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Pharmaceutical And Immunollogical Challenge Of Fungal Pathogens
Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Clinical Microbiology, Umeå Univeristy.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Incidences of fungal infections are on the rise in immunosuppressed people. Predominant causative agents for these mycoses are species of the genus Candida, including Candida albicans, Candida glabrata and Candida dublieniensis. Despite a wide range of emerging pathogens, C. albicans remains the leading cause. According to recent epidemiological studies, blood stream infections with C. albicans cause annually ~55% mortality in approximately 300,000 patients from intensive care units worldwide. Furthermore, the percentage of morbidity linked to oral, esophageal and vulvovaginal mycoses cause by C. albicans reach up to 90%. Reasons for these medical concerns are the lack of efficient diagnostics and antifungal therapy.

Here, we therefore sought to find novel antifungal strategies inspired by innate immune cells, such as neutrophils. These phagocytes are able to block the fungal pathogenicity. Neutrophils are bloodstream leukocytes serving as the first line of defense against pathogenic microbes. It has been shown that neutrophils have a strong antifungal activity by impairing the conversion of the dimorphic C. albicans from yeast to hyphal form (Y-H). Consequently, we raised the question whether other immune cells, such as mast cells, with less phagocytic cabapilities may have similar activity to neutrophils.

Mast cells are tissue-dwelling cells. Mucosal tissue is rich in mast cells and usually constitutes the entry ports for fungal pathogens into the human body. A contribution of mast cells in antifungal defense is, thus, very likely. We human explored mast cell functions upon encounter with fungal pathogens. Interestingly, human mast cells show a transient potential to impair fungal viability. To understand the mechanism behind this impairment we analyzed the human mast cell functions in more detail. We found that human mast cells challenged with C. albicans, immediately degranulate and secrete distinct cytokines and chemokines in an orchestrated manner. The chemokines secreted attract neutrophils. Mast cells moreover are able to internalize fungal cells and to ‘commit suicide’ by releasing extracellular DNA traps that ensnare the pathogen.

 

The effectiveness of future antifungals is depended on targeting the pathogen virulence with more efficiency.

The dimorphism of C. albicans is proven to be essential its virulence. Blockage of this switching ability could render the pathogen avirulent. Consequently, we screened for compounds that mimic the neutrophils anti-dimorphic activity by screening small chemical molecule libraries that block Y-H transition. The screening of big chemical libraries requires a reliable, reproducible and rapid high-throughput screening assay (HTS). We developed an HTS assay based on automated microscopy and image analysis, thereby allowing to distinguish between yeast and filamentous forms. In order to find the ideal Y-H blocker, we also evaluated the cell viability via the count of ATP levels when challenged with the respective small chemical molecules.

 

Drug development is an elaborate and expensive process. We therefore applied our screening setup to identify antidimorphic/antifungal activity in compounds from two different chemical libraries including FDA-approved drugs. The study disclosed 7 off-patent antifungal drugs that have potent antimycotic activity, including 4 neoplastic agents, 2 antipsychotic drugs and 1 antianemic medication.

In a nutshell, we aimed to mimic the anti-dimorphic/antifungal activity of neutrophils with small chemical molecules. Furthermore, we elucidated how immune cells contribute to antifungal defense to exploit these mechanisms for the development of novel antifungal therapies. Thus, this thesis provides novel tools for the discovery of more efficient compounds, identifies previously unknown antifungal aspect of off-patent FDA-approved drugs and highlights the interplay of mast cells with pathogenic fungi with the aim to define new screening strategies.

Place, publisher, year, edition, pages
Umeå Universitet , 2015. , 52 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1735
Keyword [en]
mast cells, Candida albicans, yeast to hyphal form, antifungal drugs, repurposed drugs, HTS
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-107713ISBN: 978-91-7601-308-3 (print)OAI: oai:DiVA.org:umu-107713DiVA: diva2:849183
Public defence
2015-09-25, sal Eo4, byggnad 6E, NUS, Norrlands universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2015-09-04 Created: 2015-08-27 Last updated: 2015-09-02Bibliographically approved
List of papers
1. Novel High-Throughput Screening Method for Identification of Fungal Dimorphism Blockers
Open this publication in new window or tab >>Novel High-Throughput Screening Method for Identification of Fungal Dimorphism Blockers
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2015 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 20, no 2, 285-91 p.Article in journal (Refereed) Published
Abstract [en]

Invasive mycoses have been increasing worldwide, with Candida spp. being the most prevalent fungal pathogen causing high morbidity and mortality in immunocompromised individuals. Only few antimycotics exist, often with severe side effects. Therefore, new antifungal drugs are urgently needed. Because the identification of antifungal compounds depends on fast and reliable assays, we present a new approach based on high-throughput image analysis to define cell morphology. Candida albicans and other fungi of the Candida clade switch between different growth morphologies, from budding yeast to filamentous hyphae. Yeasts are considered proliferative, whereas hyphae are required for invasion and dissemination. Thus, morphotype switching in many Candida spp. is connected to virulence and pathogenesis. It is, consequently, reasonable to presume that morphotype blockers interfere with the virulence, thereby preventing hazardous colonization. Our method efficiently differentiates yeast from hyphal cells using a combination of automated microscopy and image analysis. We selected the parameters length/width ratio and mean object shape to quantitatively discriminate yeasts and hyphae. Notably, Z' factor calculations for these parameters confirmed the suitability of our method for high-throughput screening. As a second stage, we determined cell viability to discriminate morphotype-switching inhibitors from those that are fungicidal. Thus, our method serves as a basis for the identification of candidates for next-generation antimycotics.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-101194 (URN)10.1177/1087057114552954 (DOI)000348642300012 ()25281739 (PubMedID)
Available from: 2015-03-24 Created: 2015-03-24 Last updated: 2017-12-04Bibliographically approved
2. Antifungal Application of Nonantifungal Drugs
Open this publication in new window or tab >>Antifungal Application of Nonantifungal Drugs
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2014 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 58, no 2, 1055-1062 p.Article in journal (Refereed) Published
Abstract [en]

Candida species are the cause of 60% of all mycoses in immunosuppressed individuals, leading to similar to 150,000 deaths annually due to systemic infections, whereas the current antifungal therapies either have toxic side effects or are insufficiently efficient. We performed a screening of two compound libraries, the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology collection library, for anti-Candida activity based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. From a total of 844 drugs, 26 agents showed activity against Candida albicans. Of those, 12 were standard antifungal drugs (SADs) and 7 were off-target drugs previously reported to be active against Candida spp. The remaining 7 off-target drugs, amonafide, tosedostat, megestrol acetate, melengestrol acetate, stanozolol, trifluperidol, and haloperidol, were identified with this screen. The anti-Candida activities of the new agents were investigated by three individual assays using optical density, ATP levels, and microscopy. The antifungal activities of these drugs were comparable to those of the SADs found in the screen. The aminopeptidase inhibitor tosedostat, which is currently in a clinical trial phase for anticancer therapy, displayed a broad antifungal activity against different Candida spp., including Candida glabrata. Thus, this screen reveals agents that were previously unknown to be anti-Candida agents, which allows for the design of novel therapies against invasive candidiasis.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-86824 (URN)10.1128/AAC.01087-13 (DOI)000330637500053 ()
Available from: 2014-03-17 Created: 2014-03-11 Last updated: 2017-12-05Bibliographically approved
3. Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells
Open this publication in new window or tab >>Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells
2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, 12287Article in journal (Refereed) Published
Abstract [en]

Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal, colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen. Mast cells are present in tissues prone to fungal colonization being expectedly among the first immune cells to get into contact with C. albicans. However, mast cell-fungus interaction remains a neglected area of study. Here we show that human mast cells mounted specific responses towards C. albicans. Collectively, mast cell responses included the launch of initial, intermediate and late phase components determined by the secretion of granular proteins and cytokines. Initially mast cells reduced fungal viability and occasionally internalized yeasts. C. albicans could evade ingestion by intracellular growth leading to cellular death. Furthermore, secreted factors in the supernatants of infected cells recruited neutrophils, but not monocytes. Late stages were marked by the release of cytokines that are known to be anti-inflammatory suggesting a modulation of initial responses. C. albicans-infected mast cells formed extracellular DNA traps, which ensnared but did not kill the fungus. Our results suggest that mast cells serve as tissue sentinels modulating antifungal immune responses during C. albicans infection. Consequently, these findings open new doors for understanding fungal pathogenicity.

Place, publisher, year, edition, pages
Nature Publishing Group, 2015
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-106777 (URN)10.1038/srep12287 (DOI)000358152500001 ()26192381 (PubMedID)
Available from: 2015-08-20 Created: 2015-08-07 Last updated: 2017-12-04Bibliographically approved

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