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Antimicrobial resistance has rendered many conventional therapeutic measures, such as antibiotics, ineffective. This makes the treatment of infections from pathogenic micro-organisms a major growing health, social, and economic challenge. Recently, nanomaterials, including two-dimensional (2D) materials, have attracted scientific interest as potential antimicrobial agents. Many of these studies, however, rely on the input of activation energy and lack real-world utility. In this work, we present the broad-spectrum antimicrobial activity of few-layered black phosphorus (BP) at nanogram concentrations. This property arises from the unique ability of layered BP to produce reactive oxygen species, which we harness to create this unique functionality. BP is shown to be highly antimicrobial toward susceptible and resistant bacteria and fungal species. To establish cytotoxicity with mammalian cells, we showed that both L929 mouse and BJ-5TA human fibroblasts were metabolically unaffected by the presence of BP. Finally, we demonstrate the practical utility of this approach, whereby medically relevant surfaces are imparted with antimicrobial properties via functionalization with few-layer BP. Given the self-degrading properties of BP, this study demonstrates a viable and practical pathway for the deployment of novel low-dimensional materials as antimicrobial agents without compromising the composition or nature of the coated substrate.

The fabrication of antimicrobial surfaces that exhibit enhanced activity toward a large variety of microbial species is one of the major challenges of our time. In fact, the negative effects associated with both bacterial and fungal infections are enormous, especially considering that many microbial species are developing resistance to known antibiotics. In this work, we show how a combination of a specific surface morphology and surface chemistry can create a surface that exhibits nearly 100% antimicrobial activity toward both Gram-negative and Gram-positive bacteria and fungal cells. Arrays of vertically aligned, oxygen-deficient zinc oxide (ZnO) nanowires grown on a substrate exhibit enhanced antimicrobial activity compared with surfaces containing either less defective nanowires or highly oxygen-deficient flat films. This synergistic effect between physical activity (morphology) and chemical activity (surface composition) has been shown to be responsible for the outstanding antimicrobial activity of our surfaces, especially toward notoriously resilient bacterial or fungal species. These findings provide a series of design rules for tuning the activities of antibacterial and antifungal nanomaterials. These rules constitute an excellent platform for the development of next-generation antimicrobial surfaces.

Background Fungal infections are common life-threatening diseases amongst immunodeficient individuals. Invasive fungal disease is commonly treated with an azole antifungal agent, resulting in selection pressure and the emergence of drug resistance. Antifungal resistance is associated with higher mortality rates and treatment failure, making the current clinical management of fungal disease very challenging. Clinical isolates from a variety of fungi have been shown to contain mutations in the MSH2 gene, encoding a component of the DNA mismatch repair pathway. Mutation of MSH2 results in an elevated mutation rate that can increase the opportunity for selectively advantageous mutations to occur, accelerating the development of antifungal resistance. Objectives To characterize the molecular mechanisms causing the microevolutionary emergence of antifungal resistance in msh2 mismatch repair mutants of Cryptococcus neoformans. Methods The mechanisms resulting in the emergence of antifungal resistance were investigated using WGS, characterization of deletion mutants and measuring ploidy changes. Results The genomes of resistant strains did not possess mutations in ERG11 or other genes of the ergosterol biosynthesis pathway. Antifungal resistance was due to small contributions from mutations in many genes. MSH2 does not directly affect ploidy changes. Conclusions This study provides evidence that resistance to fluconazole can evolve independently of ERG11 mutations. A common microevolutionary route to the emergence of antifungal resistance involves the accumulation of mutations that alter stress signalling, cellular efflux, membrane trafficking, epigenetic modification and aneuploidy. This complex pattern of microevolution highlights the significant challenges posed both to diagnosis and treatment of drug-resistant fungal pathogens.

Fungal infections are common life-threatening diseases amongst immunodeficient individuals. Invasive fungal disease is commonly treated with an azole antifungal agent, resulting in selection pressure and the emergence of drug resistance. Antifungal resistance is associated with higher mortality rates and treatment failure, making the current clinical management of fungal disease very challenging. Clinical isolates from a variety of fungi have been shown to contain mutations in theTo characterize the molecular mechanisms causing the microevolutionary emergence of antifungal resistance inThe mechanisms resulting in the emergence of antifungal resistance were investigated using WGS, characterization of deletion mutants and measuring ploidy changes.The genomes of resistant strains did not possess mutations inThis study provides evidence that resistance to fluconazole can evolve independently of

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.

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.

Passaging of microbes in vitro can lead to the selection of microevolved derivatives with differing properties to their original parent strains. One well characterised instance is the phenotypic differences observed between the series of strains derived from the type strain of the human pathogenic fungus Cryptococcus neoformans. A second case was reported in the close relative Cryptococcus deneoformans, in which a well-studied isolate ATCC 24067 (52D) altered its phenotypic characteristics after in vitro passaging in different laboratories. One of these derivatives, ATCC 24067A, has decreased virulence and also exhibits a hypermutator phenotype, in which the mutation rate is increased compared to wild type. In this study, the molecular basis behind the changes in the lineage of ATCC 24067 was determined by next-generation sequencing of the parent and passaged strain genomes. This analysis resulted in the identification of a point mutation that causes a D270G amino acid substitution within the exonuclease proofreading domain of the DNA polymerase delta subunit encoded by POL3. Complementation with POL3 confirmed that this mutation is responsible for the hypermutator phenotype of this strain. Regeneration of the mutation in C. neoformans, to eliminate the additional mutations present in the ATCC 24067A genetic background, demonstrated that the hypermutator phenotype of the pol3(D270G) mutant causes rapid microevolution in vitro but does not result in decreased virulence. These findings indicate that mutator strains can emerge in these pathogenic fungi without conferring a fitness cost, but the subsequent rapid accumulation of mutations can be deleterious.

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.

Discovering the genes underlying fundamental processes that enable cells to live and reproduce is a technical challenge, because loss of gene function in mutants results in organisms that cannot survive. This study describes a forward genetics method to identify essential genes in fungi, based on the propensity for Agrobacterium tumefaciens to insert T-DNA molecules into the promoters or 5' untranslated regions of genes and by placing a conditional promoter within the T-DNA. Insertions of the promoter of the GAL7 gene were made in the human pathogen Cryptococcus neoformans. Nine strains of 960 T-DNA insertional mutants screened grew on media containing galactose, but had impaired growth on media containing glucose, which suppresses expression from GAL7. T-DNA insertions were found in the homologs of IDI1, MRPL37, NOC3, NOP56, PRE3 and RPL17, all of which are essential in ascomycete yeasts Saccharomyces cerevisiae or Schizosaccharomyces pombe. Altering the carbon source in the medium provided a system to identify phenotypes in response to stress agents. The pre3 proteasome subunit mutant was further characterized. The T-DNA insertion and phenotype co-segregate in progeny from a cross, and the growth defect is complemented by the reintroduction of the wild type gene into the insertional mutant. A deletion allele was generated in a diploid strain, this heterozygous strain was sporulated, and analysis of the progeny provided additional genetic evidence that PRE3 is essential. The experimental design is applicable to other fungi and has other forward genetic applications such as to isolate over-expression suppressors or enhance the production of traits of interest.

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., Author Summary Fungal pathogen populations show patterns ranging from globally recombining to endemic and clonal. Among the most genetically and spatially restricted fungi is the highly clonal pathogen Penicillium marneffei, an endemic AIDS-associated pathogen in Southeast Asia. Previous studies have shown that P. marneffei has a pattern of extreme clonality despite the ability to disperse across wide distances and the presence of mating type genes that are required for sexual recombination. In this study we used genetic markers, comparative genomics, experimental data, and spatial models to determine the influence of sex on P. marneffei populations, and we found that although there was substantial evidence of sexual recombination, most of the recombination in natural populations was limited to sexual neighborhoods, amongst genetically similar and spatially close individuals. Based on the results of experiments and spatial models we found support for sex occurring in bamboo rats that are known to harbor P. marneffei and the pathogens sexual neighborhoods. Our study suggests that the high levels of effective clonality and endemicity found in P. marneffei may have more to do with specific host interactions than with an innate inability to generate population genetic diversity through sexual recombination.

The global prevalence of fungal diseases has escalated in the last several decades. Currently, it is estimated that fungi infect 1.7 billion people annually and result in 1.5 million deaths every year1. Deaths due to fungal infections are increasing, with mortality often exceeding 50%, further increasing to 100% if treatment is delayed1. Despite these staggering figures, the contribution of fungal infections to the global burden of disease remains under-recognised. In Australia, over a 5-year period fungal infections cost Australia an estimated $583 million2. The median cost for one invasive fungal disease (IFD) is AU$30957, increasing to AU$80291 if the patient is admitted to an intensive care unit3. Treatment of fungal infections poses significant challenges due to the small number of safe and effective antifungal drugs available and emerging antifungal drug resistance. Resistance to every class of antifungal drugs has been described and for some drug classes is extremely common4,5.

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., 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.

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.

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.

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., Author Summary Fungi that infect humans are a major health problem, especially for those with compromised immune systems. Many fungal infections are extremely difficult to cure and if left untreated are fatal. For successful infection to occur, the fungal pathogen must be able to grow by acquiring and utilising the available nutrient sources within the host whilst evading or tolerating the host’s defence systems. Expression profiling in several pathogenic fungal species has revealed that genes required for tyrosine catabolism are induced specifically in the pathogenic cell type at 37°C. As well as enabling the fungus to acquire carbon and nitrogen intermediates from proteins within the host, tyrosine is also an important precursor in the formation of two different types of melanin, which protects cells against the host’s defence systems. This study shows that the ability to catabolise tyrosine and produce tyrosine derived melanin is not required for the initial stages of fungal infection. However, a novel role for hpdA, which encodes the enzyme which catalyses the second step of tyrosine catabolism, was identified during growth in host cells.