SummaryThe human pathogen Candida albicans grows and colonizes sites that can vary markedly in pH. The pH response in C. albicans is governed in part by the Rim101p pathway. In Saccharomyces cerevisiae , Rim101p promotes alkaline responses by repressing expression of NRG1 , itself a transcriptional repressor. Our studies reveal that in C. albicans , Rim101p-mediated alkaline adaptation is not through repression of CaNRG1 . Furthermore, our studies suggest that Rim101p and Nrg1p act in parallel pathways to regulate hyphal morphogenesis, an important contributor to virulence. To determine the wild-type C. albicans transcriptional response to acidic and alkaline pH, we utilized microarrays and identified 514 pH-responsive genes. Of these, several genes involved in iron acquisition were upregulated at pH 8, suggesting that alkaline pH induces iron starvation. Microarray analysis of rim101-/-cells indicated that Rim101p does not govern transcriptional responses at acidic pH, but does regulate a subset of transcriptional responses at alkaline pH, including the iron acquisition genes. We found that rim101-/-cells are sensitive to iron starvation, which suggests that one important aspect of the Rim101p-dependent alkaline pH response is to adapt to iron starvation conditions.

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the "ear" domain of the clathrin adaptor AP-1 ␥ subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2⌬), the major Gga protein, accentuates growth and ␣-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2⌬ or a deletion of the AP-1 ␤ subunit gene (apl2⌬) alone are phenotypically normal, but cells carrying both gga2⌬ and apl2⌬ are defective in growth, ␣-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.

The ability of Candida albicans to switch cellular morphologies is crucial for its ability to cause infection. Because the cell cycle machinery participates in Saccharomyces cerevisiae filamentous growth, we characterized in detail the two C. albicans B-type cyclins, CLB2 and CLB4, to better understand the molecular mechanisms that underlie the C. albicans morphogenic switch. Both Clb2p and Clb4p levels are cell cycle regulated, peaking at G2/M and declining before mitotic exit. On hyphal induction, the accumulation of the G1 cyclin Cln1p was prolonged, whereas the accumulation of both Clb proteins was delayed when compared with yeast form cells, indicating that CLB2 and CLB4 are differentially regulated in the two morphologies and that the dynamics of cyclin appearance differs between yeast and hyphal forms of growth. Clb2p-depleted cells were inviable and arrested with hyper-elongated projections containing two nuclei, suggesting that Clb2p is not required for entry into mitosis. Unlike Clb2p-depleted cells, Clb4p-depleted cells were viable and formed constitutive pseudohyphae. Clb proteins lacking destruction box domains blocked cell cycle progression resulting in the formation of long projections, indicating that both Clb2p and Clb4p must be degraded before mitotic exit. In addition, overexpression of either B-type cyclin reduced the extent of filamentous growth. Taken together, these data indicate that Clb2p and Clb4p regulate C. albicans morphogenesis by negatively regulating polarized growth.

Candida albicans is an important pathogen of immunocompromised patients which grows with true hyphal, pseudohyphal, and yeast morphologies. The dynamics of cell cycle progression are markedly different in true hyphal relative to pseudohyphal and yeast cells, including nuclear movement and septin ring positioning. In Saccharomyces cerevisiae, two forkhead transcription factors (ScFKH1 and ScFKH2) regulate the expression of B-cyclin genes. In both S. cerevisiae and Schizosaccharomyces pombe, forkhead transcription factors also influence morphogenesis. To explore the molecular mechanisms that connect C. albicans morphogenesis with cell cycle progression, we analyzed CaFKH2, the single homolog of S. cerevisiae FKH1/FKH2. C. albicans cells lacking CaFkh2p formed constitutive pseudohyphae under all yeast and hyphal growth conditions tested. Under hyphal growth conditions levels of hyphae-specific mRNAs were reduced, and under yeast growth conditions levels of several genes encoding proteins likely to be important for cell wall separation were reduced. Together these results imply that Fkh2p is required for the morphogenesis of true hyphal as well as yeast cells. Efg1p and Cph1p, two transcription factors that contribute to C. albicans hyphal growth, were not required for the pseudohyphal morphology of fkh2 mutants, implying that Fkh2p acts in pathways downstream of and/or parallel to Efg1p and Cph1p. In addition, cells lacking Fkh2p were unable to damage human epithelial or endothelial cells in vitro, suggesting that Fkh2p contributes to C. albicans virulence.Candida albicans, an important human pathogen, is multimorphic and is able to grow in yeast, pseudohyphal, and true hyphal forms. Yeast are round-to-ovoid cells that usually undergo cell separation following cytokinesis; pseudohyphae form chains of ellipsoid daughter cells that remain attached for several divisions. Both yeast and pseudohyphae septate at a constriction between the mother and daughter cells. In contrast, true hyphal cells form long narrow filaments with parallel sides that do not separate from one another and that exhibit little, if any, constriction at the septa (41). Furthermore, in hyphal cells the first site of septation is located distal to the mother-daughter junction (53, 56). The transition between yeast, pseudohyphal, and true hyphal growth is influenced by environmental factors, such as temperature, pH, carbon source, nitrogen source, the presence of a quorum sensor, and physical contact with surfaces, and is thought to be an important contributor to virulence (reviewed in references 14, 36, and 40). Therefore, understanding the molecular mechanisms that contribute to these morphogenetic transitions is vital to understanding C. albicans pathogenicity.At least eight different transcription factors (Cph1p, Efg1p, Rbf1p, Tup1p, Czf1p, Nrg1p, Cph2p, and Rim101p) contribute to hyphal morphogenesis under different environmental conditions (reviewed in references 15 and 30) by affecting the levels of expression of specific subsets of downstream...

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1⌬) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1⌬ cells also harboring end3⌬ to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1⌬. Further comparison of ric1⌬ and ypt6⌬ cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v-and t-SNAREs, partially suppressed phenotypes of ric1⌬ and ypt6⌬ cells. SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p-and Ypt6p-dependent retrieval to the cis-Golgi network. INTRODUCTIONLocalization of proteins to appropriate membrane organelles is crucial for the functional compartmentalization of eukaryotic cells. For proteins that function in organelles of the secretory and endocytic pathways, localization requires not only targeting to the proper destination but mechanisms to maintain residence despite extensive membrane and protein flux through each organelle. Continued residence can be achieved through retention mechanisms that restrict incorporation into transport vesicles departing from an organelle and/or retrieval mechanisms that carry out vesicle-mediated return from distal sites in the pathway (Pelham and Munro, 1993;Rothman and Wieland, 1996).The Golgi apparatus in the yeast Saccharomyces cerevisiae, like its mammalian counterpart, is organized into dynamic, functionally distinct subcompartments that pose additional challenges for protein localization. Though not arranged into the cisternal stacks characteristic of the mammalian cell Golgi apparatus, yeast Golgi subcompartments can be considered functionally analogous to the mammalian cis-Golgi network (CGN), medial Golgi, and trans-Golgi (TGN) network (Graham and Emr, 1991;Preuss et al., 1992). The CGN serves as the site where endoplasmic reticulum-derived transport carriers dock and fuse and where mannose residues are first added to the core oligosaccharides of glycoproteins (Gaynor et al., 1994;Graham and Emr, 1991). Accordingly, this compartment is enriched for the t-SNARE Sed5p involved in the fusion of ER transport carriers and in the ␣-1,6 mannosyltransferase Och1p (Gaynor et al., 1994;Hardwick and Pelham, 1992). The medial Golgi compartment carries ...

Science 279:1355-1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, an S. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required for INT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes to INT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute to INT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2⌬ mutants for growth at 37°C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis of INT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene, SLA2, that is required for hyphal growth in C. albicans.

Purpose The reoperation rate for breast-conserving surgery is as high as 15–30% due to residual tumor in the surgical cavity after surgery. In vivo tumor-targeted optical molecular imaging may serve as a red-flag technique to improve intraoperative surgical margin assessment and to reduce reoperation rates. Cysteine cathepsins are overexpressed in most solid tumor types, including breast cancer. We developed a cathepsin-targeted, quenched fluorescent activity-based probe, VGT-309, and evaluated whether it could be used for tumor detection and image-guided surgery in syngeneic tumor-bearing mice. Methods Binding specificity of the developed probe was evaluated in vitro. Next, fluorescent imaging in BALB/c mice bearing a murine breast tumor was performed at different time points after VGT-309 administration. Biodistribution of VGT-309 after 24 h in tumor-bearing mice was compared to control mice. Image-guided surgery was performed at multiple time points tumors with different clinical fluorescent camera systems and followed by ex vivo analysis. Results The probe was specifically activated by cathepsins X, B/L, and S. Fluorescent imaging revealed an increased tumor-to-background contrast over time up to 15.1 24 h post probe injection. In addition, VGT-309 delineated tumor tissue during image-guided surgery with different optical fluorescent imaging camera systems. Conclusion These results indicate that optical fluorescent molecular imaging using the cathepsin-targeted probe, VGT-309, may improve intraoperative tumor detection, which could translate to more complete tumor resection when coupled with commercially available surgical tools and techniques.

Purpose: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity-based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity-based probe (VGT-309) for fluorescence-guided surgery. Experimental Design: We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery. Results: In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR range: 2.15-3.71). In the Phase 1 human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase 2 trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs= 2.83 & 7.18) in real-time to illustrate its successful clinical translation and potential to improve surgical management. Conclusions: This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors.