Sensitization of Candida albicans biofilms to various antifungal drugs by cyclosporine A

Background

Biofilms formed by Candida albicans are resistant towards most of the available antifungal drugs. Therefore, infections associated with Candida biofilms are considered as a threat to immunocompromised patients. Combinatorial drug therapy may be a good strategy to combat C. albicans biofilms.

Methods

Combinations of five antifungal drugs- fluconazole (FLC), voriconazole (VOR), caspofungin (CSP), amphotericin B (AmB) and nystatin (NYT) with cyclosporine A (CSA) were tested in vitro against planktonic and biofilm growth of C. albicans. Standard broth micro dilution method was used to study planktonic growth, while biofilms were studied in an in vitro biofilm model. A chequerboard format was used to determine fractional inhibitory concentration indices (FICI) of combination effects. Biofilm growth was analyzed using XTT-metabolic assay.

Results

MICs of various antifungal drugs for planktonic growth of C. albicans were lowered in combination with CSA by 2 to 16 fold. Activity against biofilm development with FIC indices of 0.26, 0.28, 0.31 and 0.25 indicated synergistic interactions between FLC-CSA, VOR-CSA, CSP-CSA and AmB-CSA, respectively. Increase in efficacy of the drugs FLC, VOR and CSP against mature biofilms after addition of 62.5 μg/ml of CSA was evident with FIC indices 0.06, 0.14 and 0.37, respectively.

Conclusions

The combinations with CSA resulted in increased susceptibility of biofilms to antifungal drugs. Combination of antifungal drugs with CSA would be an effective prophylactic and therapeutic strategy against biofilm associated C. albicans infections.     

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L

Background

Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies.

Methods

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide.

Results

Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 μg/ml for yeasts, 125 to 500 μg/ml for Aspergillus species, and 7.81 to 62.5 μg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 × MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 × MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 × to 8 × MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol.

Conclusions

The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.     

Characterization of the mRNA capping apparatus of Candida albicans

The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520. Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase. Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth. An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase. Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro. Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II. Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.     

Genetic, Physical, and Functional Interactions between the Triphosphatase and Guanylyltransferase Components of the Yeast mRNA Capping Apparatus

We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme). CES5 is identical to CET1, which encodes the RNA triphosphatase component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes hydrolysis of the γ phosphate of triphosphate-terminated RNA at a rate of 1 s⁻¹. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the result of increased affinity for the GTP substrate. A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary. The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth. This work highlights the potential for regulating mRNA cap formation through protein-protein interactions.     

Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus

RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel. Interactions of a sulfate in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data. Discrete surface domains mediate Cet1p homodimerization and Cet1p binding to the guanylyltransferase component of the capping apparatus. The structure and mechanism of fungal RNA triphosphatases are completely different from those of mammalian mRNA capping enzymes. Hence, RNA triphosphatase presents an ideal target for structure-based antifungal drug discovery.     

Trypanosoma brucei RNA triphosphatase. Antiprotozoal drug target and guide to eukaryotic phylogeny.

The mRNA capping apparatus of the protozoan parasite Trypanosoma brucei consists of separately encoded RNA triphosphatase and RNA guanylyltransferase enzymes. The triphosphatase TbCet1 is a member of a new family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and the malaria parasite Plasmodium falciparum. The protozoal/fungal enzymes are structurally and mechanistically unrelated to the RNA triphosphatases of metazoans and plants. These results highlight the potential for discovery of broad spectrum antiprotozoal and antifungal drugs that selectively block the capping of pathogen-encoded mRNAs. We propose a scheme of eukaryotic phylogeny based on the structure of RNA triphosphatase and its physical linkage to the guanylyltransferase component of the capping apparatus.     

Fungal Endocarditis Observed Over an 8-Year Period and a Review of the Literature

Background

Fungal endocarditis (FE) is a “modern” disease that is considered an emerging cause of infective endocarditis (IE). The most frequently identified fungal pathogens are Candida spp., which are responsible for up to two-thirds of all cases; the remaining cases are due to Aspergillus spp., Histoplasma capsulatum or, more rarely, other yeasts and moulds.

Objectives

To describe the prevalence, clinical characteristics and outcome of FE diagnosed in a single tertiary centre and review the literature concerning FE.

Design and setting

An 8-year retrospective review of the case records of patients attending a single Italian University Centre and diagnosed as having definite or probable IE as defined by the modified Duke criteria.

Results

Six patients were identified from 229 episodes of IE: five cases involved a prosthetic valve, and one a native valve of an intravenous drug user. Five cases were caused by Candida spp. (two by C. albicans, one each by C. lusitaniae, C. dubliniensis and C. glabrata) and one by Aspergillus flavus. Three patients were treated by means of surgery plus antifungal therapy; two received antifungal therapy alone. Three patients survived, but only the patient with Aspergillus endocarditis was followed up for a long time.

Conclusions

FE is difficult to diagnose but generally associated with healthcare infections. The optimal treatment is poorly characterised, and international collaborative studies are urgently needed to evaluate newer antifungal agents.     

Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

Background

Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. Natural phenolic compounds can serve as potent redox cyclers that inhibit microbial growth through destabilization of cellular redox homeostasis and/or antioxidation systems. The aim of this study was to identify benzaldehydes that disrupt the fungal antioxidation system. These compounds could then function as chemosensitizing agents in concert with conventional drugs or fungicides to improve antifungal efficacy.

Methods

Benzaldehydes were tested as natural antifungal agents against strains of Aspergillus fumigatus, A. flavus, A. terreus and Penicillium expansum, fungi that are causative agents of human invasive aspergillosis and/or are mycotoxigenic. The yeast Saccharomyces cerevisiae was also used as a model system for identifying gene targets of benzaldehydes. The efficacy of screened compounds as effective chemosensitizers or as antifungal agents in formulations was tested with methods outlined by the Clinical Laboratory Standards Institute (CLSI).

Results

Several benzaldehydes are identified having potent antifungal activity. Structure-activity analysis reveals that antifungal activity increases by the presence of an ortho-hydroxyl group in the aromatic ring. Use of deletion mutants in the oxidative stress-response pathway of S. cerevisiae (sod1Δ, sod2Δ, glr1Δ) and two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus (sakAΔ, mpkCΔ), indicates antifungal activity of the benzaldehydes is through disruption of cellular antioxidation. Certain benzaldehydes, in combination with phenylpyrroles, overcome tolerance of A. fumigatus MAPK mutants to this agent and/or increase sensitivity of fungal pathogens to mitochondrial respiration inhibitory agents. Synergistic chemosensitization greatly lowers minimum inhibitory (MIC) or fungicidal (MFC) concentrations. Effective inhibition of fungal growth can also be achieved using combinations of these benzaldehydes.

Conclusions

Natural benzaldehydes targeting cellular antioxidation components of fungi, such as superoxide dismutases, glutathione reductase, etc., effectively inhibit fungal growth. They possess antifungal or chemosensitizing capacity to enhance efficacy of conventional antifungal agents. Chemosensitization can reduce costs, abate resistance, and alleviate negative side effects associated with current antifungal treatments.     

Deletion of individual mRNA capping genes is unexpectedly not lethal to Candida albicans and results in modified mRNA cap structures

Eukaryotic mRNAs are modified at the 5′ end with a cap structure. In fungal cells, the formation of the mRNA cap structure is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and methyltransferase. Fungal capping enzymes have been proposed to be good antifungal targets because they differ significantly from their human counterparts and the genes encoding these enzymes are essential in Saccharomyces cerevisiae. In the present study, Candida albicans null mutants were constructed for both the mRNA triphosphatase-encoding gene (CET1) and the mRNA methyltransferase encoding gene (CCM1), proving that these genes are not essential in C. albicans. Heterozygous deletions were generated, but no null mutants were isolated for the guanylyltransferase-encoding gene (CGT1), indicating that this gene probably is essential in C. albicans. Whereas these results indicate that Cet1p and Ccm1p are not ideal molecular targets for development of anticandidal drugs, they do raise questions about the capping of mRNA and translation initiation in this fungus. Southern blot analysis of genomic DNA indicates that there are not redundant genes for CET1 and CCM1 and analysis of mRNA cap structures indicate there are not alternative pathways compensating for the function of CET1 or CCM1 in the null mutants. Instead, it appears that C. albicans can survive with modified mRNA cap structures.     

Emergence of Resistance to Fluconazole in <Emphasis Type="Italic">Candida albicans</Emphasis> Isolated From Vaginal Discharge

Purpose of Review

To provide information about the emergence of fluconazole resistance in Candida albicans isolated from vaginal discharge, in a global context, and to update the in vitro susceptibility profile of this species from Argentina.

Recent Findings

Vulvovaginal candidiasis is the second most common vaginal infection after vaginal bacteriosis. C. albicans remains the prevalent etiological yeast species, and despite antifungal treatment, the rate of recurrence remains high, which may be associated to antifungal resistance.

Summary

Data here presented were obtained from the study of C. albicans strains isolated from patients with clinical signs of vulvovaginal candidiasis from 1996 to 2017. Data obtained could represent the susceptibility profile of C. albicans strains circulating in Argentina and could be of potential usefulness to monitor and guide therapy, and also suggests the need for greater surveillance programs to detect fluconazole resistance over time.

     

Isothiocyanates inhibit fungal pathogens of potato in in vitro assays

Aims

This study aimed to examine the effects of seven different isothiocyanates against the growth and development of three important soil borne potato pathogens, (Colletotrichum coccodes, Rhizoctonia solani and Helminthosporium solani).

Methods

The study was carried out using an agar diffusion assay. The radial growth of fungal pathogens grown on agar containing different ITCs at a range of concentrations was compared to that of growth on control agar plates that did not contain ITCs.

Results

Results varied depending on the specific isothiocyanate incorporated into the agar. They ranged from those which showed a significant effect on fungal growth to those which appeared to have little or no effect. Where a suppressive effect was observed, due to the presence of the isothiocyanate, fungal colony growth decreased as the concentration of the incorporated isothiocyanate increased.

Conclusions

Results from this study indicate that fungal growth can be inhibited by exposure to ITCs. However the results observed are specific to the ITC structure and exposure concentration.     

Anti-Candida albicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans

Background

Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis.

Nimesulide inhibits pathogenic fungi: PGE2-dependent mechanisms

Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 μg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 μg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.     

Candidemia in Adults at a Tertiary Hospital in China: Clinical Characteristics,Species Distribution,Resistance, and Outcomes

Background

Candidemia is one of the most common nosocomial bloodstream infections. Early diagnosis and antifungal treatment improve clinical outcomes in some studies but not all, with diverse data reported from different institutions. Similarly, antifungal resistance is more common in the USA than in Europe, but there is little data regarding the microbiology and clinical course of candidemia in adult patients in Asia.

Aims

(1) To capture species distribution and drug resistance rates among Candida bloodstream isolates, (2) to describe clinical features of candidemia, and (3) to identify factors associated with all-cause mortality, with emphasis on early initiation of antifungal treatment, at a large tertiary University Hospital in China.

Methods

In this single-center retrospective study, we identified all patients with candidemia, between 2008 and 2014. Demographic and clinical characteristics, microbiological information, details of antifungal therapy and clinical outcomes were collected.

Results

We studied 166 patients. 71 (42.8%) had cancer. Candida albicans was the most frequent species (37.3%), followed by C. parapsilosis (24.1%), C. tropicalis (22.8%), and C. glabrata (14.5%). Antifungal resistance was more frequent in non-albicans strains and especially C. glabrata. Twenty patients received inappropriate treatment with all-cause mortality of 35%. The remaining 146 patients had significantly lower mortality (21.9%, P?=?0.045). Among patients who received antifungal treatment, mortality rate increased with time to appropriate antifungal therapy (AAT): 13.7%, for?<?24 h, 21.1% for 24–48 h, 23.1% for?>?48 h, and 32.4% among patients who received no AT (χ² for trend P?=?0.039). Initiating AAT more than 24 h after blood culture collection was an independent risk factor for mortality, after adjustment for other confounders (OR 7.1, 95% CI 1.3–39.4, P?=?0.024).

Conclusions

Candida albicans was the most frequent cause of candidemia at a large tertiary hospital in China, but antifungal resistance is a growing concern among non-albicans Candida species. The mortality rate of patients treated with ineffective antifungal agents based on in vitro susceptibilities was similar to that of patients who received no treatment at all, and delayed initiation of antifungal treatment was associated with increased risk of death.

     

A Case for Antifungal Stewardship

Purpose of Review

The expanding utilization of limited available antifungal agents has led to a pressing need to implement interventions to ensure appropriate usage. The global emergence of resistant, difficult-to-treat invasive fungal infections among the most vulnerable patient populations is a call to action to develop a multifaceted antifungal stewardship approach.

Recent Findings

Candida species demonstrating multi-drug resistance, including highly resistant Candida auris, are emerging threats. Azole-resistant Aspergillus fumigatus, likely initially originating in the environment, likewise presents a treatment challenge. Routine empiric and prophylactic antifungal use, though effective, further complicates this issue, with the emergence of breakthrough mold infections. Early evidence supports success with antifungal stewardship programs.

Summary

Broad antifungal stewardship approaches that optimize antifungal drug usage, facilitate provider education, and monitor fungal epidemiology are crucial steps to preserve the antifungal armamentarium. Future development of novel diagnostic and treatment strategies will further facilitate management of invasive fungal infections.