Influence of the agrochemical benomyl on Cryptococcus gattii-plant interaction in vitro and in vivo.

Cryptococcus gattii, an environmental fungus, is one of the agents of cryptococcosis. The influence of agrochemicals on fungal resistance to antifungals is widely discussed. However, the effects of benomyl (BEN) on fungal interaction with different hosts is still to be understood. Here we studied the influence of adaptation to BEN in the interaction with a plant model, phagocytes and with Tenebrio molitor. First, the strain C. gattii L24/01 non-adapted (NA), adapted (A) to BEN, and adapted with further culture on drug-free media (10p) interact with Nicotiana benthamiana, with a peak in the yeast burden on the 7th day post-inoculation. C. gattii L24/01 A and 10p provided lower fungal burden, but these strains increased cell diameter and capsular thickness after the interaction, together with decreased fungal growth. The strains NA and A showed reduced ergosterol levels, while 10p exhibited increased activity of laccase and urease. L24/01 A recovered from N. benthamiana was less engulfed by murine macrophages, with lower production of reactive oxygen species. This phenotype was accompanied by increased ability of this strain to grow inside macrophages. Otherwise, L24/01 A showed reduced virulence in the T. molitor larvae model. Here, we demonstrate that the exposure to BEN, and interaction with plants interfere in the morphophysiology and virulence of the C. gattii.

Autoantibodies Neutralizing GM-CSF in HIV-Negative Colombian Patients Infected with Cryptococcus gattii and C. neoformans.

BACKGROUND: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera collected from Colombian patients with non-HIV-associated cryptococcosis in a retrospective national cohort from 1997 to 2016. METHODS: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs against GM-CSF in 30 HIV negative adults with cryptococcosis (13 caused by C. gattii and 17 caused by C. neoformans). RESULTS: We detected neutralizing auto-Abs against GM-CSF in the sera of 10 out of 13 (77%) patients infected with C. gattii and one out of 17 (6%) patients infected with C. neoformans. CONCLUSIONS: We report eleven Colombian patients diagnosed with cryptococcosis who had auto-Abs that neutralize GM-CSF. Among these patients, ten were infected with C. gattii and only one with C. neoformans.

Adjuvant ArtinM favored the host immunity against Cryptococcus gattii infection in C57BL/6 mice.

Aim: Cryptococcus gattii causes a severe fungal infection with high mortality rate among immunosuppressed and immunocompetent individuals. Due to limitation of current antifungal treatment, new immunotherapeutic approaches are explored.Methods: This study investigated an immunization strategy utilizing heat-inactivated C. gattii with ArtinM as an adjuvant. C57BL/6 mice were intranasally immunized with heat-killed C. gattii and ArtinM was administrated either before immunization or along with HK-C. gattii. Mice were infected with C. gattii and the efficacy of the immunization protocol was evaluated.Results: Mice that received ArtinM exhibited increased levels of IL-10 and relative expression of IL-23 in the lungs, reduced fungal burden and preserved tissue integrity post-infection.Conclusion: Adjuvant ArtinM improved immunization against C. gattii infection in C57BL/6 mice.

Cryptococcus gattii is a fungus that can make lungs sick. Right now, there are no good treatments for it, so scientists are trying to find new ways to fight it. In a recent study, they tested a type of immunotherapy called ArtinM to see if it could help. When they gave ArtinM to mice, the mice got healthier and had less fungus in their lungs. This means ArtinM might be able to help fight this fungus.

Antifungal and antibiofilm effect of duloxetine hydrochloride against Cryptococcus neoformans and Cryptococcus gattii.

Cryptococcosis is an invasive mycosis caused mainly by Cryptococcus gattii and C. neoformans and is treated with amphotericin B (AMB), fluconazole and 5-fluorocytosine. However, antifungal resistance, limited and toxic antifungal arsenal stimulate the search for therapeutic strategies such as drug repurposing. Among the repurposed drugs studied, the selective serotonin reuptake inhibitors (SSRIs) have shown activity against Cryptococcus spp. However, little is known about the antifungal effect of duloxetine hydrochloride (DH), a selective serotonin and norepinephrine reuptake inhibitor (SSNRI), against C. neoformans and C. gattii. In this study, DH inhibited the growth of several C. neoformans and C. gattii strains at concentrations ranging from 15.62 to 62.50 µg/mL. In addition, DH exhibited fungicidal activity ranging from 15.62 to 250 µg/mL. In biofilm, DH treatment reduced Cryptococcus spp. biomass at a level comparable to AMB, with a significant reduction (85%) for C. neoformans biofilms. The metabolic activity of C. neoformans and C. gattii biofilms decreased significantly (99%) after treatment with DH. Scanning electron micrographs confirmed the anti-biofilm activity of DH, as isolated cells could be observed after treatment. In conclusion, DH showed promising antifungal activity against planktonic cells and biofilms of C. neoformans and C. gattii, opening perspectives for further studies with DH in vivo.

Molecular characterization of clinical and environmental isolates from the Cryptococcus neoformans/C. Gattii species complexes of Maceió, Alagoas, Brazil.

Cryptococcosis is one of the major life-threatening opportunistic/systemic fungal diseases of worldwide occurrence, which can be asymptomatic or establish pneumonia and meningoencephalitis mainly in immunosuppressed patients, caused by the Cryptococcus neoformans and C. gattii species complexes. Acquisition is by inhaling fungal propagules from avian droppings, tree hollows and decaying wood, and the association of the molecular types with geographic origin, virulence and antifungal resistance have epidemiological importance. Since data on cryptococcosis in Alagoas are limited, we sought to determine the molecular types of etiological agents collected from clinical and environmental sources. We evaluated 21 isolates previously collected from cerebrospinal fluid and from environment sources (pigeon droppings and tree hollows) in Maceió-Alagoas (Brazil). Restriction fragment length polymorphism of URA5 gene was performed to characterize among the eight standard molecular types (VNI-VNIV and VGI-VGIV). Among isolates, 66.67% (14) were assigned to C. neoformans VNI - 12 of them (12/14) recovered from liquor and 2 from a tree hollow (2/14). One isolate from pigeon droppings (4.76%) corresponded to C. neoformans VNIV, while five strains from tree hollows and one from pigeon droppings (6, 28.57%) to C. gattii VGII. VNI-type was present in clinical and environmental samples and most C. neoformans infections were observed in HIV-positive patients, while types VNIV and VGII were prevalent in environmental sources in Alagoas. This is the first molecular characterization of Cryptococcus spp. in Alagoas, our study provides additional information on the ecoepidemiology of Cryptococcus spp. in Brazil, contributing to a closer view of the endemic species.

In vitro effects of selective serotonin reuptake inhibitors on Cryptococcus gattii capsule and biofilm.

Infections caused by Cryptococcus gattii mainly affect immunocompetent individuals and the treatment presents important limitations. This study aimed to validate the efficacy of selective serotonin reuptake inhibitors (SSRI), fluoxetine hydrochloride (FLH), and paroxetine hydrochloride (PAH) in vitro against C. gattii. The antifungal activity of SSRI using the microdilution method revealed a minimal inhibitory concentration (MIC) of 31.25 µg/ml. The combination of FLH or PAH with amphotericin B (AmB) was analyzed using the checkerboard assay and the synergistic effect of SSRI in combination with AmB was able to reduce the SSRI or AmB MIC values 4-8-fold. When examining the effect of SSRI on the induced capsules, we observed that FLH and PAH significantly decreased the size of C. gattii capsules. In addition, the effects of FLH and PAH were evaluated in biofilm biomass and viability. The SSRI were able to reduce biofilm biomass and biofilm viability. In conclusion, our results indicate the use of FLH and PAH exhibited in vitro anticryptococcal activity, representing a possible future alternative for the cryptococcosis treatment.

First report of environmental isolation of Cryptococcus and Cryptococcus-like yeasts from Boyacá, Colombia.

The Cryptococcus genus comprises more than 100 species, of which C. neoformans and C. gattii are the leading cause of cryptococcosis. The distribution of C. gattii and C. neoformans species complexes has been extensively studied and widely reported globally. Other species such as Naganishia albida, Papiliotrema laurentii, and Papiliotrema flavescens have been reported as pathogenic yeasts. Since there are no reports of environmental isolation in the Boyacá region (Colombia), this study aimed to isolate and characterize Cryptococcus and Cryptococcus-like yeasts from pigeon feces, Eucalyptus, and olive trees distributed in the municipalities of Tunja and Ricaute Alto. The environmental data was recovered, and the isolations obtained were identified by microscopy, biochemical test, MALDI-TOF MS, URA5-RFLP, and sequencing of the ITS and LSU loci. For the 93 pigeon dropping samples collected in Tunja, 23 yielded to C. neoformans, 3 to N. globosa, 2 N. albida and 1 to P. laurentii. Of the 1188 samples collected from olive trees, 17 (1.43%) positive samples were identified as C. gattii species complex (4), C. neoformans species complex (2), P. laurentii (3), N. albida (2), N. globosa (5) and P. flavescens (1). Likewise, specimens of C. neoformans presented molecular type VNI and molecular type VNII; for C. gattii the molecular types found were VGIII and one VGIV by URA5-RFLP but VGIII by MALDI-TOF and sequencing of the ITS and LSU. Therefore, it can be concluded that the species of Cryptococcus, Naganishia and Papiliotrema genera, are present in the environment of Boyacá, and show a predilection for climate conditions that are typical of this region.

Glucocorticoids, COVID-19 and Cryptococcosis.

Cryptococcus neoformans and C. gattii pneumonitis could persist asymptomatically for indefinite periods, resolve, or progress to symptomatic dissemination, mainly in immunocompromised individuals (e.g., treated with corticosteroids). The symptoms of COVID-19 may range from a self-limiting illness with general symptoms, such as fever, to more severe complications, such as pneumonitis. The glucocorticoids emerged as potential for treatment of COVID-19, mainly those patients who required ventilator therapy. However, although treatment with glucocorticoids has shown benefits in patients with COVID-19, they can be dangerous due to increased risk of coinfections and superinfections caused by opportunistic pathogens such as Cryptococcus ssp. Some patients with severe COVID-19 pneumonia treated with glucocorticoids developed cryptococcal infection and died. Therefore, immunomodulatory therapy could increase the susceptibility to acute infection or reactivation of Cryptococcus ssp in COVID-19 patients, and this could be complicated once pulmonary cryptococcosis has symptoms similar to COVID-19 becomes difficult to distinguish between the two disease states and treatment.

Reduced Susceptibility to Azoles in Cryptococcus gattii Correlates with the Substitution R258L in a Substrate Recognition Site of the Lanosterol 14-α-Demethylase.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a life-threatening fungal infection affecting mostly immunocompromised patients. In fact, cryptococcal meningitis accounts for about 19% of AIDS-related deaths in the world. Because of long-term azole therapies to treat this mycosis, resistance to fluconazole leading to treatment failure and poor prognosis has long been reported for both fungal species. Among the mechanisms implicated in resistance to azoles, mutations in the ERG11 gene, encoding the azole target enzyme lanosterol 14-α-demethylase, have been described. This study aimed to establish the amino acid composition of ERG11 of Colombian clinical isolates of C. neoformans and C. gattii and to correlate any possible substitution with the in vitro susceptibility profile of the isolates to fluconazole, voriconazole, and itraconazole. Antifungal susceptibility testing results showed that C. gattii isolates are less susceptible to azoles than C. neoformans isolates, which could correlate with differences in the amino acid composition and structure of ERG11 of each species. In addition, in a C. gattii isolate with high MICs for fluconazole (64 µg/mL) and voriconazole (1 µg/mL), a G973T mutation resulting in the substitution R258L, located in substrate recognition site 3 of ERG11, was identified. This finding suggests the association of the newly reported substitution with the azole resistance phenotype in C. gattii. Further investigations are needed to determine the exact role that R258L plays in the decreased susceptibility to fluconazole and voriconazole, as well as to determine the participation of additional mechanisms of resistance to azole drugs. IMPORTANCE The fungal species Cryptococcus neoformans and C. gattii are human pathogens for which drug resistance or other treatment and management challenges exist. Here, we report differential susceptibility to azoles among both species, with some isolates displaying resistant phenotypes. Azoles are among the most commonly used drugs to treat cryptococcal infections. Our findings underscore the necessity of testing antifungal susceptibility in the clinical setting in order to assist patient management and beneficial outcomes. In addition, we report an amino acid change in the sequence of the target protein of azoles, which suggests that this change might be implicated in resistance to these drugs. Identifying and understanding possible mechanisms that affect drug affinity will eventually aid the design of new drugs that overcome the global growing concern of antifungal resistance.

Molecular typing and antifungal susceptibility of clinical isolates of Cryptococcus neoformans and Cryptococcus gattii species complexes from the National Invasive Fungal Surveillance Network of Uruguay.

Cryptococcus neoformans and C. gattii species complexes (phylum: Basidiomycota) are environmental yeasts and are the main cause of human cryptococcosis worldwide. The most recent molecular typing studies in Latin America have focused on the intertropical region. Thus, this study aimed to update the knowledge of human cryptococcosis in the South American temperate region. We obtained and analyzed 116 C. neoformans/C. gattii species complexes isolates from the Public Health Surveillance Laboratory between 2008-2013 and 2017-2021 (C. gattii species complex = 1 and C. neoformans species complex = 115). The average patient age was 45 years, with an overall male:female ratio of 3.1:1. The proportion of HIV-negative patients was significantly higher in the second study period. Restriction fragment length polymorphism typing of URA5 gene revealed that the C. neoformans species complex comprised 75.7% VNI, 2.6% VNII, 0.9% VNIII, 1.7% VNIV, 17.4% VNII/VNIV hybrids, and one C. neoformans isolate (0.9%) misidentified as VGI; the C. gattii species complex isolates comprised one VGII. The overall case fatality rate was 49.5%, with no differences in lethality between VNI and hybrid isolates. Of the four isolates responsible for episodes of reoccurrence, only one had a genotype different from the first episode. Antifungal susceptibility testing revealed that most isolates fell below the local epidemiological cut-off value. This study provides additional information for the analysis of C. neoformans/C. gattii species complexes dynamics in the South American temperate region.

This study describes the epidemiological and molecular trends of human cryptococcosis according to the public health Uruguayan surveillance network. The findings provide additional information for analyzing the Cryptococcusneoformans/C. gattii species complexes in the South American temperate region.

New miconazole-based azoles derived from eugenol show activity against Candida spp. and Cryptococcus gattii by inhibiting the fungal ergosterol biosynthesis.

This work describes the design, synthesis and antifungal activity of new imidazoles and 1,2,4-triazoles derived from eugenol and dihydroeugenol. These new compounds were fully characterized by spectroscopy/spectrometric analyses and the imidazoles 9, 10, 13 e 14 showed relevant antifungal activity against Candida sp. and Cryptococcus gattii in the range of 4.6-75.3 µM. Although no compound has shown a broad spectrum of antifungal activity against all evaluated strains, some azoles were more active than either reference drugs employed against specific strains. Eugenol-imidazole 13 was the most promising azole (MIC: 4.6 µM) against Candida albicans being 32 times more potent than miconazole (MIC: 150.2 µM) with no relevant cytotoxicity (selectivity index >28). Notably, dihydroeugenol-imidazole 14 was twice as potent (MIC: 36.4 µM) as miconazole (MIC: 74.9 µM) and more than 5 times more active than fluconazole (MIC: 209.0 µM) against alarming multi-resistant Candida auris. Furthermore, in vitro assays showed that most active compounds 10 and 13 altered the fungal ergosterol biosynthesis, reducing its content as fluconazole does, suggesting the enzyme lanosterol 14α-demethylase (CYP51) as a possible target for these new compounds. Docking studies with CYP51 revealed an interaction between the imidazole ring of the active substances with the heme group, as well as insertion of the chlorinated ring into a hydrophobic cavity at the binding site, consistent with the behavior observed with control drugs miconazole and fluconazole. The increase of azoles-resistant isolates of Candida species and the impact that C. auris has had on hospitals around the world reinforces the importance of discovery of azoles 9, 10, 13 e 14 as new bioactive compounds for further chemical optimization to afford new clinically antifungal agents.

Yeast-amoeba interaction influences murine cryptococcosis.

The virulence of Cryptococcus spp. is modulated in the natural environment through interaction with abiotic and biotic factors, and this can occasionally have implications for the progression of cryptococcosis in mammals. Hence, we evaluated whether the prior interaction of highly virulent Cryptococcus gattii strain R265 with Acanthamoeba castellanii influenced the progression of cryptococcosis. The influence of the capsule on endocytosis was evaluated using amoeba and yeast morphometrics. Mice were intratracheally infected with yeast re-isolated from the amoeba (Interaction), yeast without prior contact with the amoeba (Non-Interaction), or sterile phosphate-buffered saline (SHAM). Morbidity signs and symptoms were monitored during the survival curve, while cytokine and fungal burden measurements and histopathological analysis were performed on the 10th day post infection. Morbidity and mortality parameters in experimental cryptococcosis were influenced by the prior interaction of yeast with amoeba, which led to phenotypic changes in the cryptococcal cells, polysaccharide secretion, and their tolerance to oxidative stress. Our results suggest that a prior yeast-amoeba interaction modulates yeast virulence, which is associated with a greater tolerance to oxidative stress related to the exo-polysaccharide content and influences the progression of cryptococcal infection.

Antimalarials and amphotericin B interact synergistically and are new options to treat cryptococcosis.

Cryptococcus gattii and Cryptococcus neoformans are the main etiological agents of cryptococcosis, an invasive mycosis treated with amphotericin B, 5-fluorocytosine, and fluconazole. This limited arsenal is toxic and is associated with antifungal resistance. Cryptococcosis and malaria pathogens are eukaryotic organisms that have a high incidence in Sub-Saharan Africa. The antimalarials (ATMs) halofantrine (HAL) and amodiaquine (AQ) block Plasmodium heme polymerase, and artesunate (ART) induces oxidative stress. Considering that Cryptococcus spp. is susceptible to reactive oxygen species and that iron is essential for metabolism, the repurposing of ATMs for treating cryptococcosis was tested. ATMs reduced fungal growth, induced oxidative and nitrosative stresses, and altered ergosterol content, melanin production, and polysaccharide capsule size in C. neoformans and C. gattii, revealing a dynamic effect on fungal physiology. A comprehensive chemical-genetic analysis using two mutant libraries demonstrated that the deletion of genes involved in synthesizing components of the plasma membrane and cell wall, and oxidative stress responses are essential for fungal susceptibility to ATMs. Interestingly, the amphotericin B (AMB) fungicidal concentrations were ∼10 times lower when combined with ATMs, demonstrating a synergistic interaction. Further, the combinations showed reduced toxicity to murine macrophages. Finally, HAL+AMB and AQ+AMB efficiently reduced lethality and fungal burden in the lungs and brain in murine cryptococcosis. These findings provide perspectives for further studies with ATMs against cryptococcosis and other fungal infections.

Criptococose respiratória e cutânea em gato de vida livre da cidade de Sobral/CE / Respiratory and cutaneous Cryptococcosis in a free-range cat in the city of Sobral/CE

A criptococose é uma doença infecciosa sistêmica de origem fúngica, causada pelas espécies: Cryptococcus neoformans e Cryptococcus gattii. É considerada uma micose oportunista capaz de infectar mamíferos domésticos, animais silvestres e seres humanos, sendo classificada como uma doença zoonótica. Esse patógeno é encontrado principalmente em ambientes contaminados por fezes de pombos (Columba livia), que atuam como importantes fontes de infecção do fungo. De acordo com sua disseminação para os tecidos do organismo, a doença pode causar diferentes síndromes tanto em seres humanos como em animais síndrome respiratória, síndrome neurológica, síndrome ocular e síndrome cutânea. O diagnóstico pode ser realizado através da pesquisa de antígeno polissacarídeo circulante no soro ou líquor, por meio da prova de látex. Testes imunoenzimáticos (ELISA) também podem ser realizados para detecção de antígenos dessa levedura. Exame citológico, histopatológico e cultura fúngica para a identificação do agente tornam o diagnóstico da criptococose mais fácil. O tratamento é baseado no uso de antifúngicos e sua escolha é realizada através da avaliação dos sinais clínicos observados. Diante do exposto, o objetivo deste trabalho é relatar um caso de criptococose respiratória e cutânea em felino doméstico de vida livre da cidade de Sobral/CE.

Cryptococcosis is a systemic infectious disease of fungal origin caused by the species: Cryptococcus neoformans and Cryptococcus gattii. It is considered an opportunistic mycosis capable of infecting domestic mammals, wild animals, and humans, being classified as a zoonotic disease. This pathogen is mainly found mainly in environments contaminated by pigeon (Columba livia) feces, which act as important sources of fungal infection. According to its spread to the body's tissues, the disease can cause different syndromes in both humans and animals: respiratory syndrome, neurological syndrome, ocular syndrome, and cutaneous syndrome. The diagnosis can be made through the investigation of circulating polysaccharide antigen in serum or cerebrospinal fluid using the latex test. Immunoenzymatic tests (ELISA) can also be performed to detect yeast antigens. Cytological examination, histopathological examination, and fungal culture to identify the agent make the diagnosis of cryptococcosis easier. The treatment is based on the use of antifungals, and its choice is made through the evaluation of the observed clinical signs. In this context, this work aims to report a case of respiratory and cutaneous cryptococcosis in a free-range domestic cat in the city of Sobral/CE.

Antifungal Activity, Antibiofilm and Association Studies with O-Alkylamidoximes against Cryptococcus spp.

This is the first study that describes the antifungal and anti-biofilm potential of O-alkylamidoximes against strains of Cryptococcus neoformans and Cryptococcus gattii. In vitro tests have shown that O-alkylamidoximes are capable of inhibiting fungal growth and biofilm formation of the C. neoformans and C. gattii strains, suggesting, from molecular docking, the potential for interaction with the Hsp90. The associations between O-alkylamidoximes and amphotericin B were beneficial. Therefore, O-alkylamidoximes can be a useful alternative to contribute to the limited arsenal of drugs, since they showed a powerful action against the primary agents of Cryptococcosis.

Cryptococcosis in domestic and wild animals: A review.

Cryptococcosis is a fungal disease of public health relevance that affects numerous animal species and humans, causing respiratory and neurological impairment. Hence, we conducted a systematic review that included publications from 1975 to 2021 and covered 132 articles that addressed reports of cryptococcosis in domestic and wild animals, its main clinical manifestations, pathological findings, etiology, diagnosis, and therapeutic protocols. We found that the highest number of reports of cryptococcosis is in domestic species, especially cats. Among the wild and/or exotic animals, koalas and ferrets are the most affected, being important carriers of Cryptococcus spp. Pulmonary and neurological involvement is predominant in all species, although nonspecific clinical manifestations have been reported in various species, making clinical suspicion and diagnosis difficult. The countries with the most reports are Australia, the United States, Brazil, and Canada, with C. gattii VGI and VGII standing out. The therapies were based on azoles, amphotericin B, and 5-flucytosine, although there is no standard treatment protocol. Although, several diagnostic methods have been described, in a significant number of reports the diagnosis was made after a necropsy. Professionals are warned about diverse and nonspecific clinical manifestations in different animal species, which underlines the importance of cryptococcosis in the differential diagnosis in clinical practice. Furthermore, it is necessary to encourage the use of laboratory and molecular tools to improve the diagnosis of cryptococcosis. We also emphasize the urgent need for standardized therapeutic protocols to guide veterinary clinicians.

This review compiles studies on cryptococcosis in domestic and wild animals. Most reports occurred in cats and koalas. Pulmonary and neurological involvement was predominant in all affected species, and C. gattii VGI and VGII stood out in the etiology of the disease.

Adjuvant Pam3CSk4 does not improve the immunization against Cryptococcus gattii infection in C57BL/6 mice.

Background: Cryptococcosis is a relevant invasive fungal infection that affects immunocompromised and immunocompetent individuals when caused by Cryptococcus gattii. Host innate and adaptive immune responses can be subverted by C. gattii, that blocks the differentiation of T helper (Th) 1 and Th17 cells, which are involved in the protection against cryptococcosis. Moreover, the macrophage polarization is modulated by C. gattii infection that requires a balance in the macrophage subsets to control the C. gattii infection. Toll-like receptor (TLR) 2 agonists are important immunomodulators favoring a pro-inflammatory response with potential fungicidal activity, and TLR2 agonists have been used as adjuvants in vaccines against infections caused by bacteria or viruses. Therefore, this work aimed to evaluate the immunomodulatory effect of the tripalmitoyl lipopeptide S-glycerol cysteine (Pam3CSK4 or P3C4), a TLR2 agonist, as an adjuvant in the vaccination against C. gattii infection. Methods and Results: C57BL/6 mice were immunized with 2 × 107 inactivated yeasts of C. gattii via intranasal route on day 1, 14 and 28 (Immunized group). Immunization was associated with 1µg or 10µg of adjuvant P3C4 (Immunized+P3C4-1µg or Immunized+P3C4-10 µg), followed by C. gattii infection on day 42 after the immunization protocol. Immunized+P3C4-1 µg group had reduced levels of IgG1, IgG2a and IgA and no significant difference in the IgG and IgM anti-GXM antibody titer was detected, compared to the Immunized group. High levels of IL-17 and IL-1ß in lung tissue of mice from the Immunized+P3C4-1µg group did not promote a predominance of Th17 cells, in contrast, the frequency of TLR2+ cells was increased in immunized mice that received 1 µg of P3C4. The reduction in the relative expression of T-bet and high levels of Foxp3 detected in the lungs of the Immunized+P3C4-1µg group suggest a prevalence of regulatory T cells in the tissue, which did not contribute to the control of C. gattii infection. The immunization protocol associated with 10 µg of adjuvant P3C4 induced high levels of IL-17 in the lung tissue, whereas the levels of pro-inflammatory cytokines were downregulated. To evaluate the effect of adjuvant P3C4 in the control of C. gattii infection, quantification of the fungal burden in the lungs was performed by the CFU assay, and the groups with adjuvant P3C4 showed a pulmonary C. gattii burden that was not significantly altered when compared with the immunized group. The mice that received 1 µg of adjuvant P3C4 had a lower percentage of inflammatory infiltrate in the lungs. Conclusion: The immunomodulatory effect of P3C4, associated with the immunization protocol, plays an imbalance between pro- and anti-inflammatory response in the lungs that did not favor a protection against C. gattii infection, which is related to the immune response characterized by a suppressive/regulatory profile in the pulmonary microenvironment after C. gattii infection.

The small heat shock protein Hsp12.1 has a major role in the stress response and virulence of Cryptococcus gattii.

Cryptococcus gattii is one of the etiological agents of cryptococcosis. To achieve a successful infection, C. gattii cells must overcome the inhospitable host environment and deal with the highly specialized immune system and poor nutrients availability. Inside the host, C. gattii uses a diversified set of tools to maintain homeostasis and establish infection, such as the expression of remarkable and diverse heat shock proteins (Hsps). Grouped by molecular weight, little is known about the Hsp12 subset in pathogenic fungi. In this study, the function of the C. gattii HSP12.1 and HSP12.2 genes was characterized. Both genes were upregulated during murine infection and heat shock. The hsp12.1 Δ null mutant cells were sensitive to plasma membrane and oxidative stressors. Moreover, HSP12 deletion induced C. gattii reactive oxygen species (ROS) accumulation associated with a differential expression pattern of oxidative stress-responsive genes compared to the wild type strain. Apart from these findings, the deletion of the paralog gene HSP12.2 did not lead to any detectable phenotype. Additionally, the double-deletion mutant strain hsp12.1 Δ /hsp12.2 Δ presented a similar phenotype to the single-deletion mutant hsp12.1 Δ, suggesting a minor participation of Hsp12.2 in these processes. Furthermore, HSP12.1 disruption remarkably affected C. gattii virulence and phagocytosis by macrophages in an invertebrate model of infection, demonstrating its importance for C. gattii pathogenicity.

Staphylococcus aureus triggers a protective inflammatory response against secondary Cryptococcus gattii infection in a murine model.

Prior infections can provide protection or enhance susceptibility to a subsequent infection through microorganism's interaction or host immunomodulation. Staphylococcus aureus (SA) and Cryptococcus gattii (CG) cause lungs infection, but it is unclear how they interact in vivo. This study aimed to study the effects of the primary SA lung infection on secondary cryptococcosis caused by CG in a murine model. The mice's survival, fungal burden, behavior, immune cells, cytokines, and chemokines were quantified to evaluate murine cryptococcosis under the influence of a previous SA infection. Further, fungal-bacterial in vitro interaction was studied in a culture medium and a phagocytosis assay. The primary infection with SA protects animals from the subsequent CG infection by reducing lethality, improving behavior, and impairing the fungal proliferation within the host. This phenotype was associated with the proinflammatory antifungal host response elicited by the bacteria in the early stage of cryptococcosis. There was no direct inhibition of CG by SA, although the phagocytic activity of macrophages was reduced. Identifying mechanisms involved in this protection may lead to new approaches for preventing and treating cryptococcosis.

Genotypic Diversity and Antifungal Susceptibility of Clinical Isolates of Cryptococcus Gattii Species Complex from Argentina.

The aim of this study was to determine the genotypic diversity of 22 Cryptococcus gattii species complex clinical isolates from Argentina and to place these genotypes within the diversity of clinical, veterinary and environmental isolates from Latin America. Mating type and antifungal susceptibility of the isolates were also determined. By URA5-RFLP, nine isolates were identified as molecular type VGI, 10 as VGII, one as VGIII and two as VGIV. Multilocus sequence typing (MSLT), following the International Society for Human and Animal Mycology (ISHAM) consensus MLST scheme, was used to determine the genotypic diversity. Our results suggest that, in Argentina, VGI isolates have low genetic diversity, while VGII isolates have high genetic diversity. Both isolates identified as VGIV by URA5-RFLP were genotyped by MLST as belonging to the currently named VGVI clade. From all isolates, eight sequence types (STs) were unique for Argentina, while five STs have been reported already in other countries, being of high interest the genotypes ST20 and ST7 since they belong to the subtypes VGIIa and VGIIb, respectively, which are associated with hypervirulent strains responsible for outbreaks in North America. To note, geographical analysis showed that some genotypes may be associated with some regions in Argentina. Most isolates were MATα, but we are reporting one isolate MATa for the first time in the country. Antifungal susceptibility tests showed that itraconazole, voriconazole and posaconazole had high activity against all isolates, while amphotericin B, fluconazole and 5-fluorocytosine were the least active drugs against all studied isolates.