Kevin Alby

The Parasexual Cycle in Candida albicans Provides an Alternative Pathway to Meiosis for the Formation of Recombinant Strains

Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and α strains. The product of mating is a tetraploid a/α cell that must undergo a reductional division to return to the diploid state. Despite the presence of several “meiosis-specific” genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle.

Homothallic and heterothallic mating in the opportunistic pathogen Candida albicans

Candida albicans is the most common fungal pathogen in humans, causing both debilitating mucosal infections and potentially life-threatening systemic infections. Until recently, C. albicans was thought to be strictly asexual, existing only as an obligate diploid. A cryptic mating cycle has since been uncovered in which diploid a and α cells undergo efficient cell and nuclear fusion, resulting in tetraploid a/α mating products. Whereas mating between a and α cells has been established (heterothallism), we report here two pathways for same-sex mating (homothallism) in C. albicans. First, unisexual populations of a cells were found to undergo autocrine pheromone signalling and same-sex mating in the absence of the Bar1 protease. In both C. albicans and Saccharomyces cerevisiae, Bar1 is produced by a cells and inactivates mating pheromone α, typically secreted by α cells. C. albicans Δbar1 a cells were shown to secrete both a and α mating pheromones; α-pheromone activated self-mating in these cells in a process dependent on Ste2, the receptor for α-pheromone. In addition, pheromone production by α cells was found to promote same-sex mating between wild-type a cells. These results establish that homothallic mating can occur in C. albicans, revealing the potential for genetic exchange even within unisexual populations of the organism. Furthermore, Bar1 protease has an unexpected but pivotal role in determining whether sexual reproduction can potentially be homothallic or is exclusively heterothallic. These findings also have implications for the mode of sexual reproduction in related species that propagate unisexually, and indicate a role for specialized sexual cycles in the survival and adaptation of pathogenic fungi.

Stress-Induced Phenotypic Switching in Candida albicans

Candida albicans is both a common commensal and an opportunistic pathogen, being a prevalent cause of mucosal and systemic infections in humans. Phenotypic switching between white and opaque forms is a reversible transition that influences virulence, mating behavior, and biofilm formation. In this work, we show that a wide range of factors induces high rates of switching from white to opaque. These factors include different forms of environmental stimuli such as genotoxic and oxidative stress, as well as intrinsic factors such as mutations in DNA repair genes. We propose that these factors increase switching to the opaque phase via a common mechanism-inhibition of cell growth. To confirm this hypothesis, growth rates were artificially manipulated by varying expression of the CLB4 cyclin gene; slowing cell growth by depleting CLB4 resulted in a concomitant increase in white-opaque switching. Furthermore, two clinical isolates of C. albicans, P37005 and L26, were found to naturally exhibit both slow growth and high rates of white-opaque switching. Notably, suppression of the slow growth phenotype suppressed hyperswitching in the P37005 isolate. Based on the sensitivity of the switch to levels of the master regulator Wor1, we propose a model for how changes in cellular growth modulate white-opaque switching frequencies.

Identification of Hemagglutinin Residues Responsible for H3N2 Antigenic Drift during the 2014–2015 Influenza Season

Influenza vaccines must be updated regularly because influenza viruses continuously acquire mutations in antibody binding sites of hemagglutinin (HA). The majority of H3N2 strains circulating in the Northern Hemisphere during the 2014-2015 season are antigenically mismatched to the A/Texas/50/2012 H3N2 vaccine strain. Recent H3N2 strains possess several new HA mutations, and it is unknown which of these mutations contribute to the 2014-2015 vaccine mismatch. Here, we use reverse genetics to demonstrate that mutations in HA antigenic site B are primarily responsible for the current mismatch. Sera isolated from vaccinated humans and infected ferrets and sheep had reduced hemagglutination inhibition and in vitro neutralization titers against reverse-genetics-derived viruses possessing mutations in the HA antigenic site B. These data provide an antigenic explanation for the low influenza vaccine efficacy observed during the 2014-2015 influenza season. Furthermore, our data support the World Health Organizations decision to update the H3N2 component of future vaccine formulations.

Discovery of a phenotypic switch regulating sexual mating in the opportunistic fungal pathogen Candida tropicalis

Sexual reproduction can promote genetic diversity in eukaryotes, and yet many pathogenic fungi have been labeled as obligate asexual species. It is becoming increasingly clear, however, that cryptic sexual programs may exist in some species, and that efficient mating requires the necessary developmental switch to be triggered. In this study we investigate Candida tropicalis, an important human fungal pathogen that has been reported to be asexual. Significantly, we demonstrate that C. tropicalis uses a phenotypic switch to regulate a cryptic program of sexual mating. Thus, diploid a and α cells must undergo a developmental transition to the mating-competent form, and only then does efficient cell-cell conjugation take place resulting in the formation of stable a/α tetraploids. We show that both the phenotypic switch and sexual mating depend on the conserved transcriptional regulator Wor1, which is regulated by temperature in other fungal species. In contrast, C. tropicalis mating occurs efficiently at both 25 °C and 37 °C, suggesting that it could occur in the mammalian host and have direct consequences for the outcome of an infection. Transcriptional profiling further reveals that ≈400 genes are differentially expressed between the two phenotypic states, including the regulatory factor Wor1. Taken together, our results demonstrate that C. tropicalis has a unique sexual program, and that entry to this program is controlled via a Wor1-mediated, metastable switch. These observations have direct implications for the regulation and evolution of cryptic sexual programs in related fungal pathogens.

Comparative Evaluation of the Nanosphere Verigene RV+ Assay and the Simplexa Flu A/B & RSV Kit for Detection of Influenza and Respiratory Syncytial Viruses

ABSTRACT Using retrospective (n = 200) and prospective (n = 150) nasopharyngeal specimens, we evaluated the Nanosphere Verigene RV+ and the Focus Diagnostics Simplexa Flu A/B & RSV tests. Overall, RV+ demonstrated sensitivities and specificities of 96.6% and 100% for influenza A virus, 100% and 99.7% for influenza B virus, and 100% and 100% for respiratory syncytial virus (RSV), while the Simplexa test sensitivities and specificities were 82.8 and 99.7%, 76.2 and 100%, and 94.6 and 100%, respectively.

Interspecies pheromone signaling promotes biofilm formation and same-sex mating in Candida albicans

The opportunistic pathogen Candida albicans undergoes a parasexual mating cycle in which cells must switch from the conventional “white” form to the alternative “opaque” form to become mating competent. Pheromones secreted by opaque cells induce the formation of polarized mating projections and result in cell–cell conjugation. In contrast, white cells are unable to undergo mating, but can still respond to pheromone by expression of adhesion genes that promote biofilm formation. In this study, we have analyzed the dual ability of pheromones to activate mating by opaque cells and biofilm formation by white cells. We first show that there is considerable plasticity in interactions between the α pheromone and its receptor, Ste2, by analysis of analogs of the α pheromone. Significantly, substituted forms of α pheromone can induce a response in opaque cells and this is sufficient to drive same-sex a-a cell fusion and homothallic mating. In addition, pheromone analogs were able to induce adhesion and biofilm formation in white cells of C. albicans. Because of the observed plasticity in pheromone signaling, we subsequently tested putative pheromones from multiple Candida species and identified nonnative ligands that can induce self-mating and biofilm responses in C. albicans. Our findings demonstrate that environmental signals can initiate C. albicans parasexual reproduction and biofilm formation, and highlight the role of the pheromone-signaling apparatus in mediating these functions.

Cost Savings Realized by Implementation of Routine Microbiological Identification by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is an emerging technology for rapid identification of bacterial and fungal isolates. In comparison to conventional methods, this technology is much less labor intensive and can provide accurate and reliable results in minutes from a single isolated colony. We compared the cost of performing the bioMérieux Vitek MALDI-TOF MS with conventional microbiological methods to determine the amount saved by the laboratory by converting to the new technology. Identification costs for 21,930 isolates collected between April 1, 2013, and March 31, 2014, were directly compared for MALDI-TOF MS and conventional methodologies. These isolates were composed of commonly isolated organisms, including commonly encountered aerobic and facultative bacteria and yeast but excluding anaerobes and filamentous fungi. Mycobacterium tuberculosis complex and rapidly growing mycobacteria were also evaluated for a 5-month period during the study. Reagent costs and a total cost analysis that included technologist time in addition to reagent expenses and maintenance service agreement costs were analyzed as part of this study. The use of MALDI-TOF MS equated to a net savings of

Comparison of Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Platforms for the Identification of Gram-Negative Rods from Patients with Cystic Fibrosis

69,108.61, or 87.8%, in reagent costs annually compared to traditional methods. When total costs are calculated to include technologist time and maintenance costs, traditional identification would have cost

Sexual reproduction in the Candida clade: cryptic cycles, diverse mechanisms, and alternative functions

142,532.69, versus