Phenotypic characterization of cryptic species in the fungal pathogen Histoplasma.

Histoplasmosis is an endemic mycosis that often presents as a respiratory infection in immunocompromised patients. Hundreds of thousands of new infections are reported annually around the world. The etiological agent of the disease, Histoplasma, is a dimorphic fungus commonly found in the soil where it grows as mycelia. Humans can become infected by Histoplasma through inhalation of its spores (conidia) or mycelial particles. The fungi transition into the yeast phase in the lungs at 37°C. Once in the lungs, yeast cells reside and proliferate inside alveolar macrophages. Genomic work has revealed that Histoplasma is composed of at least five cryptic phylogenetic species that differ genetically. Three of those lineages have received new names. Here, we evaluated multiple phenotypic characteristics (colony morphology, secreted proteolytic activity, yeast size, and growth rate) of strains from five of the phylogenetic species of Histoplasma to identify phenotypic traits that differentiate between these species: Histoplasma capsulatum sensu stricto, Histoplasma ohiense, Histoplasma mississippiense, Histoplasma suramericanum, and an African lineage. We report diagnostic traits for three species. The other two species can be identified by a combination of traits. Our results suggest that (i) there are significant phenotypic differences among the cryptic species of Histoplasma and (ii) those differences can be used to positively distinguish those species in a clinical setting and for further study of the evolution of this fungal pathogen.IMPORTANCEIdentifying species boundaries is a critical component of evolutionary biology. Genome sequencing and the use of molecular markers have advanced our understanding of the evolutionary history of fungal pathogens, including Histoplasma, and have allowed for the identification of new species. This is especially important in organisms where morphological characteristics have not been detected. In this study, we revised the taxonomic status of the four named species of the genus Histoplasma, H. capsulatum sensu stricto (ss), H. ohiense, H. mississippiense, and H. suramericanum, and propose the use of species-specific phenotypic traits to aid their identification when genome sequencing is not available. These results have implications not only for evolutionary study of Histoplasma but also for clinicians, as the Histoplasma species could determine the outcome of disease and treatment needed.

Phenotypic characterization of cryptic species in the fungal pathogen Histoplasma.

Histoplasmosis is an endemic mycosis that often presents as a respiratory infection in immunocompromised patients. Hundreds of thousands of new infections are reported annually around the world. The etiological agent of the disease, Histoplasma, is a dimorphic fungus commonly found in the soil where it grows as mycelia. Humans can become infected by Histoplasma through inhalation of its spores (conidia) or mycelial particles. The fungi transitions into the yeast phase in the lungs at 37°C. Once in the lungs, yeast cells reside and proliferate inside alveolar macrophages. We have previously described that Histoplasma is composed of at least five cryptic species that differ genetically, and assigned new names to the lineages. Here we evaluated multiple phenotypic characteristics of 12 strains from five phylogenetic species of Histoplasma to identify phenotypic traits that differentiate between these species: H. capsulatum sensu stricto, H. ohiense, H. mississippiense, H. suramericanum, and an African lineage. We report diagnostic traits for two species. The other three species can be identified by a combination of traits. Our results suggest that 1) there are significant phenotypic differences among the cryptic species of Histoplasma, and 2) that those differences can be used to positively distinguish those species in a clinical setting and for further study of the evolution of this fungal pathogen.

A Radical Reimagining of Fungal Two-Component Regulatory Systems.

Bacterial two-component regulatory systems (TCSs) mediate signal transduction by transferring phosphoryl groups between sensor kinase and response regulator proteins, sometimes using intermediary histidine-phosphotransferase (Hpt) domains to form multistep phosphorelays. Because (i) almost all known fungal sensor kinases exhibit a domain architecture characteristic of bacterial TCS phosphorelays, (ii) all known fungal Hpts are stand-alone proteins suited to shuttle between cytoplasm and nucleus, and (iii) the best-characterized fungal TCS is a canonical phosphorelay, it is widely assumed that most or all fungal TCSs function via phosphorelays. However, fungi generally encode more sensor kinases than Hpts or response regulators, leading to a disparity between putative phosphorelay inputs and outputs. The simplest resolution of this paradox is to hypothesize that most fungal sensor kinases do not participate in phosphorelays. Reimagining how fungal TCSs might function leads to multiple testable predictions.

The Small Protein RmpD Drives Hypermucoviscosity in Klebsiella pneumoniae.

Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: classical strains that are a notable problem in health care settings due to multidrug resistance, and hypervirulent (hv) strains that are historically drug sensitive but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such gene, rmpA, encodes a transcriptional regulator required for maximal capsule (cps) gene expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently reported a new cps regulator, RmpC, and ΔrmpC mutants have reduced cps expression but retain HMV, suggesting that capsule production and HMV may be separable traits. Here, we report the identification of a small protein, RmpD, that is essential for HMV but does not impact capsule. RmpD is 58 residues with a putative N-terminal transmembrane domain and highly positively charged C-terminal half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans complements both ΔrmpD and ΔrmpA mutants for HMV, suggesting that RmpD is the key driver of this phenotype. The rmpD gene is located between rmpA and rmpC, within an operon regulated by RmpA. These data, combined with our previous work, suggest a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression.IMPORTANCE Capsule is a critical virulence factor in Klebsiella pneumoniae, in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscosity (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene (rmpD) required for HMV but not for capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits.

Biodiverse Histoplasma Species Elicit Distinct Patterns of Pulmonary Inflammation following Sublethal Infection.

Histoplasma is an endemic dimorphic fungus that can cause disease in healthy and immunocompromised individuals after the transition of inhaled spores into the facultative intracellular yeast form. There is substantial diversity among Histoplasma species, but it is not clear how this heterogeneity impacts the progression of pathology and cellular immune responses during acute respiratory infection, which represents the vast majority of histoplasmosis disease burden. After inoculating mice intranasally with a sublethal inoculum, we characterized the immune response to Histoplasma capsulatum (strain G186A) and Histoplasma ohiense (strain G217B) using comprehensive flow cytometric and single-cell analyses. Within 8 days after inoculation, H. ohiense induced a significantly higher infiltration of neutrophils and inflammatory monocytes into the lung compared to H. capsulatum Microscopic analysis of infected lung tissue revealed that although the total number of fungi was similar within inflamed lung lesions, we observed different species-dependent intracellular yeast distribution patterns. Inoculation with gfp-expressing strains indicated that H. ohiense, but not H. capsulatum, was associated primarily with alveolar macrophages early after infection. Interestingly, we observed a significant reduction in the total number of alveolar macrophages 12 to 16 days after H. ohiense, but not H. capsulatum infection, despite similar intracellular growth dynamics within AMJ2-C11 alveolar macrophages in vitro Together, our data suggest that H. ohiense, but not H. capsulatum, preferentially interacts with alveolar macrophages early after infection, which may lead to a different course of inflammation and resolution despite similar rates of fungal clearance.IMPORTANCE Acute pulmonary histoplasmosis in healthy individuals comprises most of the disease burden caused by the fungal pathogen Histoplasma Fungal pneumonia is frequently delayed in diagnosis and treatment due to a prolonged period of quiescence early during infection. In this study, we used a murine respiratory model of histoplasmosis to investigate how different Histoplasma species modulate lung inflammation throughout the complete course of infection. We propose that a relatively low, sublethal inoculum is ideal to model acute pulmonary histoplasmosis in humans, primarily due to the quiescent stage of fungal growth that occurs in the lungs of mice prior to the initiation of inflammation. Our results reveal the unique course of lung immunity associated with divergent species of Histoplasma and imply that the progression of clinical disease is considerably more heterogeneous than previously recognized.

Tn-Seq Analysis Identifies Genes Important for Yersinia pestis Adherence during Primary Pneumonic Plague.

Following inhalation, Yersinia pestis rapidly colonizes the lung to establish infection during primary pneumonic plague. Although several adhesins have been identified in Yersinia spp., the factors mediating early Y. pestis adherence in the lung remain unknown. To identify genes important for Y. pestis adherence during primary pneumonic plague, we used transposon insertion sequencing (Tn-seq). Wild-type and capsule mutant (Δcaf1) Y. pestis transposon mutant libraries were serially passaged in vivo to enrich for nonadherent mutants in the lung using a mouse model of primary pneumonic plague. Sequencing of the passaged libraries revealed six mutants that were significantly enriched in both the wild-type and Δcaf1Y. pestis backgrounds. The enriched mutants had insertions in genes that encode transcriptional regulators, chaperones, an endoribonuclease, and YPO3903, a hypothetical protein. Using single-strain infections and a transcriptional analysis, we identified a significant role for YPO3903 in Y. pestis adherence in the lung and showed that YPO3903 regulated transcript levels of psaA, which encodes a fimbria previously implicated in Y. pestis adherence in vitro Deletion of psaA had a minor effect on Y. pestis adherence in the lung, suggesting that YPO3903 regulates other adhesins in addition to psaA By enriching for mutations in genes that regulate the expression or assembly of multiple genes or proteins, we obtained screen results indicating that there may be not just one dominant adhesin but rather several factors that contribute to early Y. pestis adherence during primary pneumonic plague.IMPORTANCE Colonization of the lung by Yersinia pestis is a critical first step in establishing infection during primary pneumonic plague, a disease characterized by high lethality. However, the mechanisms by which Y. pestis adheres in the lung after inhalation remain elusive. Here, we used Tn-seq to identify Y. pestis genes important for adherence early during primary pneumonic plague. Our mutant enrichment strategy resulted in the identification of genes important for regulation and assembly of genes and proteins rather than adhesin genes themselves. These results reveal that there may be multiple Y. pestis adhesins or redundancy among adhesins. Identifying the adhesins regulated by the genes identified in our enrichment screen may reveal novel therapeutic targets for preventing Y. pestis adherence and the subsequent development of pneumonic plague.

Paracoccidioides Genomes Reflect High Levels of Species Divergence and Little Interspecific Gene Flow.

The fungus Paracoccidioides is a prevalent human pathogen endemic to South America. The genus is composed of five species. In this report, we use 37 whole-genome sequences to study the allocation of genetic variation in Paracoccidioides We tested three genome-wide predictions of advanced speciation, namely, that all species should be reciprocally monophyletic, that species pairs should be highly differentiated along the whole genome, and that there should be low rates of interspecific gene exchange. We find support for these three hypotheses. Species pairs with older divergences show no evidence of gene exchange, while more recently diverged species pairs show evidence of modest rates of introgression. Our results indicate that as divergence progresses, species boundaries become less porous among Paracoccidioides species. Our results suggest that species in Paracoccidioides are at different stages along the divergence continuum.IMPORTANCEParacoccidioides is the causal agent of a systemic mycosis in Latin America. Most of the inference of the evolutionary history of Paracoccidioides has used only a few molecular markers. In this report, we evaluate the extent of genome divergence among Paracoccidioides species and study the possibility of interspecific gene exchange. We find that all species are highly differentiated. We also find that the amount of gene flow between species is low and in some cases is even completely absent in spite of geographic overlap. Our study constitutes a systematic effort to identify species boundaries in fungal pathogens and to determine the extent of gene exchange among fungal species.

Fungal species boundaries in the genomics era.

Genomic data has opened new possibilities to understand how organisms change over time, and could enable the discovery of previously undescribed species. Although taxonomy used to be based on phenotypes, molecular data has frequently revealed that morphological traits are insufficient to describe biodiversity. Genomics holds the promise of revealing even more genetic discontinuities, but the parameters on how to describe species from genomic data remain unclear. Fungi have been a successful case in which the use of molecular markers has uncovered the existence of genetic boundaries where no crosses are possible. In this minireview, we highlight recent advances, propose a set of standards to use genomic sequences to uncover species boundaries, point out potential pitfalls, and present possible future research directions.

A Klebsiella pneumoniae Regulatory Mutant Has Reduced Capsule Expression but Retains Hypermucoviscosity.

The polysaccharide capsule is an essential virulence factor for Klebsiella pneumoniae in both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulent K. pneumoniae strains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S: rcsB and rmpA mutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene near rmpA The ΔrmpC strain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of the rmpA promoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of the rmpC and rmpA mutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains.IMPORTANCEKlebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.

Recent admixture between species of the fungal pathogen Histoplasma.

Hybridization between species of pathogens has the potential to speed evolution of virulence by providing the raw material for adaptation through introgression or by assembling new combinations of virulence traits. Fungal diseases are a source high morbidity, and remain difficult to treat. Yet the frequency of hybridization between fungal species has rarely been explored, and the functional role of introgressed alleles remains largely unknown. Histoplasma mississippiense and H. ohiense are sympatric throughout their range in North America and have distinct virulence strategies, making them an ideal system to examine the role introgression may play in fungal pathogens. We identified introgressed tracts in the genomes of a sample of H. mississippiense and H. ohiense isolates. We found strong evidence in each species for recent admixture, but introgressed alleles were present at low frequencies, suggesting that they were deleterious. Consistent with this, coding and regulatory sequences were strongly depleted within introgressed regions, whereas intergenic regions were enriched, indicating that functional introgressed alleles were frequently deleterious in their new genomic context. Surprisingly, we found only two isolates with substantial admixture: the H. mississippiense and H. ohiense genomic reference strains, WU24 and G217B, respectively. Our results show that recent admixture has occurred, that it is frequently deleterious and that conclusions based on studies of the H. mississippiense and H. ohiense type strains should be revisited with more representative samples from the genus.

Identification of Two Regulators of Virulence That Are Conserved in Klebsiella pneumoniae Classical and Hypervirulent Strains.

Klebsiella pneumoniae is widely recognized as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of hospital-acquired infections (urinary tract infections [UTI], pneumonia, sepsis) and community-acquired invasive infections. The genetic heterogeneity of K. pneumoniae isolates complicates our ability to understand the virulence of K. pneumoniae Characterization of virulence factors conserved between strains as well as strain-specific factors will improve our understanding of this important pathogen. The MarR family of regulatory proteins is widely distributed in bacteria and regulates cellular processes such as antibiotic resistance and the expression of virulence factors. Klebsiella encodes numerous MarR-like proteins, and they likely contribute to the ability of K. pneumoniae to respond to and survive under a wide variety of environmental conditions, including those present in the human body. We tested loss-of-function mutations in all the marR homologues in a murine pneumonia model and found that two (kvrA and kvrB) significantly impacted the virulence of K1 and K2 capsule type hypervirulent (hv) strains and that kvrA affected the virulence of a sequence type 258 (ST258) classical strain. In the hv strains, kvrA and kvrB mutants displayed phenotypes associated with reduced capsule production, mucoviscosity, and transcription from galF and manC promoters that drive expression of capsule synthesis genes. In contrast, kvrA and kvrB mutants in the ST258 strain had no effect on capsule gene expression or capsule-related phenotypes. Thus, KvrA and KvrB affect virulence in classical and hv strains but the effect on virulence may not be exclusively due to effects on capsule production.IMPORTANCE In addition to having a reputation as the causative agent for hospital-acquired infections as well as community-acquired invasive infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. Due to the rapid emergence of carbapenem resistance among K. pneumoniae strains, a better understanding of virulence mechanisms and identification of new potential drug targets are needed. This study identified two novel regulators (KvrA and KvrB) of virulence in K. pneumoniae and demonstrated that their effect on virulence in invasive strains is likely due in part to effects on capsule production (a major virulence determinant) and hypermucoviscosity. KvrA also impacts the virulence of classical strains but does not appear to affect capsule gene expression in this strain. KvrA and KvrB are conserved among K. pneumoniae strains and thus could regulate capsule expression and virulence in diverse strains regardless of capsule type.

Genome Sequences Reveal Cryptic Speciation in the Human Pathogen Histoplasma capsulatum.

Histoplasma capsulatum is a pathogenic fungus that causes life-threatening lung infections. About 500,000 people are exposed to H. capsulatum each year in the United States, and over 60% of the U.S. population has been exposed to the fungus at some point in their life. We performed genome-wide population genetics and phylogenetic analyses with 30 Histoplasma isolates representing four recognized areas where histoplasmosis is endemic and show that the Histoplasma genus is composed of at least four species that are genetically isolated and rarely interbreed. Therefore, we propose a taxonomic rearrangement of the genus.IMPORTANCE The evolutionary processes that give rise to new pathogen lineages are critical to our understanding of how they adapt to new environments and how frequently they exchange genes with each other. The fungal pathogen Histoplasma capsulatum provides opportunities to precisely test hypotheses about the origin of new genetic variation. We find that H. capsulatum is composed of at least four different cryptic species that differ genetically and also in virulence. These results have implications for the epidemiology of histoplasmosis because not all Histoplasma species are equivalent in their geographic range and ability to cause disease.

Lysosomal Degradation Is Required for Sustained Phagocytosis of Bacteria by Macrophages.

Clearance of bacteria by macrophages involves internalization of the microorganisms into phagosomes, which are then delivered to endolysosomes for enzymatic degradation. These spatiotemporally segregated processes are not known to be functionally coupled. Here, we show that lysosomal degradation of bacteria sustains phagocytic uptake. In Drosophila and mammalian macrophages, lysosomal dysfunction due to loss of the endolysosomal Cl- transporter ClC-b/CLCN7 delayed degradation of internalized bacteria. Unexpectedly, defective lysosomal degradation of bacteria also attenuated further phagocytosis, resulting in elevated bacterial load. Exogenous application of bacterial peptidoglycans restored phagocytic uptake in the lysosomal degradation-defective mutants via a pathway requiring cytosolic pattern recognition receptors and NF-κB. Mammalian macrophages that are unable to degrade internalized bacteria also exhibit compromised NF-κB activation. Our findings reveal a role for phagolysosomal degradation in activating an evolutionarily conserved signaling cascade, which ensures that continuous uptake of bacteria is preceded by lysosomal degradation of microbes.

Evaluation of the Arthritis Foundation's Camine Con Gusto Program for Hispanic Adults With Arthritis.

INTRODUCTION: Camine Con Gusto (CCG) is the Hispanic version of an evidence-based walking program for people with arthritis. This study examined CCG outcomes, feasibility, tolerability, safety, and acceptability and potential tailoring. METHOD: A pre and post 6-week evaluation was conducted in Hispanic people with arthritis. Outcomes included pain, stiffness, fatigue, functional capacity, helplessness, and self-efficacy. A formative evaluation with program participants and key stakeholders explored program tailoring. RESULTS: Participants' mean age was 46.9 years, 44.4% had a high school degree or less, 2.5% were born in United States, 60.1% spoke only Spanish, and 74.7% were female. Moderate effect sizes were found: 0.50 for pain, 0.75 for fatigue, 0.49 for stiffness, 0.33 for function, 0.26 for helplessness, and 0.24 for self-efficacy. There were 285 participants recruited with an 82% 6-week retention (feasibility), no adverse events were reported (safety), and 98% reported program satisfaction (acceptability). Recommended adaptations included simpler language, more pictures and content addressing nutrition and chronic conditions, shortened materials, and inclusion of motivational strategies. CONCLUSION: CCG showed improvement in outcomes in Hispanic individuals comparable to those noted in non-Hispanic White and Black individuals with arthritis.

Comparison of phylogenetically distinct Histoplasma strains reveals evolutionarily divergent virulence strategies.

Infection with the dimorphic fungus Histoplasma capsulatum results from the inhalation of contaminated soil. Disease outcome is variable and depends on the immune status of the host, number of organisms inhaled, and the H. capsulatum strain. H. capsulatum is divided into seven distinct clades based on phylogenetic analyses, and strains from two separate clades have been identified in North America (denoted as NAm strains). We characterized an H. capsulatum isolate (WU24) from the NAm 1 lineage in relation to two other well-characterized Histoplasma isolates, the Panamanian strain G186A and the NAm 2 strain G217B. We determined that WU24 is a chemotype II strain and requires cell wall α-(1,3)-glucan for successful in vitro infection of macrophages. In a mouse model of histoplasmosis, WU24 exhibited a disease profile that was very similar to that of strain G186A at a high sublethal dose; however, at this dose G217B had markedly different kinetics. Surprisingly, infection with a lower dose mitigated many of the differences during the course of infection. The observed differences in fungal burden, disease kinetics, symptomology, and cytokine responses all indicate that there is a sophisticated relationship between host and fungus that drives the development and progression of histoplasmosis. Importance: Histoplasmosis has a wide range of clinical manifestations, presenting as mild respiratory distress, acute respiratory infection, or a life-threatening disseminated disease most often seen in immunocompromised patients. Additionally, the outcome appears to be dependent on the amount and strain of fungus inhaled. In this study, we characterized a recent clinical H. capsulatum isolate that was collected from an HIV(+) individual in North America. In contrast to other isolates from the same lineage, this strain, WU24, infected both macrophages and wild-type mice. We determined that in contrast to many other North American strains, WU24 infection of macrophages is dependent on the presence of cell wall α-(1,3)-glucan. Surprisingly, comparison of WU24 with two previously characterized isolates revealed that many conclusions regarding relative strain virulence and certain hallmarks of histoplasmosis are dependent on the inoculum size.

Expression of Paracoccidioides brasiliensis AMY1 in a Histoplasma capsulatum amy1 mutant, relates an α-(1,4)-amylase to cell wall α-(1,3)-glucan synthesis.

In the cell walls of the pathogenic yeast phases of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum, the outer α-(1,3)-glucan layer behaves as a virulence factor. In H. capsulatum, an α-(1,4)-amylase gene (AMY1) is essential for the synthesis of this polysaccharide, hence related to virulence. An orthologous gene to H. capsulatum AMY1 was identified in P. brasiliensis and also labeled AMY1. P. brasiliensis AMY1 transcriptional levels were increased during the yeast phase, which correlates with the presence of α-(1,3)-glucan as the major yeast cell wall polysaccharide. Complementation of a H. capsulatum amy1 mutant strain with P. brasiliensis AMY1, suggests that P. brasiliensis Amy1p may play a role in the synthesis of cell wall α-(1,3)-glucan. To study some biochemical properties of P. brasiliensis Amy1p, the enzyme was overexpressed, purified and studied its activity profile with starch and amylopeptin. It showed a relatively higher hydrolyzing activity on amylopeptin than starch, producing oligosaccharides from 4 to 5 glucose residues. Our findings show that P. brasiliensis Amy1p produces maltooligosaccharides which may act as a primer molecule for the fungal cell wall α-(1,3)-glucan biosynthesis by Ags1p.

Microsatellite analysis of three phylogenetic species of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an important human systemic mycosis in Latin America. Recently, the existence of three different phylogenetic species (S1, PS2, and PS3) of P. brasiliensis was demonstrated. Despite being genetically isolated, all three species were capable of inducing disease in both humans and animals, although lower virulence has been found with the PS2 species. The available molecular methods developed to characterize and type strains have not been useful for assigning isolates to the described species, creating the need for molecular markers capable of distinguishing genetically isolated groups. Here, we describe a PCR and sequencing-based microsatellite marker system that is stable, easy to assay, adaptable to large series of isolates, and discriminatory enough to be used as a typing system in identifying the three proposed species of P. brasiliensis. In addition, this system provides an unambiguous tool for strain discrimination between two (S1 and PS2) of the three phylogenetic species.