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During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host's defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells.

Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type.

Molecular genetic approaches typically detect recombination in microbes regardless of assumed asexuality. However, genetic data have shown the AIDS-associated pathogen Penicillium marneffei to have extensive spatial genetic structure at local and regional scales, and although there has been some genetic evidence that a sexual cycle is possible, this haploid fungus is thought to be genetically, as well as morphologically, asexual in nature because of its highly clonal population structure. Here we use comparative genomics, experimental mixed-genotype infections, and population genetic data to elucidate the role of recombination in natural populations of P. marneffei. Genome wide comparisons reveal that all the genes required for meiosis are present in P. marneffei, mating type genes are arranged in a similar manner to that found in other heterothallic fungi, and there is evidence of a putatively meiosis-specific mutational process. Experiments suggest that recombination between isolates of compatible mating types may occur during mammal infection. Population genetic data from 34 isolates from bamboo rats in India, Thailand and Vietnam, and 273 isolates from humans in China, India, Thailand, and Vietnam show that recombination is most likely to occur across spatially and genetically limited distances in natural populations resulting in highly clonal population structure yet sexually reproducing populations. Predicted distributions of three different spatial genetic clusters within P. marneffei overlap with three different bamboo rat host distributions suggesting that recombination within hosts may act to maintain population barriers within P. marneffei.

Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37 degrees C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37 degrees C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37 degrees C. Loss of pakB also leads to the inappropriate production of yeast cells at 25 degrees C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.

Asexual spores (conidia) are the infectious propagules of many pathogenic fungi, and the capacity to sense the host environment and trigger conidial germination is a key pathogenicity determinant. Germination of conidia requires the de novo establishment of a polarised growth axis and consequent germ tube extension. The molecular mechanisms that control polarisation during germination are poorly understood. In the dimorphic human pathogenic fungus Penicillium marneffei, conidia germinate to produce one of two cell types that have very different fates in response to an environmental cue. At 25 degrees C, conidia germinate to produce the saprophytic cell type, septate, multinucleate hyphae that have the capacity to undergo asexual development. At 37 degrees C, conidia germinate to produce the pathogenic cell type, arthroconidiating hyphae that liberate uninucleate yeast cells. This study shows that the p21-activated kinase pakA is an essential component of the polarity establishment machinery during conidial germination and polarised growth of yeast cells at 37 degrees C but is not required for germination or polarised growth at 25 degrees C. Analysis shows that the heterotrimeric G protein alpha subunit GasC and the CDC42 orthologue CflA lie upstream of PakA for germination at both temperatures, while the Ras orthologue RasA only functions at 25 degrees C. These findings suggest that although some proteins that regulate the establishment of polarised growth in budding yeast are conserved in filamentous fungi, the circuitry and downstream effectors are differentially regulated to give rise to distinct cell types.

The mortality rates of invasive fungal infections remain high because of the limited number of antifungal drugs available and antifungal drug resistance, which can rapidly evolve during treatment. Mutations in key resistance genes such as ERG11 were postulated to be the predominant cause of antifungal drug resistance in the clinic. However, recent advances in whole genome sequencing have revealed that there are multiple mechanisms leading to the microevolution of resistance. In many fungal species, resistance can emerge through ERG11-independent mechanisms and through the accumulation of mutations in many genes to generate a polygenic resistance phenotype. In addition, genome sequencing has revealed that full or partial aneuploidy commonly occurs in clinical or microevolved in vitro isolates to confer antifungal resistance. This review will provide an overview of the mutations known to be selected during the adaptive microevolution of antifungal drug resistance and focus on how recent advances in genome sequencing technology have enhanced our understanding of this process.

Adaptation to the changing environmental conditions experienced within a host requires genetic diversity within a microbial population. Genetic diversity arises from mutations which occur due to DNA damage from exposure to exogenous environmental stresses or generated endogenously through respiration or DNA replication errors. As mutations can be deleterious, a delicate balance must be obtained between generating enough mutations for micro-evolution to occur while maintaining fitness and genomic integrity. Pathogenic microorganisms can actively modify their mutation rate to enhance adaptive micro-evolution by increasing expression of error-prone DNA polymerases or by mutating or decreasing expression of genes required for DNA repair. Strains which exhibit an elevated mutation rate are termed mutators. Mutators are found in varying prevalence in clinical populations where large-effect beneficial mutations enhance survival and are predominately caused by defects in the DNA mismatch repair (MMR) pathway. Mutators can facilitate the emergence of antibiotic resistance, allow phenotypic modifications to prevent recognition and destruction by the host immune system and enable switching to metabolic and cellular morphologies better able to survive in the given environment. This review will focus on recent advances in understanding the phenotypic and genotypic changes occurring in MMR mutators in both prokaryotic and eukaryotic pathogens.

Biofilms are assemblages of microbial cells, extracellular polymeric substances (EPS), and other components extracted from the environment in which they develop. Within biofilms, the spatial distribution of these components can vary. Here we present a fundamental characterization study to show differences between biofilms formed by Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Pseudomonas aeruginosa, and the yeast-type Candida albicans using synchrotron macro attenuated total reflectance-Fourier transform infrared (ATR-FTIR) microspectroscopy. We were able to characterise the pathogenic biofilms’ heterogeneous distribution, which is challenging to do using traditional techniques. Multivariate analyses revealed that the polysaccharides area (1200–950 cm−1) accounted for the most significant variance between biofilm samples, and other spectral regions corresponding to amides, lipids, and polysaccharides all contributed to sample variation. In general, this study will advance our understanding of microbial biofilms and serve as a model for future research on how to use synchrotron source ATR-FTIR microspectroscopy to analyse their variations and spatial arrangements.

ABSTRACT Although mutation drives evolution over long and short terms, measuring and comparing mutation rates accurately have been particularly difficult. This is especially true when mutations lead to an alteration in fitness. E. Shor, J. Schuyler, and D. S. Perlin (https://doi.org/10.1128/mBio.00120-19) present a new method to compare mutation rates across fungal strains and under different growth conditions: they employ the green fluorescent protein (GFP) as the reporter and count mutations using fluorescence-activated cell sorting (FACS). The estimates of mutation rates using the GFP-FACS approach are similar to those calculated with other reporters, and the method was used to assess if different alleles of the mismatch repair pathway gene MSH2 impact the mutation rates in the human pathogen Candida glabrata. The approach could be extended to other microbes and applications, opening the way for a better understanding of how mutation rates have impacted speciation and the emergence of antimicrobial resistance.

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.

ABSTRACT The ability to adapt to a changing environment provides a selective advantage to microorganisms. In the case of many pathogens, a large change in their environment occurs when they move from a natural setting to a setting within a human host and then during the course of disease development to various locations within that host. Two clinical isolates of the human fungal pathogen Cryptococcus neoformans were identified from a collection of environmental and clinical strains that exhibited a mutator phenotype, which is a phenotype which provides the ability to change rapidly due to the accumulation of DNA mutations at high frequency. Whole-genome analysis of these strains revealed mutations in MSH2 of the mismatch repair pathway, and complementation confirmed that these mutations are responsible for the mutator phenotype. Comparison of mutation frequencies in deletion strains of eight mismatch repair pathway genes in C. neoformans showed that the loss of three of them, MSH2, MLH1, and PMS1, results in an increase in mutation rates. Increased mutation rates enable rapid microevolution to occur in these strains, generating phenotypic variations in traits associated with the ability to grow in vivo, in addition to allowing rapid generation of resistance to antifungal agents. Mutation of PMS1 reduced virulence, whereas mutation of MSH2 or MLH1 had no effect on the level of virulence. These findings thus support the hypothesis that this pathogenic fungus can take advantage of a mutator phenotype in order to cause disease but that it can do so only in specific pathways that lead to a mutator trait without a significant tradeoff in fitness. IMPORTANCE Fungi account for a large number of infections that are extremely difficult to treat; superficial fungal infections affect approximately 1.7 billion (25%) of the general population worldwide, and systemic fungal diseases result in an unacceptably high mortality rate. How fungi adapt to their hosts is not fully understood. This research investigated the role of changes to DNA sequences in adaption to the host environment and the ability to cause disease in Cryptococcus neoformans, one of the world’s most common and most deadly fungal pathogens. The study results showed that microevolutionary rates are enhanced in either clinical isolates or in gene deletion strains with msh2 mutations. This gene has similar functions in regulating the rapid emergence of antifungal drug resistance in a distant fungal relative of C. neoformans, the pathogen Candida glabrata. Thus, microevolution resulting from enhanced mutation rates may be a common contributor to fungal pathogenesis.

ABSTRACT 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 (sakAF316L) 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.