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Category: publications

Cryopreservation of Plasmodium Sporozoites

Cryopreservation protocol for Plasmodium sporozoites.

Malaria is a deadly disease caused by the parasite, Plasmodium, and impacts the lives of millions of people around the world. Following inoculation into mammalian hosts by infected mosquitoes, the sporozoite stage of Plasmodium undergoes obligate development in the liver before infecting erythrocytes and causing clinical malaria. The most promising vaccine candidates for malaria rely on the use of attenuated live sporozoites to induce protective immune responses. The scope of widespread testing or clinical use of such vaccines is limited by the absence of efficient, reliable, or transparent strategies for the long-term preservation of live sporozoites. Here we outline a method to cryopreserve the sporozoites of various human and murine Plasmodium species. We found that the structural integrity, viability, and in vivo or in vitro infectiousness were conserved in the recovered cryopreserved sporozoites. Cryopreservation using our approach also retained the transgenic properties of sporozoites and immunization with cryopreserved radiation attenuated sporozoites (RAS) elicited strong immune responses. Our work offers a reliable protocol for the long-term storage and recovery of human and murine Plasmodium sporozoites and lays the groundwork for the widespread use of live sporozoites for research and clinical applications.

Carson Bowers, Lisa Hancox, Kristen Peissig, Justine C. Shiau, Amélie Vantaux, Benoit Witkowski, Sivchheng Phal, Steven P. Maher, John T. Harty, Dennis E. Kyle, and Samarchith P. Kurup. Pathogens 2022, 11(12), 1487; https://doi.org/10.3390/pathogens11121487

Integrative genetic manipulation of Plasmodium cynomolgi reveals MultiDrug Resistance-1 Y976F associated with increased in vitro susceptibility to mefloquine

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized P. cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using CRISPR-Cas9 increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species.

Kurt E Ward, Peter Christensen, Annie Racklyeft, Satish K Dhingra, Adeline C Y Chua, Caroline Remmert, Rossarin Suwanarusk, Jessica Matheson, Michael J Blackman, Osamu Kaneko, Dennis E Kyle, Marcus C S Lee, Robert W Moon, Georges Snounou, Laurent Rénia, David A Fidock, Bruce Russell, Pablo Bifani. J Infect Dis. 2022 Dec 7;jiac469. doi: 10.1093/infdis/jiac469. Online ahead of print.

An aphid symbiont confers protection against a specialized RNA virus, another increases vulnerability to the same pathogen

Insects often harbor heritable symbionts that provide defense against specialized natural enemies, yet little is known about symbiont protection when hosts face simultaneous threats. In pea aphids (Acyrthosiphon pisum), the facultative endosymbiont Hamiltonella defensa confers protection against the parasitoid, Aphidius ervi, and Regiella insecticola protects against aphid-specific fungal pathogens, including Pandora neoaphidis. Here we investigated whether these two common aphid symbionts protect against a specialized virus A. pisum virus (APV), and whether their anti-fungal and anti-parasitoid services are impacted by APV infection. We found that APV imposed large fitness costs on symbiont-free aphids and these costs were elevated in aphids also housing H. defensa. In contrast, APV titers were significantly reduced and costs to APV infection were largely eliminated in aphids with R. insecticola. To our knowledge, R. insecticola is the first aphid symbiont shown to protect against a viral pathogen, and only the second arthropod symbiont reported to do so. In contrast, APV infection did not impact the protective services of either R. insecticola or H. defensa. To better understand APV biology, we produced five genomes and examined transmission routes. We found that moderate rates of vertical transmission, combined with horizontal transfer through food plants, were the major route of APV spread, although lateral transfer by parasitoids also occurred. Transmission was unaffected by facultative symbionts. In summary, the presence and species identity of facultative symbionts resulted in highly divergent outcomes for aphids infected with APV, while not impacting defensive services that target other enemies. These findings add to the diverse phenotypes conferred by aphid symbionts, and to the growing body of work highlighting extensive variation in symbiont-mediated interactions.

C H V Higashi, W L Nichols, G Chevignon, V Patel, S E Allison, K L Kim, M R Strand, K M Oliver. Mol Ecol. 2022 Dec 2. doi: 10.1111/mec.16801. Online ahead of print.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Jeremy D DeBarry, Mustafa V Nural, Suman B Pakala, Vishal Nayak, Susanne Warrenfeltz, Jay Humphrey, Stacey A Lapp, Monica Cabrera-Mora, Cristiana F A Brito, Jianlin Jiang, Celia L Saney, Allison Hankus, Hannah M Stealey, Megan B DeBarry, Nicolas Lackman, Noah Legall, Kevin Lee, Yan Tang, Anuj Gupta, Elizabeth D Trippe, Robert R Bridger, Daniel Brent Weatherly, Mariko S Peterson, Xuntian Jiang, ViLinh Tran, Karan Uppal, Luis L Fonseca, Chester J Joyner, Ebru Karpuzoglu, Regina J Cordy, Esmeralda V S Meyer, Lance L Wells, Daniel S Ory, F Eun-Hyung Lee, Rabindra Tirouvanziam, Juan B Gutiérrez 1, Chris Ibegbu, Tracey J Lamb, Jan Pohl, Sarah T Pruett, Dean P Jones, Mark P Styczynski, Eberhard O Voit, Alberto Moreno, Mary R Galinski, Jessica C Kissinger. Sci Data. 2022 Nov 24;9(1):722. doi: 10.1038/s41597-022-01755-y.

Avoiding clinical trial failures in neglected tropical diseases: The example of Chagas disease

Human clinical trials are expensive, and when they fail, they create the impression that a problem is intractable, thus depressing interest in future attempts. For neglected tropical diseases, where there are likely limited numbers of “shots on goal”, such failures need to be assiduously avoided. Chagas disease drug discovery efforts have experienced more than its share of human clinical trial failures. Here Here are some guidelines, many specific for Chagas, but some which might also have application for other neglected tropical diseases. Chagas disease has major challenges (e.g., the lack of a definitive test of cure) but also has outstanding advantages, among these the unmatched multi-species natural infection systems that can be exploited to de-risk compounds before human trials. Fully utilizing these advantages while frankly acknowledging and addressing the challenges should bring better options to patients, sooner.

Rick Tarleton. Clin Infect Dis. 2022 Nov 14;ciac884. doi: 10.1093/cid/ciac884. Online ahead of print.

Evaluation of delayed LNFPIII treatment initiation protocol on improving long-term behavioral and neuroinflammatory pathology in a mouse model of Gulf War Illness

Chemical overexposures and war-related stress during the 1990-1991 Gulf War (GW) are implicated in the persisting pathological symptoms that many GW veterans continue to endure. These symptoms culminate into a disease known as Gulf War Illness (GWI) and affect about a third of the GW veteran population. Currently, comprehensive effective GWI treatment options are unavailable. Here, an established GWI mouse model was utilized to explore the (1) long-term behavioral and neuroinflammatory effects of deployment-related GWI chemicals exposure and (2) ability of the immunotherapeutic lacto-N-fucopentaose III (LNFPIII) to improve deficits when given months after the end of exposure. Male C57BL6/J mice (8-9 weeks old) were administered pyridostigmine bromide (PB) and DEET for 14 days along with corticosterone (CORT; latter 7 days) to emulate wartime stress. On day 15, a single injection of the nerve agent surrogate diisopropylfluorophosphate (DFP) was given. LNFPIII treatment began 7 months post GWI chemicals exposure and continued until study completion. A battery of behavioral tests for assessment of cognition/memory, mood, and motor function in rodents was performed beginning 8 months after exposure termination and was then followed by immunohistochemcal evaluation of neuroinflammation and neurogenesis. Within tests of motor function, prior GWI chemical exposure led to hyperactivity, impaired sensorimotor function, and altered gait. LNFPIII attenuated these motor-related deficits and improved overall grip strength. GWI mice also exhibited more anxiety-like behavior that was reduced by LNFPIII; this was test-specific. Short-term, but not long-term memory, was impaired by prior GWI exposure; LNFPIII improved this measure. In the brains of GWI mice, but not in mice treated with LNFPIII, glial activation was increased. Overall, it appears that months after exposure to GWI chemicals, behavioral deficits and neuroinflammation are present. Many of these deficits were attenuated by LNFPIII when treatment began long after GWI chemical exposure termination, highlighting its therapeutic potential for veterans with GWI.

Jessica M Carpenter, Kyle A Brown, Lukas Veltmaat, Helaina D Ludwig, Kendall B Clay, Thomas Norberg, Donald A Harn, John J Wagner, Nikolay M Filipov. Brain Behav Immun Health. 2022 Nov 8;26:100553. doi: 10.1016/j.bbih.2022.100553. eCollection 2022 Dec.

Gastrointestinal helminths increase Bordetella bronchiseptica shedding and host variation in supershedding

Co-infected hosts, individuals that carry more than one infectious agent at any one time, have been suggested to facilitate pathogen transmission, including the emergence of supershedding events. However, how the host immune response mediates the interactions between co-infecting pathogens and how these affect the dynamics of shedding remains largely unclear. We used laboratory experiments and a modeling approach to examine temporal changes in the shedding of the respiratory bacterium Bordetella bronchiseptica in rabbits with one or two gastrointestinal helminth species. Experimental data showed that rabbits co-infected with one or both helminths shed significantly more B. bronchiseptica, by direct contact with an agar petri dish, than rabbits with bacteria alone. Co-infected hosts generated supershedding events of higher intensity and more frequently than hosts with no helminths. To explain this variation in shedding an infection-immune model was developed and fitted to rabbits of each group. Simulations suggested that differences in the magnitude and duration of shedding could be explained by the effect of the two helminths on the relative contribution of neutrophils and specific IgA and IgG to B. bronchiseptica neutralization in the respiratory tract. However, the interactions between infection and immune response at the scale of analysis that we used could not capture the rapid variation in the intensity of shedding of every rabbit. We suggest that fast and local changes at the level of respiratory tissue probably played a more important role. This study indicates that co-infected hosts are important source of variation in shedding, and provides a quantitative explanation into the role of helminths to the dynamics of respiratory bacterial infections.

Nhat TD Nguyen, Ashutosh K Pathak, Isabella M Cattadori (2022) Gastrointestinal helminths increase Bordetella bronchiseptica shedding and host variation in supershedding eLife 11:e70347. https://doi.org/10.7554/eLife.70347

Cephalotane-type C20 diterpenoids from Cephalotaxus fortunei var. alpina

Seventeen new cephalotane-type diterpenoids, fortalides A-Q (1-17), along with five known analogues, were isolated from the seeds of Cephalotaxus fortunei var. alpina. Their structures were determined by extensive spectroscopic methods, as well as electronic circular dichroism (ECD) and X-ray crystallographic data analyses. Some isolates exhibited unusual structural features that were first found in cephalotane-type diterpenoids, such as the occurrence of the 7-oxabicyclo[4.1.1]octane moiety in 14 and 15 and the cis-arrangement of 3-OH and Me-19 in 9. Besides, the antiplasmodial activity of these compounds was evaluated in this study.

Zhan-Peng Ge, Bin Zhou, Flavia M Zimbres, Reagan S Haney, Qun-Fang Liu, Yan Wu, Maria B Cassera, Jin-Xin Zhao, Jian-Min Yue. Org Biomol Chem. 2022 Nov 4. doi: 10.1039/d2ob01748b

Pseudokinase NRP1 facilitates endocytosis of transferrin in the African trypanosome

Trypanosoma brucei causes human African trypanosomiasis (HAT) and nagana in cattle. During infection of a vertebrate, endocytosis of host transferrin (Tf) is important for viability of the parasite. The majority of proteins involved in trypanosome endocytosis of Tf are unknown. Here we identify pseudokinase NRP1 (Tb427tmp.160.4770) as a regulator of Tf endocytosis. Genetic knockdown of NRP1 inhibited endocytosis of Tf without blocking uptake of bovine serum albumin. Binding of Tf to the flagellar pocket was not affected by knockdown of NRP1. However the quantity of Tf per endosome dropped significantly, consistent with NRP1 promoting robust capture and/or retention of Tf in vesicles. NRP1 is involved in motility of Tf-laden vesicles since distances between endosomes and the kinetoplast were reduced after knockdown of the gene. In search of possible mediators of NRP1 modulation of Tf endocytosis, the gene was knocked down and the phosphoproteome analyzed. Phosphorylation of protein kinases forkhead, NEK6, and MAPK10 was altered, in addition to EpsinR, synaptobrevin and other vesicle-associated proteins predicted to be involved in endocytosis. These candidate proteins may link NRP1 functionally either to protein kinases or to vesicle-associated proteins.

Gaurav Kumar, Bryanna Thomas, Kojo Mensa-Wilmot. Sci Rep. 2022 Nov 3;12(1):18572. doi: 10.1038/s41598-022-22054-x.

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi infection and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections.

Juan M Bustamante, Angel M Padilla, Brooke White, Lisa D Auckland, Rachel E Busselman, Stephanie Collins, Elizabeth L Malcolm, Briana F Wilson, Ashley B Saunders, Sarah A Hamer, Rick L Tarleton. PLoS Negl Trop Dis. 2022 Oct 31;16(10):e0010688. doi: 10.1371/journal.pntd.0010688.