Measuring Laccase Activity and Melanin Production in Cryptococcus neoformans.

Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.

Just around the Corner: Advances in the Optimization of Yeasts and Filamentous Fungi for Lactic Acid Production.

Lactic acid (LA) production has seen significant progress over the past ten years. LA has seen increased economic importance due to its broadening use in different sectors such as the food, medicine, polymer, cosmetic, and pharmaceutical industries. LA production bioprocesses using microorganisms are economically viable compared to chemical synthesis and can benefit from metabolic engineering for improved productivity, purity, and yield. Strategies to optimize LA productivity in microorganisms on the strain improvement end include modifying metabolic routes, adding gene coding for lactate transporters, inducing tolerance to organic acids, and choosing cheaper carbon sources as fuel. Many of the recent advances in this regard have involved the metabolic engineering of yeasts and filamentous fungi to produce LA due to their versatility in fuel choice and tolerance of industrial-scale culture conditions such as pH and temperature. This review aims to compile and discuss metabolic engineering innovations in LA production in yeasts and filamentous fungi over the 2013-2023 period, and present future directions of research in this area, thus bringing researchers in the field up to date with recent advances.

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Genetic strategies for improving hyaluronic acid production in recombinant bacterial culture.

Hyaluronic acid (HA) is a biopolymer of repeating units of glucuronic acid and N-acetylglucosamine. Its market was valued at USD 8.9 billion in 2019. Traditionally, HA has been obtained from rooster comb-like animal tissues and fermentative cultures of attenuated pathogenic streptococci. Various attempts have been made to engineer a safe micro-organism for HA synthesis; however, the HA titres obtained from these attempts are in general still lower than those achieved by natural, pathogenic producers. In this scenario, ways to increase HA molecule length and titres in already constructed strains are gaining attention in the last years, but no recent publication has reviewed the main genetic strategies applied to improve HA production on heterologous hosts. In light of that, we hereby compile the advances made in the engineering of micro-organisms to improve HA synthesis.

Molecular epidemiology of Paracoccidiodes spp. recovered from patients with paracoccidioidomycosis in a teaching hospital from Minas Gerais State of Brazil.

INTRODUCTION: Paracoccidioidomycosis (PCM) is caused by several species of the Paracoccidioides genus which can be differentiated by interspecific genetic variations, morphology and geographic distribution. Intraspecific variability correlation with clinical and epidemiological aspects of these species still remains unclear. This study aimed to sequence the loci GP43, exon 2 and ARF of 23 clinical isolates of Paracoccidioides spp. from patients in the Southeast Region of Brazil. METHODOLOGY AND MAIN FINDINGS: GenBank was used to compare the present (23) with previous described sequences (151) that included ARF and GP43. It was identified a high polymorphism rate among the 23 isolates in comparison to the other 151. Among the isolates, 22 (95.66%) were S1/P. brasiliensis and 1 (4.34%) was identified as PS2/P. americana. A total of 45 haplotypes were found as follows: 19 from S1/P. brasiliensis (13 from the present study), 15 from P. lutzii, 6 from PS2/P. americana (1 from the present study), 3 from PS3/P. restrepiensis and 2 from PS4/P. venezuelensis. Moreover, exclusive haplotypes according to clinical origin and geographical area were found. S1/P. brasiliensis (HD = 0.655 and K = 4.613) and P. lutzii (HD = 0.649 and K = 2.906) presented the highest rate of polymorphism among all species, from which 12 isolates of the present study were clustered within S1b/P. brasiliensis. The GP43 locus showed a higher variability and was found to be the main reason for the species differentiation. CONCLUSIONS: The results herein decribed show a high intraspecific genetic variability among S1/P. brasiliensis isolates and confirm the predominance of this species in the Southeast region of Brazil. The finding of exclusive haplotypes according to clinical origin and geographical area would suggest correlation between the molecular profile with the clinical form and geographic origin of patients with PCM.

Epidemiology of paracoccidioidomycosis in Venezuela: a retrospective study from 1954 to 2019.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America. Etiological agents are Paracoccidioides species that diverge phylogenetically throughout South America. OBJECTIVES: This study aimed to document the epidemiology of PCM in Venezuela. METHODS: We have performed a retrospective cross-sectional descriptive study in 31,081 clinical records of patients from two reference centres during 65 years (1954-2019). FINDINGS: PCM diagnosis was confirmed in 745 patients. Chronic PCM was the most prevalent form (90.06% cases); 80.67% were male and the most affected age range was 41-60. Farming and construction were the most prevalent occupation and Miranda State had a higher prevalence. Lung and skin were the most affected organs, followed by oral manifestations. Direct examination, culture and serology showed a high sensibility, and no statistical difference was observed among the diagnostic tools. Out of 17 Paracoccidioides isolates genotyped from Venezuela, one was typed as Paracoccidioides americana and 16 as Paracoccidioides venezuelensis. MAIN CONCLUSIONS: Clinical manifestations observed, information about the epidemiology and molecular profile is essential not only for diagnosis but also for understanding therapeutic responses to mycotic drugs and prognosis. Therefore, it is necessary to sequence all positive isolated strains in order to confirm the dominance of P. venezuelensis in Venezuela.

Evaluation of Ogataea (Hansenula) polymorpha for Hyaluronic Acid Production.

Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by ß-1,4 and ß-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors' ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.

Epidemiology of paracoccidioidomycosis in Venezuela: a retrospective study from 1954 to 2019

BACKGROUND Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America. Etiological agents are Paracoccidioides species that diverge phylogenetically throughout South America. OBJECTIVES This study aimed to document the epidemiology of PCM in Venezuela. METHODS We have performed a retrospective cross-sectional descriptive study in 31,081 clinical records of patients from two reference centres during 65 years (1954-2019). FINDINGS PCM diagnosis was confirmed in 745 patients. Chronic PCM was the most prevalent form (90.06% cases); 80.67% were male and the most affected age range was 41-60. Farming and construction were the most prevalent occupation and Miranda State had a higher prevalence. Lung and skin were the most affected organs, followed by oral manifestations. Direct examination, culture and serology showed a high sensibility, and no statistical difference was observed among the diagnostic tools. Out of 17 Paracoccidioides isolates genotyped from Venezuela, one was typed as Paracoccidioides americana and 16 as Paracoccidioides venezuelensis. MAIN CONCLUSIONS Clinical manifestations observed, information about the epidemiology and molecular profile is essential not only for diagnosis but also for understanding therapeutic responses to mycotic drugs and prognosis. Therefore, it is necessary to sequence all positive isolated strains in order to confirm the dominance of P. venezuelensis in Venezuela.

A novel Sporothrix brasiliensis genomic variant in Midwestern Brazil: evidence for an older and wider sporotrichosis epidemic.

Sporotrichosis is a subcutaneous infection caused by fungi from the genus Sporothrix. It is transmitted by inoculation of infective particles found in plant-contaminated material or diseased animals, characterizing the classic sapronotic and emerging zoonotic transmission, respectively. Since 1998, southeastern Brazil has experienced a zoonotic sporotrichosis epidemic caused by S. brasiliensis, centred in the state of Rio de Janeiro. Our observation of feline sporotrichosis cases in Brasília (Midwestern Brazil), around 900 km away from Rio de Janeiro, led us to question whether the epidemic caused by S. brasiliensis has spread from the epicentre in Rio de Janeiro, emerged independently in the two locations, or if the disease has been present and unrecognized in Midwestern Brazil. A retrospective analysis of 91 human and 4 animal cases from Brasília, ranging from 1993 to 2018, suggests the occurrence of both sapronotic and zoonotic transmission. Molecular typing of the calmodulin locus identified S. schenckii as the agent in two animals and all seven human patients from which we were able to recover clinical isolates. In two other animals, the disease was caused by S. brasiliensis. Whole-genome sequence typing of seven Sporothrix spp. strains from Brasília and Rio de Janeiro suggests that S. brasiliensis isolates from Brasília are genetically distinct from those obtained at the epicentre of the outbreak in Rio de Janeiro, both in phylogenomic and population genomic analyses. The two S. brasiliensis populations seem to have separated between 2.2 and 3.1 million years ago, indicating independent outbreaks or that the zoonotic S. brasiliensis outbreak might have started earlier and be more widespread in South America than previously recognized.

Evaluation of Product Distribution in Chemostat and Batch Fermentation in Lactic Acid-Producing Komagataella phaffii Strains Utilizing Glycerol as Substrate.

Lactic acid is the monomeric unit of polylactide (PLA), a bioplastic widely used in the packaging, automotive, food, and pharmaceutical industries. Previously, the yeast Komagataella phaffii was genetically modified for the production of lactate from glycerol. For this, the bovine L-lactate dehydrogenase- (LDH)-encoding gene was inserted and the gene encoding the pyruvate decarboxylase (PDC) was disrupted, resulting in the GLp strain. This showed a yield of 67% L-lactic acid and 20% arabitol as a by-product in batches with oxygen limitation. Following up on these results, the present work endeavored to perform a detailed study of the metabolism of this yeast, as well as perturbing arabitol synthesis in an attempt to increase lactic acid titers. The GLp strain was cultivated in a glycerol-limited chemostat at different dilution rates, confirming that the production of both lactic acid and arabitol is dependent on the specific growth rate (and consequently on the concentration of the limiting carbon source) as well as on the oxygen level. Moreover, disruption of the gene encoding arabitol dehydrogenase (ArDH) was carried out, resulting in an increase of 20% in lactic acid and a 50% reduction in arabitol. This study clarifies the underlying metabolic reasons for arabitol formation in K. phaffii and points to ways for improving production of lactic acid using K. phaffii as a biocatalyst.

Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans.

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

A hidden battle in the dirt: Soil amoebae interactions with Paracoccidioides spp.

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae-all potential predators of fungi-grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits.

Antifungal drugs: New insights in research & development.

The need for better antifungal therapy is commonly accepted in view of the high mortality rates associated with systemic infections, the low number of available antifungal classes, their associated toxicity and the increasing number of infections caused by strains with natural or acquired resistance. The urgency to expand the range of therapeutic options for the treatment of fungal infections has led researchers in recent decades to seek alternative antifungal targets when compared to the conventional ones currently used. Although new potential targets are reported, translating the discoveries from bench to bedside is a long process and most of these drugs fail to reach the patients. In this review, we discuss the development of antifungal drugs focusing on the approach of drug repurposing and the search for novel drugs for classical targets, the most recently described gene targets for drug development, the possibilities of immunotherapy using antibodies, cytokines, therapeutic vaccines and antimicrobial peptides.

Imaging Cryptococcus spp. Capsule by Differential Interference Contrast Microscopy Using Percoll®.

The most important virulence factor in the Cryptococcus genus is the polysaccharide capsule. This genus includes several species that cause life-threatening invasive disease. An increase in capsule thickness is important during fungal infection. The capsule is usually imaged using India ink, and crucial insights on the dynamics of its growth have been obtained using capsule-binding proteins such as specific antibodies or complement. We have developed an alternative method that allows both static and time-lapse imaging of the capsule using Percoll®, a suspension of nanometric spheres that do not penetrate the capsule. Given that these particles have a higher refractive index than the capsule, the latter can be imaged by differential interference contrast (DIC) microscopy. Static observation of the capsule with DIC and Percoll® results in capsule thickness measurements that match those made with India ink. Using capsule-inducing media, a glass-bottom incubation chamber and a live-imaging system equipped for DIC microscopy, this method allows time-lapse imaging of capsule growth. In contrast with India ink staining, DIC imaging of Percoll® exclusion halos result in crisp images. The greatest advantage of this method, though, is that unlike India ink, the Percoll® particles are non-toxic and unlike opsonins they do not bind the capsule, resulting in observations of capsule growth that are free from interference of bound proteins on capsule physiology.

A Wor1-Like Transcription Factor Is Essential for Virulence of Cryptococcus neoformans.

Gti1/Pac2 transcription factors occur exclusively in fungi and their roles vary according to species, including regulating morphological transition and virulence, mating and secondary metabolism. Many of these functions are important for fungal pathogenesis. We therefore hypothesized that one of the two proteins of this family in Cryptococcus neoformans, a major pathogen of humans, would also control virulence-associated cellular processes. Elimination of this protein in C. neoformans results in reduced polysaccharide capsule expression and defective cytokinesis and growth at 37°C. The mutant loses virulence in a mouse model of cryptococcal infection and retains only partial virulence in the Galleria mellonella alternative model at 30°C. We performed RNA-Seq experiments on the mutant and found abolished transcription of genes that, in combination, are known to account for all the observed phenotypes. The protein has been named Required for cytokinesis and virulence 1 (Rcv1).

The role of Aspartyl aminopeptidase (Ape4) in Cryptococcus neoformans virulence and authophagy.

In order to survive and cause disease, microbial pathogens must be able to proliferate at the temperature of their infected host. We identified novel microbial features associated with thermotolerance in the opportunistic fungal pathogen Cryptococcus neoformans using a random insertional mutagenesis strategy, screening for mutants with defective growth at 37°C. Among several thermosensitive mutants, we identified one bearing a disruption in a gene predicted to encode the Ape4 aspartyl aminopeptidase protein. Ape4 metalloproteases in other fungi, including Saccharomyces cerevisiae, are activated by nitrogen starvation, and they are required for autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway. However, none have been previously associated with altered growth at elevated temperatures. We demonstrated that the C. neoformans ape4 mutant does not grow at 37°C, and it also has defects in the expression of important virulence factors such as phospholipase production and capsule formation. C. neoformans Ape4 activity was required for this facultative intracellular pathogen to survive within macrophages, as well as for virulence in an animal model of cryptococcal infection. Similar to S. cerevisiae Ape4, the C. neoformans GFP-Ape4 fusion protein co-localized with intracytoplasmic vesicles during nitrogen depletion. APE4 expression was also induced by the combination of nutrient and thermal stress. Together these results suggest that autophagy is an important cellular process for this microbial pathogen to survive within the environment of the infected host.

The transcriptional response of Cryptococcus neoformans to ingestion by Acanthamoeba castellanii and macrophages provides insights into the evolutionary adaptation to the mammalian host.

Virulence of Cryptococcus neoformans for mammals, and in particular its intracellular style, was proposed to emerge from evolutionary pressures on its natural environment by protozoan predation, which promoted the selection of strategies that allow intracellular survival in macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then can replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least 2-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes that were found in both groups, we focused on open reading frame (ORF) CNAG_05662, which was potentially related to sugar transport but had no determined biological function. To characterize its function, we constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (polyol transporter protein 1), is involved in the transport of 5- and 6-carbon polyols such as mannitol and sorbitol, but its presence or absence had no effect on cryptococcal virulence for mice or moth larvae. Overall, these results are consistent with the hypothesis that the capacity for mammalian virulence originated from fungus-protozoan interactions in the environment and provide a better understanding of how C. neoformans adapts to the mammalian host.

Quorum sensing-mediated, cell density-dependent regulation of growth and virulence in Cryptococcus neoformans.

UNLABELLED: Quorum sensing (QS) is a cell density-dependent mechanism of communication between microorganisms, characterized by the release of signaling molecules that affect microbial metabolism and gene expression in a synchronized way. In this study, we investigated cell density-dependent behaviors mediated by conditioned medium (CM) in the pathogenic encapsulated fungus Cryptococcus neoformans. CM produced dose-dependent increases in the growth of planktonic and biofilm cells, glucuronoxylomannan release, and melanin synthesis, important virulence attributes of this organism. Mass spectrometry revealed the presence of pantothenic acid (PA) in our samples, and commercial PA was able to increase growth and melanization, although not to the same extent as CM. Additionally, we found four mutants that were either unable to produce active CM or failed to respond with increased growth in the presence of wild-type CM, providing genetic evidence for the existence of intercellular communication in C. neoformans. C. neoformans CM also increased the growth of Cryptococcus albidus, Candida albicans, and Saccharomyces cerevisiae. Conversely, CM from Cryptococcus albidus, C. albicans, S. cerevisiae, and Sporothrix schenckii increased C. neoformans growth. In summary, we report the existence of a new QS system regulating the growth and virulence factor expression of C. neoformans in vitro and, possibly, also able to regulate growth in other fungi. IMPORTANCE: Quorum sensing is a strategy of communication used by pathogenic microorganisms to coordinate the expression of attributes necessary to cause disease. In this work, we describe a quorum sensing system in Cryptococcus neoformans, a yeast that can cause severe central nervous system infections. Adding conditioned medium--culture medium in which C. neoformans has previously grown--to fresh cultures resulted in faster growth of C. neoformans both as isolated cells and in microbial communities called biofilms. The addition of conditioned medium also increased the secretion of capsule carbohydrates and the formation of melanin pigment, two tools used by this microorganism to thrive in the host. This remarkable example of microbial communication shows that C. neoformans cells can act in unison when expressing attributes necessary to survive in the host, a finding that could point the way to improvements in the treatment of cryptococcosis.

Characterisation of the heat shock factor of the human thermodimorphic pathogen Paracoccidioides lutzii.

Thermodimorphic fungi include most causative agents of systemic mycoses, but the molecular mechanisms that underlie their defining trait, i.e. the ability to shift between mould and yeast on temperature change alone, remain poorly understood. We hypothesised that the heat shock factor (Hsf), a protein that evolved to sense thermal stimuli quickly, might play a role in this process in addition to the known regulator Drk1 and the Ryp proteins. To test this hypothesis, we characterised the Hsf from the thermodimorph Paracoccidioides lutzii (formerly Paracoccidioides brasiliensis isolate 01). We show in the present work that PlHsf possesses regulatory domains that are exclusive of the Eurotiomycetidae family, suggesting evolutionary specialisation; that it can successfully rescue the otherwise lethal loss of the native protein of Saccharomyces cerevisiae; and that its DNA-binding domain is able to recognise regulatory elements from the promoters of both Drk1 and Ryp1. An in silico screening of all 1 kb sequences upstream of P. lutzii ORFs revealed that 7% of them possess a heat shock element. This is the first description of a heat shock factor in a thermodimorphic fungus.