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Author: Donna Huber

Increasing the knowledge base on brain-eating amoeba

graduate student Cassie Russell in front of biological safety cabinet in Dennis Kyle's laboratory at the University of Georgia
Cassie Russell, a graduate student in the Department of Infectious Diseases, in her laboratory space. (Photo by Ian Bennett)

Cassie Russell, a graduate student in the Department of Infectious Diseases, was an undergraduate when she first heard of Naegleria fowleri, also known as the brain-eating amoeba. While whole lectures in her parasitology course had been dedicated to other parasites, N. fowleri was barely a mention.

“I remember maybe 15 minutes was spent on it,” said Russell. “I was shocked that was all that was known about this deadly organism.”

N. fowleri causes the acute neurological disease primary amoebic meningoencephalitis (PAM). There have been hundreds of reported cases of PAM, but only seven survivors worldwide, according to the Centers for Disease Control and Prevention.

SEM image of Naegleria fowleri
Scanning electron microscopy image of Naegleria fowleri (submitted by Cassie Russell)

“I had the opportunity to speak with families in Florida who had lost someone to Naegleria fowleri infection,” she said. “The fear they had in not knowing what was wrong with their loved one and then learning that there was very little that could be done—their stories were just heartbreaking.”After arriving at UGA, Russell was pleased to find out that N. fowleri was one of the parasites being studied in Dennis Kyle’s laboratory at the Center for Tropical and Emerging Global Diseases.

Individuals, most commonly young children, become infected when they inhale warm freshwater contaminated with N. fowleri. This typically occurs during the late summer months when people are participating in recreational activities in rivers and lakes, but it can also occur when people use unsterilized tap water in nasal irrigation devices. It is more likely to occur in the southern United States, but infection is very rare. Between 2011 and 2020 only 33 cases were reported in the United States, according the CDC.

N. fowleri is one of the most neglected of the neglected tropical diseases. However, knowledge about the parasite has been growing since the 1960s as scientists build on new data and apply new technology. Russell is doing her part and was the lead on a study recently published in Microbiology Spectrum where, for the first time, drug susceptibility was tested across 11 clinical isolates.

“Current drug treatment is a cocktail of six different drugs,” said Russell. “However, only a few isolates have been tested in the lab for susceptibility. We don’t know if some drugs work better for different strains.”

A big question facing researchers is why these drugs show effectiveness in the lab when so few real-world cases have been successfully treated. Russell suspected that other factors were at play in treatment failure, such as genetic differences among geographically distinct amoeba populations.

The 11 isolates used in the study came from patients who contracted N. fowleri in different geographic regions. Russell found that these isolates had significant differences in susceptibility to seven of the eight drugs currently used to treat the infection.

The need for effective and fast-acting treatments is especially great. PAM is almost always fatal, with death occurring about a week after the initial onset of symptoms.

Doctors are racing against the clock as there is often a delay in diagnosis: The symptoms mimic meningitis, and N. fowleri is a rare infection. The drugs used can also be pretty toxic, so identifying the safest and most effective drug treatment could significantly improve outcomes.

Russell’s findings are another stepping stone to propel N. fowleri research toward increased understanding of this parasite and ultimately better treatments. For example, she realized that there is not a gold standard for genotyping.

“Researchers could be talking about genetically different isolates but not realize it,” said Russell.

In addition to creating a genotyping standard, she has identified combinational drug studies to test for synergism as a next step. For now, though, Russell is focusing on another need in the fight against N. fowleri—diagnostics.

“Awareness, improved diagnostic techniques and faster-acting drugs are needed to improve outcomes,” she said.

 

This story first appeared at https://research.uga.edu/news/increasing-the-knowledge-base-on-brain-eating-amoeba/

Polymorphic Molecular Signatures in Variable Regions of the Plasmodium falciparum var2csa DBL3x Domain Are Associated with Virulence in Placental Malaria

The Plasmodium falciparum protein VAR2CSA allows infected erythrocytes to accumulate within the placenta, inducing pathology and poor birth outcomes. Multiple exposures to placental malaria (PM) induce partial immunity against VAR2CSA, making it a promising vaccine candidate. However, the extent to which VAR2CSA genetic diversity contributes to immune evasion and virulence remains poorly understood. The deep sequencing of the var2csa DBL3X domain in placental blood from forty-nine primigravid and multigravid women living in malaria-endemic western Kenya revealed numerous unique sequences within individuals in association with chronic PM but not gravidity. Additional analysis unveiled four distinct sequence types that were variably present in mixed proportions amongst the study population. An analysis of the abundance of each of these sequence types revealed that one was inversely related to infant gestational age, another was inversely related to placental parasitemia, and a third was associated with chronic PM. The categorization of women according to the type to which their dominant sequence belonged resulted in the segregation of types as a function of gravidity: two types predominated in multigravidae whereas the other two predominated in primigravidae. The univariate logistic regression analysis of sequence type dominance further revealed that gravidity, maternal age, placental parasitemia, and hemozoin burden (within maternal leukocytes), reported a lack of antimalarial drug use, and infant gestational age and birth weight influenced the odds of membership in one or more of these sequence predominance groups. Cumulatively, these results show that unique var2csa sequences differentially appear in women with different PM exposure histories and segregate to types independently associated with maternal factors, infection parameters, and birth outcomes. The association of some var2csa sequence types with indicators of pathogenesis should motivate vaccine efforts to further identify and target VAR2CSA epitopes associated with maternal morbidity and poor birth outcomes.

Eldin Talundzic, Stephen Scott, Simon O Owino, David S Campo, Naomi W Lucchi, Venkatachalam Udhayakumar, Julie M Moore, David S Peterson. Pathogens. 2022 Apr 28;11(5):520. doi: 10.3390/pathogens11050520

Essential Bromodomain Tc BDF2 as a Drug Target against Chagas Disease

Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein containing a bromodomain was recently described. Using the Tet-regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing, we were able to demonstrate the essentiality of TcBDF2 in T. cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results point to TcBDF2 as a druggable target against T. cruzi.

Alejandro Pezza, Luis E Tavernelli, Victoria L Alonso, Virginia Perdomo, Raquel Gabarro, Rab Prinjha, Elvio Rodríguez Araya, Inmaculada Rioja, Roberto Docampo, Felix Calderón, Julio Martin, Esteban Serra. ACS Infect Dis. 2022 Apr 28. doi: 10.1021/acsinfecdis.2c00057.

Improving in vitro continuous cultivation of Plasmodium cynomolgi, a model for P. vivax

The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Peter Christensen, Annie Racklyeft, Kurt E Ward, Jessica Matheson, Rossarin Suwanarusk, Adeline C Y Chua, Osamu Kaneko, Htin Lin Aung, Laurent Rénia, Nadia Amanzougaghene, Victor Magneron, Julien Lemaitre, Roger Le Grand, Dennis Kyle, Pablo Bifani, Gregory M Cook, Georges Snounou, Bruce Russell. Parasitol Int. 2022 Apr 22;89:102589. doi: 10.1016/j.parint.2022.102589. Online ahead of print.

Alkyne modified purines for assessment of activation of Plasmodium vivax hypnozoites and growth of pre-erythrocytic and erythrocytic stages in Plasmodium spp

Graphical Abstract

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite’s ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesized that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalyzed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.

Alona Botnar, Grant Lawrence, Steven P Maher, Amélie Vantaux, Benoît Witkowski, Justine C Shiau, Emilio F Merino, David De Vore, Christian Yang, Cameron Murray, Maria B Cassera, James W Leahy, Dennis E Kyle. Int J Parasitol. 2022 Apr 18;S0020-7519(22)00066-2. doi: 10.1016/j.ijpara.2022.03.003.

Ad libitum consumption of protein- or peptide-sucrose solutions stimulates egg formation by prolonging the vitellogenic phase of oogenesis in anautogenous mosquitoes

Background: Anautogenous mosquitoes commonly consume nectars and other solutions containing sugar but are thought to only produce eggs in discrete gonadotrophic cycles after blood-feeding on a vertebrate host. However, some anautogenous species are known to produce eggs if amino acids in the form of protein are added to a sugar solution. Unclear is how different sources of amino acids in sugar solutions affect the processes that regulate egg formation and whether responses vary among species. In this study, we addressed these questions by focusing on Aedes aegypti and conducting some comparative assays with Aedes albopictus, Anopheles gambiae, Anopheles stephensi and Culex quinquefasciatus.

Methods: Adult female mosquitoes were fed sugar solutions containing amino acids, peptides or protein. Markers for activation of a gonadotrophic cycle including yolk deposition into oocytes, oviposition, ovary ecdysteroidogenesis, expression of juvenile hormone and 20-hydroxyecdysone-responsive genes, and adult blood-feeding behavior were then measured.

Results: The five anautogenous species we studied produced eggs when fed two proteins (bovine serum albumin, hemoglobin) or a mixture of peptides (tryptone) in 10% sucrose but deposited only small amounts of yolk into oocytes when fed amino acids in 10% sucrose. Focusing on Ae. aegypti, cultures were maintained for multiple generations by feeding adult females protein- or tryptone-sugar meals. Ad libitum access to protein- or tryptone-sugar solutions protracted production of ecdysteroids by the ovaries, vitellogenin by the fat body and protease activity by the midgut albeit at levels that were lower than in blood-fed females. Females also exhibited semi-continual oogenesis and repressed host-seeking behavior.

Conclusions: Several anautogenous mosquitoes produce eggs when provided ad libitum access to protein- or peptide-sugar meals, but several aspects of oogenesis also differ from females that blood-feed.

Ruby E Harrison, Kangkang Chen, Lilith South, Ange Lorenzi, Mark R Brown, Michael R Strand. Parasit Vectors. 2022 Apr 12;15(1):127. doi: 10.1186/s13071-022-05252-4.

Polychlorinated cyclopentenes from a marine derived Periconia sp. (strain G1144)

Studies on an organic extract of a marine fungus, Periconia sp. (strain G1144), led to the isolation of three halogenated cyclopentenes along with the known and recently reported rhytidhyester D; a series of spectrometric and spectroscopic techniques were used to elucidate these structures. Interestingly, two of these compounds represent tri-halogenated cyclopentene derivatives, which have been observed only rarely from Nature. The relative and absolute configurations of the compounds were established via mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, Mosher’s esters method, optical rotation and GIAO NMR calculations, including correlation coefficient calculations and the use of both DP4+ and dJ DP4 analyses. Several of the isolated compounds were tested for activity in anti-parasitic, antimicrobial, quorum sensing inhibition, and cytotoxicity assays and were shown to be inactive.

Kristóf B Cank, Robert A Shepherd, Sonja L Knowles, Manuel Rangel-Grimaldo, Huzefa A Raja, Zoie L Bunch, Nadja B Cech, Christopher A Rice, Dennis E Kyle, Joseph O Falkinham 3rd, Joanna E Burdette, Nicholas H Oberlies. Phytochemistry . 2022 Apr 11;113200. doi: 10.1016/j.phytochem.2022.113200

Creating databases to help cure diseases worldwide

Jessica Kissinger poses for a photo in the Infectious Diseases Institute in Uganda where she is currently a US Fullbright Scholar. (Photo/Courtesy Jessica Kissinger)

Jessica Kissinger is using her expertise in biology and big data to help other scientists

 

Jessica Kissinger never set out to make databases. From the time she was a little girl, she wanted to be a biologist.

Today, the University of Georgia professor not only studies deadly pathogens like malaria and Cryptosporidium (a waterborne parasite), but also is a driving force behind worldwide, groundbreaking collaborations on novel databases. During her time at UGA, she has received nearly $40 million in federal and private grants and contracts.

These databases can crunch vast amounts of biological information at warpspeed and reveal important patterns that pave the way for new approaches to scourges such as Leishmania (common in the tropics, subtropics, and southern Europe), toxoplasmosis (a systemic disease due to one of the world’s most common parasites), and Valley Fever (a fungus born on the wind that can cause lung and systemic infections). Novel drug and vaccine targets can be developed, as well as fresh insights on life-threatening pathogens.

“Fighting infections and developing new drug and vaccine targets requires detailed knowledge of a pathogen and how it functions,” explained Kissinger, a Distinguished Research Professor in UGA’s Department of Genetics, Institute of Bioinformatics and Center for Tropical and Emerging Global Diseases.

And, like internet searches, the databases are all free. Kissinger said it’s likely that pharmaceutical companies are mining some of the information in their quest to discover new therapeutic targets.

“They don’t tell us what they’re working on,” she said. “A database itself doesn’t produce a cure. A database can, however, remove most barriers to analysis of existing data.”

Big Data paves the way for big advances in science

It once took an entire decade to sequence a single genome—and the cost was many millions. Today, researchers can sequence a genome in a single afternoon for a few thousand dollars, transforming the field of genomics. Similar astounding advances have reshaped other ‘omics’ specialties, such as proteomics (study of proteins), metabolomics (study of metabolism), transcriptomics (study of RNA), and epigenomics (the influence of the environment on gene function). These advances mark the “Big Data” era in biology.

“The power that is unleashed by big data is phenomenal,” said Kissinger, “and it’s a very exciting time in history, with major funders and visionaries all across the world forming consortia to create a kind of ideal data universe.” Like explorers trekking into a new world, they will make discoveries we might only imagine right now.

Creating a malaria database

Kissinger’s innovations began over 23 years ago, while she was a postdoctoral researcher at the University of Pennsylvania studying a single-celled parasite called Toxoplasma gondii. The parasite shares some important features with the malaria pathogen, whose genome was in the process of being sequenced.

“I rounded up genome data from all over the world on Plasmodium (the causative agent of malaria), and ran analyses and put it on a website, so I could study the genes it might share with Toxoplasma,” she recalled. “It turns out nobody had made the Plasmodium data available for searching before.”

Soon she and her adviser, David Roos, had a million-dollar grant to formally establish a malaria database, PlasmoDB, and since its launch in 1999 it has grown to include additional pathogens and received continual funding from the NIH, the most recent for up to $38.4 million to maintain what has now become the Eukaryotic Pathogen, Vector and Host Informatics Resources knowledgebase (VEuPathDB), covering 14 different pathogens as well as host responses to infections. This comprehensive database is an integrated centralized resource for data mining on over 500 organisms.

The databases collectively contain over nine terabytes (9,000 gigabytes) of data, and have been compared to a Wikipedia for molecular parasitology by the British Society for Parasitology, which noted back in 2006: “We don’t know what we would do without it!”

Each month, VEuPathDB receives over 11 million hits from an average of 36,000 unique visitors in more than 100 countries, including India, Brazil and Kenya. A related database on vectors of disease (such as ticks that carry Lyme disease) was recently merged into VEuPathDB. The merger expanded each resource and enables researchers to better explore data on vectors such as ticks and mosquitoes and the pathogens they transmit.

Powerful tools are key to analyzing data

The databases are not just strings of numbers or words. They allow visualizations and graphic interfaces. Already, research is emerging that can help direct vaccine and drug development away from proteins that hosts and pathogens share, in order to protect the cell. Scientists using the databases have discovered proteins that reduce severe malaria and other proteins that protect malaria parasites from the human fever response. They have also found proteins that help Toxoplasma penetrate host cells.

In a single year an average of 200 publications a month cite VEuPathDB, and to date there have already been 24,000 citations total. Next up: cloud-ready applications and improved integration with yet other databases. These databases “have become essential data mining and access platforms for fungal and parasite genomics research,” said microbiologist and plant pathologist Jason Stajich of the University of California at Riverside.

“Without powerful, user-friendly tools to analyze it, “Big Data” is more a curse than a blessing,” explained John Boothroyd, an immunologist and microbiologist at Stanford University School of Medicine. “VEuPathDB is just such a tool and we owe Jessica Kissinger and her colleagues an enormous thank you for their tireless and selfless efforts to first conceive and then continuously improve this absolutely essential resource.”

Grants for related projects have come from a wide array of organizations, among them the Bill & Melinda Gates Foundation, the Sloan Foundation, and the World Health Organization. One of those projects, called ClinEpiDB, is home to a multicenter study that contains data from over 22,000 children from seven different sites in South Asia and Africa. This study is the largest ever to investigate the causes of diarrhea in children in lower- to middle-income countries. Other uses of ClinEpiDB include new data on hidden signs of malaria transmission in areas where incidence is declining, or how breastfeeding protects infants from common infections.

The VEuPathDB database would be enough to secure Kissinger’s reputation in the biological sciences, but she has not stopped there. At the University of Georgia, she was a founding member of the Institute of Bioinformatics, and served as its director from 2011 to 2109. The Institute’s mission is to facilitate cutting-edge interdisciplinary research in computational biology, and the program offers both masters and doctorates. She is a key researcher helping to partner a national hub for infectious disease research by linking with Emory University in Atlanta. The two institutions have grants totaling over $45 million to work on everything from tuberculosis to HIV to malaria.

“These databases are a success beyond my wildest dreams,” said Kissinger. “They are made by biologists for other biologists and address a real-life need.”

 

This story first appeared at UGA Today.

Anopheles gambiae strain (Ag55) cultured cells originated from Anopheles coluzzii and are phagocytic with hemocyte-like gene expression

Anopheles gambiae and Anopheles coluzzii are closely related species that are predominant vectors of malaria in Africa. Recently, A. gambiae form M was renamed A. coluzzii and we now conclude on the basis of a diagnostic PCR-restriction fragment length polymorphism assay that Ag55 cells were derived from A. coluzzii. We established an Ag55 cell transcriptome, and KEGG pathway analysis showed that Ag55 cells are enriched in phagosome pathway transcripts. The Ag55 transcriptome has an abundance of specific transcripts characteristic of mosquito hemocytes. Functional E. coli bioparticle uptake experiments visualized by fluorescence microscopy and confocal microscopy and quantified by flow cytometry establish the phagocytic competence of Ag55 cells. Results from this investigation of Ag55 cell properties will guide researchers in the use and engineering of the Ag55 cell line to better enable investigations of Plasmodium, other microbes, and insecticidal toxins. Graphical abstract: Anopheles gambiae cultured Ag55 cells originated from Anopheles coluzzi, have a hemocyte-like transcriptome and are phagocytic. This article is protected by copyright. All rights reserved.

Ruchir Mishra, Gang Hua, Ujwal R Bagal, Donald E Champagne, Michael J Adang. Insect Sci. 2022 Mar 31. doi: 10.1111/1744-7917.13036.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12)

The catechol derivative RC-12 (WR 27653) (1) is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard Plasmodium cynomolgi-rhesus monkey (Macaca mulatta) model, but in a small clinical trial, it had no efficacy against Plasmodium vivax hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of 1 and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and in vitro liver-stage activity of 1 and its metabolites. Compound 1 had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the O-desmethyl, combined O-desmethyl/N-desethyl, and N,N-didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of 1 in rhesus monkeys vs that seen in humans. Indeed, the rate of metabolism was considerably lower in human liver microsomes in comparison to rhesus monkey microsomes, as was the formation of the combined O-desmethyl/N-desethyl metabolite, which was the only metabolite tested that had any activity against liver-stage P. vivax; however, it was not consistently active against liver-stage P. cynomolgi. As 1 and all but one of its identified Phase I metabolites had no in vitro activity against P. vivax or P. cynomolgi liver-stage malaria parasites, we suggest that there may be additional unidentified active metabolites of 1 or that the exposure of 1 achieved in the reported unsuccessful clinical trial of this drug candidate was insufficient to kill the P. vivax hypnozoites.

Yuxiang Dong, Yogesh Sonawane, Steven P Maher, Anne-Marie Zeeman, Victor Chaumeau, Amélie Vantaux, Caitlin A Cooper, Francis C K Chiu, Eileen Ryan, Jenna McLaren, Gong Chen, Sergio Wittlin, Benoît Witkowski, François Nosten, Kamaraj Sriraghavan, Dennis E Kyle, Clemens H M Kocken, Susan A Charman, Jonathan L Vennerstrom. ACS Omega. 2022 Mar 30;7(14):12401-12411. doi: 10.1021/acsomega.2c01099.