Augmentation of innate host defenses against opportunistic fungal pathogens

Development of invasive fungal infection is the result of the complex interaction between fungal and host factors. The outcome of infection, once it has developed, depends upon appropriate use of antifungal therapy, surgical debridement as indicated, and improvement of host defenses. Thus, there have been major efforts for development of new strategies for immunomodulation and augmentation of host defenses in prevention and treatment of invasive mycoses. These modalities include granulocyte and granulocyte-macrophage colony-stimulating factors, interferon-γ, granulocyte transfusions, immunotherapy with infusion of dendritic cells and T cells, anti-heat shock protein 90 monoclonal antibodies, long pentraxin 3, mannose-binding lectin, and deferasirox. Although major strides in our understanding of augmentation of host response to invasive fungal infections are opening up novel avenues of therapy to harness patients’ innate immune systems against these frequently lethal pathogens, well-designed clinical trials are needed to demonstrate safety and efficacy of these new approaches.     

Molecular identification and phylogenetic analysis of fungal pathogens isolated from diseased fish in Xinjiang,China

The outbreaks of fungal diseases in cultured fish have been severe in recent years, which is harmful to the healthy and sustainable development of fish farming. In this study, an investigation was conducted for significant fungal infections of 12 species of fish in four regions in Xinjiang, China, to understand the distribution of local fish fungal pathogens. Twenty-six fungal strains with pathogenicity were isolated, and the challenge experiment showed that eight strains from Changji area had high infection rate to fish eggs. Based on internal transcribed spacer sequence data and molecular analysis, the 26 strains were classified into nine different species of six fungal genera. Phylogenetic analysis showed that all strains were divided into two clades, namely Cluster 1 (contains only the genus Mucor) and Cluster 2 (consists of five small branches), and the distribution of strains from the same region was scattered in two clusters. There is no strict host selectivity for these fungi to infect fish. Mucor sp. are the main fungal pathogen of fish in these four regions, whereas Hypophthalmichthys molitrix and Carassius auratus are two types of fish that were susceptible to pathogen. In addition, the environmental adaptability experiments showed that eight highly pathogenic strains have different adaptability to the environment, and their optimum temperature and pH were 25°C and 7.0, respectively, whereas the concentration of NaCl was negatively correlated with the growth of strains. Therefore, these results indicated that the coinfection of multiple fungal pathogens in a culture region should be considered in the future study.     

Revisiting old friends: Developments in understanding <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis> pathogenesis

Histoplasma capsulatum is a dimorphic pathogenic fungus and causative agent of histoplasmosis, which is a respiratory and systemic infection that is particularly severe in immunocompromised hosts and represents the fungal homolog of tuberculosis. In highly endemic regions, the majority of individuals have been infected and carry the organism in a persistent latent form that is a danger for reactivation if host defenses are suppressed. H. capsulatum has been a model organism for intracellular pathogenesis and fungal morphogenesis for decades. New genomic information and application of approaches for molecular genetic manipulation are shedding new light on virulence mechanisms.     

Exploiting amoeboid and non-vertebrate animal model systems to study the virulence of human pathogenic fungi

Experiments with insects, protozoa, nematodes, and slime molds have recently come to the forefront in the study of host-fungal interactions. Many of the virulence factors required for pathogenicity in mammals are also important for fungal survival during interactions with non-vertebrate hosts, suggesting that fungal virulence may have evolved, and been maintained, as a countermeasure to environmental predation by amoebae and nematodes and other small non-vertebrates that feed on microorganisms. Host innate immune responses are also broadly conserved across many phyla. The study of the interaction between invertebrate model hosts and pathogenic fungi therefore provides insights into the mechanisms underlying pathogen virulence and host immunity, and complements the use of mammalian models by enabling whole-animal high throughput infection assays. This review aims to assist researchers in identifying appropriate invertebrate systems for the study of particular aspects of fungal pathogenesis.     

Modeling fimbriae mediated parasite-host interactions

Type 1 fimbriae are a known virulence factor in a number of pathogenic enterobacteriaceae, including Salmonella, Shigella and E. coli. Yet, they are also expressed by some commensal strains, notably of E. coli. One hypothesis of the role of fimbriae in commensals is that they evoke a small but tolerable host immune response in order to have the host release sialic acid, which is a valuable nutrient. Genetic evidence suggests that sialic acid down-regulates fimbriation. This has been believed to enable the cells to reduce virulence when the host response is increasing, thus avoiding a full activation of host defenses. In this article we assess the plausibility of this hypothesis using mathematical models. Our models lead us to two main conclusions: A slight activation of host defenses is only possible with a carefully tuned set of parameters, whereas under a wide range of parameters and assumptions, the model predicts the host defenses to be activated to at least half their potential in response to fimbriation. Secondly, the fact that fimbriation is suppressed by sialic acid seems irrelevant for the global qualitative properties.     

Elevated genetic variation within virulence-associated Botrytis cinerea polygalacturonase loci

Botrytis cinerea, or gray mold, is a necrotrophic fungal pathogen of hundreds of plant species. The genetic diversity of B. cinerea may contribute to its broad host range; however, the level and structure of genetic variation at pathogenesis-associated loci has not been described. B. cinerea possesses six distinct cell-wall-degrading polygalacturonases (PGs), enzymes of demonstrated importance to pathogenesis and interaction with host plant defenses. Sequencing a collection of 34 B. cinerea isolates at three PG-encoding loci, BcPG1, BcPG2, and BcPG3, revealed limited evidence of host-mediated genetic subdivision within loci, yet suggested differences in the action of evolutionary forces among loci. BcPG1 and BcPG2 are highly polymorphic, particularly when compared with previously published data from nonpathogenicity loci, whereas BcPG3 is relatively conserved. Sequence variation at BcPG1 and BcPG2 did not appear to be associated with virulence on Arabidopsis leaves; however, BcPG2 variation showed a statistically significant association with growth rate on pectin. Rather than providing evidence for host-mediated genetic subdivision at individual PG loci, our data support specialization among PGs and the potential diversification of PGs interacting directly with host defenses.     

Fungal virulence, vertebrate endothermy, and dinosaur extinction: is there a connection?

Fungi are relatively rare causes of life-threatening systemic disease in immunologically intact mammals despite being frequent pathogens in insects, amphibians, and plants. Given that virulence is a complex trait, the capacity of certain soil fungi to infect, persist, and cause disease in animals despite no apparent requirement for animal hosts in replication or survival presents a paradox. In recent years studies with amoeba, slime molds, and worms have led to the proposal that interactions between fungi and other environmental microbes, including predators, select for characteristics that are also suitable for survival in animal hosts. Given that most fungal species grow best at ambient temperatures, the high body temperature of endothermic animals must provide a thermal barrier for protection against infection with a large number of fungi. Fungal disease is relatively common in birds but most are caused by only a few thermotolerant species. The relative resistance of endothermic vertebrates to fungal diseases is likely a result of higher body temperatures combined with immune defenses. Protection against fungal diseases could have been a powerful selective mechanism for endothermy in certain vertebrates. Deforestation and proliferation of fungal spores at cretaceous-tertiary boundary suggests that fungal diseases could have contributed to the demise of dinosaurs and the flourishing of mammalian species.     

Soil propagule banks of ectomycorrhizal fungi share many common species along an elevation gradient

We conducted bioassay experiments to investigate the soil propagule banks of ectomycorrhizal (EM) fungi in old-growth forests along an elevation gradient and compared the elevation pattern with the composition of EM fungi on existing roots in the field. In total, 150 soil cores were collected from three forests on Mt. Ishizuchi, western Japan, and subjected to bioassays using Pinus densiflora and Betula maximowicziana. Using molecular analyses, we recorded 23 EM fungal species in the assayed propagule banks. Eight species (34.8 %) were shared across the three sites, which ranged from a warm–temperate evergreen mixed forest to a subalpine conifer forest. The elevation pattern of the assayed propagule banks differed dramatically from that of EM fungi on existing roots along the same gradient, where only a small proportion of EM fungal species (3.5 %) were shared across sites. The EM fungal species found in the assayed propagule banks included many pioneer fungal species and composition differed significantly from that on existing roots. Furthermore, only 4 of 23 species were shared between the two host species, indicating a strong effect of bioassay host identity in determining the propagule banks of EM fungi. These results imply that the assayed propagule bank is less affected by climate compared to EM fungal communities on existing roots. The dominance of disturbance-dependent fungal species in the assayed propagule banks may result in higher ecosystem resilience to disturbance even in old-growth temperate forests.     

水霉菌的形态及ITS区分子鉴定

研究利用形态学和分子生物学相结合的方法, 对从患病黄颡鱼病灶处分离的水霉菌株(HSY)的分类地位进行了研究。结果显示, 该菌在18—23℃均能形成游动孢子囊, 但只在20℃才大量释放游动孢子; 在15℃培养约3 周出现大量的藏卵器, 多数顶生, 形态与Saprolegnia litoralis 和Saprolegnia ferax 的均非常相似。序列比对分析显示, HSY 菌株与S. litoralis、S. bulbosa、S. oliviae、S. longicaulis、S. ferax、S. mixta 和S.anomalies 的ITS 区(包括5.8S rDNA)序列相似性均高于99%。系统发育分析显示, 所选的真菌分为3 个群,HSY 落于第一个群, MP 树显示HSY 菌株与S. litoralis、S. bulbosa、S. oliviae、S. longicaulis、S. ferax 以及S. mixta 枝系均互相平行, 而NJ 树显示其与S. ferax 和S. mixta 更近。综合形态学、ITS 区序列及系统发育分析, 将HSY 菌株暂定为多子水霉(Saprolegnia ferax)。       

Lessons from interactions within the cassava green mite fungal pathogen Neozygites tanajoae system and prospects for microbial control using Entomophthorales

Most fungal pathogens lack the capacity to search for their host but rather develop sit-and-wait strategies that favour contact with them. The success of these strategies depends upon the interactions of the pathogen with its host, the host plant and the environmental conditions, which altogether determine its transmissibility. Given the limited success that has characterized application of sustainable microbial control, particularly using Entomophthorales, interaction studies have been conducted with the entomophthoralean fungus Neozygites tanajoae, pathogenic to the cassava green mite (CGM), Mononychellus tanajoa, to help understand differences observed between laboratory and field performances of this pathogen. Reciprocal pathogen-host interactions as well as tritrophic interactions involving the host plant were studied. It was found that herbivory triggers the release of volatiles that promote sporulation of isolates of N. tanajoae, whereas the host mite avoids haloes of spores of this pathogen. However, the host mite does not avoid the pathogen when inside the mummified fungus-killed cadaver. The status of microbial control of CGM in Africa is reviewed and implications of these interactions are discussed for prospective application of microbial control using Entomophthorales.     

PCR-RFLP和多重PCR技术检测常见病原性丝状真菌的实验研究

目的建立检测常见丝状真菌感染病原菌的PCR-RFLP和多重PCR方法。方法建立以PCR技术为基础的限制片段长度多态性(RFLP)方法 ,首先用真菌通用引物扩增丝状真菌的ITS区,然后用限制性核酸内切酶对PCR产物进行酶切。用4种丝状真菌的特异性引物建立多重PCR体系,用该体系检测单模板、双模板和三模板的扩增情况,并测定该体系的特异性和敏感性。结果用PCR-RFLP技术能够鉴别5种常见丝状真菌,多重PCR能够根据扩增片段的不同鉴别菌种,在合适的反应条件下,对单模板、双模板和三模板均能扩增出目的片段。结论 PCR-RFLP和多重PCR技术能够快速鉴定丝状真菌感染病原菌,有临床应用的良好前景。     

Overexpression of mannitol dehydrogenase in zonal geranium confers increased resistance to the mannitol secreting fungal pathogen Botrytis cinerea

The sugar alcohol mannitol is a carbohydrate with well-documented roles in both metabolism and osmoprotection in plants and fungi. In addition, however, mannitol is an antioxidant, and current research suggests that pathogenic fungi can secrete mannitol into the plant’s extracellular spaces during infection to suppress reactive oxygen-mediated host defenses. In response to pathogen attack, plants have been shown to secrete the normally symplastic enzyme, mannitol dehydrogenase (MTD). Given that MTD converts mannitol to the sugar mannose, extracellular MTD may be an important defense against mannitol-secreting fungal pathogens. Previous work demonstrated that overexpression of MTD in tobacco did, in fact, provide increased resistance to the mannitol-secreting fungal pathogen Alternaria alternata. In the present work we demonstrate that the fungal pathogen Botrytis cinerea also can secrete mannitol, and that overexpression of MTD in zonal geranium (Pelargonium × hortorum) in turn provides increased resistance to B. cinerea. These results are not only an important validation of previous work, but support the idea that MTD-overexpression might be used to engineer a broad variety of plants for resistance to mannitol-secreting fungal pathogens like B. cinerea for which specific resistance is lacking.     

Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata

Our previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen-mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non-mannitol-containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol- and non-mannitol-secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non-mannitol-secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol-secreting fungal plant pathogens.     

Strain Dependent Variation of Immune Responses to A. fumigatus: Definition of Pathogenic Species

For over a century microbiologists and immunologist have categorized microorganisms as pathogenic or non-pathogenic species or genera. This definition, clearly relevant at the strain and species level for most bacteria, where differences in virulence between strains of a particular species are well known, has never been probed at the strain level in fungal species. Here, we tested the immune reactivity and the pathogenic potential of a collection of strains from Aspergillus spp, a fungus that is generally considered pathogenic in immuno-compromised hosts. Our results show a wide strain-dependent variation of the immune response elicited indicating that different isolates possess diverse virulence and infectivity. Thus, the definition of markers of inflammation or pathogenicity cannot be generalized. The profound understanding of the molecular mechanisms subtending the different immune responses will result solely from the comparative study of strains with extremely diverse properties.     

Assessment of genetic variability and relatedness among atypical Aeromonas salmonicida from marine fishes,using AFLP-fingerprinting

Atypical strains of Aeromonas salmonicida are the causal agent of atypical furunculosis or ulcer disease in various fish species, including spotted wolffish Anarhichas minor, which is a promising species in the Norwegian fish-farming industry. Isolates of atypical A. salmonicida comprise a very heterogenous group showing large variety in biochemical, molecular and virulence characteristics. The genetic variability among atypical isolates from wolffish was characterised using amplified fragment length polymorphism analysis: AFLP-fingerprinting. Additional isolates from halibut Hippoglossus hippoglossus, turbot Scophthalmus maximus, cod Gadus morhua and several salmonid fishes were included for assessment of variability and relatedness among a total of 56 atypical isolates of A. salmonicida. They were compared to reference strains of A. salmonicida subspecies and to other Aeromonas species pathogenic in fishes. AFLP-fingerprints subjected to similarity analysis yielded a grouping of the isolates into several clusters, revealing genetic heterogeneity among the isolates. There seems to be a correlation between genetic similarity among isolates and the fish host. The Icelandic isolates, mainly from cod, formed a very homogeneous subcluster, which was closely related to the wolffish isolates. All atypical isolates from spotted and common wolffish grouped together in a large cluster and appear to be very homogeneous, even though they had been isolated over a period of 8 yr at different locations in Norway. On the other hand, most of the isolates from turbot and halibut grouped together into 2 different clusters, while the 9 atypical isolates from salmonids appeared in 4 different clusters. Thus, the atypical isolates of A. salmonicida from halibut, turbot and salmonid fishes seem to be more genetically diverse than those from wolffish and cod.     

Virulence and fitness of the fungal pathogen Entomophaga maimaiga in its host Lymantria dispar, for pathogen and host strains originating from Asia, Europe, and North America

In this study, we tested (1) whether non-North American gypsy moth strains are susceptible to North American isolates of Entomophaga maimaiga and (2) the potential for erosion in the efficacy of E. maimaiga in controlling gypsy moth. We used bioassays to assess the variability in virulence (measured as time to death) as well as fitness of the pathogen (measured as spore production) in four gypsy strains challenged with six E. maimaiga isolates, using host and pathogen strains originating from Asia, Europe, and North America. We found that all E. maimaiga isolates tested were pathogenic to all strains of Lymantria dispar, regardless of the geographical origin of the fungal isolate, with at least 86% mortality for all combinations of fungal isolate and gypsy moth strain. We therefore conclude that Asian gypsy moths are susceptible to North American strains of E. maimaiga. No significant interactions between fungal isolates and gypsy moth strains with regard to time to death were found, indicating that each fungal isolate had the same overall effect on all the gypsy moth strains tested. However, fungal isolates differed significantly with regard to virulence, with a Russian isolate being the slowest to kill gypsy moth (5.1+/-0.1 days) and a Japanese isolate being the overall fastest to kill its host (4.0+/-0.1 days). Fungal isolates also differed in fitness, with variability in types of spores produced. These differences in virulence and fitness were, however, not correlated with geographical origin of the fungal isolate. Gypsy moth strains had no or only little effect on fungal virulence and fitness. Based on our studies with laboratory-reared gypsy moth strains, erosion of successful control of gypsy moth by E. maimaiga seems unlikely.     

Variation in host resistance and pathogen selective value in the interaction between Pinus sylvestris and the fungus Crumenulopsis sororia

There have been many studies of plant pathogen evolution in systems showing gene-for-gene control of host resistance. However little is known about situations, exemplified by Scots pine, Pinus sylvestris, and its fungal pathogen Crumenulopsis sororia, where variation in host resistance is quantitative. In a field experiment genetically marked isolates of C. sororia from three natural populations were reciprocally inoculated on 1- and 2-year-old branch tissue of P. sylvestris in the three sites from which they had been collected. Quantitative variation in host resistance was measured by comparing the performance of the same inocula on different host populations, individuals and tissues. The selective value of isolates derived from different populations was estimated by comparing the frequency of genotypes in lesion re-isolations with those in the initial inoculum mixtures. Host resistance varied significantly among populations, individuals within populations and between 1- and 2-year-old branch tissue of P. sylvestris. Large differences in the relative selective values of C. sororia isolates from different populations were detected. The selective value of pathogens was independent of the host population on which they were inoculated. However, their selective value did depend on the age of the tissue on which they grew. The implications of these results for modelling evolution in pathogen-host interactions that lack gene-for-gene determination of host resistance are discussed.