Tools for live-cell imaging of cytoskeletal and nuclear behavior in the unconventional yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous fungus with a wide variety of morphologies and growth modes including "typical" single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in a single cell cycle. The study of A. pullulans promises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for A. pullulans, which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semiopen manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.

Calprotectin-Mediated Zinc Chelation Inhibits Pseudomonas aeruginosa Protease Activity in Cystic Fibrosis Sputum.

Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts, including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to those grown in control cultures but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum, indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here, we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to posttranslationally inhibit zinc metalloprotease activity and thereby affect the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. IMPORTANCE The factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases, suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens.

BACKGROUND: The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. RESULTS: Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. CONCLUSION: We conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

Pseudomonas aeruginosa Ethanol Oxidation by AdhA in Low-Oxygen Environments.

Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosaIMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

Evolution of drug resistance in an antifungal-naive chronic Candida lusitaniae infection.

Management of the limited number of antimicrobials currently available requires the identification of infections that contain drug-resistant isolates and the discovery of factors that promote the evolution of drug resistance. Here, we report a single fungal infection in which we have identified numerous subpopulations that differ in their alleles of a single gene that impacts drug resistance. The diversity at this locus was markedly greater than the reported heterogeneity of alleles conferring antibiotic resistance in bacterial infections. Analysis of genomes from hundreds of Clavispora (Candida) lusitaniae isolates, through individual and pooled isolate sequencing, from a single individual with cystic fibrosis revealed at least 25 nonsynonymous mutations in MRR1, which encodes a transcription factor capable of inducing fluconazole (FLZ) resistance in Candida species. Isolates with high-activity Mrr1 variants were resistant to FLZ due to elevated expression of the MDR1-encoded efflux pump. We found that high Mrr1-regulated Mdr1 activity protected against host and bacterial factors, suggesting drug resistance can be selected for indirectly and perhaps explaining the Mrr1 heterogeneity in this individual who had no prior azole exposure. Regional analysis of C. lusitaniae populations from the upper and lower lobes of the right lung suggested intermingling of subpopulations throughout. Our retrospective characterization of sputum and lung populations by pooled sequencing found that alleles that confer FLZ resistance were a minority in each pool, possibly explaining why they were undetected before unsuccessful FLZ therapy. New susceptibility testing regimes may detect problematical drug-resistant subpopulations in heterogeneous single-species infections.

Profiling of Bacterial and Fungal Microbial Communities in Cystic Fibrosis Sputum Using RNA.

Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect 42 bacterial and fungal genera or species common in cystic fibrosis (CF) sputum and demonstrated the taxon specificity of these probes, as well as a linear response over more than 3 logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString, and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. Compared at the genus level, the relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and with further development, may provide quick and easily interpreted diagnostic information on diverse bacteria and fungi at the genus or species level.IMPORTANCE Here we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.

Use of RNA-Protein Complexes for Genome Editing in Non-albicans Candida Species.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome modification systems have greatly facilitated the genetic analysis of fungal pathogens. In CRISPR-Cas9 genome editing methods designed for use in Candida albicans, DNAs that encode the necessary components are expressed in the target cells. Unfortunately, expression constructs that work efficiently in C. albicans are not necessarily expressed well in other pathogenic species within the genus Candida or the related genus Clavispora. To circumvent the need for species-specific expression constructs, we implemented an expression-free CRISPR genome editing system and demonstrated its successful use in three different non-albicans Candida species: Candida (Clavispora) lusitaniae, Candida glabrata, and Candida auris. In CRISPR-Cas9-mediated genome editing methods, a targeted double-stranded DNA break can be repaired by homologous recombination to a template designed by the investigator. In this protocol, the DNA cleavage is induced upon transformation of purified Cas9 protein in complex with gene-specific and scaffold RNAs, referred to as RNA-protein complexes (RNPs). In all three species, the use of RNPs increased both the number of transformants and the percentage of transformants in which the target gene was successfully replaced with a selectable marker. We constructed mutants defective in known or putative catalase genes in C. lusitaniae, C. glabrata, and C. auris and demonstrated that, in all three species, mutants were more susceptible to hydrogen peroxide than the parental strain. This method, which circumvents the need for expression of CRISPR-Cas9 components, may be broadly useful in the study of diverse Candida species and emergent pathogens for which there are limited genetic tools. IMPORTANCE Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems.