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Author: Donna Huber

Conservation of Ancient Genetic Pathways for Intracellular Persistence Among Animal Pathogenic Bordetellae

Animal and human pathogens of the genus Bordetella are not commonly considered to be intracellular pathogens, although members of the closely related classical bordetellae are known to enter and persist within macrophages in vitro and have anecdotally been reported to be intracellular in clinical samples. B. bronchiseptica, the species closest to the ancestral lineage of the classical bordetellae, infects a wide range of mammals but is known to have an alternate life cycle, persisting, replicating and disseminating with amoeba. These observations give rise to the hypothesis that the ability for intracellular survival has an ancestral origin and is common among animal-pathogenic and environmental Bordetella species. Here we analyzed the survival of B. bronchiseptica and defined its transcriptional response to internalization by murine macrophage-like cell line RAW 264.7. Although the majority of the bacteria were killed and digested by the macrophages, a consistent fraction survived and persisted inside the phagocytes. Internalization prompted the activation of a prominent stress response characterized by upregulation of genes involved in DNA repair, oxidative stress response, pH homeostasis, chaperone functions, and activation of specific metabolic pathways. Cross species genome comparisons revealed that most of these upregulated genes are highly conserved among both the classical and non-classical Bordetella species. The diverse Bordetella species also shared the ability to survive inside RAW 264.7 cells, with the single exception being the bird pathogen B. avium, which has lost several of those genes. Knock-out mutations in genes expressed intracellularly resulted in decreased persistence inside the phagocytic cells, emphasizing the importance of these genes in this environment. These data show that the ability to persist inside macrophage-like RAW 264.7 cells is shared among nearly all Bordetella species, suggesting that resisting phagocytes may be an ancient mechanism that precedes speciation in the genus and may have facilitated the adaptation of Bordetella species from environmental bacteria to mammalian respiratory pathogens.

Israel Rivera, Bodo Linz, Kalyan K. Dewan, Longhuan Ma, Christopher A. Rice, Dennis E. Kyle and Eric T. Harvill. Front Microbiol. 2019 Dec 11;10:2839. doi: 10.3389/fmicb.2019.02839. eCollection 2019.

Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.

The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing “tried and true” methods of our lab who pioneered the first use of GECI’s in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

Stephen A. Vella, Abigail Calixto, Beejan Asady, Zhu-Hong Li, Silvia N. J. Moreno. Methods Mol Biol. 2020;2071:187-207. doi: 10.1007/978-1-4939-9857-9_11.

The genomes of two parasitic wasps that parasitize the diamondback moth

Background

Parasitic insects are well-known biological control agents for arthropod pests worldwide. They are capable of regulating their host’s physiology, development and behaviour. However, many of the molecular mechanisms involved in host-parasitoid interaction remain unknown.

Results

We sequenced the genomes of two parasitic wasps (Cotesia vestalis, and Diadromus collaris) that parasitize the diamondback moth Plutella xylostella using Illumina and Pacbio sequencing platforms. Genome assembly using SOAPdenovo produced a 178 Mb draft genome for C. vestalis and a 399 Mb draft genome for D. collaris. A total set that contained 11,278 and 15,328 protein-coding genes for C. vestalis and D. collaris, respectively, were predicted using evidence (homology-based and transcriptome-based) and de novo prediction methodology. Phylogenetic analysis showed that the braconid C. vestalis and the ichneumonid D. collaris diverged approximately 124 million years ago. These two wasps exhibit gene gains and losses that in some cases reflect their shared life history as parasitic wasps and in other cases are unique to particular species. Gene families with functions in development, nutrient acquisition from hosts, and metabolism have expanded in each wasp species, while genes required for biosynthesis of some amino acids and steroids have been lost, since these nutrients can be directly obtained from the host. Both wasp species encode a relative higher number of neprilysins (NEPs) thus far reported in arthropod genomes while several genes encoding immune-related proteins and detoxification enzymes were lost in both wasp genomes.

Conclusions

We present the annotated genome sequence of two parasitic wasps C. vestalis and D. collaris, which parasitize a common host, the diamondback moth, P. xylostella. These data will provide a fundamental source for studying the mechanism of host control and will be used in parasitoid comparative genomics to study the origin and diversification of the parasitic lifestyle.

Min Shi, Zhizhi Wang, Xiqian Ye, Hongqing Xie, Fei Li, Xiaoxiao Hu, Zehua Wang, Chuanlin Yin, Yuenan Zhou, Qijuan Gu, Jiani Zou, Leqing Zhan, Yuan Yao, Jian Yang, Shujun Wei, Rongmin Hu, Dianhao Guo, Jiangyan Zhu, Yanping Wang, Jianhua Huang, Francesco Pennacchio, Michael R. Strand & Xuexin Chen. The genomes of two parasitic wasps that parasitize the diamondback moth. BMC Genomics 20893 (2019) doi:10.1186/s12864-019-6266-0.

Chagas Disease Drug Discovery: Multiparametric Lead Optimization against Trypanosoma cruzi in Acylaminobenzothiazole Series

Acylaminobenzothiazole hits were identified as potential inhibitors of Trypanosoma cruzi replication, a parasite responsible for Chagas disease. We selected compound 1 for lead optimization, aiming to improve in parallel its anti-T. cruzi activity (IC50 = 0.63 μM) and its human metabolic stability (human clearance = 9.57 mL/min/g). A total of 39 analogues of 1 were synthesized and tested in vitro. We established a multiparametric structure-activity relationship, allowing optimization of antiparasite activity, physicochemical parameters, and ADME properties. We identified compound 50 as an advanced lead with an improved anti-T. cruzi activity in vitro (IC50 = 0.079 μM) and an enhanced metabolic stability (human clearance = 0.41 mL/min/g) and opportunity for the oral route of administration. After tolerability assessment, 50 demonstrated a promising in vivo efficacy.

Charlotte Fleau, Angel Padilla, Juan Miguel-Siles, Maria T. Quesada-Campos, Isabel Saiz-Nicolas, Ignacio Cotillo, Juan Cantizani Perez, Rick L. Tarleton, Maria Marco, Gilles Courtemanche. J Med Chem. 2019. doi: 10.1021/acs.jmedchem.9b01429.

Field Relevant Variation in Ambient Temperature Modifies Density-Dependent Establishment of Plasmodium falciparum Gametocytes in Mosquitoes

The relationship between Plasmodium falciparum gametocyte density and infections in mosquitoes is central to understanding the rates of transmission with important implications for control. Here, we determined whether field relevant variation in environmental temperature could also modulate this relationship. Anopheles stephensi were challenged with three densities of P. falciparum gametocytes spanning a ~10-fold gradient, and housed under diurnal/daily temperature range (“DTR”) of 9°C (+5°C and -4°C) around means of 20, 24, and 28°C. Vector competence was quantified as the proportion of mosquitoes infected with oocysts in the midguts (oocyst rates) or infectious with sporozoites in the salivary glands (sporozoite rates) at peak periods of infection for each temperature to account for the differences in development rates. In addition, oocyst intensities were also recorded from infected midguts and the overall study replicated across three separate parasite cultures and mosquito cohorts. While vector competence was similar at 20 DTR 9°C and 24 DTR 9°C, oocyst and sporozoite rates were also comparable, with evidence, surprisingly, for higher vector competence in mosquitoes challenged with intermediate gametocyte densities. For the same gametocyte densities however, severe reductions in the sporozoite rates was accompanied by a significant decline in overall vector competence at 28 DTR 9°C, with gametocyte density per se showing a positive and linear effect at this temperature. Unlike vector competence, oocyst intensities decreased with increasing temperatures with a predominantly positive and linear association with gametocyte density, especially at 28 DTR 9°C. Oocyst intensities across individual infected midguts suggested temperature-specific differences in mosquito susceptibility/resistance: at 20 DTR 9°C and 24 DTR 9°C, dispersion (aggregation) increased in a density-dependent manner but not at 28 DTR 9°C where the distributions were consistently random. Limitations notwithstanding, our results suggest that variation in temperature could modify seasonal dynamics of infectious reservoirs with implications for the design and deployment of transmission-blocking vaccines/drugs.

Ashutosh K. Pathak, Justine C. Shiau, Matthew B. Thomas and Courtney C. Murdock, Front Microbiol. 2019 Nov 15;10:2651. doi: 10.3389/fmicb.2019.02651. eCollection 2019.

Cultivation-assisted genome of Candidatus Fukatsuia symbiotica; the enigmatic ‘X-type’ symbiont of aphids

Heritable symbionts are common in terrestrial arthropods and often provide beneficial services to hosts. Unlike obligate, nutritional symbionts that largely persist under strict host control within specialized host cells, heritable facultative symbionts exhibit large variation in within-host lifestyles and services rendered with many retaining the capacity to transition among roles. One enigmatic symbiont, Candidatus Fukatsuia symbiotica, frequently infects aphids with reported roles ranging from pathogen, defensive symbiont, mutualism exploiter and nutritional co-obligate symbiont. Here we used an in vitro culture-assisted protocol to sequence the genome of a facultative strain of Fukatsuia from pea aphids (Acyrthosiphon pisum). Phylogenetic and genomic comparisons indicate that Fukatsuia is an aerobic heterotroph, which together with Regiella insecticola and Hamiltonella defensa form a clade of heritable facultative symbionts within the Yersiniaceae (Enterobacteriales). These three heritable facultative symbionts largely share overlapping inventories of genes associated with housekeeping functions, metabolism, and nutrient acquisition, while varying in complements of mobile DNA. One unusual feature of Fukatsuia is its strong tendency to occur as a co-infection with H. defensa. However, the overall similarity of gene inventories among aphid heritable facultative symbionts suggest that metabolic complementarity is not the basis for co-infection, unless playing out on a H. defensa strain-specific basis. We also compared the pea aphid Fukatsuia with a strain from the aphid Cinara confinis (Lachninae) where it is reported to have transitioned to co-obligate status to support decaying Buchnera function. Overall the two genomes are very similar with no clear genomic signatures consistent with such a transition, which suggests co-obligate status in C. confinis was a recent event.

V Patel, G Chevignon, A Manzano-Marín, J W Brandt, M R Strand, J A Russell, K M Oliver. 2019 Cultivation-assisted genome of Candidatus Fukatsuia symbiotica; the enigmatic ‘X-type’ symbiont of aphids. Genome Biology and Evolution, evz252, https://doi.org/10.1093/gbe/evz252

Sharing the Knowledge: NIH Award Supports Expanded Genomics Data Resource

By: Alan Flurry

A team led by scientists at the University of Pennsylvania and University of Georgia provides thousands of researchers around the world with access to the Eukaryotic Pathogen Genomics Database (EuPathDB.org), a collection of resources for analyzing large-scale datasets associated with microbial pathogens. These include the parasites responsible for malaria, sleeping sickness, and toxoplasmosis; the fungi responsible for thrush, aspergillosis and Valley Fever; and many other important diseases. In parallel, a team led by investigators at the University of Notre Dame has been responsible for similar resources covering invertebrate vectors of disease (VectorBase.org), including the mosquitoes transmitting malaria, Zika, and yellow fever, the ticks responsible for Lyme disease and Rocky Mountain Spotted Fever, and others.

To ensure that this important work continues, the National Institute of Allergy and Infectious Diseases, a part of the National Institutes of Health, has awarded a new contract to integrate these resources, worth up to $7.2 million in 2019-2020. The five‐year award for this project, rebranded as VEuPathDB.org (The Eukaryotic Pathogen, Host & Vector Genomics Resource) could total as much as $38.4 million if all associated options are exercised.

The patterns revealed by such “Big Data” provide insight into important diseases, permit the development of diagnostic methods, and define drug and vaccine targets. But to be useful, these immense datasets must be sensibly organized and made conveniently accessible to the researchers worldwide. The integrated VEuPathDB database hosts data on thousands of genomes, representing hundreds of species, along with extensive information on isolate provenance, gene function and the like.

The award is based at Penn, and directed by David S Roos, E Otis Kendall Professor of Biology in the School of Arts & Sciences. Key subcontracts include the University of Georgia (Joint PI Jessica C. Kissinger, Distinguished Professor of Genetics and Bioinformatics in the Franklin College of Arts and Sciences and the Center for Tropical and Emerging Global Diseases), University of Notre Dame (Joint PI Mary Ann McDowell, Associate Professor of Biological Sciences at the Eck Institute for Global Health).  Additional co-investigators include Professors Christian Stoeckert of Penn’s Perelman School of Medicine, Mark Caddick of the University of Liverpool, George K Christophides of Imperial College London, and Paul Flicek, Associate Director of the EMBL-EBI (European Bioinformatics Institute).

“It is wonderful to see the continued investment by NIH, the Wellcome Trust and others in resources that make performing much needed global research on infectious diseases both easier and better,” Kissinger said. “Datasets are larger and more complex than ever due to significant advances in technology. These breakthroughs create challenges for making the resulting data truly accessible and usable by the average researcher.  We strive to remove barriers, integrate diverse data and accelerate the speed with which new hypotheses can be generated and ideas tested both in silico and in the lab.”

“A critical aspect of this now joint program will be its accessibility throughout the world, empowering any infectious disease investigator to interrogate these highly complex databases in comprehensible and productive ways,” said Dan Colley, UGA professor of microbiology and member of the CTEGD who has conducted extensive research on n schistosomiasis in western Kenya. “These databases have led, and the merged data base will lead, to the design of new drugs and studies on how to better control and eliminate these major public health challenges, such as malaria, toxoplasmosis, yellow fever, eastern equine encephalitis and Lyme disease.”

“Since its conception, corresponding with the release of the first parasite genomes, EuPathDB has been a transformative tool in our search for a better understanding of human disease and parasite biology,” said Stephen Hadjuk, Professor Emeritus of biochemistry & molecular biology at UGA whose lab investigates trypanosomes, the causative agent of an human African sleeping sickness. “Today, it’s difficult to imagine any serious research on parasites and host pathology that doesn’t rely, at least to some extent, on EuPathDB. The decision to incorporate the vectors database into the eukaryotic pathogens database was brilliant, and makes this is an exciting new chapter in the EuPathDB story.”

“Innumerable investigators, including my own laboratory, rely on daily access to the high quality genomic and functional datasets made available by the VEuPathDB Project,” says Keith Gull, Professor of Molecular Microbiology at Oxford University.  “Sustainable support for such resources is imperative if we are to capitalize on the promise of modern technologies for scientific discovery and translational application.”  Joe Heitman, James B Duke Professor / Chair of Molecular Genetics & Microbiology at Duke University agrees: “Inclusion of fungal pathogens under the BRC umbrella has greatly enhanced our ability to study important human mycoses.  Cross-species comparisons provide insights into the biology and pathogenesis of these fascinating organisms, which can be deadly – but can also serve as workhorses for valuable biotechnology development.”

Originally published at https://www.franklin.uga.edu/news/stories/2019/sharing-knowledge-nih-award-supports-expanded-genomics-data-resource

Modeling approaches to predicting persistent hotspots in SCORE studies for gaining control of schistosomiasis mansoni in Kenya and Tanzania

BACKGROUND:

Some villages, labeled “persistent hotspots (PHS),” fail to respond adequately in regard to prevalence and intensity of infection to mass drug administration (MDA) for schistosomiasis. Early identification of PHS, for example, before initiating or after a year or two of MDA could help guide programmatic decision-making.

METHODS:

In a study with multiple rounds of MDA, data collected prior to the third MDA were used to predict PHS. We assessed six predictive approaches using data from before MDA and after 2 rounds of annual MDA from Kenya and Tanzania.

RESULTS:

Generalized linear models with variable selection possessed relatively stable performance compared to tree-based methods. Models applied to Kenya data alone or combined data from Kenya and Tanzania could reach over 80% predictive accuracy, while predicting PHS for Tanzania was challenging. Models developed from one country and validated in another failed to achieve satisfactory performance. Several Year 3 variables were identified as key predictors.

CONCLUSIONS:

Statistical models applied to Year 3 data could help predict PHS and guide program decisions, with infection intensity, prevalence of heavy infections (≥400 eggs/gram of feces), and total prevalence being particularly important factors. Additional studies including more variables and locations could help in developing generalizable models.

Ye Shen, Meng-Hsuan Sung, Charles H King, Sue Binder, Nupur Kittur, Christopher C Whalen, Daniel G Colley. J Infect Dis. 2019 Oct 17. pii: jiz529. doi: 10.1093/infdis/jiz529