Repurposing benzimidazoles to fight Cryptococcus

The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high rates of mortality and toxic or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Benzimidazoles are potentially attractive antifungal compounds that were introduced in clinical practice nearly 60 years ago to treat helminthic infections. In addition to being safe, their cost of treatment is extraordinarily low. Several studies suggested benzimidazoles as promising anticryptococcal agents combining low-cost and high antifungal efficacy. So far, anti-cryptococcal activities were demonstrated for 16 different benzimidazoles. In particular, albendazole, mebendazole, flubendazole, and fenbendazole have potent in vitro antifungal activity against C. neoformans and C. gattii. Mice lethally infected with C. neoformans and treated with fenbendazole had 100 % survival when the drug was administered intranasally. In this review, we discuss the potential of benzimidazoles as potential anti-cryptococcal agents, including a general literature overview, most recent findings, mechanism of antifungal action, costs, toxicity, and antifungal potential in vivo.     

A proteomic‐based approach for the identification of immunodominant Cryptococcus neoformans proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life‐threatening meningoencephalitis in immune compromised patients. Previous, studies in our laboratory have shown that prior exposure to an IFN‐γ‐producing C. neoformans strain (H99γ) elicits protective immunity against a second pulmonary C. neoformans challenge. Here, we characterized the antibody response produced in mice protected against experimental pulmonary C. neoformans infection compared to nonprotected mice. Moreover, we evaluated the efficacy of using serum antibody from protected mice to detect immunodominant C. neoformans proteins. Protected mice were shown to produce significantly more C. neoformans‐specific antibodies following a second experimental pulmonary cryptococcal challenge compared to nonprotected mice. Immunoblot analysis of C. neoformans proteins resolved by 2‐DE using serum from nonprotected mice failed to show any reactivity. In contrast, serum from protected mice was reactive with several cryptococcal protein spots. Analysis of these spots by capillary HPLC‐ESI‐MS/MS identified several cryptococcal proteins shown to be associated with the pathogenesis of cryptococcosis. Our studies demonstrate that mice immunized with C. neoformans strain H99γ produce antibodies that are immune reactive against specific cryptococcal proteins that may provide a basis for the development of immune based therapies that induce protective anticryptococcal immune responses.     

Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans

Aims: To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. Methods and Results: Silkworms reared at 37°C died after an injection of viable serotype A C. neoformans fungus into the haemolymph. The serotype A C. neoformans, which is known to have higher mammal pathogenicity than the serotype D, was also more virulent against the silkworm. Furthermore, the deletion mutants of genes gpa1, pka1 and cna1, which are genes known to be necessary for the pathogenesis in mammals, showed an increase in the number of fungal cells necessary to kill half of the silkworm population (LD₅₀ value). Antifungal drugs, amphotericin B, flucytosine, fluconazole and ketoconazole, showed therapeutic effects in silkworms infected with C. neoformans. However, amphotericin B was not therapeutically effective when injected into the silkworm intestine, comparable to the fact that amphotericin B is not absorbed by the intestine in mammals. Conclusions: The silkworm–C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. Significance and Impact of the Study: The silkworm infection model has various advantages for screening antifungal drug candidates. We can also elucidate the cryptococcal pathogenesis and evaluate the in vivo pharmacokinetics and toxicity of each drug.     

Improved potency and reduced toxicity of the antifungal peptoid AEC5 through submonomer modification

As proteolytically stable peptidomimetics, peptoids could serve as antifungal agents to supplement a therapeutic field wrought with toxicity issues. We report the improvement of an antifungal peptoid, AEC5, through an iterative structure-activity relationship study. A sarcosine scan was used to first identify the most pharmacophorically important peptoid building blocks of AEC5, followed by sequential optimization of each building block. The optimized antifungal peptoid from this study, β-5, has improved potency towards Cryptococcus neoformans and decreased toxicity towards mammalian cells. For example, the selectivity ratio for C. neoformans over mammalian fibroblasts was improved from 8 for AEC5 to 37 for β-5.     

Biofilm Formation by Cryptococcus neoformans Under Distinct Environmental Conditions

Cryptococcus neoformans is an opportunistic fungal pathogen with a propensity to infect the central nervous system of immune compromised individuals causing life-threatening meningoencephalitis. Cryptococcal biofilms have been described as a protective niche against microbial predators in nature and shown to enhance resistance against antifungal agents and specific mediators of host immune responses. Based on the potential importance of cryptococcal biofilms to its survival in the human host and in nature, these studies were designed to investigate those factors that mediate biofilm formation by C. neoformans. We observed that C. neoformans preferentially grew as planktonic cells when cultured under specific conditions designed to mimic growth within host tissues (37°C, neutral pH, and ~5% CO₂) or phagocytes (37°C, acidic pH, and ~5% CO₂) and as biofilms when cultured under conditions such as those encountered in the external environment (25–37°C, neutral pH, and ambient CO₂). Altogether, our studies suggest that conditions similar to those observed in its natural habitat may be conducive to biofilm formation by C. neoformans.     

Identification and characterization of Cryptococcus neoformans protein fractions that induce protective immune responses

Cryptococcus neoformans, the main causative agent of cryptococcosis, is a fungal pathogen that causes life‐threatening meningoencephalitis in immunocompromised patients. To date, there is no vaccine or immunotherapy approved to treat cryptococcosis. Cell‐ and antibody‐mediated immune responses collaborate to mediate optimal protection against C. neoformans infections. Accordingly, we identified cryptococcal protein fractions capable of stimulating cell‐ and antibody‐mediated immune responses and determined their efficacy to elicit protection against cryptococcosis. Proteins were extracted from C. neoformans and fractionated based on molecular mass. The fractions were then evaluated by immunoblot analysis for reactivity to serum extracted from protectively immunized mice and in cytokine recall assays for their efficacy to induce pro‐inflammatory and Th1‐type cytokine responses associated with protection. MS analysis revealed a number of proteins with roles in stress response, signal transduction, carbohydrate metabolism, amino acid synthesis, and protein synthesis. Immunization with select protein fractions containing immunodominant antigens induced significantly prolonged survival against experimental pulmonary cryptococcosis. Our studies support using the combination of immunological and proteomic approaches to identify proteins that elicit antigen‐specific antibody and Th1‐type cytokine responses. The immunodominant antigens that were discovered represent attractive candidates for the development of novel subunit vaccines for treatment and/or prevention of cryptococcosis.     

Toward Developing a Universal Treatment for Fungal Disease Using Radioimmunotherapy Targeting Common Fungal Antigens

Background

Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall.

Methods

MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth (²¹³Bi) using the chelator CHXA”. B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium (¹⁸⁸Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls.

Results

²¹³Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80–100%. The ²¹³Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing.

Conclusion

Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

     

Hydroxyaldimines as potent in vitro anticryptococcal agents

Cryptococcosis, a fungal infection that affects both immunocompromised and immunocompetent individuals, contributes to increasing indices of mortality and morbidity. The development of resistance by Cryptococcus spp., the limited number of commercial antifungal drugs and the various side effects of these drugs cause the treatment of cryptococcosis to be a challenge. The in vitro anticryptococcal activity of nine hydroxyaldimines was evaluated against 24 strains of Cryptococcus spp. Antifungal susceptibility was evaluated using a broth microdilution assay following the Clinical and Laboratory Standards Institute guidelines, using fluconazole as a positive control. Parameters such as the minimum inhibitory concentration and the minimum fungicidal concentration (MIC and MFC, respectively) were also determined. Antiproliferative activity on the normal cell line VERO was assessed 48 h post‐compound exposure to determine the selectivity index (SI) of the hydroxyaldimines and fluconazole. All hydroxyaldimines were active against Cryptococcus spp. strains. Compounds 3A9 and 3B7 were the most potent against the Cryptococcus gattii and Cryptococcus neoformans strains. Selectivity indices also revealed that 3B10, 3C3, 3D3 and 3D9 are good candidates for in vivo studies. The in vitro anticryptococcal activity of hydroxyaldimines against various strains of C. gattii and C. neoformans indicates the potential of this class of molecules as lead compound for the development of selective and efficient anticryptococcal agents.

Significance and Impact of the Study

The effectiveness of hydroxyaldimines for inhibition of Cryptococcus spp. growth and their low toxicity against healthy monkey kidney epithelial cells makes them promising lead compounds for the design of new anticryptococcal agents.     

Cryptococcus neoformans: in vivo protection of mice by pretreatment with pyran copolymer

Synthetic polyanions have been shown to alter host resistance to infection. The anticryptococcal effect of pyran copolymer was assessed in vivo and in vitro. Pretreatment with pyran copolymer significantly extended mean survival in mice lethally infected with Cryptococcus neoformans when compared to untreated animals (p < 0.01). The anticryptococcal effect of peritoneal exudate cells (PEC) elicited by 10% thioglycollate or pyran copolymer (25 mg/kg) was assessed in vitro. Initial percent phagocytosis of both encapsulated and non-encapsulated isolates of C. neoformans was greatest in the pyran elicited PEC. Significant killing of C. neoformans in vitro was observed only in pyran-activated PEC cultures combined with non-encapsulated cells of C. neoformans, although pyran PEC did inhibit initial growth of phagocytized encapsulated yeast cells. The protection of pyran copolymer pretreated mice from infection with C. neoformans, but the absence of significant killing of encapsulated yeast in vitro suggest a complex mechanism of host defense which may involve an activation of the reticuloendothelial system by pyran copolymer.     

Combatting the evolution of antifungal resistance in Cryptococcus neoformans

Fungal infections are a global concern and the evolution of intrinsic resistance to current antifungals presents an alarming problem. For Cryptococcus neoformans, a human fungal pathogen of primarily immunocompromised individuals, resistance toward treatment strategies demands alternative approaches. Given the prevalence of virulence factor production during cryptococcal infection, an emerging and important field of research encompasses the development of novel antivirulence therapies proposed to improve host immune responses and promote fungal clearance. To accomplish this task, information regarding the presence and role of virulence factors, the mechanisms of action within the host, and the ability to influence fungal susceptibility to antifungals is pertinent. Research into mechanisms of antifungal resistance for C. neoformans is limited but extrapolation from successful studies in other fungal species can improve our understanding of mechanisms employed by C. neoformans and suggest targeted strategies to enhance our ability to combat the pathogen. In this Review, we highlight antifungal therapy options against Cryptococcus, explore current knowledge of underlying mechanisms promoting resistance, and present new opportunities for novel and effective strategies to overcome fungal infections and reduce, or possibly even reverse, the effects of resistance evolution.     

Real‐time in vivo imaging of fungal migration to the central nervous system

Recent technical advances have afforded valuable new insights into the pathogenesis of fungal infections in the central nervous system (CNS), which continue to cause devastating complications, particularly in immunocompromised individuals. To cause CNS mycosis, organisms such as Cryptococcus neoformans become blood borne and progress through a series of pathogenic checkpoints that culminate in fungal replication in the brain. Critical steps include fungal arrest in the vasculature of the brain, interaction and signalling of the fungal and endothelial cells leading to transmigration with subsequent parenchymal invasion and fungal replication in the CNS. Previous studies that made use of in vitro and ex vivo approaches contributed greatly to our understanding of brain invasion by fungi. However, the knowledge gained from previous studies relied on in vitro models that did not account for vascular haemodynamics. For this reason, more refined approaches that model blood flow and vascular anatomy are required, andultimately studying fungal invasion and dissemination in vivo. Indeed, in vivo imaging (also known as intravital imaging) has emerged as a valuable technique to probe host–pathogen interactions. In this review, with a focus on C. neoformans, we will provide an overview of the applications of the prior techniques and recent advances, their strengths and limitations in characterizing the migration of fungi into the brain, and unanswered questions that may provide new directions for research.     

Melanization decreases the susceptibility of Cryptococcus neoformans to enzymatic degradation

Cryptococcus neoformans is a free-living fungus that is primarily found in soils contaminated with avian excreta. Recent studies have shown that C. neoformans can synthesize melanins or melanin-like compounds in avian excreta. Melanization has been associated with protection of C. neoformans against harsh environmental conditions, such as ultraviolet radiation and extremes of temperature. In this study we examined whether melanization can protect C. neoformans against enzymatic degradation. Our results demonstrated that in vitro melanization decreases the susceptibility of C. neoformans to hydrolytic enzymes. This suggests a role for melanin in protection of C. neoformans against enzymatic degradation by antagonistic microbes in the environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features

In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.     

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.     

Development of non-natural flavanones as antimicrobial agents

With growing concerns over multidrug resistance microorganisms, particularly strains of bacteria and fungi, evolving to become resistant to the antimicrobial agents used against them, the identification of new molecular targets becomes paramount for novel treatment options. Recently, the use of new treatments containing multiple active ingredients has been shown to increase the effectiveness of existing molecules for some infections, often with these added compounds enabling the transport of a toxic molecule into the infecting species. Flavonoids are among the most abundant plant secondary metabolites and have been shown to have natural abilities as microbial deterrents and anti-infection agents in plants. Combining these ideas we first sought to investigate the potency of natural flavonoids in the presence of efflux pump inhibitors to limit Escherichia coli growth. Then we used the natural flavonoid scaffold to synthesize non-natural flavanone molecules and further evaluate their antimicrobial efficacy on Escherichia coli, Bacillus subtilis and the fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus. Of those screened, we identified the synthetic molecule 4-chloro-flavanone as the most potent antimicrobial compound with a MIC value of 70 µg/mL in E. coli when combined with the inhibitor Phe-Arg-ß-naphthylamide, and MICs of 30 µg/mL in S. cerevesiae and 30 µg/mL in C. neoformans when used alone. Through this study we have demonstrated that combinatorial synthesis of non-natural flavonones can identify novel antimicrobial agents with activity against bacteria and fungi but with minimal toxicity to human cells.     

T Cell-Dependent Activation of Macrophages and Enhancement of Their Phagocytic Activity in the Lungs of Mice Inoculated with Heat-Killed Cryptococcus neoformans: Involvement of IFN-γ and Its Protective Effect against Cryptococcal Infection

Previous investigations have demonstrated that macrophages play a critical role in the first-line cellular defense mechanism against infection with Cryptococcus neoformans. In the present study, to elucidate the way in which anticryptococcal activity of macrophages is regulated at the site of infection, pulmonary intraparenchymal macrophages were directly analyzed for expression of their surface molecules and their phagocytic activities against the organism, and the effects of depletion of T cells and endogenous IFN-γ in vivo on these parameters were examined. In the lungs of mice intratracheally inoculated with heat-killed C. neoformans, macrophages were activated, as indicated by augmented expression of MHC class II, intercellular adhesion molecule-1 (ICAM-1) and Fc receptor (FcR), and about two-thirds of macrophages were found to have ingested an average of 3.77 ± 0.12 yeast cells per macrophage. In mice depleted of both CD4⁺ and CD8⁺ T cells by injecting the specific monoclonal antibodies (mAbs) or anti-IFN-γ mAb, not only augmentation of the expression of macrophage activation markers but also phagocytosis of C. neoformans was significantly reduced. These results suggest that anticryptococcal activity of macrophages is regulated by IFN-γ endogenously produced by T cells. Additionally, treatment with IFN-γ were shown to significantly prolong the survival time of mice infected with viable C. neoformans. Additionally, preimmunization with heat-killed C. neoformans significantly prolonged the survival time of mice which received the following infection.     

Synthesis and Biological Evaluation of Novel Carbazole Hybrids as Promising Antimicrobial Agents

Two series of carbazole analogs of 8‐methoxy‐N‐substituted‐9H‐carbazole‐3‐carboxamides (series 1) and carbazolyl substituted rhodanines (series 2) were synthesized through facile synthetic routes. All the final compounds from these two series were evaluated for their preliminary in vitro antifungal and antibacterial activity against four fungal (Candida albicans, Cryptococcus neoformans, Cryptococcus tropicalis and Aspergillus niger) and four bacterial (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa) strains, respectively. Among the tested compounds, three compounds of series 1 displayed promising antifungal and antibacterial activity, especially against C. neoformans and S. aureus. In addition, one compound of series 1 displayed notable antimicrobial activity (MIC: 6.25 μg/mL) against clinical isolates of C. albicans and C. neoformans (MIC: 12.5 μg/mL). From the second series, four compounds exhibited significant antifungal and antibacterial activity, especially against C. neoformans and S. aureus. The most active compound of series 2 displayed a prominent antimicrobial activity against C. neoformans (MIC: 3.125 μg/mL) and S. aureus (MIC: 1.56 μg/mL), respectively.