fate of GFP-expressing S. aureus (GFP-S. aureus) in the house fly alimentary canal with microscopy and by culture of whole flies and excreta. Furthermore, the concurrent expression of the antimicrobial peptide gene defensin was measured in the crop, proventriculus, midgut, and fat body. As soon as 4 h postingestion (PI), GFP-S. aureus were visualized as cocci or diplococci in the hindgut and rectum of flies fed ≈10(5) colony forming units. Bacteria persisted up to 6 h PI but significantly decreased. Excretion of viable GFP-S. aureus peaked at 2 h PI and, although significantly less, continued up to 4 h PI. defensin was highly upregulated locally in the alimentary canal and systemically in fat body at 2, 4, and 6 h PI making this study the first to report, to our knowledge, an epithelial and systemic response to a bacterium with lysine-type peptidoglycan in flies exposed via feeding. While flies harbored S. aureus for up to 6 h PI, the highest probability of vectoring biologically relevant amounts of bacteria occurred 0–2 h PI. The combined effects of excretion, digestion and antimicrobial effectors likely contribute to loss of ingested bacteria. Nonetheless, house flies are relevant vectors for S. aureus up to 2 h PI and environmental reservoirs up to 6 h PI

Pseudomonas aeruginosa in Musca domestica L.: temporospatial examination of bacteria population dynamics and house fly antimicrobial responses.

House flies associate with microbes throughout their life history. Bacteria ingested by adult flies enter the alimentary canal and face a hostile environment including antimicrobial defenses. Because the outcome of this interaction impacts bacterial survival and dissemination, our primary objective was to understand the temporospatial dynamics of fly-bacteria associations. We concurrently examined the temporospatial fate of GFP-expressing Pseudomonas aeruginosa (GFP-P. aeruginosa) in the house fly alimentary canal along with antimicrobial peptide (AMP) expression. Motile, viable GFP-P. aeruginosa were found in all regions of the alimentary canal and were culturable throughout the observation period (2-24 h). A significant decrease in recoverable bacteria occurred between 2 and 12 h, followed by an increase between 12 and 24 h. qRT-PCR analysis showed expression of the AMPs cecropin, diptericin, and defensin both locally (gut) and systemically. Furthermore, mRNA of all AMPs were expressed throughout gut tissues, with some tissue-specific temporal variation. Interestingly, fluctuation in recoverable P. aeruginosa was associated with AMP protein expression in the gut (immunofluorescent signal detection), but not with mRNA (qRTPCR). In regards to vector competence, flies excreted GFP-P. aeruginosa throughout the 24 h period, serving as both reservoirs and disseminators of this bacterium. Collectively, our data show flies can harbor and disseminate P. aeruginosa, and that the interactions of fly defenses with bacteria can influence vector competence

No More Monkeying Around: Primate Malaria Model Systems are Key to Understanding Plasmodium vivax Liver-Stage Biology, Hypnozoites, and Relapses

Plasmodium vivax is a human malaria parasite responsible for significant morbidity worldwide and potentially death. This parasite possesses formidable liver-stage biology that involves the formation of dormant parasites known as hypnozoites. Hypnozoites are capable of activating weeks, months, or years after a primary blood-stage infection causing relapsing bouts of illness. Elimination of this dormant parasitic reservoir will be critical for global malaria eradication. Although hypnozoites were first discovered in 1982, few advancements have been made to understand their composition and biology. Until recently, in vitro models did not exist to study these forms and studying them from human ex vivo samples was virtually impossible. Today, non-human primate models and modern systems biology approaches are poised as tools to enable the in-depth study of P. vivax liver-stage biology, including hypnozoites and relapses. Non-human primate liver-stage model systems for P. vivax and the related simian malaria species P. cynomolgi are discussed along with perspectives regarding metabolite biomarker discovery, putative roles of extracellular vesicles, and relapse immunobiology

No more monkeying around: primate malaria model systems are key to understanding Plasmodium vivax liver-stage biology, hypnozoites, and relapses

Plasmodium vivax is a human malaria parasite responsible for significant morbidity worldwide and potentially death. This parasite possesses formidable liver-stage biology that involves the formation of dormant parasites known as hypnozoites. Hypnozoites are capable of activating weeks, months, or years after a primary blood-stage infection causing relapsing bouts of illness. Elimination of this dormant parasitic reservoir will be critical for global malaria eradication. Although hypnozoites were first discovered in 1982, few advancements have been made to understand their composition and biology. Until recently, in vitro models did not exist to study these forms and studying them from human ex vivo samples was virtually impossible. Today, non-human primate (NHP) models and modern systems biology approaches are poised as tools to enable the in-depth study of P. vivax liver-stage biology, including hypnozoites and relapses. NHP liver-stage model systems for P. vivax and the related simian malaria species P. cynomolgi are discussed along with perspectives regarding metabolite biomarker discovery, putative roles of extracellular vesicles, and relapse immunobiology

Temporospatial antimicrobial peptide detection in the alimentary canal of house flies fed GFP-<i>P. aeruginosa</i>.

<p>Flies (n = 12 in each of three replicates) were fed an average of 1.05×10⁵ CFU (SD = 3.37×10⁴) bacteria, and at 4, 6, and 8 h post-ingestion flies (n = 4 per replicate) were dissected to remove the alimentary canal. Control flies (n = 4) were fed sterile BHI broth and dissected at 2 h PI. Immunofluorescent microscopy was used to detect the antimicrobial peptides (AMPs) Cecropin (A, B), Defensin (C, D) and Diptericin (E,F). AMPs were detected in the midgut in flies fed bacteria at 6 h PI (B, D, F) by immunofluorescent microscopy, but not in control flies (A, C, E). AMPs also were detected in the midgut at 8 h PI, and in other tissues (not shown, discussed in the text). L, gut lumen. Scale bar = 10 µm.</p

Tissue-specific expression of antimicrobial peptide genes in the alimentary canal of flies that ingested GFP-<i>P. aeruginosa</i>.

<p>House flies (n = 30 in each of two replicates) were fed an average 6.6×10⁴ CFU GFP-<i>P. aeruginosa</i> and at 2, 6, 10, 12, and 24 h post-ingestion, flies (n = 5 per replicate) were dissected to separate the alimentary canal tissues (proventriculus, crop, midgut, hindgut), which were pooled within replicate/time point for qRT-PCR analysis. Fold changes <i>cecropin</i> (A), <i>defensin</i> (B), and <i>diptericin</i> (C) expression were calculated using the REST-MCS© by calibrating to AMP expression levels in unfed adult flies and using the reference gene <i>rps18</i>. Mean log₂-fold changes in expression are shown, and error bars are standard error. Different letters represent significant differences between mean AMP expression levels across tissues within the indicated time point (P≤0.05). Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079224#pone.0079224.s004" target="_blank">Table S3</a> for REST-MCS analysis of tissue-specific AMP expression for each replicate.</p

GFP-<i>P. aeruginosa</i> persisted and proliferated within the house fly.

<p>House flies (n = 20 in each of three replicates) were fed an average of 3.60×10⁵ CFU (SD = 1.75×10⁵) bacteria. At 2, 6, 10, 12, and 24 h post-ingestion, GFP-<i>P. aeruginosa</i> were enumerated from whole flies (n = 4 per replicate) by culture on selective media. Details on culture methods and statistical analysis are in the text. Different letters represent significant differences between mean CFUs recovered from flies (P≤0.04), and error bars are standard error.</p