A multifunctional, synthetic Gaussia princeps luciferase reporter for live imaging of Candida albicans infections

Peer reviewedPublisher PD

Gymnemic acids inhibit hyphal growth and virulence in Candida albicans

Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs) as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine

Cell Surface Expression of Nrg1 Protein in Candida auris

Candida auris is an emerging antifungal resistant human fungal pathogen increasingly reported in healthcare facilities. It persists in hospital environments, and on skin surfaces, and can form biofilms readily. Here, we investigated the cell surface proteins from C. auris biofilms grown in a synthetic sweat medium mimicking human skin conditions. Cell surface proteins from both biofilm and planktonic control cells were extracted with a buffer containing β-mercaptoethanol and resolved by 2-D gel electrophoresis. Some of the differentially expressed proteins were excised and identified by mass spectrometry. C. albicans orthologs Spe3p, Tdh3p, Sod2p, Ywp1p, and Mdh1p were overexpressed in biofilm cells when compared to the planktonic cells of C. auris. Interestingly, several proteins with zinc ion binding activity were detected. Nrg1p is a zinc-binding transcription factor that negatively regulates hyphal growth in C. albicans. C. auris does not produce true hypha under standard in vitro growth conditions, and the role of Nrg1p in C. auris is currently unknown. Western blot analyses of cell surface and cytosolic proteins of C. auris against anti-CalNrg1 antibody revealed the Nrg1p in both locations. Cell surface localization of Nrg1p in C. auris, an unexpected finding, was further confirmed by immunofluorescence microscopy. Nrg1p expression is uniform across all four clades of C. auris and is dependent on growth conditions. Taken together, the data indicate that C. auris produces several unique proteins during its biofilm growth, which may assist in the skin-colonizing lifestyle of the fungus during its pathogenesis

Interaction of Candida albicans Biofilms with Antifungals: Transcriptional Response and Binding of Antifungals to Beta-Glucans ▿ †

Candida albicans can form biofilms that exhibit elevated intrinsic resistance to various antifungal agents, in particular azoles and polyenes. The molecular mechanisms involved in the antifungal resistance of biofilms remain poorly understood. We have used transcript profiling to explore the early transcriptional responses of mature C. albicans biofilms exposed to various antifungal agents. Mature C. albicans biofilms grown under continuous flow were exposed for as long as 2 h to concentrations of fluconazole (FLU), amphotericin B (AMB), and caspofungin (CAS) that, while lethal for planktonic cells, were not lethal for biofilms. Interestingly, FLU-exposed biofilms showed no significant changes in gene expression over the course of the experiment. In AMB-exposed biofilms, 2.7% of the genes showed altered expression, while in CAS-exposed biofilms, 13.0% of the genes had their expression modified. In particular, exposure to CAS resulted in the upregulation of hypha-specific genes known to play a role in biofilm formation, such as ALS3 and HWP1. There was little overlap between AMB- or CAS-responsive genes in biofilms and those that have been identified as AMB, FLU, or CAS responsive in C. albicans planktonic cultures. These results suggested that the resistance of C. albicans biofilms to azoles or polyenes was due not to the activation of specific mechanisms in response to exposure to these antifungals but rather to the intrinsic properties of the mature biofilms. In this regard, our study led us to observe that AMB physically bound C. albicans biofilms and beta-glucans, which have been proposed to be major constituents of the biofilm extracellular matrix and to prevent azoles from reaching biofilm cells. Thus, enhanced extracellular matrix or beta-glucan synthesis during biofilm growth might prevent antifungals, such as azoles and polyenes, from reaching biofilm cells, thus limiting their toxicity to these cells and the associated transcriptional responses

Isolation and Characterization of VceC Gain-of-Function Mutants That Can Function with the AcrAB Multiple-Drug-Resistant Efflux Pump of Escherichia coli

VceC is the outer membrane component of the major facilitator (MF) VceAB-VceC multiple-drug-resistant (MDR) efflux pump of Vibrio cholerae. TolC is the outer membrane component of the resistance-nodulation-division AcrAB-TolC efflux pump of Escherichia coli. Although these proteins share little amino acid sequence identity, their crystal structures can be readily superimposed upon one another. In this study, we have asked if TolC and VceC are interchangeable for the functioning of the AcrAB and VceAB pumps. We have found that TolC can replace VceC to form a functional VceAB-TolC MDR pump, but VceC cannot replace TolC to form a functional AcrAB-VceC pump. However, we have been able to isolate gain-of-function (gof) VceC mutants which can functionally interface with AcrAB. These mutations map to four different amino acids located at the periplasmic tip of VceC. Chemical cross-linkage experiments indicate that both wild-type and gof mutant VceC can physically interact with the AcrAB complex, suggesting that these gof mutations are not affecting the recruitment of VceC to the AcrAB complex but rather its ability to functionally interface with the AcrAB pump

Evidence that Clostridium difficile TcdC Is a Membrane-Associated Protein

Clostridium difficile produces two toxins, A and B, which act together to cause pseudomembraneous colitis. The genes encoding these toxins, tcdA and tcdB, are part of the pathogenicity locus, which also includes tcdC, a putative negative regulator of the toxin genes. In this study, we demonstrate that TcdC is a membrane-associated protein in C. difficile

Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile▿

Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by ΦCD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in ΦCD119 lysogens. During this study we found that repR, a putative repressor gene of ΦCD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in ΦCD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen

Fungal strains used in this study.

<p>Fungal strains used in this study.</p

Effect of GAs on <i>C. albicans</i> infection in <i>Caenorhabditis elegans</i> and mammalian cells.

<p>(A) Rescue of <i>C. albicans</i> infected <i>C. elegans</i> from death by GAs. Larvae of <i>C. elegans</i> fed with yeast cells of <i>C. albicans</i> were incubated in RPMI medium with or without GAs (40 µg/ml) in a 96 well microtiter plate and incubated at 30°C for 2–4 days. Arrow in the top left panel (-GAs) shows the growth of <i>C. albicans</i> hyphae from the dead worms while addition of GAs (+GAs) prevent growth of hyphae from the worms’ body and hence worms survival (top right panel). Small round structures in the background are <i>C. albicans</i> yeast cells. Inset of <i>C. elegans</i> from GAs treated well shows confocal microscopic image of <i>C. elegans</i> containing <i>C. albicans</i> yeast cells in the worm’s gut (arrow). Scale bar (inset) = 10 µm. Bar graph at lower left panel indicates the % worms surviving after 4 days of exposure to GAs or to AMB. Survival of worms was determined by their movements and absence of hyphal growth from worms using microscope. Error bars indicate SD from the averages of 3 independent experiments. (B) GAs are non hemolytic and nontoxic to mammalian cells. Hemolytic assay was performed on tryptic soy agar plate containing human red blood cells (hRBC, 5%) (left side). A diagrammatic representation with sample identity is shown on the right side. Different fractions containing GAs [<i>G. sylvestre</i> extract, GE 1 mg/ml, 4 µl; fraction #194 (4 µl); and purified GAs (40 µg/ml, 4 µl)] were diluted in PBS and spotted on hRBC-agar. Positive controls including actively growing <i>Staphylococcus aureus</i> cells (2 µl) or PBS containing Triton X-100 (1%) (Tri-X) were also spotted on the blood agar medium as controls. Plates were incubated for 24–48 h at 37°C and the results were recorded by image capture. White clear halos around spots indicate hemolytic activity. GAs are not toxic to mammalian kidney epithelial cells (far right sector). Napthaquinone (NAP, 50 µg/ml) killed the kidney epithelial cells whereas solvent control (-GAs), amphotericin B (+AMB) or test compounds (+GAs; 40 µg/ml) did not. Scale bar = 10 µm.</p

Effect of GAs on germination of <i>Aspergillus fumigatus</i> spores.

<p>(A) A conidiospore suspension was incubated in RPMI medium at 37°C with (40 µg/ml) or without GAs for 15 h under static condition in microtiter wells. Percentage of germination was calculated at least from nine different fields from triplicate wells. A spore is considered germinated when the length of the germ tube is twice or more the size of a spore. Scale bars = 25 µm. (B) Table showing the impact of GAs on <i>A. fumigatus</i> spore germination and germ tube lengths. The lengths of germ tubes were measured by using µScope software (µScope Essential) and shown ± SD.</p