Overexpression of TUF1 restores respiratory growth and fluconazole sensitivity to a Cryptococcus neoformans vad1Δ mutant

The yeast-like fungus Cryptococcus neoformans favours respiration as a mechanism of energy production, and thus depends heavily on mitochondrial function. Previous studies of a C. neoformans vad1Δ mutant revealed reduced expression of the mitochondrial elongation factor TUF1 and defects in glycerol utilization, consistent with mitochondrial dysfunction. In this study, we found that in trans expression of TUF1 in the vad1Δ mutant suppressed the mitochondrial defects, including growth on respiration-dependent carbon sources and fluconazole resistance, associated with VAD1 deletion. Tetracycline, an inhibitor of mitochondrial translation, was found to confer resistance to fluconazole in the wild-type and vad1Δ mutant, whereas the fluconazole susceptibility of the TUF1-overexpressing strain was unaffected by tetracycline treatment. In the presence of fluconazole, the vad1Δ mutant exhibited increased activation of the global transcriptional regulator Sre1. TUF1 overexpression failed to alter cleavage of Sre1 in response to fluconazole in the vad1Δ mutant, suggesting that TUF1 repression in the vad1Δ mutant is distal to Sre1, or that it occurs through an independent pathway

Mapping Antimicrobial Stewardship in Undergraduate Medical, Dental, Pharmacy, Nursing and Veterinary Education in the United Kingdom.

OBJECTIVES: To investigate the teaching of antimicrobial stewardship (AS) in undergraduate healthcare educational degree programmes in the United Kingdom (UK). PARTICIPANTS AND METHODS: Cross-sectional survey of undergraduate programmes in human and veterinary medicine, dentistry, pharmacy and nursing in the UK. The main outcome measures included prevalence of AS teaching; stewardship principles taught; estimated hours apportioned; mode of content delivery and teaching strategies; evaluation methodologies; and frequency of multidisciplinary learning. RESULTS: 80% (112/140) of programmes responded adequately. The majority of programmes teach AS principles (88/109, 80.7%). 'Adopting necessary infection prevention and control precautions' was the most frequently taught principle (83/88, 94.3%), followed by 'timely collection of microbiological samples for microscopy, culture and sensitivity' (73/88, 82.9%) and 'minimisation of unnecessary antimicrobial prescribing' (72/88, 81.8%). The 'use of intravenous administration only to patients who are severely ill, or unable to tolerate oral treatment' was reported in ~50% of courses. Only 32/88 (36.3%) programmes included all recommended principles. DISCUSSION: Antimicrobial stewardship principles are included in most undergraduate healthcare and veterinary degree programmes in the UK. However, future professionals responsible for using antimicrobials receive disparate education. Education may be boosted by standardisation and strengthening of less frequently discussed principles

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

Inhibition of nucleotide biosynthesis potentiates the antifungal activity of amphotericin B.

The polyene antifungal agent Amphotericin B exhibits potent and broad spectrum fungicidal activity. However, high nephrotoxicity can hinder its administration in resource poor settings. Quantification of early fungicidal activity in studies of HIV patients with cryptococcosis demonstrate that 5-Fluorocytosine therapy in combination with Amphotericin B results in faster clearance than with Amphotericin B alone. In vitro synergy between the two drugs has also been reported but the mechanism by which 5-Fluorocytosine synergizes with Amphotericin B has not been delineated. In this study we set out to investigate the effect of genetic mutation or pharmacologic repression of de novo pyrimidine and purine biosynthesis pathways on the Amphotericin B susceptibility of Cryptococcus neoformans. We demonstrate that a ura- derivative of wild type Cryptococcus neoformans strain H99 is hypersensitive to Amphotericin B. This sensitivity is remediated by re-introduction of a wild type URA5 gene, but not by addition of exogenous uracil to supplement the auxotrophy. Repression of guanine biosynthesis by treatment with the inosine monophosphate dehydrogenase inhibitor, mycophenolic acid, was synergistic with Amphotericin B as determined by checkerboard analysis. As in Cryptococcus neoformans, a ura(-) derivative of Candida albicans was also hypersensitive to Amphotericin B, and treatment of Candida albicans with mycophenolic acid was likewise synergistic with Amphotericin B. In contrast, neither mycophenolic acid nor 5-FC had an effect on the Amphotericin B susceptibility of Aspergillus fumigatus. These studies suggest that pharmacological targeting of nucleotide biosynthesis pathways has potential to lower the effective dose of Amphotericin B for both C. neoformans and C. albicans. Given the requirement of nucleotide and nucleotide sugars for growth and pathogenesis of Cryptococcus neoformans, disrupting nucleotide metabolic pathways might thus be an effective mechanism for the development of novel antifungal drugs

Tools for Assessing Translation in Cryptococcus neoformans

Cryptococcus neoformans is a ubiquitous environmental fungus capable of establishing an infection in a human host. Rapid changes in environments and exposure to the host immune system results in a significant amount of cellular stress, which is effectively combated at the level of translatome reprogramming. Repression of translation following stress allows for the specific reallocation of limited resources. Understanding the mechanisms involved in regulating translation in C. neoformans during host infection is critical in the development of new antifungal drugs. In this review, we discuss the main tools available for assessing changes in translation state and translational output during cellular stress

CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in Cryptococcus neoformans

Stress adaptation is fundamental to the success of Cryptococcus neoformans as a human pathogen and requires a reprogramming of the translating pool of mRNA. This reprogramming begins with the regulated degradation of mRNAs encoding the translational machinery. The mechanism by which these mRNAs are specified has not been determined. This study has identified a cis element within a G-quadruplex structure that binds two C. neoformans homologues of cellular nucleic acid binding protein (CNBP). These proteins regulate the polysome association of the target mRNA but perform functions related to sterol homeostasis which appear independent of ribosomal protein mRNAs. The presence of two CNBP homologues in C. neoformans suggests a diversification of function of these proteins, one of which appears to regulate sterol biosynthesis and fluconazole sensitivity.In Cryptococcus neoformans, mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3′ untranslated regions (UTRs) of RP transcripts regulated by mRNA decay, and a single, significant motif, GGAUG, was identified. Znf9, a small zinc knuckle RNA binding protein identified by mass spectrometry, was found to interact specifically with the RPL2 3′-UTR probe. A second, homologous protein, Gis2, was identified in the genome of C. neoformans and also bound the 3′-UTR probe, and deletion of both genes resulted in loss of binding in cell extracts. The RPL2 3′ UTR contains four G-triplets (GGG) that have the potential to form a G-quadruplex, and temperature gradient gel electrophoresis revealed a potassium-dependent structure consistent with a G-quadruplex that was abrogated by mutation of G-triplets. However, deletion of G-triplets did not abrogate the binding of either Znf9 or Gis2, suggesting that these proteins either bind irrespective of structure or act to prevent structure formation. Deletion of both GIS2 and ZNF9 resulted in a modest increase in basal stability of the RPL2 mRNA which resulted in an association with higher-molecular-weight polysomes under unstressed conditions. The gis2Δ mutant and gis2Δ znf9Δ double mutant exhibited sensitivity to cobalt chloride, fluconazole, and oxidative stress, and although transcriptional induction of ERG25 was similar to that of the wild type, analysis of sterol content revealed repressed levels of sterols in the gis2Δ and gis2Δ znf9Δ double mutant, suggesting a role in translational regulation of sterol biosynthesis

Perturbations in nucleotide biosynthesis sensitize <i>Candida albicans</i> to AmB.

<p>E-Test analysis for AmB susceptibility of (A) <i>C. albicans</i> wt and <i>ura</i>⁻ mutant on AM3 medium (B) <i>C. albicans</i> wt on AM3 medium alone or supplemented with 3.25 µg/mL MPA (C) <i>C. albicans</i> wt and <i>ura</i>⁻ mutant supplemented with 20 µM uracil or 20 µM uridine.</p

A <i>C. neoformans ura</i>⁻ strain is hypersensitive to AmB.

<p>(A) E-Test for AmB susceptibility of <i>C. neoformans</i> wt, <i>ura</i>⁻ mutant and <i>ura</i>⁻ strain complemented with the wild type <i>URA5</i> gene on AM3 medium (B) E-Test for AmB susceptibility of <i>C. neoformans</i> wt and <i>ura</i>⁻ mutant on AM3 medium supplemented with 20 µM uracil and 20µM uridine.</p