Osmiotrophs
Organisms that secrete enzymes into the surrounding substrate and absorb digested nutrients (fungi)
Conidia
vegetative spores formed by fungi
Asexual life cycle of molds
Dispersal -> conidia -> (swelling and apical growth) -> Germling -> (elongation and branching) -> hyphae -> mycelium -> condidophore (conidia-carrier - releases spores)
fungal mycobiota of the human skin
Mostly Malassezia species (11 identified)
fungal mycobiota of the human lung
Pneumocystis jiroveci
Structure of fungal cell wall
from inner-most to outer -most:
plasma membrane Chitin layer B-1,3 glucan B-1,6 glucan outermost layer differs between species, but often contains mannan (mannoproteins in Candida)
90% of the cell wall is carbohydrate
Clec7A
Dectin-1, recognizes B1,3 glucan
During what process are fungal antigens most likely to be exposed?
During the budding of the yeast, underlying proteins and B-glucans are exposed that can be targeted antigenically. Otherwise, they are hidden beneath the carbohydrate cell wall.
How does the immune system differentiate between shed (soluble) and particulate B-glucan from fungi?
Dectin-1 requires cross-linking for activation, as it is blocked by CD45 phosphatase.
Mutations in which genes lead to redisposition to candidiasis?
Dectin-1 CARD9 Phagocyte oxidase IL-23R STAT-3 STAT-1 IL-17 IL-17RA or, autoantibodies to IL-17, IL-22
What are the two layers of sensing of C albicans on the skin?
In the epidermis, yeast forms of C albicans are sensed by Langerhan cells, which use Dectin-1 to initiate Skin Th17 responses, while dermal (deeper) hyphal infections activate CD103+ DCs that use TLR2 to initiate Th1 responses.
Heterokaryon Incompatibility
When fungal hyphae fuse, they can share their genetic material. If certain loci differ between the two ‘parent’ strains, they must be compatible hets. If they are uncompatably different, the cell undergoes regulated cell death.
What is the function of BIR1 in Aspergillus and what seems to activate its expression?
BIR1 is a negative regulator of programmed cel death, ie., its expression leads to increased resistance to cell death. It seems to be activated in the context of oxidative bursts conferred by neutrophils.
Blocking of BIR1 leads to increased fungal clearance.
Fungal antigen that elicits the strongest immune response
B-1,3 glucan (is even stronger when simultaneously detected with mannan)
Non-lytic expulsion
An actin-dependent mechanism used by microorganisms to escape from within a macrophage. Both the macrophage and the microorganism are viable after expulsion takes place. Non-lytic expulsion is also known as vomocytosis.
What is unique about the synthesis of B1,3 glucans and chitin in the fungal cell wall?
Polysaccharides such as chitin and glucan are synthesized at the plasma membrane (PM) by transmembrane enzymatic complexes that are targeted to the PM in an
inactive form via secretory vesicles and then activated after insertion into the PM (see below). This is in contrast to mannans and other glycoconjugates that are
synthesized in the endoplasmic reticulum and Golgi, where they may be conjugated to cell wall proteins, and then brought to the cell wall by the classical secretory
route via secretory vesicles
What is the substrate used by synthases to insert nascent polysaccharides into the cell wall of fungi?
UDP sugars
What is the next step for a nascent polysaccharide that is inserted into the fungal cell wall?
In the cell wall, polysaccha-
rides can then hydrogen-bond together or be cross-
linked or branched by enzymes that reside in the cell wall
What is the substrate for chitin synthesis in fungi cell wall?
UDP-N-acetylglucosamin
What is the makeup of chitin in fungal cel wall?
Chitin is composed of linear chains of β-(1,4)
N-acetylglucosamine and represents the most ancestral
structural polysaccharide in the fungal cell wall
target of the echinocandin family of antifungal drugs
The Fks/Gsc subunits of the PM-bound glucan synthase in fungi that makes B1,3 glucan chains for insertion into the cell wall
PfAP2-G
“Master switch” for sexual differentiation in Plasmodium falciparum/ (“Pf” stands for the organism, AP2-G is the transcription factor)
Malaria parasite genus/species
Plasmodium falciparum
How is sexual differentiation controlled in P. falciparum at an epigenetic level?
heterochromatin maintenance at the ap2-g locus keeps P falciparum at asexual replication. When heterochromatin maintenance is impaired, low level ap2-g expression can induce its own promotion through a positive feedback loop, inducing sexual differentiation.
Which phospholipid precursors suppress sexual commitment in P. falciparum?
lysophosphatidylcholine (lysoPC) and choline, both precursors to phosphocholine headgroup.
SAM / SAH
S-adenosylmethionine (SAM) and S-adenosyl homocysteine (SAH) - SAM is the major source of methyl groups for transferring via methyltransferases, and is used by both PMT for P-cho synthesis and histone methyltransferases for heterochromatin maintenance.
Which phospholipid accounts for the vast majority of phospholipid increase during a P falciparum infection of RBC?
Phosphatidylcholine (PtdCho)
Which are the two pathways that P falciparum can de novo synthesize phosphatidylcholine?
Always derived from phosphocholine, which can be either generated from serine via phospho-ethanolamine or scavenged from extracellular choline and LysoPC.
phosphoethanolamine methyltransferase (PMT)
generates phosphocholine by transferring three consecutive methyl-groups from the methyl donor S-adenosylmethionine (SAM) onto P-etn
How is PMT regulated in P falciparum?
High levels of phosphocholine (PMT’s product) inhibit PMT activity/levels.
SAMS
SAM synthase, malaria parasite enzym that is the sole producer of SAM from methionine.
3-DZA (3-deaza-adenosine)
potent inhibitor of SAH hydrolase, which converts SAH into adenine and homocysteine (treatment with 3-DZA will lead to increased levels of SAH)
Which histone modification is the most potent regulator of gene expression in P falciparum?
H3K9me3 (silencing)
How does low SAM levels in P falciparum lead to sexual differentiation commitment?
SAM is the only source for methyltransferase enzymes that are needed for heterochromtin maintenance. In the absence of SAM substrate, heterochromatin cannot remain faithfully methylated, and low level gene expression results. This includes the typically silenced ap2-g locus, which promotes its own expression and leads to sexual commitment in P falciparum.
Which two survival processes do sexual and asexual reproduction in P falciparum contribute to?
asexual reproduction is required to maintain sufficient levels of parasite within the host, whereas sexual production of gametocytes is required for the transmission to mosquito hosts.
schizogony
replicative process of parasites wherein continuous rounds of nuclear repli- cation produce a single multi-nucleated cell that gives rise to 16–30 new merozoite progeny through a single round of cell division near the end of the lytic cycle
What recognizes H3K9me3 histone modifications?
heterochromatin protein 1 (HP1)
How does LysoPC alter sexual commitment in P falciparum?
LysoPC suppresses sexual commitent. LysoPC acts as a substrate for phosphatidylcholine synthesis, freeing up SAM (used in the alternative phosphatidylcholine pathway) as a substrate for histone methyltransferases that can maintain heterochromatin.
Rodent malaria genus
Plasmodium berghei
Why is rodent Plasmodium resistant to the sexual commitment suppression effects of LysoPC?
Rodent Plasmodium species have lost PMT gene, meaning that they are unable to utilize LysoPC as a substrate for phosphatidylcholine. To compensate, these parasite have an expansion of transporters that allow for extracellular phosphatidylcholine import.
How do high SAH levels suppress sexual committment in P falciparum?
High levels of SAH generally inhibit meyhtltransferase activity, which leads to loss of heterochromatin maintenance in the genome.
What is the medicine and biological way to define parasites?
Medicine: Pathogen that is not a bacterium, virus, or fungus.
Biological: An organism living in association with another (usually larger) organism (the host) benefitting at the host’s expense.
What are the four/five types of motility in protozoa?
Flagellated amoeboid gliding ciliated non-motile
Definitive Host for parasite
Definitive host is the host wherein the parasite undergoes sexual replication. This is, for example, mosquitos for malaria.
Intermediate Host for parasite
Intermediate hosts are hosts wherein the parasite does not undergo sexual replication, but it is required for other life stages that are crucial for the life cycle of the parasite.
Accidental Host for parasite
Accidental hosts are hosts wherein no integral portions of a parasite’s lifecycle occur; the parasite will likely die out within that host. Brain-eating amoeba in humans is one example
What are different ways in which parasites can cause disease?
Immune evasion (malaria trying to avoid the spleen leads to blood clots, compared to toxoplasma, which activate teh immune response purposefully)
Tranmisssion (diarrheal parasites need the disease for transmission)
Immune mediated (think COVID for example)
Metamonads
Trichomas and Giardia.
Trichonomas vaginalis
Single stage, human-specific
lack mitochondria, have hydrogenosome instead (uses pH for energy)
Evades immune response by antibody proteases.
Giardia lamblia
single mammalian host (beaver), produces very stable cysts that pass with the feces.
Antigenetic variation of surface proteins.
Adheres to intestinal lining using flagella to create suction.
Kinetoplastids
Trypanosoma and Leishmania
2nd most medically relevant
large family, huge range,
single mitochondrial organelle called kinetoplast
Kinetoplast
Single mitochondrion in kinetoplasts that is located at the base of the flagellum. kDNA is very large catenated disk of dozens of maxi-circles. During replication, it has to be untangled, replicated, and re-tangled. High RNA editing (each minicircle contribute to the editing of transcripts that comes from the maxi-circles)
Polycistronic transcription
Whole chromosome is transcribed at once and transcript is cut and polyadenylated individually. Hardly any epigenetic regulation on the DNA level.
Why is ‘sleeping sickness’ almost always lethal?
African trypanosomes have extremely high antigenic variation (VSG protein), and often sweep their antigens backwards and degrade bound antibodies. Thus, the adaptive response can never ‘catch up’
Only one VSG gene is active at any time
Leishmania target cell in humans
Live inside macrophage lysosomes (intracellular pathogen)
Transmitted by sandfly.
Apicomplexa parasites
Plasmodium, Toxoplasma, Cryptosporidium
Most important group medically. Every species are parasitic, all obligate intracellular at one point.
What is the unique biology of the Apicoplast?
It is derived from two endosymbiotic events and thus has four membranes surrounding it - first was a cyanobacteria engulfed by an algae, followed by the engulfment of both by an apicomplex parasite
This parasite is therefore susceptible to some antibacterial antibiotics due to this organelle.
What is unique about the intracellular invasion of apicomplexa parasites?
They will invade a cell membrane and invaginate the plasma membrane, while using a ‘molecular sieve’ to remove any transmembrane proteins - thus, the resulting vacuole is ‘naked’ and cannot interact with any intracellular proteins (prevention of lysosomic fusion)
What is one reason that toxoplasma has such a broad host range?
Toxoplasma inserts its own receptor for invasion into the host cell - therefore, it does not require a matching host cell surface receptor
Schizogony
Type of replication in malaria parasites. Nuclei continue to divide along side essential organelles without division of the plasma membrane. After there are ~30 nuclei, all separate into individual cells (somehow)
Dormant hypnozoites
Certain malaria parasites (P. vivax and P. ovale) have dormant liver stages called hypnozoites that can reactivate and cause relapse up to 10 years later after initial infection
How long is the RBC lytic cycle for malaria parasites?
48 hours. This causes the periodic fever spikes - synchronous bursts causes bursts of cytokines in the blood. The fever response controls the synchronicity.
What is the quality control of RBCs conferred by the spleen?
The spleen filters RBCs and ensures that they are sufficiently pliable in order to squeeze through the capillaries. RBCs with tons of parasites obviously fail this test, so malaria must find a way to avoid the spleen.
How does malaria avoid being cleared by the spleen?
Malaria makes surface proteins that are trafficked to the plasma membrane of the iRBC that interact with endothelial cells - cytoadhesion - that prevents their circulation. This is where most of the pathogenesis of malaria arises from, apart from anemia from hemolysis.
Why does toxoplasma want to activate the immune system?
Toxoplasma has a highly invariable surface antigens - infects recruited DCs and changes DCs to be more motile to disseminate to other tissues. It ‘wants’ to be controlled by the immune system - forms cysts when targeted by the immune system.
Tachyzoite
Toxoplasma form - fast replication and host cell lysis (1 day), resulting in tissue destruction
Disseminates by infecting monocytes
Invariant SAG1 surface coat
SAG1 is highly immunogenic
Bradyzoite
Toxoplasma form
Slow replication, long term stability (months)
Forms protective cyst wall
No SAG1 expression
What triggers the tachyzoite to cradyzoite conversion in toxoplasma?
Nitric oxide, TNFa, IFNg
What determines where in the world that we can find malaria
- Definitive host range (mosquitos)
- High enough temperature for replication to be fast enough in mosquitos before they die
- Infected people
Tuft cells
A very rare chemo-sensing epithelial cell type that is spread throughout the gastrointestinal and respiratory tracts. Tuft cells secrete the alarmin IL-25 in response to parasitic infections and perhaps other stimuli and are thought to be important sentinels for and innate initiators of the type 2 response.
Goblet cells
A specialized, mucus-secreting epithelial cell found throughout the gastrointestinal and respiratory tracts and across mucosal surfaces. Goblet cells and their secreted mucins are critical for protecting mucosal surfaces, maintaining barrier integrity and removing large extracellular irritants and debris.
Alarmins
considered to be some of the earliest of the type 2 cytokines to be secreted following tissue damage, and they have the capacity to both initiate and propagate a fulminant type 2 immune response
Include IL-25, IL-33, and thymic stromal lymphopoietin (TSLP)
What are the specific ligands from microbial communities?
TLR and NLR ligands Adenosine SCFAs PSA SlpA metabolites
ILC2 phenotype in the lung
Lin-CD25+CD127+IL-33R+
What do all kinetoplastids have in common in their transmission?
All kinetoplastids are transmitted by an insect host at some point in their lifecycle.
What is unique about trypanosome genetics (besides kinetoplast)?
Genome is polycistronic - has one promoter that encodes the entire genome, which is then spliced and polyadenylated by other means.
How do malaria parasites sense that they are in a host fly?
drop in temperature, xanthurenic acid (in the gut of flies), change in pH.
VSG in trypanosomes
Variant Surface glycoprotein that covers the entire outer surface of trypanosomes. Highly variable and used to thwart the immune system by expressing different types.
How is antigenic diversity maintained in a chronic trypanosome infection?
There are a large reservior of silent. VSG genes. Many are encoded on small, circular minichromosomes (~100) and many more are encoded sub-telemeromically (~1000). These genes can be converted in a similar manner to V(D)J recombination where different parts of different VSG genes are spliced together, or a new VSG gene is switched out altogether.
What is the only point of endo- and exocytosis in trypanosomes?
The flagellar pocket (targeted by innate immune system ApoL1)
SRA
Serum Resistance Associated gene evolved by some species of trypanosomes to prevent ApoL1 from lysing their lysosome, thus being able to infect humans.
Eflornithine
failed cancer drug that kills trypanosomes efficiently. PRoduction was ceased in 1995 by Aventis. In 2001, they resumed production because it was discovered that it was also a hair removal treatment ($$$)
Artemisinin
Discovered by Tu Youyou (Nobel Prize). Malaria treatment drug that targets free iron (digested by malaria from hemoglobin) Iron bound by hemoglobin in the blood is not affected. Has a very short halflife (90 minutes) that some parasites have evolved resistance to through alteration of their cell cycle length.
Digestive vacuole in malaria parasites
Vacuole wherein hemoglobin is digested, freeing up amino acids for the parasite. The iron that is released is crystallized into hemozoin, which is targeted by quinine and other treatment drugs and stored in the digestive vacuole (also makes them magnetic).
Hemozoin
crystallized iron from digested hemoglobin in malaria infections (stored in digestive vacuole of malaria)
Why can only immature malaria iRBCs be identified in the circulation
More mature iRBCs are swelled and distorted and thus need to avoid clearance by the spleen, thus they cytoadhere to the endothelium.
PfEMP1
Surface protein receptor that is expressed on the PM of iRBCs and binds to endothelial receptors in order to cytoadhere. Malaria is able to undergo significant antigenic variation of this protein to avoid immune detection.
var genes
genes encoding different forms of PfEMP1 proteins in malaria parasites. Under epigenetic control (so, no recombination).
uORF
upstream reading frame of a particular var gene in malaria that encodes for a PfEMP1 protein that targets the placenta (var2csa). uORF disallows transcription of var2csa unless in a pregnant woman. Also, var2csa serves as a ‘node’ to switch to between variations.
How does adding additional choline lead to changes in var gene expression in malaria parasites?
Choline is upstream of the CDP pathway, which uses the CDP pathway for lipid synthesis without the need for SAM for methyl groups. This increases the SAM pool that is available for the methylation used by HMTs to trigger histone modification and gene expression.
