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Candida albicans adaption to host microenvironments drives immune evasion
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
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. Here we describe how mast cells acted as tissue sentinels and modulated initial antifungal immune responses. Mast cells response was able to reduce fungal viability and signaled for neutrophil infiltration to the tissue. Upon chemokine sensing circulating neutrophils are rapid infiltrating to the mucosal to help fight infection. A high number of infiltrating cells coupled with the formation of multicellular structures such as biofilm comes with induction of hypoxic and anoxic micro niches. We found that a persistence anoxia hampered neutrophil responses by affecting fungi sensing and consequent antifungal due to cell wall masking. Adaption to low oxygen seems is important for a successful host infection. Hypoxic and anoxic environments do not allow neutrophils to efficiently produce ROS. Neutrophil oxidative burst is essential for antifungal activity and many fungal pathogens evolved antioxidative factors to mediate survival during infection. We reasoned that targeting of fungal redox balances could be a new therapy approach. We have tested tempol, a redox-cycling nitroxide Tempol as a new antifungal drug. Tempol proved an efficient compound in our testing. We found that Tempol affected fundamental pathways for fungal homeostases such as glycolysis and steroid biosynthesis. Additionally, Tempol helped curve fungal infectivity in a mouse model and leads for an enhanced immune system cytokine profile in human blood. The results obtained proposed tempol as a valid new antifungal compound and open new opportunities for the future development of therapies. Efficient antifungal therapies are still urgent since only 6 classes of antimycotics exist and all with few restricted fungal targets. Since primarily fungal infections affect patients with other immunosuppressive conditions, which are undergoing treatment, we reasoned that repurposing drugs could offer clinical benefits. We performed a screening of two US Food and Drug Administration (FDA)–approved compound libraries for compounds with anti-Candida activity. From 844 drugs, 26 agents showed activity against C. albicans. We identified 7 new off-target drugs all with potent anti- C. albicans activity. The use of these new drugs could be prophylactic or to treat both conditions simultaneously offering, therefore the intended benefit.

Overall, in this thesis work, we have focused on the sensing clearing and management of fungal pathogens. These findings open new doors for understanding better fungal pathogenicity and purpose valid new antifungal compounds that pave the way for future development of therapies.

Abstract [sv]

Den mänskliga kroppen är ständigt utsatt för angrepp från mikrober i vår miljö. Varje dag träffar vår kropp på organismer som potentiellt kan infektera oss, så kallade patogener så som bakterier, virus, parasiter och svampar. Det är immunsystemets uppgift att befria oss från oönskade mikroorganismer. Vårt immunförsvar är mycket effektivt vilket framgår av att trots den ständiga närvaron av mikroorganismer så blir vi sällan allvarligt sjuka. Men, människor med nedsatt immunförsvar kan utveckla allvarliga infektioner från opportunister patogener så som svampen Candida albicans. C. albicans är en viktig orsak till sjukdom och död över hela världen. Syftet med denna avhandling är att bättre förstå samspelet mellan dessa svampar och vårt immunförsvar. Vi fokuserar på det medfödda immunförsvaret och försöker identifiera de delar som är nödvändiga för kontroll under en infektion. Vi studerar också hur svamparna utvecklar mekanismer för att möta värdorganismens försvar och vad som reglerar dessa mekanismer. Det medfödda immunförsvaret finns i hela kroppen och består bland annat av vävnader - huden och slemhinnorna - och en mängd olika celltyper. Mastcellerna är en del av det medfödda immunförsvaret och finns utspridda på kroppens ytor mot omvärlden. Främst finns de i huden, tarmens slemhinnor och luftvägarna och det är också här som patogena svampar förekommer i kroppen. Mastcellerna är därför strategiskt placerade för att snabbt kunna möta infekterande mikroorganismer. Här har vi visat att mastceller är kroppens vaktposter och mycket viktiga vid svampinfektioner, men de kan bara tillfälligt kontrollera C. albicans. Mastcellerna aktiveras när de möter C. albicans och släpper omedelbart ut inflammatoriska mediatorer som är lagrade i cellernas granula. Denna degranulering kan också rekrytera fler immunceller så som neutrofiler. De kan även släppa ut strukturer som kallas extracellulära fällor (MCETs), som fångar in inkräktarna för nedbrytning. Med dessa metoder kan Mastcellerna ta upp och oskadliggöra invaderande mikroorganismer, men här har även C. albicans utvecklat ett eget försvar mot Mastcellerna. När C. albicans tas upp av mastcellerna kan de undkomma avdödning och utnyttjar då istället den skyddade intracellulära miljön för döda mastcellerna inifrån.

Neutrofiler är också en del av det medfödda immunförsvaret och de är den vanligaste typen av vita blodkroppar. Dessa celler cirkulerar i blodet men reagerar snabbt på hot och migrerar då från blodet in i vävnaden där bekämpandet av svamparna sker. De är oftast först på plats vid en infektion.

I det andra projektet har vi tittat närmare på neutrofilers försvar mot C. albicans infektioner och hur detta skiljer sig mellan syrerika och syrefattiga miljöer. Vi fann att neutrofilernas försvarsförmåga är kraftigt minskad under syrefattiga förhållanden. Både formationen av neutrofila extracellulära fällor (NETs) och även fagocytering, eller förtäring är kraftigt nedsatta under dessa förhållanden och detta kan ha en stor betydelse för immunförsvarets förmåga att bekämpa C. albicans infektioner. Bekämpandet av mikroorganismer är en aktiv process, driven av neutrofilernas snabba migration från blodet ut till vävnaden där mycket av syret förbrukas. I syrefattiga miljöer kan C. albicans också ändra cellväggens komposition och det ändrar i sin tur hur immunförsvaret reagerar mot dem.

Produktion av reaktiva syreradikaler är en av de mest påverkade försvarsmekanismerna i syrefattig miljö. Neutrofilerna använder reaktiva syreradikaler som en robust och effektiv kontroll-mekanism mot svamppatogener. I det tredje projektet undersöker vi om vi kan förhindra spridning av svampceller genom att påverka redoxpotentialen. Vi använder Tempol som är ett ämne med bevisad påverkan på metabolism och cellväggens komposition och har även inflammatorisk effekt. Behandling av svampcellerna med Tempol gav en framgångsrik anti-Candida effekt som karakteriserades med hjälp av RNA-sekvensering och metaboliska tester.

Idag finns det ett mycket stort behov av nya och bättre läkemedel mot systemisk svampinfektion. Opportunistiska svampinfektioner är ett stort hot mot patienter med nedsatt immunförsvar e.g patienter med leukemi eller som fått organtransplantationer. I projekt nummer fyra tänker vi oss att dessa patienter kanske skulle kunna får endast ett läkemedel mot både sjukdomarna. Därför har vi undersökt 844 godkänt-FDA läkemedlen för effekt mot C. albicans. Vi identifierat 7 nya molekyler med god effekt mot C. albicans. Vi har karakteriserad dessa läkemedlen och visat att de är lika effektiva som kommersiellt tillgängliga läkemedel.

Sammanfattningsvis visar våra resultat nödvändigheten till en fördjupad förståelse om immunceller och svampars samspel i mikromiljöer, så som de syrefattiga. En mer detaljerad förståelse kan därmed bidra till att utveckla bättre diagnostiska och terapeutiska verktyg för svampinfektioner.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2018. , p. 64+4
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1977
Keywords [en]
fungi, candida albicans, neutrophils, mast cells, immune evasion, mycoses, infectious diseases
National Category
Basic Medicine
Research subject
Immunology; Infectious Diseases; Molecular Immunology
Identifiers
URN: urn:nbn:se:umu:diva-151598ISBN: 978-91-7601-919-1 (print)OAI: oai:DiVA.org:umu-151598DiVA, id: diva2:1246321
Public defence
2018-10-05, Major Groove, Universitetssjukhuset 6L, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2018-09-14 Created: 2018-09-07 Last updated: 2018-09-19Bibliographically approved
List of papers
1. 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, article id 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: 2018-09-07Bibliographically approved
2. Evasion of immune surveillance under low oxygen enhances Candida albicans survival
Open this publication in new window or tab >>Evasion of immune surveillance under low oxygen enhances Candida albicans survival
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Human colonizers have evolved to sense and adapt environmental cues and nutrient availability. Oxygen is a constantly changing environmental parameter within different host tissues upon different types of infection. We describe how C. albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of neutrophils to the site of infection contributes to a low oxygen milieu. A persistent low oxygen milieu did not affect viability nor metabolism of polymorphonuclear leukocytes (PMNs), however, disturbed anti-Candida responses. PMNs were not able to efficiently phagocytize, produce ROS or release extracellular DNA traps. This failure to respond was caused by C. albicans β-glucan cell wall masking upon exposure to low oxygen which hindered PAMP sensing by dectin-1. Overall, this contributed to immune evasion which in turn enhanced fungal survival. The effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen condition. Finally, low oxygen adaptation increased the pathogenicity and successful colonization of C. albicans in vivo which we confirmed using a Caenorhabditis elegans infection model. 

National Category
Basic Medicine
Research subject
Immunology; Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-151595 (URN)
Available from: 2018-09-07 Created: 2018-09-07 Last updated: 2018-09-07
3. Stable Redox-Cycling Nitroxide Tempol has Antifungal and Immune-modulatory Properties
Open this publication in new window or tab >>Stable Redox-Cycling Nitroxide Tempol has Antifungal and Immune-modulatory Properties
Show others...
2019 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1843Article in journal (Refereed) Published
Abstract [en]

Invasive mycoses remain underdiagnosed and difficult to treat. Hospitalized individuals with compromised immunity increase in number and constitute the main risk group for severe fungal infections. Current antifungal therapy is hampered by slow and insensitive diagnostics and frequent toxic side effects of standard antifungal drugs. Identification of new antifungal compounds with high efficacy and low toxicity is therefore urgently required. We investigated the antifungal activity of tempol, a cell-permeable nitroxide. To narrow down possible mode of action we used RNA-seq technology and metabolomics to probe for pathways specifically disrupted in the human fungal pathogen Candida albicans due to tempol administration. We found genes upregulated which are involved in iron homeostasis, mitochondrial stress, steroid synthesis, and amino acid metabolism. In an ex vivo whole blood infection, tempol treatment reduced C. albicans colony forming units and at the same time increased the release of pro-inflammatory cytokines, such as interleukin 8 (IL-8, monocyte chemoattractant protein-1, and macrophage migration inhibitory factor). In a systemic mouse model, tempol was partially protective with a significant reduction of fungal burden in the kidneys of infected animals during infection onset. The results obtained propose tempol as a promising new antifungal compound and open new opportunities for the future development of novel therapies.

Keywords
antifungal activity, redox active, immunomodulators, candidiasis, Candida albicans, Candida glabrata
National Category
Microbiology in the medical area
Research subject
Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-151596 (URN)10.3389/fmicb.2019.01843 (DOI)000481763300001 ()31481939 (PubMedID)
Funder
Swedish Research Council, 2014-02281The Kempe Foundations, 1453
Note

Originally included in thesis in manuscript form 

Available from: 2018-09-07 Created: 2018-09-07 Last updated: 2019-10-11Bibliographically approved
4. Antifungal Application of Nonantifungal Drugs
Open this publication in new window or tab >>Antifungal Application of Nonantifungal Drugs
Show others...
2014 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 58, no 2, p. 1055-1062Article 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: 2018-09-12Bibliographically approved

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