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Category: publications

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.

Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruzi MPC1 (TcMPC1) and TcMPC2 with 3×c-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1-KO and TcMPC2-KO epimastigotes showed reduced O2 consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O2 consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca2+ uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC-KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.

IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual T. cruzi MPC1 (TcMPC1) and TcMPC2 knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.

Raquel S. NegreirosNoelia LanderMiguel A. ChiurilloAnibal E. VercesiRoberto Docampo.

Synthesis of Mono- and Bisperoxide-Bridged Artemisinin Dimers to Elucidate the Contribution of Dimerization to Antimalarial Activity

During the past decade, artemisinin as an antimalarial has been in the spotlight, in part due to the Nobel Prize in Physiology or Medicine awarded to Tu Youyou. While many studies have been completed detailing the significant increase in activity resulting from the dimerization of natural product artemisinin, activity increases unaccounted for by the peroxide bridge have yet to be researched. Here we outline the synthesis and testing for antimalarial activity of artemisinin dimers in which the peroxide bridge in one-half of the dimer is reduced, resulting in a dimer with one active and one deactivated artemisinin moiety.

Cynthia L Lichorowic, Yingzhao Zhao, Steven P Maher, Vivian Padín-Irizarry, Victoria C Mendiola, Sagan T de Castro, Jacob A Worden, Debora Casandra, Dennis E Kyle, Roman Manetsch. ACS Infect Dis. 2021 Apr 1. doi: 10.1021/acsinfecdis.1c00066

Ca2+ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V‐H+‐ATPase in Toxoplasma gondii

Ca2+ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca2+ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work we studied the role of proton transport in cytosolic Ca2+ homeostasis and the initiation of Ca2+ signaling. We used a T. gondii mutant of the V-ATPase, a pump previously shown to transport protons to the extracellular medium, control intracellular pH and membrane potential and we show that proton gradients are important for maintaining resting cytosolic Ca2+ at physiological levels and for Ca2+ influx. Proton transport was also important for Ca2+ storage by acidic stores and, unexpectedly, the endoplasmic reticulum. Proton transport impacted the amount of polyphosphate (polyP), a phosphate polymer that binds Ca2+ and concentrate in acidocalcisomes. This was supported by the co-localization of the vacuolar transporter chaperone 4 (VTC4), the catalytic subunit of the VTC complex that synthesizes polyP, with the V-ATPase in acidocalcisomes. Our work show that proton transport regulate plasma membrane Ca2+ transport and control acidocalcisome polyP and Ca2+ content impacting Ca2+ signaling and downstream stimulation of motility and egress in T. gondii.

Andrew J Stasic, Eric J Dykes, Ciro D Cordeiro, Stephen A Vella, Mojtaba S Fazli, Shannon Quinn, Roberto Docampo, Silvia N J Moreno. Mol Microbiol. 2021 Apr 1. doi: 10.1111/mmi.14722

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU’s) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established.

Padilla AM, Yao PY, Landry TJ, Cooley GM, Mahaney SM, Ribeiro I, VandeBerg JL. Tarleton RL. (2021) High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA. PLoS Negl Trop Dis 15(3): e0009141.

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Logan Tillery, Kayleigh Barrett, Jenna Goldstein, Jared W Lassner, Bram Osterhout, Nathan L Tran, Lily Xu, Ryan M Young, Justin Craig, Ian Chun, David M Dranow, Jan Abendroth, Silvia L Delker, Douglas R Davies, Stephen J Mayclin, Brandy Calhoun, Madison J Bolejack, Bart Staker, Sandhya Subramanian, Isabelle Phan, Donald D Lorimer, Peter J Myler, Thomas E Edwards, Dennis E Kyle, Christopher A Rice, James C Morris, James W Leahy, Roman Manetsch, Lynn K Barrett, Craig L Smith, Wesley C Van Voorhis (2021) Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba. PLoS ONE 16(3): e0241738.