Epidemiology, management and outcomes of Cryptococcus gattii infections: A 22-year cohort.

BACKGROUND: Cryptococcus gattii is a globally endemic pathogen causing disease in apparently immune-competent hosts. We describe a 22-year cohort study from Australia's Northern Territory to evaluate trends in epidemiology and management, and outcome predictors. METHODS: A retrospective cohort study of all C. gattii infections at the northern Australian referral hospital 1996-2018 was conducted. Cases were defined as confirmed (culture-positive) or probable. Demographic, clinical and outcome data were extracted from medical records. RESULTS: 45 individuals with C. gattii infection were included: 44 Aboriginal Australians; 35 with confirmed infection; none HIV positive out of 38 tested. Multifocal disease (pulmonary and central nervous system) occurred in 20/45 (44%). Nine people (20%) died within 12 months of diagnosis, five attributed directly to C. gattii. Significant residual disability was evident in 4/36 (11%) survivors. Predictors of mortality included: treatment before the year 2002 (4/11 versus 1/34); interruption to induction therapy (2/8 versus 3/37) and end-stage kidney disease (2/5 versus 3/40). Prolonged antifungal therapy was the standard approach in this cohort, with median treatment duration being 425 days (IQR 166-715). Ten individuals had adjunctive lung resection surgery for large pulmonary cryptococcomas (median diameter 6cm [range 2.2-10cm], versus 2.8cm [1.2-9cm] in those managed non-operatively). One died post-operatively, and 7 had thoracic surgical complications, but ultimately 9/10 (90%) treated surgically were cured compared with 10/15 (67%) who did not have lung surgery. Four patients were diagnosed with immune reconstitution inflammatory syndrome which was associated with age <40 years, brain cryptococcomas, high cerebrospinal fluid pressure, and serum cryptococcal antigen titre >1:512. CONCLUSION: C. gattii infection remains a challenging condition but treatment outcomes have significantly improved over 2 decades, with eradication of infection the norm. Adjunctive surgery for the management of bulky pulmonary C. gattii infection appears to increase the likelihood of durable cure and likely reduces the required duration of antifungal therapy.

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.

Promising whole-cell vaccines against cryptococcosis.

Cryptococcosis is a mycosis caused by Cryptococcus neoformans and C. gattii species complexes. Although this infection is potentially lethal, no prophylactic vaccine is yet commercially available, and the immune memory that enables prevention is still under investigation. These pathogens have a capsule layer for immune evasion and a sophisticated mechanism to advance the infection, and it is expected that these characteristics will make it difficult to develop prophylactic vaccines and to decipher the protective immunity. The current vaccine studies are focused on subunit, mRNA, DNA, and viral vector vaccines, with whole-cell vaccines also proving successful against cryptococcal infections. Cryptococcal whole-cell vaccines have been composed of highly immunostimulating strains with low-pathogenicity that are modified by genetic recombination technology. Examples include the whole-cell vaccines H99γ, sgl1∆, fbp1∆, znf2oe , cda1/2/3∆, cap59∆, and cap60∆. Some of these whole-cell vaccines were found to be highly effective in prolonging life and suppressing the fungal burden after an infection challenge in mice, and to be cross-reactive to C. neoformans, C. gattii, and other fungal pathogens. Furthermore, for some vaccines, the protective effect can be retained even in an immunocompromised host depleted of CD4+ T cells. These findings have provided new insights into protective immunity that should aid in vaccine development. In this review, we highlight the upsides and downsides of whole-cell vaccines against cryptococcosis.

[A case of Cryptococcus gattii pneumonia].

Cryptococcus gattii often causes meningitis, but rarely causes pulmonary infections. Here, we reported a patient with asymptomatic pulmonary cryptococcosis caused by Cryptococcus gattii. The patient presented to the thoracic surgery department with an isolated pulmonary nodule that had been present for three years and underwent a thoracoscopic pulmonary wedge resection. Postoperative pathology was consistent with Cryptococcus gattii infection. Although the incidence of Cryptococcus gattii infection is lower than that of Cryptococcus neoformans, the neurological involvement is common and has severe complications. In this report, the risk factors, symptoms, diagnosis, treatment, and prognosis of Cryptococcus gattii pneumonia were discussed to improve clinical awareness of this disease.

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.

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.

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.

Characteristics and prognostic risk factors of patients with sequence type 5 lineage-associated cryptococcosis in China.

OBJECTIVES: Cryptococcus neoformans sequence type 5 (ST5) lineage could infect immunocompetent hosts and cause a significant medical burden. We sought to identify characteristics and prognostic risk factors of ST5 lineage-associated cryptococcosis. METHODS: Multilocus sequence typing and antifungal susceptibility testing were conducted for Cryptococcus isolates. The clinical and laboratory characteristics of cryptococcosis patients were investigated. The multivariable logistic regression identified variables independently associated with 30-day mortality in patients with ST5 lineage-associated cryptococcosis without HIV. RESULTS: The infection rate of the ST5 isolates was 89.4% (370/414) in China. The proportion of ST5 isolates with nonwild-type minimum inhibitory concentrations to amphotericin B, 5-flucytosine, voriconazole, posaconazole, itraconazole, and fluconazole were 0%, 5.4%, 0.3%, 1.4%, 0.3%, and 8.1%, respectively. The ST5 lineage-infected group exhibited significantly higher blood platelet count, lower blood cryptococcal antigen (CrAg) titer, lower cerebrospinal fluid (CSF) CrAg titer than the non-ST5 lineage-infected group, and lower hemoglobin and lower CSF CrAg titer than the Cryptococcus gattii isolates-infected group. Seven baseline parameters, including underlying disease, dyskinesia, anemia, high peripheral blood neutrophils, low platelet count, high CSF fungal burden, and high CSF opening pressure, were associated independently with the 30-day mortality of patients with ST5 lineage-associated cryptococcosis without HIV. CONCLUSION: Our study has provided an understanding of the ST5 lineage associated with cryptococcosis.

Reversible deafness and blindness in Cryptococcus gattii meningitis with a ventriculoperitoneal shunt: A case report and literature review.

Cryptococcus gattii (C. gattii) has been considered a leading cause of meningitis in immunocompetent hosts in tropical and subtropical regions. Visual loss is common but hearing impairment is relatively infrequent in C. gattii meningitis. Notably, there have been limited studies on the etiology, and especially therapy of auditory and ocular complications associated with C. gattii meningitis. Here we report a case of reversible deafness and blindness treated with a ventriculoperitoneal shunt (VPS) surgery in C. gattii meningitis. This case indicated that elevated intracranial pressure (ICP) may play a role in the concurrent hearing and vision impairments associated with C. gattii meningitis and the early VPS surgery after the initiation of the antifungal therapy may effectively improve both hearing and vision in this condition.

Factors Influencing the Nitrogen-Source Dependent Flucytosine Resistance in Cryptococcus Species.

Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes. IMPORTANCE 5-FC is a commonly used antifungal drug for treatment of cryptococcosis caused by Cryptococcus neoformans and C. gattii species complexes. When various amino acids were used as the sole nitrogen source for growth, we found lineage dependent differences in 5-FC susceptibility. Deletion of the classical cytosine permease (FCY2) and deaminase (FCY1) genes caused increased 5-FC resistance in all tested nitrogen sources in C. neoformans but not in C. gattii. Furthermore, we demonstrate that the two GATA family transcription factor genes GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance by regulating the expression of the paralogs of cytosine permease and deaminase genes. Our study not only identifies the new function of paralogs of the cytosine permease and deaminase and the role of their regulatory transcription factors but also denotes the differences in the mechanism of 5-FC resistance among the two etiologic agents of cryptococcosis under different nitrogen sources.

Effects of combination of Cryptococcus gattii and IFN-γ, IL-4 or IL-27 on human bronchial epithelial cells.

BACKGROUND: Airway epithelial cells are crucial for the establishment of cryptococcosis. In experimental cryptococcosis, the Th2 immune response is associated with host susceptibility, while Th1 cells are associated with protection. The absence of IL-27 receptor alpha in mice favor the increase Cryptococcus neoformans burden in the lung. Here, we evaluated the effects of the combination of IL-4, IFN-γ or IL-27 with C. gattii on human bronchial epithelial cells (BEAS-2B). METHODS: BEAS-2B were stimulated with IL-4, IFN-γ or IL-27 (100 ng/mL) and/or live yeast forms of C. gattii (multiplicities of infection (MOI) of 1-100) and vice-versa, as well as with heat-killed cells of C. gattii for 24 h. RESULTS: None of the C. gattii MOIs had cytotoxic effects on BEAS-2B when compared to control. The cells stimulated by cytokines (IL-4, IFN-γ or IL-27) followed by live yeast forms of C. gattii (MOI of 100) infection and vice-versa demonstrated a reduction in IL-6, IL-8 and/or CCL2 production and activation of STAT6 (induced by IL-4) and STAT1 (induced by IL-27 or IFN-γ) when compared to cells stimulated with C. gattii, IL-4, IFN-γ or IL-27. In the combination of cytokines and heat-killed cells of C. gattii, no inhibition of these inflammatory parameters was observed. The growth of C. gattii was increased while the phagocytosis of live yeast forms of C. gattii in the BEAS-2B were reduced in the presence of IL-4, IFN-γ or IL-27. Conclusion The association of live yeast forms, but not heat-killed yeast forms, of C. gattii with IL-4, IFN-γ or IL-27 induced an anti-inflammatory effect.

Avaliação dos efeitos antifúngicos de fármacos inibidores seletivos da recaptação de serotonina em Cryptococcus gattii / Evaluation of the antifungal effects of selective serotonin reuptake inhibitors in Cryptococcus gattii

A criptococose é uma infecção fúngica que acomete tanto indivíduos imunocomprometidos como imunocompetentes. O arsenal antifúngico é limitado, existem relatos de desenvolvimento de resistência fúngica a esses compostos e também alta toxicidade ao paciente. O reposicionamento de fármacos dos inibidores seletivos da recaptação de serotonina (ISRS) tem mostrado ação contra espécies fúngicas, tornando estes compostos alternativas a serem estudadas para o tratamento das infecções criptocócicas. Assim, o objetivo deste estudo foi avaliar os efeitos antifúngicos em cápsula, biofilme e células planctônicas de Cryptococcus gattii (cepa ATCC 56990 e isolado clínico 5) dos fármacos ISRS, cloridrato de fluoxetina (CF) e cloridrato de paroxetina (CP). Para isso, foi utilizada a técnica de microdiluição em caldo de acordo com European Committee on Antimicrobial Susceptibility Testing (EUCAST) para determinar a Concentração Inibitória Mínima (CIM), sendo a CIM de 31,25 µg/mL determinada para os fármacos CF e CP e ambos os fármacos demonstraram ação fungicida (CIM/CFM = 1). Em seguida foi verificado atividade sinérgica dos fármacos CF e CP combinados com anfotericina B (AmB), como resultado encontramos três concentrações sinérgicas, para CF reduzindo 8 e 4x em relação ao valor de CIM, para CP reduzindo 4x em relação ao valor de CIM e para AmB houve redução de 4x em relação ao valor de CIM. Posteriormente, o efeito dos fármacos mencionados foi avaliado na redução da biomassa do biofilme, por meio da técnica de cristal violeta. Nas concentrações 10x (312,5 µg/mL) e 20x (625 µg/mL) CIM de observou-se a redução da biomassa do biofilme de C. gattii em 57,72% a 76,66% para CF e redução entre 42,69 a 68,03% para CP. Além disso, em concentrações sub- inibitórias, ambos fármacos reduziram o tamanho da cápsula da levedura em até 62,46% para CF e 58,94% para CP. Também foi analisada a viabilidade do biofilme pela contagem de unidades formadoras de colônia/mL, após tratamento com CF e CP 20x valor de CIM e foi observada redução entre 1,21 a 1,446 log na viabilidade do biofilme (p< 0,0001). Ainda, o efeito dos fármacos em biofilme foi avaliado quanto ao efeito na atividade metabólica pelo ensaio XTT nas concentrações 10x e 20x CIM de ambos os fármacos foi possível observar redução entre 39,05% a 84,62% para CF e redução entre 56,99% a 67,64% para CP. Assim, os fármacos avaliados apresentaram potencial antifúngico frente C. gattii em todos os ensaios in vitro, podendo ser considerados novas alternativas para o tratamento deste patógeno (AU)

Cryptococcosis is a fungal infection that affects both immunocompromised and immunocompetent individuals. The antifungal arsenal is limited, there are reports of the development of fungal resistance to these compounds and also high toxicity to the patient. The drug repositioning of selective serotonin reuptake inhibitors (SSRIs) has shown action against fungal species, making these compounds alternatives to be studied for the treatment of cryptococcal infections. Thus, the aim of this study was to evaluate the antifungal effects on capsule, biofilm and planktonic cells of Cryptococcus gattii (ATCC strain 56990 and clinical isolate 5) of the SSRI drugs, fluoxetine hydrochloride (FLH) and paroxetine hydrochloride (PAH). For this, the broth microdilution technique was used according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) to determine the Minimum Inhibitory Concentration (MIC), and the MIC of 31.25 µg/mL was determined for the drugs (FLH) and PAH and both drugs demonstrated fungicidal action (MIC/CFM = 1).Then it was verified synergistic activity of the drugs FLH and PAH combined with amphotericin B (AmB), as a result we found three synergistic concentrations, for FLH reducing 8 and 4x compared to the MIC value, for PAH reducing 4x compared to the MIC value and for AmB there was 4x reduction compared to the MIC value. Subsequently, the effect of the mentioned drugs was evaluated in the reduction of biofilm biomass by means of the crystal violet technique. At 10x (312.5 µg/mL) and 20x (625 µg/mL) MIC concentrations of it was observed the reduction of C. gattii biofilm biomass by 57.72% to 76.66% for FLH and reduction between 42.69 to 68.03% for PAH. Furthermore, at sub-inhibitory concentrations, both drugs reduced the yeast capsule size by up to 62.46% for FLH and 58.94% for PAH. The biofilm viability was also analyzed by counting colony forming units/mL, after treatment with FC and CP 20x the MIC value and a reduction between 1.21 to 1.446 log in biofilm viability was observed (p< 0.0001). Also, the effect of the drugs in biofilm was evaluated as the effect on the metabolic activity by the XTT assay in concentrations 10x and 20x MIC of both drugs was possible to observe reduction between 39.05% to 84.62% for FC and reduction between 56.99% to 67.64% for CP. Thus, the drugs evaluated showed antifungal potential against C. gattii in all in vitro assays, and may be considered new alternatives for the treatment of this pathogen.(AU)

The Cryptococcus gattii species complex: Unique pathogenic yeasts with understudied virulence mechanisms.

Members of Cryptococcus gattii/neoformans species complex are the etiological agents of the potentially fatal human fungal infection cryptococcosis. C. gattii and its sister species cause disease in both immunocompetent and immunocompromised hosts, while the closely related species C. neoformans and C. deneoformans predominantly infect immunocompromised hosts. To date, most studies have focused on similarities in pathogenesis between these two groups, but over recent years, important differences have become apparent. In this review paper, we highlight some of the major phenotypic differences between the C. gattii and neoformans species complexes and justify the need to study the virulence and pathogenicity of the C. gattii species complex as a distinct cryptococcal group.

Cryptococcus escapes host immunity: What do we know?

Cryptococcus is an invasive fungus that seriously endangers human life and health, with a complex and well-established immune-escaping mechanism that interferes with the function of the host immune system. Cryptococcus can attenuate the host's correct recognition of the fungal antigen and escape the immune response mediated by host phagocytes, innate lymphoid cells, T lymphocytes, B lymphocytes with antibodies, and peripheral cytokines. In addition, the capsule, melanin, dormancy, Titan cells, biofilm, and other related structures of Cryptococcus are also involved in the process of escaping the host's immunity, as well as enhancing the ability of Cryptococcus to infect the host.

Cryptococcus neoformans- and Cryptococcus gattii-specific IgG, IgA and IgM differ among children and adults with and without cryptococcosis from Colombia.

Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg) cause cryptococcosis, a life-threatening systemic mycosis of global distribution affecting mainly immunocompromised adults. Although a humoral response occurs during cryptococcosis, the role of antibody production against this mycosis is not fully understood. We aimed to determine total and specific antibodies against cryptococcal protein antigens in sera from people with and without a diagnosis of cryptococcosis from Colombia. Using ELISA, total and specific levels of immunoglobulin (Ig)G, IgA and IgM were determined in sera from children and adults with (n = 109) and without (n = 119) cryptococcosis. Specific antibodies were those binding Cn- and Cg-protein antigens. In general, the mean of the total IgG production was higher in cryptococcosis patients than in controls (13 942.32 vs. 6459.91 µg/ml), while levels of IgA (488.13 vs. 1564.53 µg/ml) and IgM (775.69 vs. 1014.72 µg/ml) were higher in controls than in cryptococcosis patients (P ≤ .05). In patients with cryptococcosis, total IgG, IgA and IgM levels were higher in HIV + compared with HIV- (P ≤ .05). Specific antibodies tended to be higher in cryptococcosis patients than in controls and in adults than in children, with a positive correlation between antibody reactivity and age. All immunoglobulins were more reactive against Cn-proteins than Cg-proteins. Overall, a positive weak correlation between total and specific antibodies was found, although not always statistically significant. In patients with cryptococcosis from Colombia, the levels of immunoglobulins, total and specific, differ with respect to people without cryptococcosis. Variations in antibody production among adults and children with cryptococcosis and between Cn- and Cg-protein antigens were as well established. Our findings encourage further studies to determine the role of humoral immunity for host defense against cryptococcosis.

Differential IgG, IgA, and IgM production and their reactivity with cryptococcal proteins, both among children and adults with and without a diagnosis of cryptococcosis from Colombia, lead to reappraise the study of the potential role of antibody production as host defense against this fungal infection.

Discovery of Novel 7-Hydroxy-5-oxo-4,5-dihydrothieno[3,2-b]pyridine-6-carboxamide Derivatives with Potent and Selective Antifungal Activity against Cryptococcus Species.

Cryptococcus neoformans and Cryptococcus gattii can cause fatal invasive infections, especially in immunocompromised patients. However, few antifungal drugs are available to help treat cryptococcosis. In this study, by compound library screening, we presented the first report of hit compound P163-0892, which had potent in vitro and in vivo antifungal activity against Cryptococcus spp. In vitro tests showed that P163-0892 was not cytotoxic and had highly selective and strong antifungal activities against Cryptococcus spp. with MIC values less than 1 µg/mL. Synergism of P163-0892 and fluconazole was also observed in vitro. The in vivo antifungal efficacy of P163-0892 was assessed in a wax moth larval fungal infection model, and treatment with 10 mg/kg P163-0892 caused a significant reduction in fungal burden and significant extension of the survival time. Taken together, our data indicate that the hit compound P163-0892 warrants further investigation as a novel anti-Cryptococcus agent.

MDA5 signaling induces type 1 IFN- and IL-1-dependent lung vascular permeability which protects mice from opportunistic fungal infection.

Lungs balance threat from primary viral infection, secondary infection, and inflammatory damage. Severe pulmonary inflammation induces vascular permeability, edema, and organ dysfunction. We previously demonstrated that poly(I:C) (pICLC) induced type 1 interferon (t1IFN) protected mice from Cryptococcus gattii (Cg) via local iron restriction. Here we show pICLC increased serum protein and intravenously injected FITC-dextran in the lung airspace suggesting pICLC induces vascular permeability. Interestingly, pICLC induced a pro-inflammatory signature with significant expression of IL-1 and IL-6 which depended on MDA5 and t1IFN. Vascular permeability depended on MDA5, t1IFN, IL-1, and IL-6. T1IFN also induced MDA5 and other MDA5 signaling components suggesting that positive feedback contributes to t1IFN dependent expression of the pro-inflammatory signature. Vascular permeability, induced by pICLC or another compound, inhibited Cg by limiting iron. These data suggest that pICLC induces t1IFN which potentiates pICLC-MDA5 signaling increasing IL-1 and IL-6 resulting in leakage of antimicrobial serum factors into lung airspace. Thus, induced vascular permeability may act as an innate defense mechanism against opportunistic fungal infection, such as cryptococcosis, and may be exploited as a host-directed therapeutic target.

An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii.

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus.