Natural Killer Cell Dependent Within-Host Competition Arises during Multiple MCMV Infection: Consequences for Viral Transmission and Evolution

It is becoming increasingly clear that many diseases are the result of infection from multiple genetically distinct strains of a pathogen. Such multi-strain infections have the capacity to alter both disease and pathogen dynamics. Infection with multiple strains of human cytomegalovirus (HCMV) is common and has been linked to enhanced disease. Suggestions that disease enhancement in multi-strain infected patients is due to complementation have been supported by trans-complementation studies in mice during co-infection of wild type and gene knockout strains of murine CMV (MCMV). Complementation between naturally circulating strains of CMV has, however, not been assessed. In addition, many models of multi-strain infection predict that co-infecting strains will compete with each other and that this competition may contribute to selective transmission of more virulent pathogen strains. To assess the outcome of multi-strain infection, C57BL/6 mice were infected with up to four naturally circulating strains of MCMV. In this study, profound within-host competition was observed between co-infecting strains of MCMV. This competition was MCMV strain specific and resulted in the complete exclusion of certain strains of MCMV from the salivary glands of multi-strain infected mice. Competition was dependent on Ly49H⁺ natural killer (NK) cells as well as the expression of the ligand for Ly49H, the MCMV encoded product, m157. Strains of MCMV which expressed an m157 gene product capable of ligating Ly49H were outcompeted by strains of MCMV expressing variant m157 genes. Importantly, within-host competition prevented the shedding of the less virulent strains of MCMV, those recognized by Ly49H, into the saliva of multi-strain infected mice. These data demonstrate that NK cells have the strain specific recognition capacity required to meditate within-host competition between strains of MCMV. Furthermore, this within-host competition has the capacity to shape the dynamics of viral shedding and potentially select for the transmission of more virulent virus strains.     

The use of fluorescent fatty acid analogs as labels in trematode experimental infections

We examined the utility of fluorescent fatty acid analog dyes for labeling larval trematodes to use in experimental infections. Our goals were to identify two dyes that label larval trematodes belonging to the species Maritrema novaezealandensis and Coitocaecum parvum, determine if the dyes influence survival and infectivity of larval trematodes and/or host mortality, and if larval trematodes labeled with alternative dyes could be distinguished post-infection. The two dyes tested, BODIPY FL C₁₂ and BODIPY 558/568 C₁₂, successfully labeled all treated larval trematodes, did not influence cercariae survival or infectivity, and did not influence host mortality in either host-parasite system. All larval parasites were fluorescent and distinguishable after 5 days in amphipod intermediate hosts. In addition, larval Acanthoparyphium sp. were strongly fluorescent with both dyes after 5 weeks within cockle hosts. This method should be extremely useful for experimental studies using trematode-host systems as models for addressing a range of ecological and evolutionary questions.     

Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy

Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D.     

Regression-Based Ranking of Pathogen Strains with Respect to Their Contribution to Natural Epidemics

Genetic variation in pathogen populations may be an important factor driving heterogeneity in disease dynamics within their host populations. However, to date, we understand poorly how genetic diversity in diseases impact on epidemiological dynamics because data and tools required to answer this questions are lacking. Here, we combine pathogen genetic data with epidemiological monitoring of disease progression, and introduce a statistical exploratory method to investigate differences among pathogen strains in their performance in the field. The method exploits epidemiological data providing a measure of disease progress in time and space, and genetic data indicating the relative spatial patterns of the sampled pathogen strains. Applying this method allows to assign ranks to the pathogen strains with respect to their contributions to natural epidemics and to assess the significance of the ranking. This method was first tested on simulated data, including data obtained from an original, stochastic, multi-strain epidemic model. It was then applied to epidemiological and genetic data collected during one natural epidemic of powdery mildew occurring in its wild host population. Based on the simulation study, we conclude that the method can achieve its aim of ranking pathogen strains if the sampling effort is sufficient. For powdery mildew data, the method indicated that one of the sampled strains tends to have a higher fitness than the four other sampled strains, highlighting the importance of strain diversity for disease dynamics. Our approach allowing the comparison of pathogen strains in natural epidemic is complementary to the classical practice of using experimental infections in controlled conditions to estimate fitness of different pathogen strains. Our statistical tool, implemented in the R package StrainRanking, is mainly based on regression and does not rely on mechanistic assumptions on the pathogen dynamics. Thus, the method can be applied to a wide range of pathogens.     

Natural,Persistent Oscillations in a Spatial Multi-Strain Disease System with Application to Dengue

Many infectious diseases are not maintained in a state of equilibrium but exhibit significant fluctuations in prevalence over time. For pathogens that consist of multiple antigenic types or strains, such as influenza, malaria or dengue, these fluctuations often take on the form of regular or irregular epidemic outbreaks in addition to oscillatory prevalence levels of the constituent strains. To explain the observed temporal dynamics and structuring in pathogen populations, epidemiological multi-strain models have commonly evoked strong immune interactions between strains as the predominant driver. Here, with specific reference to dengue, we show how spatially explicit, multi-strain systems can exhibit all of the described epidemiological dynamics even in the absence of immune competition. Instead, amplification of natural stochastic differences in disease transmission, can give rise to persistent oscillations comprising semi-regular epidemic outbreaks and sequential dominance of dengue''s four serotypes. Not only can this mechanism explain observed differences in serotype and disease distributions between neighbouring geographical areas, it also has important implications for inferring the nature and epidemiological consequences of immune mediated competition in multi-strain pathogen systems.     

The impact of cross-immunity, mutation and stochastic extinction on pathogen diversity

We examine the dynamics of antigenically diverse infectious agents using a mathematical model describing the transmission dynamics of arbitrary numbers of pathogen strains, interacting via cross-immunity, and in the presence of mutations generating new strains and stochastic extinctions of existing ones. Equilibrium dynamics fall into three classes depending on cross-immunity, transmissibility and host population size: systems where global extinction is likely, stable single-strain persistence, and multiple-strain persistence with stable diversity. Where multi-strain dynamics are stable, a diversity threshold region separates a low-prevalence, low-diversity region of parameter space from a high-diversity, high-prevalence region. The location of the threshold region is determined by the reproduction number of the pathogen and the intensity of cross-immunity, with the sharpness of the transition being determined by the manner in which immunity accrues with repeated infections. Host population size and cross-immunity are found to be the most decisive factors in determining pathogen diversity. While the model framework developed is simplified, we show that it can capture essential aspects of the complex evolutionary dynamics of pathogens such as influenza.     

Characterization of a BODIPY-labeled {fl}uorescent fatty acid analogue. Binding to fatty acid-binding proteins, intracellular localization, and metabolism

The BODIPY-labeled fatty acid analogues are a useful addition to the tools employed to study the cellular uptake and metabolism of lipids. In this study, we show that BODIPY FL C₁₆ binds to purified liver and intestinal fatty acid-binding proteins with high affinity at a site similar to that for the physiological fatty acid oleic acid. Further, in human intestinal Caco-2 cells BODIPY FL C₁₆ co-localizes extensively with mitochondria, endoplasmic reticulum/Golgi, and L-FABP. Virtually no esterification of BODIPY FL C₁₆ was observed under the experimental conditions employed. We conclude that BODIPY FL C₁₆ may be a useful tool for studying the distribution and function of FABPs in a cellular environment.     

Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins

Three BODIPY GTPgammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP molecules were synthesized as possible fluorescent probes to study guanine nucleotide binding spectroscopically. Binding to G(alphao) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-fold, respectively. Binding of GTPgammaS and GppNHp analogs to G(alphao) is of high affinity (K(D) 11, 17, 55, and 110 nM, respectively) and reaches a stable plateau while fluorescence of BODIPY FL GTP shows a transient increase which returns to baseline. Furthermore, BODIPY FL GTPgammaS shows varying affinities for alpha(o), alpha(s), alpha(i1), and alpha(i2) (6, 58, 150, and 300 nM). The affinities of BODIPY FL GppNHp for all four G(alpha) subunits are 10-fold lower than for BODIPY FL GTPgammaS. Half-times for the fluorescence increase are consistent with known GDP release rates for those proteins. Enhancement of fluorescence upon binding the G(alpha) subunit is most likely due to a rotation around the gamma-thiol (GTPgammaS) or the 3' ribose-hydroxyl (GppNHp) bond to relieve the quenching of BODIPY fluorescence by the guanine base. Binding to G(alpha) exposes the BODIPY moiety to the external environment, as seen by an increase in sodium iodide quenching. The visible excitation and emission spectra and high fluorescence levels of these probes permit robust real-time detection of nucleotide binding.     

Microspectroscopic imaging of nodulation factor-binding sites on living Vicia sativa roots using a novel bioactive fluorescent nodulation factor.

A novel bioactive fluorescent nodulation (Nod) factor, NodRlv-IV(BODIPY FL-C16), has been synthesized by attaching a BODIPY FL-C16 acyl chain to the primary amino group of chitotetraose deacetylated at the nonreducing terminus by recombinant NodB. The binding of the fluorescent Nod factor to root systems of Vicia sativa was investigated with fluorescence spectral imaging microscopy (FSPIM) and fluorescence ratio imaging microscopy (FRIM). Spatially resolved fluorescence spectra of living and labeled Vicia sativa root systems were measured by FSPIM. Strong autofluorescence, inherent to many plant systems when excited at 488 nm, was corrected for by utilizing the difference in fluorescence emission spectra of the autofluorescence and NodRlv-IV(BODIPY FL-C16). A methodology is presented to break down the in situ fluorescence emission spectra into spatially resolved autofluorescence and BODIPY FL fluorescence spectra. Furthermore, an FRIM method was developed for correcting autofluorescence in fluorescence micrographs for this system. After autofluorescence correction it was shown that NodRlv-IV(BODIPY FL-C16) was concentrated in the root hairs, but was also bound to other parts of the root surface.     

A slow-fast dynamic decomposition links neutral and non-neutral coexistence in interacting multi-strain pathogens

Understanding the dynamics of multi-type microbial ecosystems remains a challenge, despite advancing molecular technologies for diversity resolution within and between hosts. Analytical progress becomes difficult when modelling realistic levels of community richness, relying on computationally-intensive simulations and detailed parametrisation. Simplification of dynamics in polymorphic pathogen systems is possible using aggregation methods and the slow-fast dynamics approach. Here, we develop one new such framework, tailored to the epidemiology of an endemic multi-strain pathogen. We apply Goldstone’s idea of slow dynamics resulting from spontaneously broken symmetries to study direct interactions in co-colonization, ranging from competition to facilitation between strains. The slow-fast dynamics approach interpolates between a neutral and non-neutral model for multi-strain coexistence, and quantifies the asymmetries that are important for the maintenance and stabilisation of diversity.     

Eukaryotic expression and purification of anti-epilepsy peptide of Buthus martensii Karsch and its protein interactions

To investigate the structure–function relationships of intestinal fatty acid-binding protein (I-FABP) in cellular fatty acid (FA) trafficking, we compared the distribution of a fluorescent FA analog (BODIPY FL C16) in Cos-1 cells transiently transfected with the wild type protein (wt I-FABP) to that of a variant deleted of the alpha helical domain (HL I-FABP). In vector-only cells, BODIPY fluorescence was distributed throughout the cytoplasm. In the absence of added FA, wt I-FABP was found largely in the perinuclear region with some cytoplasmic staining as well. Addition of BODIPY FL C16 to transfected cells showed that the fluorescent FA was essentially completely colocalized with the protein in the cytoplasmic and perinuclear regions as well as in cytoplasmic clusters that are not observed in the absence of wt I-FABP. For HL I-FABP, the distribution of the protein in the absence of FA was diffusely cytoplasmic, in marked contrast to the wt protein. Addition of BODIPY led to less extensive colocalization than that observed for wt I-FABP. In particular, no localization to the perinuclear region was found. Organelle colocalization studies showed that both proteins colocalized with mitochondria and endoplasmic reticulum/golgi markers, but little with a lysosomal marker. The perinuclear localization for wt I-FABP and BODIPY did not show colocalization with any of the markers tested. Taken together, these results indicate that I-FABP binds FA in vivo and that the helical domain may be important for targeting I-FABP to a perinuclear domain but not, perhaps, to the endoplasmic reticulum, golgi apparatus or mitochondria.     

Populations of a susceptible amphibian species can grow despite the presence of a pathogenic chytrid fungus

Disease can be an important driver of host population dynamics and epizootics can cause severe host population declines. Batrachochytrium dendrobatidis (Bd), the pathogen causing amphibian chytridiomycosis, may occur epizootically or enzootically and can harm amphibian populations in many ways. While effects of Bd epizootics are well documented, the effects of enzootic Bd have rarely been described. We used a state-space model that accounts for observation error to test whether population trends of a species highly susceptible to Bd, the midwife toad Alytes obstetricans, are negatively affected by the enzootic presence of the pathogen. Unexpectedly, Bd had no negative effect on population growth rates from 2002-2008. This suggests that negative effects of disease on individuals do not necessarily translate into negative effects at the population level. Populations of amphibian species that are susceptible to the emerging disease chytridiomycosis can persist despite the enzootic presence of the pathogen under current environmental conditions.     

FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.     

Batrachochytrium dendrobatidis infection dynamics in the Columbia spotted frog Rana luteiventris in north Idaho, USA

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) is contributing to amphibian declines worldwide. Temperature plays an important role in both pathogen growth and host immune function, but little is known about seasonal dynamics of Bd infection in north temperate regions. Our objective was to increase understanding of Bd disease ecology by investigating patterns of Bd infection of Columbia spotted frogs Rana luteiventris across seasons, age classes, and sexes in north Idaho, USA. We collected skin swabs from 223 R. luteiventris in spring, summer, and fall 2009 at 7 ponds in the Palouse region and quantified Bd zoospores for each sample using quantitative PCR. Across seasons, Bd prevalence of adults was higher in summer than in spring or fall, suggesting that individuals may be clearing low-level infections over the summer. Among age classes, all but one late stage tadpole (Gosner stage 43-45) tested negative for Bd. Conversely, 100% of metamorphs tested positive for Bd and had the highest Bd loads of all age classes, suggesting they may be the most vulnerable age class. Adult R. luteiventris had high infection prevalence (> 60%) in all seasons, indicating that Bd infection is maintained within populations and that adults likely serve as disease reservoirs across seasons. Among adults, we also found weak evidence for females having higher infection prevalence than males. Further laboratory and field studies are needed to determine whether there are individual and population impacts from Bd on R. luteiventris and other amphibians in north Idaho.     

Urinary corticosterone metabolites and chytridiomycosis disease prevalence in a free-living population of male Stony Creek frogs (Litoria wilcoxii)

The emerging amphibian disease chytridiomycosis, which is caused by the fungal pathogen (Batrachochytrium dendrobatidis, Bd), has caused mass mortalities of native amphibian populations globally. There have been no previous studies on the relationships between stress hormones in free-living amphibians and Bd infections. In this study, we measured urinary corticosterone metabolite concentrations and Bd infections within free-living populations of male Stony Creek frog (Litoria wilcoxii) in Queensland, Australia. Prevalence of Bd zoospores from frog skin swabs was quantified using a real-time quantitative PCR technique. A urinary corticosterone enzyme-immunoassay (EIA) was validated using adrenocorticotropic hormone (ACTH) challenge. Urinary corticosterone concentrations of male frogs increased within 1-2 days after ACTH challenge and returned to baseline levels within 3 days post-ACTH injection. None of the frogs showed any rise in urinary corticosterone after saline injections. Individual male frogs showed either low or high baseline corticosterone concentrations. Male frogs identified as positive for Bd infection had significantly higher baseline urinary corticosterone concentrations in comparison to Bd negative male frogs. Urinary corticosterone EIA provides a reliable indication of stress in this frog species and this non-invasive physiological tool can be used to further assess the dynamics of Bd infections and physiological stress responses in other native amphibians.     

New fluorescent macrolide derivatives for studying interactions of antibiotics and their analogs with the ribosomal exit tunnel

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.     

Analysis of symmetries in models of multi-strain infections

In mathematical studies of the dynamics of multi-strain diseases caused by antigenically diverse pathogens, there is a substantial interest in analytical insights. Using the example of a generic model of multi-strain diseases with cross-immunity between strains, we show that a significant understanding of the stability of steady states and possible dynamical behaviours can be achieved when the symmetry of interactions between strains is taken into account. Techniques of equivariant bifurcation theory allow one to identify the type of possible symmetry-breaking Hopf bifurcation, as well as to classify different periodic solutions in terms of their spatial and temporal symmetries. The approach is also illustrated on other models of multi-strain diseases, where the same methodology provides a systematic understanding of bifurcation scenarios and periodic behaviours. The results of the analysis are quite generic, and have wider implications for understanding the dynamics of a large class of models of multi-strain diseases.     

Effects of fluorescent probe structure on the dynamics at cysteine-34 within bovine serum albumin: evidence for probe-dependent modulation of the cybotactic region.

We have prepared a series of bovine serum albumins (BSA) that have been site-selectively labeled at cysteine-34 with one of four different sulfhydryl-selective boron dipyrromethene difluoride (BODIPY) fluorescent probes (BODIPY FL IA, BODIPY FL C(1) IA, BODIPY 530/550 IA, and BODIPY 493/503 MB). We determine how the choice of extrinsic probe structure dictates the recovered BSA-BODIPY dynamics under thermal (10-80 degrees C) and chemical (0-5M guanidine hydrochloride) denaturation conditions. The results of these experiments show that the global protein dynamics are sensed equally by each fluorescent probe; however, the probe itself influences the local probe dynamics within the cybotactic region that surrounds cysteine-34. Thus, it seems inappropriate to think of these extrinsic fluorescent probes as passive, nonparticipatory viewers of local protein dynamics.     

Effects of nutrient supplementation on host‐pathogen dynamics of the amphibian chytrid fungus: a community approach

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Fungal disease and temperature alter skin microbiome structure in an experimental salamander system

Pathogens compete with host microbiomes for space and resources. Their shared environment impacts pathogen–microbiome–host interactions, which can lead to variation in disease outcome. The skin microbiome of red‐backed salamanders (Plethodon cinereus) can reduce infection by the pathogen Batrachochytrium dendrobatidis (Bd) at moderate infection loads, with high species richness and high abundance of competitors as putative mechanisms. However, it is unclear if the skin microbiome can reduce epizootic Bd loads across temperatures. We conducted a laboratory experiment to quantify skin microbiome and host responses (P. cinereus: n = 87) to Bd at mimicked epizootic loads across temperatures (13, 17 and 21°C). We quantified skin microbiomes using 16S rRNA gene metabarcoding and identified operational taxonomic units (OTUs) taxonomically similar to culturable bacteria known to kill Bd (anti‐Bd OTUs). Prior to pathogen exposure, temperature changed the microbiome (OTU richness decreased by 12% and the abundance of anti‐Bd OTUs increased by 18% per degree increase in temperature), but these changes were not predictive of disease outcome. After exposure, Bd changed the microbiome (OTU richness decreased by 0.1% and the abundance of anti‐Bd OTUs increased by 0.2% per 1% increase in Bd load) and caused high host mortality across temperatures (35/45: 78%). Temperature indirectly impacted microbiome change and mortality through its direct effect on pathogen load. We did not find support for the microbiome impacting Bd load or host survival. Our research reveals complex host, pathogen, microbiome and environmental interactions to demonstrate that during epizootic events the microbiome will be unlikely to reduce pathogen invasion, even for putatively Bd‐resistant species.