David R. Soll

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

The Ins and Outs of DNA Fingerprinting the Infectious Fungi

DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies.

Cutting Edge: Candida albicans Hyphae Formation Triggers Activation of the Nlrp3 Inflammasome

The proinflammatory cytokine IL-1β plays an important role in antifungal immunity; however, the mechanisms by which fungal pathogens trigger IL-1β secretion are unclear. In this study we show that infection with Candida albicans is sensed by the Nlrp3 inflammasome, resulting in the subsequent release of IL-1β. The ability of C. albicans to switch from a unicellular yeast form into a filamentous form is essential for activation of the Nlrp3 inflammasome, as C. albicans mutants incapable of forming hyphae were defective in their ability to induce macrophage IL- 1β secretion. Nlrp3-deficient mice also demonstrated increased susceptibility to infection with C. albicans, which is consistent with a key role for Nlrp3 in innate immune responses to the pathogen C. albicans.

A characterization of pH-regulated dimorphism in Candida albicans

When cells of the dimorphic yeast Candida albicans are grown to stationary phase in defined liquid medium at 25°C, they accumulate as singlets in Gl of the cell cycle. When these pluripotent, stationary phase singlets are released into fresh medium at 37°C, they synchronously evaginate after an average period of 135 to 140 minutes and form either buds or mycelia, depending upon the pH of the medium into which they are released. This method of dimorphic regulation offers the distinct advantage of comparability and serves as a very precise method for temporal comparisons of molecular and cytological events related to the establishment of the alternate growth phenotypes. In the present report, we have carefully examined the effects of individually varying pH or temperature on the length of the pre-evagination period, the population synchrony for evagination, and the phenotype of daughter cells. Exact phenotypic transition points, optima, and upper limits are defined for both temperature and pH. In addition, a method of pH-regulated dimorphism is developed in which the original temperature shift is removed from the inductive process. Finally, a common transition phenotype is described for cells reverting from the initial mycelial to budding phenotype when either pH or temperature traverse their respective transition points. The advantages as well as limitations of pH-regulated dimorphism are discussed in detail.

TOS9 Regulates White-Opaque Switching in Candida albicans

ABSTRACT In Candida albicans, the a1-α2 complex represses white-opaque switching, as well as mating. Based upon the assumption that the a1-α2 corepressor complex binds to the gene that regulates white-opaque switching, a chromatinimmunoprecipitation-microarray analysis strategy was used to identify 52 genes that bound to the complex. One of these genes, TOS9, exhibited an expression pattern consistent with a “master switch gene.” TOS9 was only expressed in opaque cells, and its gene product, Tos9p, localized to the nucleus. Deletion of the gene blocked cells in the white phase, misexpression in the white phase caused stable mass conversion of cells to the opaque state, and misexpression blocked temperature-induced mass conversion from the opaque state to the white state. A model was developed for the regulation of spontaneous switching between the opaque state and the white state that includes stochastic changes of Tos9p levels above and below a threshold that induce changes in the chromatin state of an as-yet-unidentified switching locus. TOS9 has also been referred to as EAP2 and WOR1.

Natural Defenses against Candida Colonization Breakdown in the Oral Cavities of the Elderly

Candida colonization of the oral cavity increases in the elderly. A major predisposing condition is denture use, which also increases in the elderly. To test whether the increase in colonization is age-related in a fashion independent of denture use, we analyzed the frequency (incidence) of carriage, the intensity of carriage, the multiplicity of species, and the genetic relatedness of strains in the oral cavities of 93 test subjects separated into the three age groups: 60 to 69 yr, 70 to 79 yr, and ≥ 80 yr. Each age group was further subdivided into subjects with and without dentures, and into males and females. The results demonstrate that the frequency of carriage, the intensity of carriage, and multispecies carriage all increase as a function of age and differ according to gender, in both cases independent of denture use, suggesting that the natural suppression of yeast carriage in the oral cavity breaks down in the elderly. In addition, it is demonstrated that Candida glabrata colonizes the oral cavities of elderly individuals without dentures only after 80 yr of age, suggesting that there are age-related compromising conditions other than denture use in this most elderly age group.

A role for myosin VII in dynamic cell adhesion

BACKGROUND The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.

A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium

Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP‐binding proteins (GbpA–GbpD) are implicated in cGMP signalling. GbpA and GbpB are homologous proteins with a Zn2+‐hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP‐phosphodiesterase activity and a 10‐fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA−B− cells is associated with increased myosin II phosphorylation compared with wild‐type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras‐GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP‐binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and GbpD, or mutants lacking both guanylyl cyclases. Thus, a novel cGMP signalling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process.

Opaque cells signal white cells to form biofilms in Candida albicans

Upon homozygosis from a/α to a/a or α/α, Candida albicans must still switch from the ‘white’ to ‘opaque’ phenotype to mate. It was, therefore, surprising to discover that pheromone selectively upregulated mating‐associated genes in mating‐incompetent white cells without causing G1 arrest or shmoo formation. White cells, like opaque cells, possess pheromone receptors, although their distribution and redistribution upon pheromone treatment differ between the two cell types. In speculating about the possible role of the white cell pheromone response, it is hypothesized that in overlapping white a/a and α/α populations in nature, rare opaque cells, through the release of pheromone, signal majority white cells of opposite mating type to form a biofilm that facilitates mating. In support of this hypothesis, it is demonstrated that pheromone induces cohesiveness between white cells, minority opaque cells increase two‐fold the thickness of majority white cell biofilms, and majority white cell biofilms facilitate minority opaque cell chemotropism. These results reveal a novel form of communication between switch phenotypes, analogous to the inductive events during embryogenesis in higher eukaryotes.

THE USE OF COMPUTERS IN UNDERSTANDING HOW ANIMAL CELLS CRAWL

Amoeboid cell motility is a complex three-dimensional process which involves pseudopod expansion, cellular translocation, and, in some cases, pseudopod retraction and complex interactions between the ventral surface of the pseudopod and substratum. In order to quantify the basic behavior of amoeboid cells and the dynamics of pseudopod extension and retraction, sophisticated two-dimensional and three-dimensional computer-assisted motion analysis systems have been developed which reconstruct digitized images and compute motility and dynamics morphology parameters. These systems provide a wealth of information of how amoeboid cells crawl and they have begun to be utilized (1) to elucidate the basic rules of amoeboid movement, (2) to identify the behavioral defects of cytoskeletal mutants, and (3) to elucidate the mechanism of chemotaxis. In addition, these systems represent powerful tools for analyzing the effects of drugs on cell behavior, most notably that of white blood cells and neoplastic cells. Since computer-assisted motion analysis is a relatively young field, the technologies are still evolving and have been underutilized in most areas involving cell motility. This review, which includes a description of these technologies and examples of their application, will hopefully serve as an impetus for expanded use.