Genome-wide transcriptional profiling of the cyclic AMP-dependent signaling pathway during morphogenic transitions of Candida albicans

Candida albicans is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment, C. albicans transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of C. albicans by studying genes encoding adenylate cyclase-associated protein (CAP1) and high-affinity phosphodiesterase (PDE2) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. Microbiol. 50:391-409, 2003; and Y. S. Bahn and P. Sundstrom, J. Bacteriol. 183:3211-3223, 2001). However, little is known about the downstream targets of the cAMP signaling pathway that are responsible for morphological transitions and the expression of virulence factors. Here, microarrays were probed with RNA from strains with hypoactive (cap1/cap1 null mutant), hyperactive (pde2/pde2 null mutant), and wild-type cAMP signaling pathways to provide insight into the molecular mechanisms of virulence that are regulated by cAMP and that are related to the morphogenesis of C. albicans. Genes controlling metabolic specialization, cell wall structure, ergosterol/lipid biosynthesis, and stress responses were modulated by cAMP during hypha formation. Phenotypic traits predicted to be regulated by cAMP from the profiling results correlated with the relative strengths of the mutants when tested for resistance to azoles and subjected to heat shock stress and oxidative/nitrosative stress. The results from this study provide important insights into the role of the cAMP signaling pathway not only in morphogenic transitions of C. albicans but also for adaptation to stress and for survival during host infections.     

Effect of cyclic AMP on RNA and protein synthesis in Candida albicans.

Sequence analysis by the automated Edman degradation shows that dopamine $\text{β}$-hydroxylase (dopamine $\text{β}$-monooxygenase; EC 1.14.17.1) from bovine adrenal medulla contains equal amounts of NH₂-terminal alanine and serine residues. The sequence data are in agreement with the proposal that this enzyme consists of two types of polypeptide chains which are identical in the NH₂-terminal ends, except that one of the chains lack the NH₂-terminal tripeptide Ser-Ala-Thr.     

Defining pheromone-receptor signaling in Candida albicans and related asexual Candida species

Candida albicans is an important human fungal pathogen in which sexual reproduction is under the control of the novel white-opaque switch. Opaque cells are the mating-competent form, whereas white cells do not mate but can still respond to pheromones, resulting in biofilm formation. In this study, we first define the domains of the α-pheromone receptor Ste2 that are necessary for signaling in both white and opaque forms. Both cell states require the IC loop 3 (IC3) and the C-terminal tail of Ste2 for the cellular response, whereas the first IC loop (IC1) of Ste2 is dispensable for signaling. To also address pheromone-receptor interactions in related species, including apparently asexual Candida species, Ste2 orthologues were heterologously expressed in Candida albicans. Ste2 receptors from multiple Candida clade species were functional when expressed in C. albicans, whereas the Ste2 receptor of Candida lusitaniae was nonfunctional. Significantly, however, expression of a chimeric C. lusitaniae Ste2 receptor containing the C-terminal tail of Ste2 from C. albicans generated a productive response to C. lusitaniae pheromone. This system has allowed us to characterize pheromones from multiple Candida species and indicates that functional pheromone-receptor couples exist in fungal species that have yet to be shown to undergo sexual mating.     

白念珠菌MAPK信号通路研究进展

白念珠菌是人类最常见的条件致病菌。促分裂素原活化蛋白激酶（MAPK链）是真核生物信号传递网络中的重要途径之一,在基因表达调控和细胞质功能活动中发挥关键作用。在白念珠菌中主要有4条MAPK途径：Mkcl途径、Cekl途径、Cek2途径和HOG途径。其中HOG途径在白念珠菌MAPK信号通路起着重要的作用。对于白念珠菌MAPK信号通路的作用及相关调控机制的了解,可以为寻找新的药物作用靶点,治疗念珠菌病提供帮助。     

Role of actin cytoskeletal dynamics in activation of the cyclic AMP pathway and HWP1 gene expression in Candida albicans

Changes in gene expression during reversible bud-hypha transitions of the opportunistic fungal pathogen Candida albicans permit adaptation to environmental conditions that are critical for proliferation in host tissues. Our previous work has shown that the hypha-specific adhesin gene HWP1 is up-regulated by the cyclic AMP (cAMP) signaling pathway. However, little is known about the potential influences of determinants of cell morphology on HWP1 gene expression. We found that blocking hypha formation with cytochalasin A, which destabilizes actin filaments, and with latrunculin A, which sequesters actin monomers, led to a loss of HWP1 gene expression. In contrast, high levels of HWP1 gene expression were observed when the F-actin stabilizer jasplakinolide was used to block hypha formation, suggesting that HWP1 expression could be regulated by actin structures. Mutants defective in formin-mediated nucleation of F-actin were reduced in HWP1 gene expression, providing genetic support for the importance of actin structures. Kinetic experiments with wild-type and actin-deficient cells revealed two distinct phases of HWP1 gene expression, with a slow, actin-independent phase preceding a fast, actin-dependent phase. Low levels of HWP1 gene expression that appeared to be independent of stabilized actin and cAMP signaling were detected using indirect immunofluorescence. A connection between actin structures and the cAMP signaling pathway was shown using hyper- and hypomorphic cAMP mutants, providing a possible mechanism for up-regulation of HWP1 gene expression by stabilized actin. The results reveal a new role for F-actin as a regulatory agent of hypha-specific gene expression at the bud-hypha transition.     

Interrogating cyclic AMP signaling using optical approaches

Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.     

A cyclic AMP-independent protein kinase from Candida albicans.

A cyclic AMP-independent protein kinase which phosphorylates casein was purified to homogeneity from Candida albicans by affinity and ion-exchange chromatography. This protein kinase exhibits maximal activity with casein as substrate and is not stimulated by cyclic AMP or cyclic GMP. The Mr of the purified enzyme is 115,000, as determined by h.p.l.c. It migrates as a single band on gel electrophoresis and has three non-identical subunits, of Mr 44,000, 28,500 and 26,000, as determined by SDS/polyacrylamide-gel electrophoresis. This enzyme is insensitive to heparin, but is inhibited by polyamines. Furthermore, it is sensitive to thermal denaturation and to thiol reagents.     

Quantitative proteome and acidic subproteome profiling of Candida albicans yeast-to-hypha transition

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.     

ADP-ribosyl cyclase couples to cyclic AMP signaling in the cardiomyocytes

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR) from beta-NAD(+). In this study, we examined the molecular basis of which beta-adrenergic receptor (betaAR) stimulation induces cADPR formation and characterized cardiac ADPR-cyclase. The results revealed that isoproterenol-mediated increase of [Ca(2+)](i) in rat cardiomyocytes was blocked by pretreatment with a cADPR antagonistic derivative 8-Br-cADPR, a PKA inhibitor H89 or high concentration of ryanodine. Moreover, incubation of ventricular lysates with isoproterenol, forskolin or cAMP resulted in activation of ADPR-cyclase that was inhibited by pretreatment with H89. Supporting the observations, the cADPR antagonist and H89 blocked 8-CPT-cAMP, a cell-permeant cAMP analog-induced increase in [Ca(2+)](i) but not cGMP-mediated increase. Characterization of partially purified cardiac ADPR-cyclase showed a molecular mass of approximately 42 kDa and no cross-activity with CD38 antibodies, and the enzyme activity was inhibited by Zn(2+) but not dithiothreitol. Microinjection of the enzyme into rat cardiomyocytes increased the level of [Ca(2+)](i) in a concentration-dependent manner. The enzyme-mediated increase of [Ca(2+)](i) was blocked by the cADPR antagonist. These findings suggest that betaAR-mediated regulation of [Ca(2+)](i) in rat cardiomyocytes is primed by activation of cardiac ADPR-cyclase via cAMP/PKA signaling and that cardiac ADPR-cyclase differs from CD38 in biochemical and immunological properties.     

Changes in cyclic nucleotide levels and dimorphic transition in Candida albicans.

The relationship between the levels of cyclic nucleotides and dimorphic transition in Candida albicans was examined. The results showed that cells of this pathogenic fungus contained both cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP), the concentration of the latter being about one-tenth that of the former in stationary-phase cells of the yeast form. Our results further indicated that germ tube formation induced by incubation at 40 degrees C followed a rise in cAMP concentration in the cell with no accompanying change in cGMP content. Cysteine, which suppressed germination, also reversed the increase in intracellular cAMP concentration. Dibutyryl cAMP (1 MM) significantly promoted germination in proline medium at temperatures of 32 to 34 degrees C. These results suggested that cAMP was one of the controlling factors in the morphological transition in Candida albicans.