Gregory M. Gauthier

Dimorphism in fungal pathogens of mammals, plants, and insects.

The capacity for pathogenic fungi to change morphology during their lifecycle is widespread. However, relatively few fungi are considered dimorphic, which refers to the ability to switch between two morphologies, yeast and hyphae (Table 1). These pathogens can be roughly subdivided into thermal (morphologic switch induced by temperature) and non-thermal dimorphic fungi (Table 1). Table 1 Dimorphic fungal pathogens. Worldwide, the thermally dimorphic fungi cause several million human infections each year. In the United States, Histoplasma capsulatum and Coccidioides spp. are estimated to infect 500,000 and 150,000 persons annually, respectively [1,2]. The incidence of coccidioidomycosis has recently increased and its endemic range has extended beyond the Southwest to include eastern Washington state [3]. Although the thermally dimorphic fungi typically infect healthy hosts, these pathogens account for 5.3% of fungal infections in solid organ transplant recipients [4]. Immunosuppressed patients are at risk for respiratory failure and extrapulmonary dissemination [4]. Phytopathogenic dimorphic fungi have had a major impact on urban landscapes and agriculture. Ophiostoma ulmi, which caused the first Dutch elm disease epidemic, has been replaced by a more virulent species, Ophiostoma novo-ulmi, which has destroyed millions of elm trees in the US and Europe [5]. Taphrina deformans, the etiologic agent of peach leaf curl, results in economic losses of

Crossover fungal pathogens: the biology and pathogenesis of fungi capable of crossing kingdoms to infect plants and humans.

2.5–3 million in the US [6]. Although Ustilago maydis, which causes corn smut, is not a major agricultural threat, the galls (i.e., huitlacoche) caused by infection are eaten as a delicacy [7]. Entomopathogenic fungi have been utilized to control insects harmful to agriculture and to study how pathogens control host behavior [8,9].

The dynamic genome and transcriptome of the human fungal pathogen Blastomyces and close relative Emmonsia

The outbreak of fungal meningitis associated with contaminated methylprednisolone acetate has thrust the importance of fungal infections into the public consciousness. The predominant pathogen isolated from clinical specimens, Exserohilum rostratum (teleomorph: Setosphaeria rostrata), is a dematiaceous fungus that infects grasses and rarely humans. This outbreak highlights the potential for fungal pathogens to infect both plants and humans. Most crossover or trans-kingdom pathogens are soil saprophytes and include fungi in Ascomycota and Mucormycotina phyla. To establish infection, crossover fungi must overcome disparate, host-specific barriers, including protective surfaces (e.g. cuticle, skin), elevated temperature, and immune defenses. This review illuminates the underlying mechanisms used by crossover fungi to cause infection in plants and mammals, and highlights critical events that lead to human infection by these pathogens. Several genes including veA, laeA, and hapX are important in regulating biological processes in fungi important for both invasive plant and animal infections.

Calcium binding by the essential virulence factor BAD-1 of Blastomyces dermatitidis.

Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in Blastomyces.

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

BAD-1 (Blastomyces adhesin 1), a 120-kDa protein of Blastomyces dermatitidis, functions as an adhesin, immune modulator, and essential virulence factor. Structurally, BAD-1 is composed of a short N-terminal region, a core of 30 tandem repeats critical for virulence, and a C-terminal epidermal growth factor domain that binds the protein to yeast cell surface chitin. Each of the 30 acidic residue-rich tandem repeats contains a sequence that resembles the calcium-binding loop of the EF-hand domain found in many calcium-binding proteins. Here, we investigated the binding of calcium by BAD-1 and its biological significance. Yeast washed with double distilled H2O released surface-bound BAD-1, but EGTA washes were an order of magnitude more efficient, suggesting an interaction between BAD-1 and calcium. Immobilized BAD-1 was stained with ruthenium red dye, an indicator of calcium-binding proteins. In equilibrium dialysis, BAD-1 bound 45Ca2+ with an affinity of 0.41 × 10-5 m and a capacity of 27 calcium/mol. Mass spectrometry confirmed this capacity. Elevated [Ca2+] diminished BAD-1 solubility. Upon deletion of its C-terminal epidermal growth factor-like domain, BAD-1 resisted aggregation by elevated [Ca2+] but retained its affinity and capacity for calcium. Removing 20 copies of the tandem repeat, however, sharply reduced the capacity of BAD-1 for calcium. Growth of the bad-1 null yeast was inhibited by 5 mm EGTA, and re-expression of BAD-1 in trans or the addition of exogenous purified BAD-1 restored growth. Thus, BAD-1 is a high capacity calcium-binding protein. This property contributes to the structure and function of BAD-1, as well as to B. dermatitidis acquisition of calcium from the environment.

Transmission of Babesia microti Parasites by Solid Organ Transplantation.

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.

Fungal Morphology, Iron Homeostasis, and Lipid Metabolism Regulated by a GATA Transcription Factor in Blastomyces dermatitidis.

Infection with this parasite should be included in differential diagnosis of fever and anemia after blood transfusion or organ transplantation.

Discordant Influence of Blastomyces dermatitidis Yeast-Phase-Specific Gene BYS1 on Morphogenesis and Virulence

In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0–48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

Paracoccidioides brasiliensis presents metabolic reprogramming and secretes a serine proteinase during murine infection

ABSTRACT Blastomyces dermatitidis is a thermally induced dimorphic fungus capable of causing lung and systemic infections in immunocompetent animal hosts. With the publication of genomic sequences from three different strains of B. dermatitidis and the development of RNA interference as a gene-silencing tool, it has become possible to easily ascertain the virulence and morphological effects of knocking down the expression of candidate genes of interest. BYS1 (Blastomyces yeast-phase-specific 1), first identified by Burg and Smith, is expressed at high levels in yeast cells and is undetectable in mold. The deduced protein sequence of BYS1 has a putative signal sequence at its N terminus, opening the possibility that the BYS1-encoded protein is associated with the yeast cell wall. Herein, strains of B. dermatitidis with silenced expression of BYS1 were engineered and tested for morphology and virulence. The silenced strains produced rough-surfaced cultures on agar medium and demonstrated a propensity to form pseudohyphal cells on prolonged culture in vitro and in vivo, as measured in the mouse lung. Tests using a mouse model of blastomycosis with either yeast or spore inocula showed that the bys1-silenced strains were as virulent as control strains. Thus, although silencing of BYS1 alters morphology at 37°C, it does not appear to impair the pathogenicity of B. dermatitidis.

Peritonitis caused by Blastomyces dermatitidis in a kidney transplant recipient: case report and literature review

ABSTRACT Paracoccidoides brasiliensis and Paracoccidioides lutzii, the etiologic agents of paracoccidioidomycosis, cause disease in healthy and immunocompromised persons in Latin America. We developed a method for harvesting P. brasiliensis yeast cells from infected murine lung to facilitate in vivo transcriptional and proteomic profiling. P. brasiliensis harvested at 6 h post-infection were analyzed using RNAseq and LC-MSE. In vivo yeast cells had 594 differentially expressed transcripts and 350 differentially expressed proteins. Integration of transcriptional and proteomic data indicated that early in infection (6 h), P. brasiliensis yeast cells underwent a shift in metabolism from glycolysis to β-oxidation, upregulated detoxifying enzymes to defend against oxidative stress, and repressed cell wall biosynthesis. Bioinformatics and functional analyses also demonstrated that a serine proteinase was upregulated and secreted in vivo. To our knowledge this is the first study depicting transcriptional and proteomic data of P. brasiliensis yeast cells upon 6 h post-infection of mouse lung.