Fungi and protozoa

Fungi

Fungi is a separate kingdom, they are ALL Eukaryotic organism. Based of the morphology, we can classify two types of fungi: filamentous or hyphal form (MOLD) and single celled or budding form (YEAST). We can classified fungi also in another classification based on the pathology that are able to cause in human: in this case we can have mold and yeast, but also YEAST LIKE FUNGI (ex. Candida albicans) and DIMORPHIC FUNGI. In general, yeasts are all unicellular, molds are multicellular. Another characteristic is that all fungi lack chloroplast. They can be:

Saprophytes: they require dead or decaying organic matter;

Symbionts: they live in relationship with other living organisms that derive mutual benefit;

Commensals: they interact with other living organisms taking advantage of nutrients without causing damage to the host;

Parasites: they live at the expense of a host to whom they cause harm.

Other characteristic and consequent classification:

Mode of nutrition: Fungi are organotrophic heterotrophs, mostly Fungi are saprophytic, and some are parasitic (as we mention before).

Environment: Fungi grow best in acidic environment (tolerate acidic pH). Fungi can tolerate high sugar concentration and dry condition. Most of the fungi are Obligate aerobes (molds) and few are facultative anaerobes (yeasts). Optimum temperature of growth for most saprophytic fungi is 20-30°C while 30-37°C for parasitic fungi. Growth rate of fungi is slower than that of bacteria.

They have two different types of reproduction:

sexual reproduction (very few) and sexual spores are completely different, they have a lot more infrastructure. We can classify different types of sexual spores due to their formation and structure.

asexual reproduction, (spore named conidi). Asexual reproduction could happen with budding (tipo gemmazione) or we can have a fission.

The main characteristic of fungi cell membrane is the presence of Ergosterol that is slightly different from Cholesterol. It’s important because is a drug target. Some fungi contain a polysaccharidic capsule, similar to the one of the bacteria and is a virulence factor and is typical of only one fungus that is the Cryptococcus. This is the schematic representation of the cell wall. The cell wall is made with layer of different molecules. Mainly we have the chitin microfibrils. So we have chitin that is a polymer of N-acetylglucosamine, then we have the glucan, that is a polymer of the D-glucose, and the mannan. Some of the molecules that are present at the level of the cell wall are important for adhesive molecules. So, the fungi used these molecules to recognize the receptors that are present in the host cells and to adhere to the host cells. These are mainly the peptidomannans and galactomannans. Based on the structure, we can classify fungi in yeast, mold and dimorphous.

Yeasts are always unicellular. They are characterized by a thallus that is made by isolated rounded cells, rounded or oval cells that are called blastospores.

The molds are multicellular and are composed of Hyphae which may have cross-walls or septa or may lack septa (coenocytic). Macroscopically appears as cottony/woolly/velvety/granular growth on the culture media. Microscopically appears as thread-like filamentous Hyphae (2-10 microns) seen in tissues and in culture. Examples: Aspergillus fumigatus, Penicillium notatum.

The dimorphous are called in this way because they can be in the form of yeast or mold based on the temperature shift, so they are in the saprophytic phase (mold) at 25°C, but they are in the parasitic phase (yeast) at 37°C. Example: Histoplasma capsulatum.

Yeast cellular fungi are unicellular fungi that reproduce by budding or by fission. They macroscopically appear as pasty colonies on the culture media, microscopically appears as spherical or oval structure; filamentous structures may be seen due to the chains of elongated budding cells joined end to end (pseudo hyphae) in tissues and in culture. An example is candida albicans.

The filamentous fungi are multicellular, they are form by hyphae. More hyphae together can form the mycelium. The vegetative mycelium is the one that is important for the nutrition and it’s important for the collection of nutrients. The aerial mycelium is the outside part. The spores are in aerial hyphe.

Where we can find the fungi? We say that can be related to human symptoms, means that we live with them and they do not cause any pathology in humans, at least in some specific places. We can find fungi more or less everywhere. They can infect several niches in the human host. Some fungi, like Aspergillus, can infect more parts of the body, like brain, lungs, bones. Another common fungus is Candida Albicans, which can be present in genitals, nails, skin, eyes, esophagus. Another common fungus is Cryptococcus, which can infect brain, skin and lungs. These types of microorganisms can cause different pathologies, some of them can be very critical like aspergillus fumigatus, candida auris and albicans, cryptococcus neoformans. Some of them are present in the environment (Green spot), some of them are commensals (orange). When our immune system is compromised, candida albicans become pathogenic. Fusarium, histoplasma, candida tropicalis and candida parapsilosis are considered at high risk. The ones that are considered medium risk are cryptococcus gattii, pneumocystis ecc. Disease in humans is an accidental event in the life cycle of almost all pathogenic fungi. They can cause:

EXOGENOUS MYCOSIS: the infectious agent comes from the environment (spores and conidia). Contact with skin, hair, wounds. In visceral mycoses the entry route is airborne, almost never digestive. They are never pathogenic if they are ingested with food. Very important.

ENDOGENOUS MYCOSIS: commensal agent of the upper respiratory tract, skin or mucous membranes. The infection can spread via the blood and lymphatic system to other organs and tissues, giving rise to single or multiple infections. Example: Candida albicans.

FUNGAL VIRULENCE FACTORS are:

production of enzymes that damage cell membranes: keratinase, phenoloxidase, proteinase (aspartyl-proteinase, metallo-proteinase, serine-proteinase), phospholipase. These are exoenzymes. Melanin in Cryptococcus neoformans.

Size of the fungal thallus that is often larger than the phagocytic cells (filamentous fungi).

ability to grow at 37°C.

dimorphism that facilitates multiplication in the host.

presence of capsule (only Cryptococcus).

production of “mycotoxins” (gliotoxin, fumigatoxin).

CLASSIFICATION OF MYCOSIS (Based on location)

Superficial mycoses: infections limited to the outer layer of the skin and hair (Pityriasis versicolor, Tinea nigra, Piedra blanca, Piedra nera).

Cutaneous mycoses: infections that deeply invade the skin and appendages (dermatophytosis, candidiasis of the skin and visible mucous membranes).

Subcutaneous mycoses: infections of the dermis, subcutaneous tissue, cornea, muscle, (sporotrichosis, mycetomas).

SYSTEMIC MYCOSIS: by inhalation of soil spores: histoplasmosis, coccidioidomycosis, blastomycosis, paracoccidioidomycosis. The spores can reach internal zones of the body.

OPPORTUNISTIC MYCOSIS: candidiasis, cryptococcosis, aspergillosis, zygomycosis. These mycoses usually appear when the host is immunocompromised (for example HIV patients). It’s very difficult to treat these mycoses.

TREATMENT

Can we cure these diseases? In theory yes, because we have got some compounds and drugs that can be used to cure fungus infection. Fluconazole for example is one of the most famous drugs that is used to cure mycosis and the target of this drug is ergosterol’s syntesis. We have got different types of molecules that can target the cytoplasmic membrane, the replication of the genomic material, the cell wall etc.

PROTOZOA

The word “protozoa” came from Greek and the meaning is “first animal”. The phylum protozoa is very ancient. THEY ARE UNICELLULAR! Protozoa do not have a cell wall and therefore can have a variety of shapes. Body is unicellular performs all functions of the body. The cytoplasm is divided into two regions: ectoplasm and endoplasm. The body is either bilaterally, radial symmetry or asymmetry. Protozoa prefer living in moist and aquatic habitats. Their cysts can be found in the bleakest parts of the ecosphere. There are both Heterotrophic and Autotrophic based on their type of nutrition. They live freely, may be parasitic or symbiotic. Respiration is by diffusion. Movement is by pseudopodia, flagella, cilia. The reproduction can be asexual or sexual. Asexual methods of reproduction are: Budding, Binary Fission and Schizogony or Multiple Fission. Sexual Methods are: Conjugation and Gametogony. Excretion is via contractile vacuoles or by diffusion. Protozoa are classified into four main types based on their motility:

Flagellates: they move by help of Flagella (a tail-like structure). The movement is whip like. Examples: Tripanosoma, Trichomonas

Ciliates: Ciliates protozoa have movement through cilia (fine hair like structure attached with their body).

Sarcodina: Major locomotor organelles in Sarcodina is pseudopodia (Pseudo means false, podia mean Foot). Example: amoeba.

Sporozoates: they move only during particular stages of the life Cycle (Plasmodium: gliding motility)

What she wants to highlight is that, as already mentioned, the protozoa have got a very complex life cycle that involved one or more than one host. So, we have got the host, for example, human, like in this case, and then we have got what is called, kind of vector, because most of these protozoa are transmitted to the vectors. So, different type of host can be subdivided in definitive host and intermediated host. Which one is the definitive and which one is the intermediate host? The difference is based on which one of the two the parasite can have the sexual reproduction. Remember that the parasite can multiply with asexual reproduction and the sexual reproduction. The sexual reproduction happens only in one of the two hosts. The one where the parasite can sexually reproduce is the definitive host. In the case of malaria, the definitive host is the mosquitoes. It's not the human, because the parasite is able to reproduce via sexual reproduction only in mosquitoes. In the human host, the division is by mitosis. Instead, in the mosquitoes, it's through meiosis.

Toxoplasma gondii is another protozoa and has got more than two hosts, because the Toxoplasma can reproduce in the human host, in the cat and in the pig. Again, within these three, which one is the definitive host? The cat. The cat is the definitive host because the toxoplasma has got the sexual reproduction only in the cat. So, Toxoplasma is polyxenic (needs more than two hosts). Regarding the route of transmission, this can be oral-fecal route (Giardia, Entamoeba, Toxoplasma gondii. *REMEMBER “THE 7 F”: fluids, food, feces, fingers, fomites, fornication, flies); percutaneous route or bites of arthropod vectors (Plasmodium); Air route (Naegleria); Sexual route (Trichomonas); Transplacental route (contaminated or raw meat can have this protozoon, that’s the reason why pregnant women can’t eat raw or uncooked meat, unless the woman has already had Toxoplasma).

MAIN INFECTIONS

toxoplasmosis

amoebiasis

malaria

trichomoniasis

intestinal worms (pinworms, tapeworms, etc.) ELMINTS, NON-PROTOZOA

+ many others, especially in tropical countries.

There are four phyla of protozoa that are clinically important:

Sarcomasticophora

Ciliophora

Microspora.

Apycomplexa. It contains different type of parasites like Cryptosporidium, Plasmodium and Toxoplasma. Let’s focus on Plasmodium. There are five species of plasmodium that are dangerous for human: Vivax, Ovale, Malariae, Falciparum (the most dangerous), Knowlesi (zoonotic disease, is maily present in scimpanzee, but tribes in contact with scimpanzee rarely contract this protozoon). It has two hosts: mosquitoes (main host) and human. Mosquitoes are the most dangerous animals in the world. Only few species can host the plasmodium, the most common in Africa are of the Anopheles genre. We start with a mosquito already infected by plasmodium. At the end of plasmodium cycle (sporozoites), it can be found at the level of salivary glands of the mosquito. FEMALE MOSQUITOES bites human because need blood for the eggs. During this process, few sporozoites (10-100) enter in human body. Sometimes, the sporozoites are deposit at the level of the derma, but, as we mentioned before, they can move through the derma and enter the blood strain. Once the sporozoites are in the blood stream, they arrive in the liver. In the liver, they enter in hepatic cells (“bond theory” that explains how sporozoites move within the liver: Sporozoites enter in the liver, enter in a hepatic cell, if it doesn’t like them, they move to another cell. When they find the right cell, they stop and proliferate within the cell using asexual reproduction. So, the parasite in the liver cells grow until it reaches the final stage: SCHIZONT. Schizont has got a huge vacuole within the hepatic cell that contains a lot (thousands)of merozoites. At certain point, when the parasites are mature, the hepatic cells break, they free merozoites which they enter in the blood stream and start to infect the red blood cells. This is the final target within the human host. THE SYMPTOMATOLOGY STARTS HERE. At the level of exo-erythrocytic stage, the disease is still completely asymptomatic. The parasites can grow and mature within hepatic cells up two weeks. So, we have got the clinical symptomatology, fever and so on, only when the parasite reaches the red blood cells. The single plasmid that escaped from the liver enters the red blood cells. This is the initial stage of the parasite replication in the red blood cells. The parasite is so small and has got a peculiar form (a ring, in fact this phase is called “ring stage”). We can visualize the parasite within the red blood cell with a gram staining: the pink one is the nucleus; the violet one is the cytoplasm. From the ring stage, the parasite starts to grow and starts to occupy the entire volume of the cell (trophozoite stage, then schizont stage, the last stage). In the schizont stage, again, every single dot is a single merozoite. The parasite reproduces within the red blood cells (asexual reproduction). When the schizont is fully mature, the red blood cells collapses and free merozoites in the blood stream, they survive in the blood stream for like 20 minutes (because plasmodium is an obligated intracellular parasite). Free merozoites enter again in other red cells end they restart the erythrocytic cycle. Potentially, this cycle can last until there are blood cells, but in human is impossible because we started to get sick earlier. Occasionally, the ring stage develop a different structure called gametocytes. There are two types of gametocytes: female and male. They became the sexual form of the parasite that will be responsible for sexual reproduction within the mosquitoes. In P. Falciparum, gametocytes have a banana shape. These gametocytes, within the human host, after few days they will die. A person that is infected has got a mixture of red blood cells: some with the ring stage, some with trophozoite stage, some with the schizont and some with male and female gametocytes. What’s happen when a non-infected mosquito bites an infected human? She collects the blood of the human host, and, consequentially, collect all the different forms of the parasites. The mosquito needs blood because inside the red blood cells there is Hemoglobin, a very big molecule and the mosquito use the amino acids from the degradation of Hb and the blood’s liquids to grow the eggs. In the gut of the mosquitoes, the two gametocytes will develop, and they will form microgametes and macrogametes. Microgametes contain a flagellum, so this is a motile stage. The microgametes will move within the stomach of the mosquitoes looking for macrogametes for sexual reproduction. So, the fertilization forms the zygote. The zygote then develops in ookinetes (long shape). The ookinete is diploid. From the inside of the gut, the ookinetes can cross the wall of the gut and localize in the external matrix of the gut and form a round structure called oocyst. At this stage, the parasite can reproduce again (ASEXUAL). Within a single oocyst we can find like 10000 sporozoites. Once the ookinetes are completely mature, they break and free sporozoites that reach the salivary glands of the mosquito.

Toxoplasma gondii can be found in the muscular tissue of several animals like pigs. Toxoplasma forms cyst in muscular tissue and, if we eat infected raw meat, we are getting in contact with toxoplasma. Within the human host, toxoplasma generates a very bad disease because initially is asymptomatic or similar to a mild flu. The problem is that the parasite has got a different form like plasmodium, and a certain point, the parasite tries to escape from our immune system and arrive to the brain, where the immune system is very weak. The toxoplasma begins to create oocysts at the level of our brain that remain in the brain for the rest of human life. Most of the time nothing happens, but if we are immunocompromised (ex. HIV infection or transplantation), the cyst reactivates and start the active form of the disease. We don’t have many drugs to fight this disease, so most of the time the patient will die. WE CAN’T DESTROY OOCYST IN THE BRAIN. Other example of parasites that remain forever: Trichinella spiralis. Viruses: Herpes or varicella.