PATHOGENIC FUNGI Flashcards
- What are the reasons for the dramatic increase in fungal infections during the past decades?
underlying diseases (cancer, AIDS)
- immunosuppressive therapy
- disturbance of the own microbiota (e.g. by broad-spectrum antibiotics)
Opportunistic fungal infections have risen dramatically in the past decades because of the growing number of susceptible persons. They are now by far the medically most important fungal infections.
Susceptible people could be those going through underlying diseases like cancer or AIDS, immunosupressive therapy or going through disturbance of the own microbiota (e.g. by broad-spectrum antibiotics).
- Which are the most important pathogenic fungi for humans?
Candida albicans and other Candida species
- Cryptococcus
- Aspergillus fumigatus
- What kind of infections can pathogenic fungi cause in humans
We have two types of fungal infections (mycoses):
- Superficial infections of the skin. These affect a large part of the human population, but they are not life-threatening. Microsporum, trichophyton and Epidermophyton.
- Systemic infections. Here the fungi will spread throughout the body and disseminate to internal organs. Such infections are severe and often deadly. Histoplasmosis.
- Opportunistic mycoses: infections caused by normally non pathogenic fungi that take advantage of the situation. They cause infection in weakened patients life underlying diseases, under immunotherapy therapy and those who have disturbed microbiota.
- Which characteristics of pathogenic fungi contribute to their virulence?
- adherence to host tissues (lectin-like adhesions, protein-protein interactions and covalent interactions with host proteins)
- Biofilm formation
- iron uptake mechanisms
- other mechanisms like hyphal growth and opaque-white switching
- How can pathogenic fungi acquire iron from the host?
Iron is an essential micronutrient for almost all organisms. It is insoluble in its oxidized form (Fe3+) and withheld from microbial pathogens by the host in storage proteins such as ferritin and transferrin. Pathogenic fungi have developed different strategies to obtain iron in host niches where it is restricted. And the way they get it is by reductive iron uptake.
- ) Extracellular reduction of Fe3+ to soluble Fe2+ by surface-localized iron reductases
- ) Reoxidization by a multicopper ferroxidase and internalization by a high-affinity Fe3+ permease
The high-affinity, reductive iron uptake mechanism is essential for the virulence of C. albicans. In addition, C. albicans can also transport heme from haemoglobin (presumably after lysis of erythrocytes) into the cell with specialized carrier proteins. It can also use heme that can be taking into the cell by carrier proteins.
In contrast, Aspergillus fumigatus, similar to many bacterial pathogens, synthesizes and secretes siderophores (small molecules that bind iron with high affinity and remove it from host proteins) and transports the iron-bound siderophores back into the cell to obtain iron from the host. Siderophore-mediated iron uptake is essential for the virulence of A. fumigatus.
- What are the different morphologies of Candida albicans and how do they affect its interactions with the host?
Mofphologies: hyphae form and yeast form.
The switch from the budding yeast morphology to filamentous growth is important for the ability of C. albicans to invade tissue. Hyphae express genes encoding additional adhesins, secreted enzymes, and also a toxin (candidalysin) that damages host cells.
The fact that it can switch from yeast form to hyphae form is really special, and C. albicans can do this. Hyphae is the invasive form. They express genes that are not expressed in the yeast form. They also secrete enzymes that degrade certain tissue structures, even toxins.
Hyphal growth is also induced after phagocytosis of yeast cells by macrophages. This enables C. albicans to escape from and kill the macrophage (if the macrophage is not faster and kills its prey). This is induced by many environmental factors that will induce this switch. For example the increase of temperature. Or a change in pH. It depends on a combination of signals that will make them decide to switch its form.
C. albicans mutants that are defective in hyphal growth are avirulent. The pictures show liver sections after an experimental infection of mice. The wild-type strain forms hyphae that invade into the tissue, whereas a nonfilamentous mutant in which the genes encoding the transcription factors Cph1 and Efg1 (you do not need to remember the name) were deleted remains on the surface. They do not enter into the tissue. The transcription factors also control many other things, but you can see this correlation.
- Which drugs are used to treat fungal infections and what is their mechanism of action?
Flucytosine:
This is the chemical stucture of flucytosine. It is an analog of the base cytosine that contains a fluor atom at C5 of the pyrimidine ring.
5FC cannot freely diffuse into cells and has to be taken up by a cytosine permease.
Flucytosine is a prodrug and not toxic by itself. It is first deaminated to 5-fluorouracil (5FU) and then converted to the nucleotide 5-fluor uridine monophosphate (5FUMP).
5FUMP is further phosphorylated to 5FUTP which, just like UTP, is incorporated into cellular RNAs. The production of aberrant RNAs is toxic to the cells. 5FUMP is also converted to 5dUMP, which irreversibly inhibits thymidylate synthase and thereby DNA synthesis.
Unfortunately, fungi rapidly develop resistance to flucytosine by preventing its transport into the cell (mutations in cytosine permease) or its intracellular conversion to 5FUMP (mutations in cytosine deaminase or uracil phosphoribosyltransferase). Such mutations are not problematic for the fungi, because they can synthesize nucleotides de novo and do not depend on the salvage pathway.
Resistance to flucytosine is so common that it is used only in combination with other drugs (e.g. for treating cryptococcal meningitis).
But why is flucytosine not toxic to our own cells?
Our cells do not contain cytosine deaminase and do not convert the prodrug into its toxic form.
Amphotericin B:
This is the stucture of amphotericin B, the only polyene that is used to treat systemic fungal infections (others, such as nystatin, are used to treat superficial mycoses).
Amphotericin B sequesters ergosterol in fungal membranes and thereby impairs membrane function. It also forms pores in the membranes, which causes cellular leakage. It is a fungicidal drug (it kills the fungal cells).
Resistence to polyenes is rare, but is observed in some fungi (Aspergillus terreus, Candida lusitaniae, Candida auris).
Alterations in sterol biosynthesis (decreased ergosterol content) can result in reduced susceptibility to amphotericin B.
The main problem with amphotericin B is that its selectivity for ergosterol is limited. Less binding sites for the target maybe. The drug is also highly toxic to human cells and has severe side effects.
Echinocandins:
Echinocandins are cyclic hexapeptides with a lipid side chain. Besides caspofungin, which is shown here, anidulafungin and micafungin are in clinical use.
The echinocandins inhibit the enzyme β-1,3-glucan synthase, which synthesizes the major cell wall component β-1,3-glucan from UDP-glucose.
Echinocandins are active against Candida and Aspergillus, but not against Cryptococcus.
Mutations in β-1,3-glucan synthase that abolish drug binding are the main cause of acquired echinocandin resistance.
Azoles:
Azole antifungals contain an azole ring with two (imidazoles) or three (triazoles) nitrogen atoms. The most widely used drug of this class is the triazole fluconazole.
Azole antifungals inhibit ergosterol biosynthesis by targeting the enzyme sterol 14α-demethylase.
This results in ergosterol depletion and production of toxic sterols from the accumulated lanosterol.
Fluconazole is a fungistatic drug that inhibits growth, but does not kill the fungal cells. This gives the pathogens the opportunity to develop resistance.
C. albicans (but also other Candida species and pathogenic fungi) can acquire fluconazole resistance by various mechanisms. The most important mechanisms are illustrated in the following slides.
- How can fungi become resistant to antimycotic drugs?
- resistance mechanisms:
Fluconazole is taken up into the cells by an unknown transporter and binds to its target enzyme sterol 14α-demethylase (Erg11, encoded by the ERG11 gene), thereby inhibiting ergosterol biosynthesis in susceptible cells.
a. Mutations in the target enzyme result in reduced drug binding
b. Overexpression of the ERG11 gene increases the amount of enzyme. Maybe there is not enough drug to inhibit this.
c. Overexpression of genes encoding multidrug efflux pumps (Cdr1/Cdr2 or Mdr1) reduces the intracellular drug concentration. ABC binding casettes. Major facilitators don’t use ATP, but the proton gradient to take up proteins into the cell along the concentration gradient, and also to take things out of the cell.
Highly drug-resistant C. albicans isolates usually exhibit a combination of several resistance mechanisms. This is illustrated in the following slides, which show the molecular analysis of serial C. albicans isolates from different infection episodes in an AIDS patient suffering from oropharyngeal candidiasis. The initial infections were successfully treated with fluconazole, but the strain (which was not eradicated by the drug) became increasingly resistant over time, and the fluconazole therapy of subsequent symptomatic infections eventually failed. (look at the two following slides which show the examples and explanations). This is all done with the same strain.
- How is drug resistance in Candida albicans transcriptionally regulated?
Slide 111.
