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

Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice

Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax, responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection

KAF156 is an antimalarial clinical candidate with potential for use in prophylaxis, treatment, and prevention of disease transmission

Renewed global efforts toward malaria eradication have highlighted the need for novel antimalarial agents with activity against multiple stages of the parasite life cycle. We have previously reported the discovery of a novel class of antimalarial compounds in the imidazolopiperazine series that have activity in the prevention and treatment of blood stage infection in a mouse model of malaria. Consistent with the previously reported activity profile of this series, the clinical candidate KAF156 shows blood schizonticidal activity with 50% inhibitory concentrations of 6 to 17.4 nM against P. falciparum drug-sensitive and drug-resistant strains, as well as potent therapeutic activity in a mouse models of malaria with 50, 90, and 99% effective doses of 0.6, 0.9, and 1.4 mg/kg, respectively. When administered prophylactically in a sporozoite challenge mouse model, KAF156 is completely protective as a single oral dose of 10 mg/kg. Finally, KAF156 displays potent Plasmodium transmission blocking activities both in vitro and in vivo. Collectively, our data suggest that KAF156, currently under evaluation in clinical trials, has the potential to treat, prevent, and block the transmission of malaria

MAM 2020: Growing Strong on the 20th Anniversary

Review of presentations at MAM202

Contribution of Horizontal Gene Transfer to Virulence and Antibiotic Resistance in Pathogens Transmitted in Healthcare Facilities.

Healthcare-associated infections (HAIs) are a significant cause of morbidity and mortality in the United States and cost up to $4.5 billion annually. Horizontal gene transfer (HGT) contributes to the evolution and emergence of pathogenic strains causing these infections by allowing DNA to be shared among diverse bacteria. This dissertation identifies and investigates the molecular mechanisms that affect HGT between bacteria and the factors that govern HGT at the patient level. Catheter-associated urinary tract infection (caUTI), the most common HAI, is often polymicrobial. Using comparative genomics, this study identified ICEPm1, a genomic island shared among Proteus mirabilis, Providencia stuartii, and Morganella morganii, common agents of polymicrobial caUTI. We show that this island is an integrative and conjugative element that is self-transmissible between clinical strains at a frequency of 1.35 x 10-5 and that transfer is dependent on an integrase and a type IV secretion system encoded within the element. ICEPm1 also encodes an adhesin and known iron-acquisition system; therefore ICEPm1 may provide a fitness advantage during colonization of the catheterized urinary tract. In support of this, I observed that ICEPm1 was present in 39/39 P. mirabilis urinary isolates screened, while distribution was heterogeneous (15/23) among P. mirabilis commensal strains. Because proximity is necessary for HGT to occur, we investigated risk factors for co-colonization with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) among residents of long-term care facilities. Co-colonization with these organisms is of interest because of the emergence of vancomycin-resistant S. aureus (VRSA); attributable to HGT of the vanA gene cluster from VRE to MRSA. Because MRSA remains susceptible to vancomycin the emergence of vancomycin-resistant S. aureus is alarming for healthcare professionals. We show that wounds, indwelling devices and functional disability are risk factors for co-colonization with MRSA and VRE; an event that precedes VRSA emergence. This dissertation provides insight into the mechanisms of transfer of mobile elements and therefore dissemination of virulence and antibiotic resistance genes. It also describes risk factors for co-colonization with MRSA and VRE within a patient, an event that is necessary for HGT to occur in vivo.Ph.D.Epidemiological ScienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86421/1/eflanner_1.pd

Parasite of the Month: Plasmodium vivax

Plasmodium vivax is a eukaryotic human pathogen and the most frequent and widely distributed cause of the recurring disease malaria. P. vivax is one of five species of malaria parasites that commonly infect humans. Although less virulent than Plasmodium falciparum, P. vivax malaria infections can lead to severe disease and death. The disease is transmitted to humans by female anopheline mosquitoes during acquisition of a blood meal. An obligate liver stage of development precedes blood stage replication which accounts for all disease-associated morbidity and mortality and chloroquine is still used to treat the disease. Approximately 2.5 billion people are at risk of infection with P. vivax, and it is found mainly in Asia and Latin America where it accounts for 65% of malaria cases. In Africa, the widespread lack of the Duffy antigen in the population has constrained transmission. A dormant liver stage form, known as the hypnozoite can reactivate and causes disease relapses and the majority of blood stage infections are caused by relapse. The hypnozoite is thus a challenge for malaria eradication campaigns and primaquine and tafenoquine are the only drugs known to target the hypnozoite. However, these therapies are inadequate since primaquine cannot be administered to pregnant women and people with commonly occurring glucose-6-phosphate dehydrogenase deficiency and tafenoquine cannot be administered to children. There is no effective P. vivax vaccine