CARD9-Associated Dectin-1 and Dectin-2 Are Required for Protective Immunity of a Multivalent Vaccine against Coccidioides posadasii Infection.

Coccidioides species are fungal pathogens that can cause a widely varied clinical manifestation from mild pulmonary symptom to disseminated, life-threatening disease. We have previously created a subunit vaccine by encapsulating a recombinant coccidioidal Ag (rCpa1) in glucan-chitin particles (GCPs) as an adjuvant-delivery system. The GCP-rCpa1 vaccine has shown to elicit a mixed Th1 and Th17 response and confers protection against pulmonary coccidioidomycosis in mice. In this study, we further delineated the vaccine-induced protective mechanisms. Depletion of IL-17A in vaccinated C57BL/6 mice prior to challenge abrogated the protective efficacy of GCP-rCpa1 vaccine. Global transcriptome and Ingenuity Pathway Analysis of murine bone marrow-derived macrophages after exposure to this vaccine revealed the upregulation of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) that are associated with activation of C-type lectin receptors (CLR) Dectin-1- and Dectin-2-mediated CARD9 signaling pathway. The GCP formulation of rCpa1 bound soluble Dectin-1 and Dectin-2 and triggered ITAM signaling of corresponding CLR reporter cells. Furthermore, macrophages that were isolated from Dectin-1 -/-, Dectin-2 -/-, and CARD9 -/- mice significantly reduced production of inflammatory cytokines in response to the GCP-rCpa1 vaccine compared with those of wild-type mice. The GCP-rCpa1 vaccine had significantly reduced protective efficacy in Dectin-1 -/-, Dectin-2 -/-, and CARD9 -/- mice that showed decreased acquisition of Th cells in Coccidioides-infected lungs compared with vaccinated wild-type mice, especially Th17 cells. Collectively, we conclude that the GCP-rCpa1 vaccine stimulates a robust Th17 immunity against Coccidioides infection through activation of the CARD9-associated Dectin-1 and Dectin-2 signal pathways.

Screening Repurposing Libraries for Identification of Drugs with Novel Antifungal Activity.

Fungal organisms are ubiquitous in nature, and progress of modern medicine is creating an expanding number of severely compromised patients susceptible to a variety of opportunistic fungal infections. These infections are difficult to diagnose and treat, leading to high mortality rates. The limited antifungal arsenal, the toxicity of current antifungal drugs, the development of resistance, and the emergence of new multidrug-resistant fungi, all highlight the urgent need for new antifungal agents. Unfortunately, the development of a novel antifungal is a rather long and expensive proposition, and no new classes of antifungal agents have reached the market in the last 2 decades. Drug repurposing, or finding new indications for old drugs, represents a promising alternative pathway to drug development that is particularly appealing within the academic environment. In the last few years, there has been a growing interest in repurposing approaches in the antifungal arena, with multiple groups of investigators having performed screenings of different repurposing libraries against different pathogenic fungi in search for drugs with previously unrecognized antifungal effects. Overall, these repurposing efforts may lead to the fast deployment of drugs with novel antifungal activity, which can rapidly bring benefits to patients, while at the same time reducing health care costs.

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

Activity of anti-CR3-RP polyclonal antibody against biofilms formed by Candida auris, a multidrug-resistant emerging fungal pathogen.

Fungal biofilm has remained a serious medical problem that complicates treatment of mycoses. In particular, once biofilms are formed, they display high levels of resistance against most common antifungals. Candida auris is currently considered as a serious emerging fungal pathogen frequently exhibiting high levels of resistance to antifungals. Recent studies have confirmed that C. auris shares similarity with Candida albicans in regards to virulence-associated proteins involved in adherence and biofilm development. Complement receptor 3-related protein (CR3-RP) is one of the key surface antigens expressed by Candida species during biofilm formation. Here, we have investigated the presence of this cell surface moiety on the surface of C. auris, as well as the potential of anti-CR3-RP polyclonal antibody (Ab) to inhibit biofilm formation by this emerging fungal pathogen. Using indirect immunofluorescence and ELISA, we were able to confirm the presence of CR3-RP in C. auris cells within biofilms. Further, not only anti-CR3-RP Ab was able to inhibit biofilm formation by multiple C. auris strains when added during the adherence phase, but it also demonstrated activity against C. auris 24-h pre-formed biofilms, which compared favorably to levels of inhibition achieved by treatment with current conventional antifungals fluconazole, amphotericin B, and caspofungin. Overall, our data demonstrate the presence of this antigen on the surface of C. auris and points to the potential of anti-CR3-RP Ab in eradication of biofilms formed by this novel fungal pathogen.

Screening a Repurposing Library for Inhibitors of Multidrug-Resistant Candida auris Identifies Ebselen as a Repositionable Candidate for Antifungal Drug Development.

Since its original isolation in 2009, Candida auris has spread across the globe as a causative agent of invasive candidiasis. C. auris is typically intrinsically resistant to fluconazole and can also be resistant to echinocandins and even amphotericin B. Thus, there is an urgent need to find new treatment options against this emerging pathogen. To address this growing problem, we performed a screen of the Prestwick Chemical library, a repurposing library of 1,280 small molecules, consisting mostly of approved off-patent drugs, in search of those with activity against a multidrug-resistant C. auris isolate. Our initial screen, using standardized susceptibility testing methodologies, identified nine miscellaneous compounds with no previous clinical indication as antifungals or antiseptics that displayed activity against C. auris Confirmation and follow-up studies identified ebselen as the drug displaying the most potent activity, with 100% inhibition of growth detected at concentrations as low as 2.5 µM. We further evaluated the ability of ebselen to inhibit C. auris biofilm formation and examined the effects of combination therapies of ebselen with clinically used antifungals. We extended our studies to different C. auris strains with various susceptibility patterns and also confirmed its antifungal activity against Candida albicans and clinical isolates of multiple other Candida species. Furthermore, ebselen displayed a broad spectrum of antifungal actions on the basis of its activity against a variety of medically important fungi, including yeasts and molds. Overall, our results indicate the promise of ebselen as a repositionable agent for the treatment of candidiasis and possibly other mycoses and, in particular, for the treatment of infections refractory to conventional treatment with current antifungals.

Screening the Pathogen Box for Identification of Candida albicans Biofilm Inhibitors.

Candida albicans remains the main causative agent of candidiasis, one of the most frequent nosocomial infections, with unacceptably high mortality rates. Biofilm formation is a major risk factor for invasive candidiasis, as Candida biofilms display high-level resistance to most antifungal agents. In this work we have screened the Pathogen Box chemical library (Medicines for Malaria Venture [MMV], Switzerland) in search for inhibitors of C. albicans biofilm formation. Our initial screen identified seven hits, and additional dose-response assays confirmed the biofilm-inhibitory activity of six of these small molecules. Three compounds, MMV688768, MMV687273, and MMV687807, were also able to reduce the metabolic activity of cells within preformed biofilms. Interestingly, the most potent of these, compound MMV688768, displayed increased antibiofilm activity compared to its activity against planktonic cultures, indicating that it may affect processes with a predominant role during the biofilm mode of growth. This compound demonstrated a high selectivity index when its antibiofilm activity was compared with its toxicity in liver hepatocellular cells. In vitro combination assays showed a synergistic interaction between compound MMV688768 and fluconazole against preformed biofilms. Overall, our results indicate that this compound may constitute a potential candidate for further clinical development.

Screening a Commercial Library of Pharmacologically Active Small Molecules against Staphylococcus aureus Biofilms.

It is now well established that bacterial infections are often associated with biofilm phenotypes that demonstrate increased resistance to common antimicrobials. Further, due to the collective attrition of new antibiotic development programs by the pharmaceutical industries, drug repurposing is an attractive alternative. In this work, we screened 1,280 existing commercially available drugs in the Prestwick Chemical Library, some with previously unknown antimicrobial activity, against Staphylococcus aureus, one of the commonly encountered causative pathogens of burn and wound infections. From the primary screen of the entire Prestwick Chemical Library at a fixed concentration of 10 µM, 104 drugs were found to be effective against planktonic S. aureus strains, and not surprisingly, these were mostly antimicrobials and antiseptics. The activity of 18 selected repurposing candidates, that is, drugs that show antimicrobial activity that are not already considered antimicrobials, observed in the primary screen was confirmed in dose-response experiments. Finally, a subset of nine of these drug candidates was tested against preformed biofilms of S. aureus We found that three of these drugs, niclosamide, carmofur, and auranofin, possessed antimicrobial activity against preformed biofilms, making them attractive candidates for repurposing as novel antibiofilm therapies.

Examination of the pathogenic potential of Candida albicans filamentous cells in an animal model of haematogenously disseminated candidiasis.

The opportunistic fungal pathogen Candida albicans is an increasingly common threat to human health. Candida albicans grows in several morphologies and mutant strains locked in yeast or filamentous forms have attenuated virulence in the murine model of disseminated candidiasis. Thus, the ability to change shape is important for virulence. The transcriptional repressors Nrg1p and Tup1p are required for normal regulation of C. albicans morphology. Strains lacking either NRG1 or TUP1 are constitutively pseudohyphal under yeast growth conditions, and display attenuated virulence in the disseminated model. To dissect the relative importance of hyphae and pseudohyphae during an infection, we used strains in which the morphological transition could be externally manipulated through controlled expression of NRG1 or TUP1. Remarkably, hyphal form inocula retain the capacity to cause disease. Whilst induction of a pseudohyphal morphology through depletion of TUP1 did result in attenuated virulence, this was not due to a defect in the ability to escape the bloodstream. Instead, we observed that pseudohyphal cells are cleared from tissues much more efficiently than either hyphal (virulent) or yeast form (avirulent) cells, indicating that different C. albicans morphologies have distinct interactions with host cells during an infection.

In Vitro Activity of Miltefosine against Candida albicans under Planktonic and Biofilm Growth Conditions and In Vivo Efficacy in a Murine Model of Oral Candidiasis.

The generation of a new antifungal against Candida albicans biofilms has become a major priority, since biofilm formation by this opportunistic pathogenic fungus is usually associated with an increased resistance to azole antifungal drugs and treatment failures. Miltefosine is an alkyl phospholipid with promising antifungal activity. Here, we report that, when tested under planktonic conditions, miltefosine displays potent in vitro activity against multiple fluconazole-susceptible and -resistant C. albicans clinical isolates, including isolates overexpressing efflux pumps and/or with well-characterized Erg11 mutations. Moreover, miltefosine inhibits C. albicans biofilm formation and displays activity against preformed biofilms. Serial passage experiments confirmed that miltefosine has a reduced potential to elicit resistance, and screening of a library of C. albicans transcription factor mutants provided additional insight into the activity of miltefosine against C. albicans growing under planktonic and biofilm conditions. Finally, we demonstrate the in vivo efficacy of topical treatment with miltefosine in the murine model of oropharyngeal candidiasis. Overall, our results confirm the potential of miltefosine as a promising antifungal drug candidate, in particular for the treatment of azole-resistant and biofilm-associated superficial candidiasis.

Effect of silver nanoparticles on Candida albicans biofilms: an ultrastructural study.

BACKGROUND: Candida albicans is the most common pathogenic fungus isolated in bloodstream infections in hospitalized patients, and candidiasis represents the fourth most common infection in United States hospitals, mostly due to the increasing numbers of immune- and medically-compromised patients. C. albicans has the ability to form biofilms and morphogenetic conversions between yeast and hyphal morphologies contribute to biofilm development and represent an essential virulence factor. Moreover, these attached communities of cells are surrounded by a protective exopolymeric matrix that effectively shelters Candida against the action of antifungals. Because of dismal outcomes, novel antifungal strategies, and in particular those targeting biofilms are urgently required. As fungi are eukaryotic, research and development of new antifungal agents has been difficult due to the limited number of selective targets, also leading to toxicity. RESULTS: By microwave-assisted techniques we obtained pure 1 nm spherical silver nanoparticles ideal for their potential biological applications without adding contaminants. A phenotypic assay of C. albicans demonstrated a potent dose-dependent inhibitory effect of silver nanoparticles on biofilm formation, with an IC50 of 0.089 ppm. Also silver nanoparticles demonstrated efficacy when tested against pre-formed C. albicans biofilms resulting in an IC50 of 0.48 ppm. The cytotoxicity assay resulted in a CC50 of 7.03 ppm. The ultrastructural differences visualized under SEM with silver nanoparticles treatment were changes in the surface appearance of the yeast from smooth to rough thus indicating outer cell wall damage. On the fungal pre-formed biofilm true hyphae was mostly absent, as filamentation was inhibited. TEM measurement of the cell-wall width of C. albicans after treatment resulted in significant enlargement (206 ± 11 nm) demonstrating membrane permeabilization. CONCLUSIONS: Our results demonstrate that silver nanoparticles are potent inhibitors of C. albicans biofilm formation. SEM observations are consistent with an overall loss of structure of biofilms mostly due to disruption of the outer cell membrane/wall and inhibition of filamentation.TEM indicates the permeabilization of the cell wall and subsequent disruption of the structural layers of the outer fungal cell wall. The anti-biofilm effects are via cell wall disruption.

Ppg1, a PP2A-type protein phosphatase, controls filament extension and virulence in Candida albicans.

Candida albicans, a major human fungal pathogen, is the primary cause of invasive candidiasis in a wide array of immunocompromised patients. C. albicans virulence requires the ability to undergo a reversible morphological transition from yeast to filaments in response to a variety of host environmental cues. These cues are sensed by the pathogen and activate multiple signal transduction pathways to induce filamentation. Reversible phosphorylation events are critical for regulation of many of these pathways. While a variety of protein kinases are known to function as components of C. albicans filamentous growth signal transduction pathways, considerably little is known about the role of phosphatases. Here we demonstrate that PPG1, encoding a putative type 2A-related protein phosphatase, is important for C. albicans filament extension, invasion, and virulence in a mouse model of systemic candidiasis. PPG1 is also important for downregulation of NRG1, a key transcriptional repressor of C. albicans filamentous growth, and is shown to affect the expression of several filament-specific target genes. An epistasis analysis suggests that PPG1 controls C. albicans filamentation via the cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway. We demonstrate that Ppg1 possesses phosphatase activity and that a ppg1 catalytic mutant shows nearly equivalent filamentation, invasion, and virulence defects compared to those of a ppg1Δ/Δ strain. Overall, our results suggest that phosphatases, such as Ppg1, play critical roles in controlling and fine-tuning C. albicans filament extension and virulence as well as signal transduction pathways, transcriptional regulators, and target genes associated with these processes.

Effect of silver nanoparticle geometry on methicillin susceptible and resistant Staphylococcus aureus, and osteoblast viability.

Orthopedic implant failure as a result of bacterial infection affects approximately 0.5-5% of patients. These infections are often caused by Staphylococcus aureus which is capable of attaching and subsequently forming a biofilm on the implant surface, making it difficult to eradicate with systemic antibiotics. Further, with the emergence of antibiotic-resistant bacteria, alternative treatments are necessary. Silver nanoparticles have received much attention for their broad spectrum antibacterial activity which has been reported to be both size and shape dependent. The purpose of this study was therefore to evaluate the effect of three different geometries on their effect on microbial susceptibility as well as evaluate their effect on bone cell viability. Silver nanoparticles of spherical, triangular and cuboid shapes were synthesized by chemical reduction methods. The susceptibility of S. aureus and methicillin-resistant S. aureus was evaluated a 24 h period and determined using a colorimetric assay. Further, the viability of human fetal osteoblast (hFOB) cells in the presence of the silver nanoparticles was evaluated over a period of 7 days by AlmarBlue fluorescence assay. hFOB morphology was also evaluated by light microscopy imaging. Results indicated that silver nanoparticle geometry did not have an effect on microbiota susceptibility or hFOB viability. However, high concentrations of silver nanoparticles (0.5 nM) conferred significant susceptibility towards the bacteria and significantly reduced hFOB viability. It was also found that the hFOBs exhibited an increasingly reduced viability to lower silver nanoparticle concentrations with an increase in exposure time.

In vitro study of sequential fluconazole and caspofungin treatment against Candida albicans biofilms.

Candida albicans biofilms are generally considered to be resistant to azole antifungal agents but susceptible to echinocandins. We demonstrate that in a sequential therapy regimen, treatment with fluconazole first followed by caspofungin leads to a significant decrease of the efficacy of this echinocandin. Cellular stress responses induced by high fluconazole concentrations and mediated by Hsp90 and calcineurin play an important role in this phenomenon.

Drug susceptibility of matrix-encapsulated Candida albicans nano-biofilms.

The rise in the use of biomedical devices and implants has seen a concomitant surge in the advent of device-related nosocomial (hospital-acquired) infections of bacterial and fungal origins. The most common nosocomial fungal infection is candidiasis caused mainly by Candida albicans biofilms. Candidiasis is associated with an unacceptably high mortality rate, and there is an urgent need for the discovery of new antifungal drugs that prevent or control biofilm formation. To this end, we recently developed an ultra-high-throughput microarray platform consisting of nano-scale biofilms of C. albicans encapsulated in collagen or alginate hydrogel matrices for antifungal drug screening. Here, we report that the choice of matrix influences the apparent susceptibility of C. albicans to the common antifungal drugs, amphotericin B, and caspofungin. While amphotericin B is equally effective against biofilms grown in collagen and alginate matrices, caspofungin is effective only against biofilms grown only in alginate, but not in collagen. We demonstrate differences in the distribution of the drugs in the two matrices may contribute to the susceptibility of C. albicans nano-biofilms. In a larger context, our results highlight the importance of the choice of matrix as a parameter in 3D cell encapsulation, and suggest a screening strategy to predict drug performance in vivo.

Overcoming antifungal resistance.

Fungal infections have become one of the major causes of morbidity and mortality in immunocompromised patients. Despite increased awareness and improved treatment strategies, the frequent development of resistance to the antifungal drugs used in clinical settings contributes to the increasing toll of mycoses. Although a natural phenomenon, antifungal drug resistance can compromise advances in the development of effective diagnostic techniques and novel antifungals. In this review, we will discuss the advent of cellular-micro- arrays, microfluidics, genomics, proteomics and other state-of-the art technologies in conquering antifungal drug resistance.

Expression of UME6, a key regulator of Candida albicans hyphal development, enhances biofilm formation via Hgc1- and Sun41-dependent mechanisms.

Biofilm formation is associated with the ability of Candida albicans, the major human fungal pathogen, to resist antifungal therapies and grow on tissues, catheters, and medical devices. In order to better understand the relationship between C. albicans morphology and biofilm formation, we examined biofilms generated in response to expression of UME6, a key filament-specific transcriptional regulator. As UME6 levels rise, C. albicans cells are known to transition from yeast to hyphae, and we also observed a corresponding increase in the level of biofilm formation in vitro. In addition to forming a biofilm, we observed that a C. albicans strain expressing constitutive high levels of UME6 promoted tissue invasion in a reconstituted human three-dimensional model of oropharyngeal candidiasis. Confocal microscopy indicated that both the top and bottom layers of the biofilm generated upon high-level constitutive UME6 expression consist primarily of hyphal cells. UME6-driven biofilm formation was reduced upon deletion of Hgc1, a cyclin-related protein important for hyphal development, as well as Sun41, a putative cell wall glycosidase. Constitutive high-level UME6 expression was also able to completely bypass both the filamentation and biofilm defects of a strain deleted for Efg1, a key transcriptional regulator of these processes. Finally, we show that both Sun41 and Efg1 affect the ability of UME6 to induce certain filament-specific transcripts. Overall, these findings indicate a strong correlation between increased C. albicans hyphal growth and enhanced biofilm formation and also suggest functional relationships between UME6 and other regulators of biofilm development.

Candidalysin: An unlikely aide for fungal gut commensalism.

Fungi colonize the mammalian gastrointestinal (GI) tract and can adopt both commensal and opportunistic lifestyles. In a recent issue of Nature, Liang et al. unraveled the complex interplay between Candida morphotypes and the gut bacterial microbiota and described a key role for candidalysin in gut colonization.1.

Identification and characterization of Cryptococcus neoformans protein fractions that induce protective immune responses.

Cryptococcus neoformans, the main causative agent of cryptococcosis, is a fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised patients. To date, there is no vaccine or immunotherapy approved to treat cryptococcosis. Cell- and antibody-mediated immune responses collaborate to mediate optimal protection against C. neoformans infections. Accordingly, we identified cryptococcal protein fractions capable of stimulating cell- and antibody-mediated immune responses and determined their efficacy to elicit protection against cryptococcosis. Proteins were extracted from C. neoformans and fractionated based on molecular mass. The fractions were then evaluated by immunoblot analysis for reactivity to serum extracted from protectively immunized mice and in cytokine recall assays for their efficacy to induce pro-inflammatory and Th1-type cytokine responses associated with protection. MS analysis revealed a number of proteins with roles in stress response, signal transduction, carbohydrate metabolism, amino acid synthesis, and protein synthesis. Immunization with select protein fractions containing immunodominant antigens induced significantly prolonged survival against experimental pulmonary cryptococcosis. Our studies support using the combination of immunological and proteomic approaches to identify proteins that elicit antigen-specific antibody and Th1-type cytokine responses. The immunodominant antigens that were discovered represent attractive candidates for the development of novel subunit vaccines for treatment and/or prevention of cryptococcosis.

High-throughput screening of a collection of known pharmacologically active small compounds for identification of Candida albicans biofilm inhibitors.

Candida albicans is the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited and is particularly ineffective against C. albicans biofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors of C. albicans biofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics, or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents that inhibit C. albicans biofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents, which are urgently needed.