Study on the mechanisms of action of berberine combined with fluconazole against fluconazole-resistant strains of Talaromyces marneffei.

Talaromyces (Penicillium) marneffei (T. marneffei) is a thermally dimorphic fungus that can cause opportunistic systemic mycoses. Our previous study demonstrated that concomitant use of berberine (BBR) and fluconazole (FLC) showed a synergistic action against FLC-resistant T. marneffei (B4) in vitro. In this paper, we tried to figure out the antifungal mechanisms of BBR and FLC in T. marneffei FLC-resistant. In the microdilution test, the minimum inhibitory concentration (MIC) of FLC was 256 µg/ml before FLC and BBR combination, and was 8 µg/ml after combination, the partial inhibitory concentration index (FICI) of B4 was 0.28. After the treatments of BBR and FLC, the studies revealed that (i) increase reactive oxygen species (ROS), (ii) reduce ergosterol content, (iii) destroy the integrity of cell wall and membrane, (iv) decrease the expression of genes AtrF, MDR1, PMFCZ, and Cyp51B however ABC1 and MFS change are not obvious. These results confirmed that BBR has antifungal effect on T. marneffei, and the combination with FLC can restore the susceptibility of FLC-resistant strains to FLC, and the reduction of ergosterol content and the down-regulation of gene expression of AtrF, Mdr1, PMFCZ, and Cyp51B are the mechanisms of the antifungal effect after the combination, which provides a theoretical basis for the application of BBR in the treatment of Talaromycosis and opens up new ideas for treatment of Talaromycosis.

A global call for talaromycosis to be recognised as a neglected tropical disease.

Talaromycosis (penicilliosis) is an invasive mycosis that is endemic in tropical and subtropical Asia. Talaromycosis primarily affects individuals with advanced HIV disease and other immunosuppressive conditions, and the disease disproportionally affects people in low-income and middle-income countries, particularly agricultural workers in rural areas during their most economically productive years. Approximately 17 300 talaromycosis cases and 4900 associated deaths occur annually. Talaromycosis is highly associated with the tropical monsoon season, when flooding and cyclones can exacerbate the poverty-inducing potential of the disease. Talaromycosis can present as localised or disseminated disease, the latter causing cutaneous lesions that are disfiguring and stigmatising. Despite up to a third of diagnosed cases resulting in death, talaromycosis has received little attention and investment from regional and global funders, policy makers, researchers, and industry. Diagnostic and treatment modalities remain extremely insufficient, however control of talaromycosis is feasible with known public health strategies. This Viewpoint is a global call for talaromycosis to be recognised as a neglected tropical disease to alleviate its impact on susceptible populations.

The novel Dbl homology/BAR domain protein, MsgA, of Talaromyces marneffei regulates yeast morphogenesis during growth inside host cells.

Microbial pathogens have evolved many strategies to evade recognition by the host immune system, including the use of phagocytic cells as a niche within which to proliferate. Dimorphic pathogenic fungi employ an induced morphogenetic transition, switching from multicellular hyphae to unicellular yeast that are more compatible with intracellular growth. A switch to mammalian host body temperature (37 °C) is a key trigger for the dimorphic switch. This study describes a novel gene, msgA, from the dimorphic fungal pathogen Talaromyces marneffei that controls cell morphology in response to host cues rather than temperature. The msgA gene is upregulated during murine macrophage infection, and deletion results in aberrant yeast morphology solely during growth inside macrophages. MsgA contains a Dbl homology domain, and a Bin, Amphiphysin, Rvs (BAR) domain instead of a Plekstrin homology domain typically associated with guanine nucleotide exchange factors (GEFs). The BAR domain is crucial in maintaining yeast morphology and cellular localisation during infection. The data suggests that MsgA does not act as a canonical GEF during macrophage infection and identifies a temperature independent pathway in T. marneffei that controls intracellular yeast morphogenesis.

Antifungal Activity and Molecular Mechanisms of Partial Purified Antifungal Proteins from Rhinacanthus nasutus against Talaromyces marneffei.

Antifungal proteins (AFPs) are able to inhibit a wide spectrum of fungi without significant toxicity to the hosts. This study examined the antifungal activity of AFPs isolated from a Thai medicinal plant, Rhinacanthus nasutus, against the human pathogenic fungus Talaromycesmarneffei. This dimorphic fungus causes systemic infections in immunocompromised individuals and is endemic in Southeast Asian countries. The R. nasutus crude protein extract inhibited the growth of T. marneffei. The anti-T. marneffei activity was completely lost when treated with proteinase K and pepsin, indicating that the antifungal activity was dependent on a protein component. The total protein extract from R. nasutus was partially purified by size fractionation to ≤10, 10-30, and ≥30 kDa fractions and tested for the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). All fractions showed anti-T. marneffei activity with the MIC and MFC values of 32 to 128 µg/mL and >128 µg/mL, respectively. In order to determine the mechanism of inhibition, all fractions were tested with T. marneffei mutant strains affected in G-protein signaling and cell wall integrity pathways. The anti-T. marneffei activity of the 10-30 kDa fraction was abrogated by deletion of gasA and gasC, the genes encoding alpha subunits of heterotrimeric G-proteins, indicating that the inhibitory effect is related to intracellular signaling through G-proteins. The work demonstrates that antifungal proteins isolated from R. nasutus represent sources for novel drug development.

Adaptation to Industrial Stressors Through Genomic and Transcriptional Plasticity in a Bioethanol Producing Fission Yeast Isolate.

Schizosaccharomyces pombe is a model unicellular eukaryote with ties to the basic research, oenology and industrial biotechnology sectors. While most investigations into S. pombe cell biology utilize Leupold's 972h- laboratory strain background, recent studies have described a wealth of genetic and phenotypic diversity within wild populations of S. pombe including stress resistance phenotypes which may be of interest to industry. Here we describe the genomic and transcriptomic characterization of Wilmar-P, an S. pombe isolate used for bioethanol production from sugarcane molasses at industrial scale. Novel sequences present in Wilmar-P but not in the laboratory S. pombe genome included multiple coding sequences with near-perfect nucleotide identity to Schizosaccharomyces octosporus sequences. Wilmar-P also contained a ∼100kb duplication in the right arm of chromosome III, a region harboring ght5+, the predominant hexose transporter encoding gene. Transcriptomic analysis of Wilmar-P grown in molasses revealed strong downregulation of core environmental stress response genes and upregulation of hexose transporters and drug efflux pumps compared to laboratory S. pombe Finally, examination of the regulatory network of Scr1, which is involved in the regulation of several genes differentially expressed on molasses, revealed expanded binding of this transcription factor in Wilmar-P compared to laboratory S. pombe in the molasses condition. Together our results point to both genomic plasticity and transcriptomic adaptation as mechanisms driving phenotypic adaptation of Wilmar-P to the molasses environment and therefore adds to our understanding of genetic diversity within industrial fission yeast strains and the capacity of this strain for commercial scale bioethanol production.

Laboratory Maintenance and Growth of Talaromyces marneffei.

Talaromyces marneffei is an important opportunistic human pathogen endemic to Southeast Asia. It is one of a number of pathogenic fungi that exhibits thermally controlled dimorphism. At 25°C, T. marneffei grows in a multicellular, filamentous hyphal form that can differentiate to produce dormant spores called conidia. These conidia are the likely infectious agent. At 37°C, T. marneffei grows as a uninucleate yeast that divides by fission. The yeast cells are the pathogenic form of this fungus. The protocols described here explain how to grow T. marneffei in the two vegetative growth forms in vitro, grow yeast cells inside mammalian macrophages, produce conidial stocks, and store strains both short and long term. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Growth of the vegetative hyphal form on solid medium Alternate Protocol 1: Growth of the vegetative hyphal form in liquid suspension Basic Protocol 2: Growth of the vegetative yeast form on solid medium Alternate Protocol 2: Growth of the vegetative yeast form in liquid suspension Basic Protocol 3: Growth for production of dormant conidia Support Protocol: Preparation of Miracloth filter tubes Basic Protocol 4: Growth of Talaromyces marneffei in mammalian macrophages Basic Protocol 5: Storage of Talaromyces marneffei strains Alternate Protocol 3: Lyophilization of Talaromyces marneffei strains.

ß-glucan-dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. ß-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.

A unique aspartyl protease gene expansion in Talaromyces marneffei plays a role in growth inside host phagocytes.

Aspartyl proteases are a widely represented class of proteolytic enzymes found in eukaryotes and retroviruses. They have been associated with pathogenicity in a range of disease-causing microorganisms. The dimorphic human-pathogenic fungus Talaromyces marneffei has a large expansion of these proteases identified through genomic analyses. Here we characterize the expansion of these genes (pop - paralogue of pep) and their role in T. marneffei using computational and molecular approaches. Many of the genes in this monophyletic family show copy number variation and positive selection despite the preservation of functional regions and possible redundancy. We show that the expression profile of these genes differs and some are expressed during intracellular growth in the host. Several of these proteins have distinctive localization as well as both additive and epistatic effects on the formation of yeast cells during macrophage infections. The data suggest that this is a recently evolved aspartyl protease gene family which affects intracellular growth and contributes to the pathogenicity of T. marneffei.

A genome-wide analysis of carbon catabolite repression in Schizosaccharomyces pombe.

BACKGROUND: Optimal glucose metabolism is central to the growth and development of cells. In microbial eukaryotes, carbon catabolite repression (CCR) mediates the preferential utilization of glucose, primarily by repressing alternate carbon source utilization. In fission yeast, CCR is mediated by transcriptional repressors Scr1 and the Tup/Ssn6 complex, with the Rst2 transcription factor important for activation of gluconeogenesis and sexual differentiation genes upon derepression. Through genetic and genome-wide methods, this study aimed to comprehensively characterize CCR in fission yeast by identifying the genes and biological processes that are regulated by Scr1, Tup/Ssn6 and Rst2, the core CCR machinery. RESULTS: The transcriptional response of fission yeast to glucose-sufficient or glucose-deficient growth conditions in wild type and CCR mutant cells was determined by RNA-seq and ChIP-seq. Scr1 was found to regulate genes involved in carbon metabolism, hexose uptake, gluconeogenesis and the TCA cycle. Surprisingly, a role for Scr1 in the suppression of sexual differentiation was also identified, as homothallic scr1 deletion mutants showed ectopic meiosis in carbon and nitrogen rich conditions. ChIP-seq characterised the targets of Tup/Ssn6 and Rst2 identifying regulatory roles within and independent of CCR. Finally, a subset of genes bound by all three factors was identified, implying that regulation of certain loci may be modulated in a competitive fashion between the Scr1, Tup/Ssn6 repressors and the Rst2 activator. CONCLUSIONS: By identifying the genes directly and indirectly regulated by Scr1, Tup/Ssn6 and Rst2, this study comprehensively defined the gene regulatory networks of CCR in fission yeast and revealed the transcriptional complexities governing this system.

In Vitro Susceptibility of Berberine Combined with Antifungal Agents Against the Yeast Form of Talaromyces marneffei.

Talaromyces (Penicillium) marneffei can cause fatal disseminated infection in immunocompromised hosts. However, therapeutic strategies for the mycosis are limited. Reports of the other fungi suggest that berberine, a component of traditional herb, inhibitors interact with antifungal agents to improve the treatment outcomes. In the study, we evaluated the in vitro efficacy of berberine in combination with conventional antifungal agents against the pathogenic yeast form of T. marneffei. We demonstrate the synergistic effect of combination of berberine with fluconazole (52.38%), itraconazole (66.67%), voriconazole (71.43%), amphotericin B (71.43%) or caspofungin (52.38%) of T. marneffei strains, respectively. Time-kill curves confirmed the synergistic interaction, and no antagonistic was observed in all of the combinations. In conclusion, berberine could enhance the efficacy of conventional antifungal agents against the yeast form of T. marneffei in vitro. The results indicated berberine might have a potential role in combination therapy for talaromycosis.

Calcineurin A Is Essential in the Regulation of Asexual Development, Stress Responses and Pathogenesis in Talaromyces marneffei.

Talaromyces marneffei is a common cause of infection in immunocompromised patients in Southeast Asia and Southern China. The pathogenicity of T. marneffei depends on the ability of the fungus to survive the cytotoxic processes of the host immune system and grow inside host macrophages. These mechanisms that allow T. marneffei to survive macrophage-induced death are poorly understood. In this study, we examined the role of a calcineurin homolog (cnaA) from T. marneffei during growth, morphogenesis and infection. Deletion of the cnaA gene in T. marneffei resulted in a strain with significant defects in conidiation, germination, morphogenesis, cell wall integrity, and resistance to various stressors. The ΔcnaA mutant showed a lower minimal inhibitory concentration (MIC) against caspofungin (16 µg/ml to 2 µg/ml) and micafungin (from 32 µg/ml to 4 µg/ml) compared with the wild-type. These results suggest that targeting calcineurin in combination with echinocandin treatment may be effective for life-threatening systemic T. marneffei infection. Importantly, the cnaA mutant was incapable of adapting to the macrophage environment in vitro and displayed virulence defects in a mouse model of invasive talaromycosis. For the first time, a role has been shown for cnaA in the morphology and pathogenicity of a dimorphic pathogenic filamentous fungus.

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo.

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei's opportunity for macrophage parasitism and thereby enhancing this pathogen's exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

Talaromyces marneffei simA Encodes a Fungal Cytochrome P450 Essential for Survival in Macrophages.

Fungi are adept at occupying specific environmental niches and often exploit numerous secondary metabolites generated by the cytochrome P450 (CYP) monoxygenases. This report describes the characterization of a yeast-specific CYP encoded by simA ("survival in macrophages"). Deletion of simA does not affect yeast growth at 37°C in vitro but is essential for yeast cell production during macrophage infection. The ΔsimA strain exhibits reduced conidial germination and intracellular growth of yeast in macrophages, suggesting that the enzymatic product of SimA is required for normal fungal growth in vivo. Intracellular ΔsimA yeast cells exhibit cell wall defects, and metabolomic and chemical sensitivity data suggest that SimA may promote chitin synthesis or deposition in vitro. In vivo, ΔsimA yeast cells subsequently lyse and are degraded, suggesting that SimA may increase resistance to and/or suppress host cell biocidal effectors. The results suggest that simA synthesizes a secondary metabolite that allows T. marneffei to occupy the specific intracellular environmental niche within the macrophage. IMPORTANCE This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth.

Extensive Metabolic Remodeling Differentiates Non-pathogenic and Pathogenic Growth Forms of the Dimorphic Pathogen Talaromyces marneffei.

Fungal infections are an increasing public health problem, particularly in immunocompromised individuals. While these pathogenic fungi show polyphyletic origins with closely related non-pathogenic species, many undergo morphological transitions to produce pathogenic cell types that are associated with increased virulence. However, the characteristics of these pathogenic cells that contribute to virulence are poorly defined. Talaromyces marneffei grows as a non-pathogenic hyphal form at 25°C but undergoes a dimorphic transition to a pathogenic yeast form at 37°C in vitro and following inhalation of asexual conidia by a host. Here we show that this transition is associated with major changes in central carbon metabolism, and that these changes are correlated with increased virulence of the yeast form. Comprehensive metabolite profiling and 13C-labeling studies showed that hyphal cells exhibited very active glycolytic metabolism and contain low levels of internal carbohydrate reserves. In contrast, yeast cells fully catabolized glucose in the mitochondrial TCA cycle, and store excess glucose in large intracellular pools of trehalose and mannitol. Inhibition of the yeast TCA cycle inhibited replication in culture and in host cells. Yeast, but not hyphae, were also able to use myo-inositol and amino acids as secondary carbon sources, which may support their survival in host macrophages. These analyses suggest that T. marneffei yeast cells exhibit a more efficient oxidative metabolism and are capable of utilizing a diverse range of carbon sources, which contributes to their virulence in animal tissues, highlighting the importance of dimorphic switching in pathogenic yeast.

A Plastic Vegetative Growth Threshold Governs Reproductive Capacity in Aspergillus nidulans.

Ontogenetic phases separating growth from reproduction are a common feature of cellular life. Long recognized for flowering plants and animals, early literature suggests this life-history component may also be prevalent among multicellular fungi. We establish the basis of developmental competence-the capacity to respond to induction of asexual development-in the filamentous saprotroph Aspergillus nidulans, describing environmental influences, including genotype-by-environment interactions among precocious mutants, gene expression associated with wild type and precocious competence acquisition, and the genetics of competence timing. Environmental effects are consistent with a threshold driven by metabolic rate and organism density, with pH playing a particularly strong role in determining competence timing. Gene expression diverges significantly over the competence window, despite a lack of overt morphological change, with differentiation in key metabolic, signaling, and cell trafficking processes. We identify five genes for which mutant alleles advance competence timing, including the conserved GTPase RasB (AN5832) and ambient pH sensor PalH (AN6886). In all cases examined, inheritance of competence timing is complex and non-Mendelian, with F1 progeny showing highly variable transgressive timing and dominant parental effects with a weak contribution from progeny genotype. Competence provides a new model for nutrient-limited life-cycle phases, and their elaboration from unicellular origins. Further work is required to establish the hormonal and bioenergetic basis of the trait across fungi, and underlying mechanisms of variable inheritance.

Differentially regulated high-affinity iron assimilation systems support growth of the various cell types in the dimorphic pathogen Talaromyces marneffei.

Iron is a key trace element important for many biochemical processes and its availability varies with the environment. For human pathogenic fungi iron acquisition can be particularly problematical because host cells sequester free iron as part of the acute-phase response to infection. Fungi rely on high-affinity iron uptake systems, such as reductive iron assimilation (RIA) and siderophore-mediated iron uptake (non-RIA). These have been extensively studied in pathogenic fungi that exist outside of host cells, but much less is known for intracellular fungal pathogens. Talaromyces marneffei is a dimorphic fungal pathogen endemic to Southeast Asia. In the host T. marneffei resides within macrophages where it grows as a fission yeast. T. marneffei has genes of both iron assimilation systems as well as a paralogue of the siderophore biosynthetic gene sidA, designated sidX. Unlike other fungi, deletion of sidA or sidX resulted in cell type-specific effects. Mutant analysis showed that T. marneffei yeast cells also employ RIA for iron acquisition, providing an additional system in this cell type that differs substantially from hyphal cells. These data illustrate the specialized iron acquisition systems used by the different cell types of a dimorphic fungal pathogen and highlight the complexity in siderophore-biosynthetic pathways amongst fungi.

Two-Component Signaling Regulates Osmotic Stress Adaptation via SskA and the High-Osmolarity Glycerol MAPK Pathway in the Human Pathogen Talaromyces marneffei.

For successful infection to occur, a pathogen must be able to evade or tolerate the host's defense systems. This requires the pathogen to first recognize the host environment and then signal this response to elicit a complex adaptive program in order to activate its own defense strategies. In both prokaryotes and eukaryotes, two-component signaling systems are utilized to sense and respond to changes in the external environment. The hybrid histidine kinases (HHKs) at the start of the two-component signaling pathway have been well characterized in human pathogens. However, how these HHKs regulate processes downstream currently remains unclear. This study describes the role of a response regulator downstream of these HHKs, sskA, in Talaromyces marneffei, a dimorphic human pathogen. sskA is required for asexual reproduction, hyphal morphogenesis, cell wall integrity, osmotic adaptation, and the morphogenesis of yeast cells both in vitro at 37°C and during macrophage infection, but not during dimorphic switching. Comparison of the ΔsskA mutant with a strain in which the mitogen-activated protein kinase (MAPK) of the high-osmolarity glycerol pathway (SakA) has been deleted suggests that SskA acts upstream of this pathway in T. marneffei to regulate these morphogenetic processes. This was confirmed by assessing the amount of phosphorylated SakA in the ΔsskA mutant, antifungal resistance due to a lack of SakA activation, and the ability of a constitutively active sakA allele (sakA(F316L) ) to suppress the ΔsskA mutant phenotypes. We conclude that SskA regulates morphogenesis and osmotic stress adaptation in T. marneffei via phosphorylation of the SakA MAPK of the high-osmolarity glycerol pathway. IMPORTANCE This is the first study in a dimorphic fungal pathogen to investigate the role of a response regulator downstream of two-component signaling systems and its connection to the high-osmolarity glycerol pathway. This study will inspire further research into the downstream components of two-component signaling systems and their role during pathogenic growth.

Talaromyces marneffei laccase modifies THP-1 macrophage responses.

Talaromyces (Penicillium) marneffei is an emerging opportunistic pathogen associated with HIV infection, particularly in Southeast Asia and southern China. The rapid uptake and killing of T. marneffei conidia by phagocytic cells along with the effective induction of an inflammatory response by the host is essential for disease control. T. marneffei produces a number of different laccases linked to fungal virulence. To understand the role of the various laccases in T. marneffei, laccase-encoding genes were investigated. Targeted single, double and triple gene deletions of laccases encoding lacA, lacB, and lacC showed no significant phenotypic effects suggesting redundancy of function. When a fourth laccase-encoding gene, pbrB, was deleted in the ΔlacA ΔlacB ΔlacC background, the quadruple mutant displayed delayed conidiation and the conidia were more sensitive to H2O2, sodium dodecyl sulfate (SDS), and antifungal agents than wild-type and other transformants. Conidia of the quadruple mutant showed marked differences in their interaction with the human monocyte cell line, THP-1 such that phagocytosis was significantly higher when compared with the wild-type at one and 2 hours of incubation while the phagocytic index was significantly different from 15 to 120 minutes. In addition, killing of the quadruple mutant by THP-1 cells was more efficient at 2 and 4 hours of incubation. The levels of the proinflammatory cytokines TNF-α, IL-1ß and IL-6 from THP-1 cells infected with the quadruple mutant were also significantly increased in comparison with wild-type. The results demonstrate that production of laccases by T. marneffei actually promotes the pathogen's resistance to innate host defenses.

Organism-wide studies into pathogenicity and morphogenesis in Talaromyces marneffei.

Organism-wide approaches examining the genetic mechanisms controlling growth and proliferation have proven to be a powerful tool in the study of pathogenic fungi. For many fungal pathogens techniques to study transcription and protein expression are particularly useful, and offer insights into infection processes by these species. Here we discuss the use of approaches such as differential display, suppression subtractive hybridization, microarray, RNA-seq, proteomics, genetic manipulation and infection models for the AIDS-defining pathogen Talaromyces marneffei. Together these methods have broadened our understanding of the biological processes, and genes that underlie them, which are involved in switching between the saprophytic and pathogenic states of T. marneffei, the maintenance of these two specialized cell types and its ability to cause disease.

Fungal dimorphism: the switch from hyphae to yeast is a specialized morphogenetic adaptation allowing colonization of a host.

The ability of pathogenic fungi to switch between a multicellular hyphal and unicellular yeast growth form is a tightly regulated process known as dimorphic switching. Dimorphic switching requires the fungus to sense and respond to the host environment and is essential for pathogenicity. This review will focus on the role of dimorphism in fungi commonly called thermally dimorphic fungi, which switch to a yeast growth form during infection. This group of phylogenetically diverse ascomycetes includes Talaromyces marneffei (recently renamed from Penicillium marneffei), Blastomyces dermatitidis (teleomorph Ajellomyces dermatitidis), Coccidioides species (C. immitis and C. posadasii), Histoplasma capsulatum (teleomorph Ajellomyces capsulatum), Paracoccidioides species (P. brasiliensis and P. lutzii) and Sporothrix schenckii (teleomorph Ophiostoma schenckii). This review will explore both the signalling pathways regulating the morphological transition and the transcriptional responses necessary for intracellular growth. The physiological requirements of yeast cells during infection will also be discussed, highlighting recent advances in the understanding of the role of iron and calcium acquisition during infection.