Parameter estimation and sensitivity analysis in an agent-based model of Leishmania major infection

Computer models of disease take a systems biology approach toward understanding host-pathogen interactions. In particular, data driven computer model calibration is the basis for inference of immunological and pathogen parameters, assessment of model validity, and comparison between alternative models of immune or pathogen behavior. In this paper we describe the calibration and analysis of an agent-based model of Leishmania major infection. A model of macrophage loss following uptake of necrotic tissue is proposed to explain macrophage depletion following peak infection. Using Gaussian processes to approximate the computer code, we perform a sensitivity analysis to identify important parameters and to characterize their influence on the simulated infection. The analysis indicates that increasing growth rate can favor or suppress pathogen loads, depending on the infection stage and the pathogen's ability to avoid detection. Subsequent calibration of the model against previously published biological observations suggests that L. major has a relatively slow growth rate and can replicate for an extended period of time before damaging the host cell.     

Molecular and infection biology of the horse pathogen Rhodococcus equi

The soil actinomycete Rhodococcus equi is a pulmonary pathogen of young horses and AIDS patients. As a facultative intracellular bacterium, R. equi survives and multiplies in macrophages and establishes its specific niche inside the host cell. Recent research into chromosomal virulence factors and into the role of virulence plasmids in infection and host tropism has presented novel aspects of R. equi infection biology and pathogenicity. This review will focus on new findings in R. equi biology, the trafficking of R. equi -containing vacuoles inside host cells, factors involved in virulence and host resistance and on host–pathogen interaction on organismal and cellular levels.     

Migrate or evolve: options for plant pathogens under climate change

Findings on climate change influence on plant pathogens are often inconsistent and context dependent. Knowledge of pathogens affecting agricultural crops and natural plant communities remains fragmented along disciplinary lines. By broadening the perspective beyond agriculture, this review integrates cross‐disciplinary knowledge to show that at scales relevant to climate change, accelerated evolution and changing geographic distribution will be the main implications for pathogens. New races may evolve rapidly under elevated temperature and CO₂, as evolutionary forces act on massive pathogen populations boosted by a combination of increased fecundity and infection cycles under favourable microclimate within enlarged canopy. Changing geographic distribution will bring together diverse lineages/genotypes that do not share common ecological niche, potentially increasing pathogen diversity. However, the uncertainty of model predictions and a lack of synthesis of fragmented knowledge remain as major deficiencies in knowledge. The review contends that the failure to consider scale and human intervention through new technology are major sources of uncertainty. Recognizing that improved biophysical models alone will not reduce uncertainty, it proposes a generic framework to increase focus and outlines ways to integrate biophysical elements and technology change with human intervention scenarios to minimize uncertainty. To synthesize knowledge of pathogen biology and life history, the review borrows the concept of ‘fitness’ from population biology as a comprehensive measure of pathogen strengths and vulnerabilities, and explores the implications of pathogen mode of nutrition to fitness and its interactions with plants suffering chronic abiotic stress under climate change. Current and future disease management options can then be judged for their ability to impair pathogenic and saprophytic fitness. The review pinpoints improving confidence in model prediction by minimizing uncertainty, developing management strategies to reduce overall pathogen fitness, and finding new sources of data to trawl for climate signatures on pathogens as important challenges for future research.     

Entamoeba histolytica: a snapshot of current research and methods for genetic analysis

Entamoeba histolytica represents one of the leading causes of parasitic death worldwide. Although identified as the causative agent of amebiasis since 1875, the molecular mechanisms by which the parasite causes disease are still not fully understood. Studying Entamoeba reveals insights into a eukaryotic cell that differs in many ways from better-studied model organisms. Thus, much can be learned from this protozoan parasite on evolution, cell biology, and RNA biology. In this review we discuss selected research highlights in Entamoeba research and focus on the development of molecular biological techniques to study this pathogen. We end by highlighting some of the many questions that remain to be answered in order to fully understand this important human pathogen.     

Biodiversity of Borrelia burgdorferi strains in tissues of Lyme disease patients

Plant and animal biodiversity are essential to ecosystem health and can provide benefits to humans ranging from aesthetics to maintaining air quality. Although the importance of biodiversity to ecology and conservation biology is obvious, such measures have not been applied to strains of an invasive bacterium found in human tissues during infection. In this study, we compared the strain biodiversity of Borrelia burgdorferi found in tick populations with that found in skin, blood, synovial fluid or cerebrospinal fluid of Lyme disease patients. The biodiversity of B. burgdorferi strains is significantly greater in tick populations than in the skin of patients with erythema migrans. In turn, strains from skin are significantly more diverse than strains at any of the disseminated sites. The cerebrospinal fluid of patients with neurologic Lyme disease harbored the least pathogen biodiversity. These results suggest that human tissues act as niches that can allow entry to or maintain only a subset of the total pathogen population. These data help to explain prior clinical observations on the natural history of B. burgdorferi infection and raise several questions that may help to direct future research to better understand the pathogenesis of this infection.     

Targeted gene deletion in Aspergillus fumigatus using microbial machinery and a recyclable marker

The emerging invasive fungal pathogen Aspergillus fumigatus causes very serious infections among immunocompromised patient populations. While the genome of this pathogen has been sequenced, a major barrier to better understanding the complex biology of this eukaryotic organism is a lack of tools for efficient genetic manipulation. To improve upon this, we have generated a new gene deletion system for A. fumigatus using yeast recombinational cloning and Agrobacterium tumefaciens mediated transformation (ATMT) employing a recyclable marker system. This system reduced the time for generating a gene deletion strain in our hands by two-thirds (12 weeks to 3 weeks) using minimal human labor, and we demonstrate that it can be used to efficiently generate multiple gene deletions within a single strain.     

Infection of Drosophila melanogaster by Tubulinosema kingi: stage-specific susceptibility and within-host proliferation

Despite its importance as a model organism very little is known about the interaction between Drosophila and its microsporidian pathogens. Here we report on the relative susceptibility of Drosophila melanogaster life history stages to infection by Tubulinosema kingi, and on patterns of pathogen proliferation. We find that only larvae can be infected, and that this susceptibility decreases with larval age. Following infection, the pathogen shows little subsequent proliferation in larvae, a limited amount in pupae while it replicates greatly in adults. We present evidence that the host launches a cellular immune response after infection with the pathogen, although its effectiveness remains to be demonstrated.     

CO2和温度升高情况下白粉菌侵染对西葫芦生长特性的影响

植物病害是降低植物产量和产品品质的重要因素,但对其在气候变化情景下如何影响植物的研究鲜见报道。利用封闭式人工气候室模拟不同环境处理,探讨了大气CO₂浓度增加和温度升高情况下白粉菌(Podosphaera xanthii)侵染对西葫芦(Cucurbita pepo)生长发育的影响。结果表明,单独CO₂加富(EC)增强了西葫芦光合作用(P<0.05),促进了植株生长和果实生产;CO₂浓度和温度同时升高(ECT)也促进了光合作用(P<0.05),加速了植株器官发育,但限制了叶片叶绿素合成和叶片面积生长,最终明显降低了植株地上部分干物质积累和果实产量(P<0.05)。和对照相比,EC处理下白粉菌的生长繁殖没有明显变化,但由于西葫芦植株的抗病性有所改善,植株病情指数略有下降;而ECT处理下白粉菌的发育繁殖明显改善,P. xanthii菌落规模和产孢能力极大提高(P<0.01),植物病情指数显著加重(P<0.01),作物严重减产(P<0.01)。可见,在未来以CO₂浓度和温度升高为特征的气候变化条件下,白粉菌倾向于加重对西葫芦的侵染。这个结论对其他葫芦科白粉病的防治管理也有借鉴作用。     

Mainstreaming Caenorhabditis elegans in experimental evolution

Experimental evolution provides a powerful manipulative tool for probing evolutionary process and mechanism. As this approach to hypothesis testing has taken purchase in biology, so too has the number of experimental systems that use it, each with its own unique strengths and weaknesses. The depth of biological knowledge about Caenorhabditis nematodes, combined with their laboratory tractability, positions them well for exploiting experimental evolution in animal systems to understand deep questions in evolution and ecology, as well as in molecular genetics and systems biology. To date, Caenorhabditis elegans and related species have proved themselves in experimental evolution studies of the process of mutation, host–pathogen coevolution, mating system evolution and life-history theory. Yet these organisms are not broadly recognized for their utility for evolution experiments and remain underexploited. Here, we outline this experimental evolution work undertaken so far in Caenorhabditis, detail simple methodological tricks that can be exploited and identify research areas that are ripe for future discovery.     

A trip in the “New Microbiology” with the bacterial pathogen Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen causing an opportunistic disease called listeriosis. This bacterium invades and replicates in most cell types, due to its multiple strategies to exploit host molecular mechanisms. Research aiming at unravelling Listeria invasion and intracellular lifestyle has led to a number of key discoveries in infection biology, cell biology and also microbiology. In this review, we report on our most recent advances in understanding the intimate crosstalk between the bacterium and its host, resulting from in-depth studies performed over the past five years. We specifically highlight new concepts in RNA-based regulation in bacteria and discuss important findings in cell biology, including a new role for clathrin and an atypical mitochondrial fragmentation mechanism. We also illustrate the notion that bacterial infection regulates host gene expression at the chromatin level, contributing to an emerging field called patho-epigenetics. This review corresponds to the lecture given by one of us (P.C.) on the occasion of the 2014 FEBS|EMBO Woman in Science Award.     

Exosomes and other microvesicles in infection biology: organelles with unanticipated phenotypes

The release of exosomes and other microvesicles by diverse prokaryotic and eukaryotic cells and organisms was first appreciated early in the 20th century. The functional properties of these organelles, however, have only recently been the focus of rigorous investigation. In this review, we discuss the release of microvesicles of varying complexity by diverse microbial pathogens. This includes vesicle secretion by Gram-negative bacteria, eukaryotic parasites of the kinetoplast lineage and opportunistic fungal pathogens of both the ascomycetes and basidiomycetes lineages. We also discuss vesicle release from mammalian cells brought about as a result of infection with bacteria, viruses and prions. In addition, we review the evidence showing that in their specific microenvironments, release of these organelles from diverse pathogens contributes to pathogenesis. Germane to this and based upon recent findings with Leishmania, we propose a model whereby exosome release by an intracellular pathogen serves as a general mechanism for effector molecule delivery from eukaryotic pathogen to host cell cytosol. These new findings linking exosomes and other microvesicles to infection biology have important implications for understanding the immune response to infection and for the design of research strategies aimed at the development of novel therapeutics and vaccines.     

Predator modulation of plant pathogen spread through induced changes in vector development rates

1. Predation on vectors of pathogens can indirectly influence infection spread. In addition to the consumptive aspect of predation, non-consumptive, predator-induced changes in various vector traits can lead to trait-mediated indirect effects on pathogen spread, potentially operating in various directions and magnitudes. 2. A widespread non-consumptive effect of predation is the alteration of individual prey development rates. Yet, the implications of this phenomenon for the spread of vector-borne plant pathogens have not been studied. It is hypothesized that the epidemiological effects of predator-induced changes in vector development rate depend on the pattern in which the transmission biology of the vector changes along its ontogeny. 3. A general epidemiological model was developed that considers the role of predation in the infection dynamics of a plant pathogen, while incorporating vector stage structure to allow for variation in its development rate. 4. By contrasting scenarios that represent typical plant disease systems, this study confirms that the magnitude of the effect of altered development rate on infection prevalence depends on the disparity between juvenile and adult vectors in their pathogen transmission potential. 5. The model also reveals that the effect of predator-induced change in development rate can impact pathogen spread counterintuitively. Specifically, slowing down vector development can result in increased pathogen prevalence due to apparent competition between infected and uninfected vector populations. 6. More detailed, stage-specific studies of non-consumptive predator effects on vectors are likely to advance our understanding of plant disease ecology, and the development of more effective biological control practices in agriculture.     

Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis

Bipolaris papendorfii has been reported as a fungal plant pathogen that rarely causes opportunistic infection in humans. Secondary metabolites isolated from this fungus possess medicinal and anticancer properties. However, its genetic fundamental and basic biology are largely unknown. In this study, we report the first draft genome sequence of B. papendorfii UM 226 isolated from the skin scraping of a patient. The assembled 33.4 Mb genome encodes 11,015 putative coding DNA sequences, of which, 2.49% are predicted transposable elements. Multilocus phylogenetic and phylogenomic analyses showed B. papendorfii UM 226 clustering with Curvularia species, apart from other plant pathogenic Bipolaris species. Its genomic features suggest that it is a heterothallic fungus with a putative unique gene encoding the LysM-containing protein which might be involved in fungal virulence on host plants, as well as a wide array of enzymes involved in carbohydrate metabolism, degradation of polysaccharides and lignin in the plant cell wall, secondary metabolite biosynthesis (including dimethylallyl tryptophan synthase, non-ribosomal peptide synthetase, polyketide synthase), the terpenoid pathway and the caffeine metabolism. This first genomic characterization of B. papendorfii provides the basis for further studies on its biology, pathogenicity and medicinal potential.     

Host persistence or extinction from emerging infectious disease: insights from white-nose syndrome in endemic and invading regions

Predicting species'' fates following the introduction of a novel pathogen is a significant and growing problem in conservation. Comparing disease dynamics between introduced and endemic regions can offer insight into which naive hosts will persist or go extinct, with disease acting as a filter on host communities. We examined four hypothesized mechanisms for host–pathogen persistence by comparing host infection patterns and environmental reservoirs for Pseudogymnoascus destructans (the causative agent of white-nose syndrome) in Asia, an endemic region, and North America, where the pathogen has recently invaded. Although colony sizes of bats and hibernacula temperatures were very similar, both infection prevalence and fungal loads were much lower on bats and in the environment in Asia than North America. These results indicate that transmission intensity and pathogen growth are lower in Asia, likely due to higher host resistance to pathogen growth in this endemic region, and not due to host tolerance, lower transmission due to smaller populations, or lower environmentally driven pathogen growth rate. Disease filtering also appears to be favouring initially resistant species in North America. More broadly, determining the mechanisms allowing species persistence in endemic regions can help identify species at greater risk of extinction in introduced regions, and determine the consequences for disease dynamics and host–pathogen coevolution.     

Homothallic sexual reproduction of Pustula helianthicola and germination of oospores

Sunflower white blister rust has become an important disease in many countries with intensive cultivation of the important oil crop. The biology of the pathogen is still partly unclear, particular with respect to its sexual reproduction and primary mode of infection. Zoospores released from sporangia of Pustula helianthicola were isolated individually and used for the inoculation of sunflower in order to generate unithallic, genetically homogenous infections. Single zoospore inoculation of young seedlings resulted in mitotic sporulation within subepidermal blisters on cotyledons and true leaves after approximately 2 weeks. Three weeks postinoculation, the infected plants started forming oospores, hence indicating homothallic sexual reproduction of the pathogen. The development of oogonia and antheridia was studied using light and fluorescence microscopy. Oospores were isolated from infected plant tissue and used for infection and germination studies. Microscopic observation of isolated oospores showed germination that formed sessile vesicle-like structures, germ sporangia or only germ tubes. The rate of germination reached approximately 40 %. Germination was not dependant on a resting phase after oospore formation. Oospores applied to the above ground parts of sunflower seedlings lead to infections within a similar time frame as was achieved with mitotic sporangia. The results underline the importance of oospores for primary infection at the beginning of the season and for long-distance dispersal of the pathogen with sunflower seeds contaminated by oospores.     

Fruit flies learn to avoid odours associated with virulent infection

While learning to avoid toxic food is common in mammals and occurs in some insects, learning to avoid cues associated with infectious pathogens has received little attention. We demonstrate that Drosophila melanogaster show olfactory learning in response to infection with their virulent intestinal pathogen Pseudomonas entomophila. This pathogen was not aversive to taste when added to food. Nonetheless, flies exposed for 3 h to food laced with P. entomophila, and scented with an odorant, became subsequently less likely to choose this odorant than flies exposed to pathogen-laced food scented with another odorant. No such effect occurred after an otherwise identical treatment with an avirulent mutant of P. entomophila, indicating that the response is mediated by pathogen virulence. These results demonstrate that a virulent pathogen infection can act as an aversive unconditioned stimulus which flies can associate with food odours, and thus become less attracted to pathogen-contaminated food.     

Phylogeny and life cycle of the zoonotic pathogen Vibrio vulnificus

Vibrio vulnificus is a zoonotic pathogen able to cause diseases in humans and fish that occasionally result in sepsis and death. Most reviews about this pathogen (including those related to its ecology) are clearly biased towards its role as a human pathogen, emphasizing its relationship with oysters as its main reservoir, the role of the known virulence factors as well as the clinic and the epidemiology of the human disease. This review tries to give to the reader a wider vision of the biology of this pathogen covering aspects related to its phylogeny and evolution and filling the gaps in our understanding of the general strategies that V. vulnificus uses to survive outside and inside its two main hosts, the human and the eel, and how its response to specific environmental parameters determines its survival, its death, or the triggering of an infectious process.     

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.     

Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243

Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin 'tails' and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle.