Candida infections frequently involve drug-resistant biofilm growth on device surfaces. Glucan synthase gene FKS1 has been linked to triazole resistance in Candida biofilms. We tested the impact of FKS1 modulation on susceptibility to additional antifungal classes. Reduction of FKS1 expression rendered biofilms more susceptible to amphotericin B, anidulafungin, and flucytosine. Increased resistance to anidulafungin and amphotericin B was observed for biofilms overexpressing FKS1. These findings suggest that Candida biofilm glucan sequestration is a multidrug resistance mechanism.In hospital settings, Candida spp. often cause disease by adhering to the surface of a medical device and adapting to a biofilm lifestyle (7,10). Biofilms consist of cells attached to a surface and embedded in a protective matrix produced by the organisms (5). C. albicans biofilm cells are phenotypically distinct, and their ability to survive exposure to high antifungal concentrations presents a serious therapeutic dilemma (1,2,11,14,(19)(20)(21). Biofilm cells exhibit up to 1,000-fold-increased resistance relative to free-floating, or planktonic, cells (3,9,12,18).Glucan synthesis by Fks1p has been implicated in C. albicans biofilm resistance to the azole drug fluconazole (17). FKS1 disruption was found to reduce manufacture and deposition of ␤-1,3-glucan in the biofilm matrix, resulting in susceptibility to fluconazole. The matrix glucan was shown to sequester the triazole, preventing it from reaching its target. The mechanism is biofilm specific and has been studied only for the triazoles.The purpose of this study was to determine the role of FKS1 in C. albicans biofilm resistance to other available antifungal drug classes. We chose to study three strains with differing expressions of FKS1 and concomitant variations in matrix glucan. The strains included a heterozygous deletion mutant (FKS1/fks1⌬), an FKS1 overexpression mutant (TDH3-FKS1) with one FKS1 allele under the control of TDH3 promoter and one allele intact, and a reference strain (4, 17). Finally, because FKS1 is essential in C. albicans, a conditional TET-FKS1 mutant was also included (22). The TET-FKS1 strain has one allele deleted and one allele under the control of a tetracycline-or doxycycline-repressible promoter. An echinocandin (anidulafungin), flucytosine, and amphotericin B deoxycholate were selected for their different mechanisms of action.For biofilm antifungal susceptibility testing, C. albicans biofilms were grown in 96-well polystyrene plates as previously described (16,20). Wells were inoculated with 10 6 cells/ml in RPMI medium-MOPS (morpholinepropanesulfonic acid). After an adherence period (6 or 24 h), biofilms were washed with phosphate-buffered saline (PBS). Fresh media and antifungals were applied, and plates were incubated for an additional 24 h at 37°C. The concentration ranges included those above and below the planktonic MIC values and included 0.001 to 0.125 g/ml anidulafungin, 0.03 to 8 g/ml flucytosine, and 0.008 to 2 g/ml amphotericin B deoxycho...

Candida spp. infect medical devices, such as venous and urinary catheters, by adhering to the surface and forming a community of drug-resistant cells surrounded by a matrix. The ability to measure drug activity during this biofilm mode of growth is of interest for the investigation of resistance mechanisms and novel antifungal therapies. The tetrazolium salt (XTT) reduction assay is the test most commonly used to estimate viable biofilm growth and to examine the impact of biofilm therapies. The primary goal of the current experiments was to identify assay variables that affect the XTT assay result in order to improve assay reproducibility, sensitivity, and throughput for the study of antifungal activity. The species used in the current studies included Candida albicans, C. parapsilosis, and C. glabrata. The assay variables that were studied included the impact of culture conditions, the duration of biofilm growth, the timing and frequency of drug administration, the XTT source and concentration, and the duration of XTT incubation. The conditions that impacted the assay readout and altered assay sensitivity included the duration of biofilm growth, the frequency of drug dosing, and the duration of XTT incubation. Several factors were found to reduce time and assay expense, including the elimination of washing steps, the shortening of incubation times, and the use of lower XTT concentrations. A description of assay pitfalls and troubleshooting is included. Recognition of these technical variables should allow investigators to better design reproducible biofilm therapeutic studies.The most clinically important phenotype of Candida biofilm cells is their remarkable resistance to antifungal drugs (1,4,17,29,39). Cells in this environment can survive up to 1,000-foldhigher concentrations of antifungals than nonbiofilm, planktonic cells. Because antifungal drugs typically are not effective against biofilm organisms, the recommended therapy for Candida biofilm infection of a medical device includes device removal, which is associated with increased procedural morbidity and health care expenditures (25). Novel drug targets and the development of new antifungal agents for the treatment of these recalcitrant infections are therefore of interest.The use of the tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) reduction assay to study Candida biofilms has been pioneered by labs in the mycology community (7,10,13,33,38). It is the method most commonly utilized for quantitative measurement of Candida biofilm mass, growth, and response to drug therapy (1,9,12,18,20,26,28,37). Other techniques used to assay the biofilm cell burden include [ 3 H]leucine incorporation, fluorescein diacetate, crystal violet staining, viable counts, dry weight measurements, and imaging using confocal or electron microscopy (5,8,10,35,36). The XTT assay has become the preferred tool due to the rapidity of the assay, the ability to use a high-throughput format (e.g., a 96-well plate), and more im...

The pleiotropic chemokine CXCL12 is involved in diverse physiological and pathophysiological processes, including embryogenesis, hematopoiesis, leukocyte migration, and tumor metastasis. It is known to engage the classical receptor CXCR4 and the atypical receptor ACKR3. Differential receptor engagement can transduce distinct cellular signals and effects as well as alter the amount of free, extracellular chemokine. CXCR4 binds both monomeric and the more commonly found dimeric forms of CXCL12, whereas ACKR3 binds monomeric forms. Here, we found that CXCL12 also bound to the atypical receptor ACKR1 (previously known as Duffy antigen/receptor for chemokines or DARC). In vitro nuclear magnetic resonance spectroscopy and isothermal titration calorimetry revealed that dimeric CXCL12 bound to the extracellular N terminus of ACKR1 with low nanomolar affinity, whereas the binding affinity of monomeric CXCL12 was orders of magnitude lower. In transfected MDCK cells and primary human Duffy-positive erythrocytes, a dimeric, but not a monomeric, construct of CXCL12 efficiently bound to and internalized with ACKR1. This interaction between CXCL12 and ACKR1 provides another layer of regulation of the multiple biological functions of CXCL12. The findings also raise the possibility that ACKR1 can bind other dimeric chemokines, thus potentially further expanding the role of ACKR1 in chemokine retention and presentation.

The human chemokine family consists of 46 protein ligands that induce chemotactic cell migration by activating a family of 23 G protein-coupled receptors. The two major chemokine subfamilies, CC and CXC, bind distinct receptor subsets. A sequence motif defining these families, the X position in the CXC motif, is not predicted to make significant contacts with the receptor, but instead links structural elements associated with binding  and activation. Here we use comparative analysis of chemokine NMR structures, structural modeling, and molecular dynamic simulations that suggested the X position reorients the chemokine N-terminus. Using CXCL12 as a model CXC chemokine, deletion of the X residue (P10) had little to no impact on the folded chemokine structure but diminished CXCR4 agonist activity as measured by ERK phosphorylation, chemotaxis, and Gi/o-mediated cAMP inhibition. Functional impairment was attributed to over 100-fold loss of CXCR4 binding affinity. Binding to the other CXCL12 receptor, ACKR3, was diminished  nearly 500-fold.  Deletion of P10 had little effect on CXCL12 binding to the CXCR4 N-terminus, a major component of the chemokine-GPCR interface. Replacement of the X residue with the most frequent amino acid at this position (P10Q) had an intermediate effect between wild-type and P10del in each assay, with ACKR3 having a higher tolerance for this mutation. This work shows that the X residue helps to position the CXCL12 N-terminus for optimal docking into the orthosteric pocket of CXCR4 and suggests that the CC/CXC motif contributes directly to receptor selectivity by orienting the chemokine N-terminus in a subfamily-specific direction.

The chemokine network is comprised of a family of signal proteins that encode messages for cells displaying chemokine G-protein coupled receptors (GPCRs). The diversity of effects on cellular functions, particularly directed migration of different cell types to sites of inflammation, is enabled by different combinations of chemokines activating signal transduction cascades on cells displaying a combination of receptors. These signals can contribute to autoimmune disease or be hijacked in cancer to stimulate cancer progression and metastatic migration. Thus far, three chemokine receptor-targeting drugs have been approved for clinical use: Maraviroc for HIV, Plerixafor for hematopoietic stem cell mobilization, and Mogalizumab for cutaneous T-cell lymphoma. Numerous compounds have been developed to inhibit specific chemokine GPCRs, but the complexity of the chemokine network has precluded more widespread clinical implementation, particularly as anti-neoplastic and anti-metastatic agents. Drugs that block a single signaling axis may be rendered ineffective or cause adverse reactions because each chemokine and receptor often have multiple context-specific functions. The chemokine network is tightly regulated at multiple levels, including by atypical chemokine receptors (ACKRs) that control chemokine gradients independently of G-proteins. ACKRs have numerous functions linked to chemokine immobilization, movement through and within cells, and recruitment of alternate effectors like β-arrestins. Atypical chemokine receptor 1 (ACKR1), previously known as the Duffy antigen receptor for chemokines (DARC), is a key regulator that binds chemokines involved in inflammatory responses and cancer proliferation, angiogenesis, and metastasis. Understanding more about ACKR1 in different diseases and populations may contribute to the development of therapeutic strategies targeting the chemokine network.