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Author: Donna Huber

Toll9 from Bombyx mori functions as a pattern recognition receptor that shares features with Toll-like receptor 4 from mammals

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spätzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)-like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2-like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9-TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4-MD-2-LPS pathway.

Ruonan Zhang, Xiaofeng Li, Jie Zhang, Yanjun Li, Yuan Wang, Yuhang Song, Feifei Ren, Huiyu Yi, Xiaojuan Deng, Yangjin Zhong, Yang Cao, Michael R Strand, Xiao-Qiang Yu, Wanying Yang. Proc Natl Acad Sci U S A. 2021 May 11;118(19):e2103021118. doi: 10.1073/pnas.2103021118.

Lacto-N-fucopentaose-III (LNFPIII) ameliorates acute aberrations in hippocampal synaptic transmission in a Gulf War Illness animal model

Approximately one-third of Persian Gulf War veterans are afflicted by Gulf War Illness (GWI), a chronic multisymptom condition that fundamentally presents with cognitive deficits (i.e., learning and memory impairments) and neuroimmune dysfunction (i.e., inflammation). Factors associated with GWI include overexposures to neurotoxic pesticides and nerve agent prophylactics such as permethrin (PM) and pyridostigmine bromide (PB), respectively. GWI-related neurological impairments associated with PB-PM overexposures have been recapitulated in animal models; however, there is a paucity of studies assessing PB-PM-related aberrations in hippocampal synaptic plasticity and transmission that may underlie behavioral impairments. Importantly, FDA-approved neuroactive treatments are currently unavailable for GWI. In the present study, we assessed the efficacy of an immunomodulatory therapeutic, lacto-N-fucopentaose-III (LNFPIII), on ameliorating acute effects of in vivo PB-PM exposure on synaptic plasticity and transmission as well as trophic factor/cytokine expression along the hippocampal dorsoventral axis. PB-PM exposure resulted in hippocampal synaptic transmission deficits 48 h post-exposure, a response that was ameliorated by LNFPIII coadministration, particularly in the dorsal hippocampus (dH). LNFPIII coadministration also enhanced synaptic transmission in the dH and the ventral hippocampus (vH). Notably, LNFPIII coadministration elevated long-term potentiation in the dH. Further, PB-PM exposure and LNFPIII coadministration uniquely altered key inflammatory cytokine and trophic factor production in the dH and the vH. Collectively, these findings demonstrate that PB-PM exposure impaired hippocampal synaptic responses 48 h post-exposure, impairments that differentially manifested along the dorsoventral axis. Importantly, LNFPIII ameliorated GWI-related electrophysiological deficits, a beneficial effect indicating the potential efficacy of LNFPIII for treating GWI.

Kyle A Brown, Collin J Preston, Jessica M Carpenter, Helaina D Ludwig, Thomas Norberg, Donald A Harn, Nikolay M Filipov, John J Wagner. Brain Res. 2021 May 4;147513. doi: 10.1016/j.brainres.2021.147513.

A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia

Mitochondrial genome content and structure vary widely across the eukaryotic tree of life, with protists displaying extreme examples. Apicomplexan and dinoflagellate protists have evolved highly reduced mitochondrial genome sequences, mtDNA, consisting of only three cytochrome genes and fragmented rRNA genes. Here, we report the independent evolution of fragmented cytochrome genes in Toxoplasma and related tissue coccidia and evolution of a novel genome architecture consisting minimally of 21 sequence blocks (SBs) totaling 5.9 kb that exist as nonrandom concatemers. Single-molecule Nanopore reads consisting entirely of SBs ranging from 0.1 to 23.6 kb reveal both whole and fragmented cytochrome genes. Full-length cytochrome transcripts including a divergent coxIII are detected. The topology of the mitochondrial genome remains an enigma. Analysis of a cob point mutation reveals that homoplasmy of SBs is maintained. Tissue coccidia are important pathogens of man and animals, and the mitochondrion represents an important therapeutic target. The mtDNA sequence has been elucidated, but a definitive genome architecture remains elusive.

Sivaranjani Namasivayam, Rodrigo P Baptista, Wenyuan Xiao, Erica M Hall, Joseph S Doggett, Karin Troell, Jessica C Kissinger. Genome Res. 2021 May;31(5):852-865. doi: 10.1101/gr.266403.120.

Baseline Mapping of Neglected Tropical Diseases in Africa: The Accelerated WHO/AFRO Mapping Project

Mapping is a prerequisite for effective implementation of interventions against neglected tropical diseases (NTDs). Before the accelerated WHO/AFRO NTD Mapping Project was initiated in 2014, mapping efforts in many countries were frequently carried out in an ad hoc and nonstandardized fashion. In 2013, there were at least 2,200 different districts (of the 4,851 districts in the WHO African region) that still required mapping, and in many of these districts, more than one disease needed to be mapped. During its 3-year duration from January 2014 through the end of 2016, the project carried out mapping surveys for one or more NTDs in at least 2,500 districts in 37 African countries. At the end of 2016, most (90%) of the 4,851 districts had completed the WHO-required mapping surveys for the five targeted Preventive Chemotherapy (PC)-NTDs, and the impact of this accelerated WHO/AFRO NTD Mapping Project proved to be much greater than just the detailed mapping results themselves. Indeed, the AFRO Mapping Project dramatically energized and empowered national NTD programs, attracted donor support for expanding these programs, and developed both a robust NTD mapping database and data portal. By clarifying the prevalence and burden of NTDs, the project provided not only the metrics and technical framework for guiding and tracking program implementation and success but also the research opportunities for developing improved diagnostic and epidemiologic sampling tools for all 5 PC-NTDs-lymphatic filariasis, onchocerciasis, schistosomiasis, soil-transmitted helminthiasis, and trachoma.

Maria P Rebollo, Adiele Nkasiobi Onyeze, Alexandre Tiendrebeogo, Mutale Nsakashalo Senkwe, Benido Impouma, Kisito Ogoussan, Honorat G M Zouré, Kebede Deribe, Jorge Cano, Ekoue Boniface Kinvi, Andrew Majewski, Eric A Ottesen, Patrick Lammie. Am J Trop Med Hyg. 2021 Apr 26;tpmd201538. doi: 10.4269/ajtmh.20-1538

Some conditions apply: Systems for studying Plasmodium falciparum protein function

Plasmodium falciparum life cycle
Fig 1. Conditional protein knockdown used throughout the Plasmodium falciparum life cycle.

Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our ability to study essential proteins and pathways in Plasmodium falciparum, the parasite associated with the most severe malaria cases. However, recent years have seen major leaps forward in the ability to genetically manipulate P. falciparum parasites and conditionally control protein expression/function. The conditional knockdown systems used in P. falciparum target all 3 components of the central dogma, allowing researchers to conditionally control gene expression, translation, and protein function. Here, we review some of the common knockdown systems that have been adapted or developed for use in P. falciparum. Much of the work done using conditional knockdown approaches has been performed in asexual, blood-stage parasites, but we also highlight their uses in other parts of the life cycle and discuss new ways of applying these systems outside of the intraerythrocytic stages. With the use of these tools, the field’s understanding of parasite biology is ever increasing, and promising new pathways for antimalarial drug development are being discovered.

Heather M Kudyba, David W Cobb, Joel Vega-Rodríguez, Vasant Muralidharan. PLoS Pathog. 2021 Apr 22;17(4):e1009442. doi: 10.1371/journal.ppat.1009442. eCollection 2021 Apr.

Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome

The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.

Catherine Sullenberger, Benjamin Hoffman, Justin Wiedeman, Gaurav Kumar, Kojo Mensa-Wilmot. PLoS One. 2021 Apr 16;16(4):e0249908. doi: 10.1371/journal.pone.0249908.

Survival of Salmonella and Shiga Toxin-producing Escherichia coli and Changes in Indigenous Microbiota During Fermentation of Kombucha Made from Home-brewing Kits

Survival and growth of Salmonella and Shiga toxin-producing Escherichia coli (STEC) in kombucha prepared from four brands of commercially available kombucha kits intended for use by home brewers were investigated. Changes in microbiota responsible for fermentation were also determined. An initial population of Salmonella (6.77 log CFU/mL) decreased to below the detection limit (0.30 log CFU/mL) within 10 d in kombucha prepared from two of the four test brands. Populations of 1.85 and 1.20 log CFU/mL were detected in two brands fermented for 14 d. An initial population of STEC (7.02 log CFU/mL) decreased to <0.30 log CFU/mL in two of the four brands within 14 d; 0.20 and 0.87 log CFU/mL were detected in kombucha prepared from the other two brands. Salmonella and STEC increased in populations within 1 d in three brands of base tea used to prepare kombucha, and were stable throughout 14 d of incubation. Both pathogens steadily declined in base tea prepared from one brand of kombucha kit. Inactivation of the pathogens occurred as the pH of kombuchas decreased, but a clear correlation between rates of inactivation and decrease in pH was not evident when comparing kombuchas prepared from the four kits. Growth and peak populations of mesophilic aerobic microorganisms, yeasts, lactic acid bacteria, and acetic acid bacteria varied, depending on the kombucha kit brand. There was not strong evidence to correlate the behavior of Salmonella and STEC with any of these groups of indigenous microbiota. Results of this study show that the ability of Salmonella and STEC to survive in kombucha and base tea used to prepare kombucha is dependent on inherent differences in commercially available kombucha kits intended for use in home settings. Strict application of hygienic practices with the goal of preventing contamination with Salmonella or STEC is essential for reducing the risk of illness associated the consumption of kombucha.

Sheridan S. Brewer, Courtney A. Lowe, Larry R. Beuchat, Ynes R. Ortega; Survival of Salmonella and Shiga Toxin-producing Escherichia coli and Changes in Indigenous Microbiota During Fermentation of Kombucha Made from Home-brewing Kits. J Food Prot 2021; doi:

EdU incorporation to assess cell proliferation and drug susceptibility in Naegleria fowleri

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2′-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro EdA (5’ethynyl-2′-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

Emma V. TrothDennis E. Kyle

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

Yin Wang, Jai Hoon Eum, Ruby E. Harrison, Luca Valzania, Xiushuai Yang, Jena A. Johnson, Derek T. Huck, Mark R. Brown, Michael R. Strand Proceedings of the National Academy of Sciences Apr 2021, 118 (15) e2101080118; DOI: 10.1073/pnas.2101080118

Estimating true prevalence of Schistosoma mansoni from population summary measures based on the Kato-Katz diagnostic technique

Background: The prevalence of Schistosoma mansoni infection is usually assessed by the Kato-Katz diagnostic technique. However, Kato-Katz thick smears have low sensitivity, especially for light infections. Egg count models fitted on individual level data can adjust for the infection intensity-dependent sensitivity and estimate the ‘true’ prevalence in a population. However, application of these models is complex and there is need for adjustments that can be done without modelling expertise. This study provides estimates of the ‘true’ S. mansoni prevalence from population summary measures of observed prevalence and infection intensity using extensive simulations parametrized with data from different settings in sub-Saharan Africa.

Methodology: An individual-level egg count model was applied to Kato-Katz data to determine the S. mansoni infection intensity-dependent sensitivity for various sampling schemes. Observations in populations with varying forces of transmission were simulated, using standard assumptions about the distribution of worms and their mating behavior. Summary measures such as the geometric mean infection, arithmetic mean infection, and the observed prevalence of the simulations were calculated, and parametric statistical models fitted to the summary measures for each sampling scheme. For validation, the simulation-based estimates are compared with an observational dataset not used to inform the simulation.

Principal findings: Overall, the sensitivity of Kato-Katz in a population varies according to the mean infection intensity. Using a parametric model, which takes into account different sampling schemes varying from single Kato-Katy to triplicate slides over three days, both geometric and arithmetic mean infection intensities improve estimation of sensitivity. The relation between observed and ‘true’ prevalence is remarkably linear and triplicate slides per day on three consecutive days ensure close to perfect sensitivity.

Conclusions/significance: Estimation of ‘true’ S. mansoni prevalence is improved when taking into account geometric or arithmetic mean infection intensity in a population. We supply parametric functions and corresponding estimates of their parameters to calculate the ‘true’ prevalence for sampling schemes up to 3 days with triplicate Kato-Katz thick smears per day that allow estimation of the ‘true’ prevalence.

Bärenbold O, Garba A, Colley DG, Fleming FM, Assaré RK, Tukahebwa EM, et al. (2021) Estimating true prevalence of Schistosoma mansoni from population summary measures based on the Kato-Katz diagnostic technique. PLoS Negl Trop Dis 15(4): e0009310.