Turning Giardia lamblia’s smile upside down

By KW

Gut-wrenching abdominal pain. Foul-smelling flatulence. Projectile vomiting. Explosive diarrhea. You feel as if you are paying some sort of penance for taking two weeks off from work. But on the bright side, you’ve got a fantastic tan from your holiday in Costa Rica. Well done, it looks like you have a typical case of giardiasis! You probably picked up the microbe by drinking contaminated water or by unwittingly ingesting fecal matter containing the resilient, hardy cysts of the parasite Giardia lamblia.

This scenario is something that occurs so often in travellers, that it has earned the nickname traveller’s or backpacker’s diarrhea. Giardiasis is also sometimes known as “beaver fever” because hikers often contract the parasite by drinking from streams that have been in contact with beaver or muskrat feces containing the Giardia cysts. It is, however, not a disease relegated to the underdeveloped, underprivileged parts of the world. G. lamblia is one of the most common causes of gastroenteritis all across the planet; there’s an estimated 280 million worldwide cases each year (Ankarklev). Besides polluted rivers and communal swimming pools, this parasite can also be picked up in children’s daycares, and indeed, children are especially susceptible to giardiasis because of their immature immune systems.

Giardia is a teardrop-shaped organism with
a smile on its face that inhabits your gut.

Giardia belongs to the earliest diverging branch of eukaryotes (Prucca, Rivero, Lujan). But they lack organelles that normally all eukaryotic cells have, namely mitochondria, Golgi complex, lysosomes, and intronic regions within their genes (Prucca, Lujan). They have two nuclei, four pairs of flagella, and are teardrop shaped, which is fitting, since the effects of this organism on your body might just reduce you to tears.

It only takes as few as 10 individual cysts to initiate infection (Mahon et al). When Giardia cysts are ingested, the acidic pH of the stomach induces them to excyst (Mahon et al). Once they reach the duodenum of the small intestine, they hit the proverbial jackpot. That’s where they mature into trophozoites and feast upon the buffet of nutrients and carbohydrates. They achieve this by latching onto the endothelial layer of your intestinal wall with their infamous sucking disks. With these disks, they will siphon out nutrients and vitamins from your body. At this time, they are also replicating asexually to give rise to more parasites that can join in on the fun. Perhaps that’s why Giardia under the microscope look like smiley faces. Alas, the celebration comes to an end in the colon and they begin to encyst once again before being expelled in the host’s feces (Prucca, Lujan).

TEM of Giardia on the microvilli of small intestine.

Unlike for Giardia, it isn’t all fun and games for the host. As a result of infection, you may suffer weight loss due to malnutrition and severe dehydration. G. lamblia infection can leave permanent scarring on the walls of the small intestine. Luckily, antibiotics such as metronidazole and tinidazole are potent drugs commonly used in the clinic to get rid of Giardia; untreated, the symptoms may last for up to a month until the body naturally removes the parasite from the gut (NHS).

Your immune system senses these free-loading, unwanted intruders and it’s getting ready to kick them out. Your white blood cells are secreting chemicals that increase inflammation and plasma cells are producing tons of antibodies that are tailor-made for the destruction of Giardia. Unfortunately, something peculiar happens.

The rapidly dividing trophozoites in your intestine seem to be immune to your immune system. How’s this?

Giardia has a cunning trick up its sleeve. It’s called antigenic variation. Giardia lamblia contains around 200 genes scattered across its ten chromosomes that encode variable surface proteins (VSPs). These proteins are rich in the amino acid cysteine, share a highly conserved C-terminus that allows the protein to be embedded in the plasma membrane, and each one has a distinct motif at the N-terminus that differentiates it from other proteins (Bruderer). Giardia has a wardrobe full of different VSPs to choose from, and it will pick just one of them to wear on its cell surface.

The VSPs are immunogenic, meaning that the host recognizes these proteins as foreign and will mount an antigen-specific response to it. By going through periodic and random costume changes, the host’s antibodies will fail to recognize and tag it for obliteration. Research suggests that it switches its VSP coat every six to sixteen generations of binary fission (Prucca et al).

Antibodies that have been generated against the previous VSP coat will not bind to the new coat. Thus, more time has to be spent in priming another antibody response against the new antigen, by which point, the parasite may have already gone through another antigen switch. Through this mechanism, the protozoan can hide in plain sight and the infection literally goes on ad nauseam.

So how does the parasite regulate protein production such that only one VSP is present at the cell surface? Since nobody wants to have persisting diarrhea due to a chronic Giardia infection, this was an important question to ask. A research group in Argentina headed by Dr. Hugo Lujan sought to find an answer in hopes of helping the millions of Giardia sufferers who are stuck on the toilet. They found that Giardia transcribes all of its VSP encoding genes into mRNA. But due to RNA interference (RNAi), all of these transcripts are degraded except for the one that is to be expressed on the cell surface (Prucca, Slavin, et al). RNA interference is typically a method of getting rid of foreign RNA, from viruses, for example (Prucca, Saura et al). Therefore, Lujan’s finding was interesting because it showed that RNAi could be used to regulate its own gene expression.

Structure of Giardia’s VSPs expressed at the

cell surface. Mechanism of variation seems to

be guided by RNA interference enzymes like

 Dicer, RNA dependent RNA polymerase 

(RdRP, and Argonaute (AGO).

Using techniques such as Northern blotting and PCR, Lujan’s group identified some of the proteins responsible for limiting the expression of the entire VSP repertoire, namely RNA-dependent RNA polymerase (RdRP) and an enzyme aptly called Dicer. The mode of action of RdRP was to add complementary, anti-sense RNA to the VSP message to create double stranded RNA. Next, Dicer would cleave the dsRNAs into tiny 20-30 nucleotide long pieces. When the machinery involved in RNA degradation was silenced, Giardia subsequently expressed multiple VSPs on its cell surface (Prucca, Slavin, et al). It could no longer change its outfit because it was now wearing them all. As far as the immune system was concerned though, Giardia was rendered naked and vulnerable because with all its VSPs exposed, antibodies could be generated against each one.

In a later paper published in 2010, Lujan and his team of parasitologists infected gerbils with this unique strain of Giardia expressing multiple VSPs and a normal control strain expressing only one. They also immunized a different group of gerbils with purified VSP proteins. Two months later, the biologists challenged them with infectious Giardia. The control group was able to resist infection from Giardia expressing a VSP that it had already been exposed to, but was susceptible to reinfection by parasites with distinct surface proteins. Remarkably, the gerbils that had been inoculated with multiple VSPs were protected from the nasty parasites (Prucca, Saura, et al). The animals had fewer cysts in their feces than the control gerbils and histology staining showed no evidence of tissue damage to the intestinal walls from Giardia.

Lujan’s findings suggest that eliminating Giardia’s RNA interference pathway can be exploited to combat the pathogen and that inoculating the host with different proteins can confer immunity to the parasite upon future infection. Additionally, the research could guide vaccine development against G. lamblia and possibly other parasites that also go through antigen switching. The phenomenon of antigenic variation and immune system evasion is a common theme in microbiology. Other eukaryotic pathogens go through antigenic variation such as Plasmodium falciparum, the causative agent of malaria, and Trypanosoma brucei, which causes African sleeping sickness. Lujan’s research gives hope that mysteries surrounding the mechanisms of antigenic variation in other pathogens can be solved.

Giardia lamblia may rank as one of the most sinister and unusual eukaryotic parasites in evolutionary history. Its unconventional cell biology yet straightforward life cycle also make it a rather curious organism to study. A vaccine for humans is still a long shot; more investigations into the methods of disrupting its ability to switch antigens need to be done. In the meantime, people will have to be smart about what goes inside their bodies unless they don’t mind hosting a gala in their gut for Giardia.

References:

Ankarklev, Johan, Jon Jerlström-Hultqvist, Emma Ringqvist, Karin Troell, and Staffan G. Svärd. "Behind the Smile: Cell Biology and Disease Mechanisms of Giardia Species." Nature Reviews Microbiology (2010)

Bruderer, Papanicolau, Castro, and Kohler. "Variant Cysteine-rich Surface Proteins of Giardia Isolates from Human and Animal Sources." Infect. Immun 61.7 (1993): 2937-944

"Giardiasis ." Giardiasis. National Health Service, n.d. http://www.nhs.uk/Conditions/Giardiasis/Pages/Introduction.aspx

Mahon, Lehman, Manuselis. Textbook of Diagnostic Microbiology. 4^th edition. Pp. 230-245; 845-847.

Prucca, César G., Fernando D. Rivero, and Hugo D. Luján. "Regulation of Antigenic Variation in Giardia lamblia." Annual Review of Microbiology 65.1 (2011): 611-30. Print.

Prucca, Cesar G., and Hugo D. Lujan. "Antigenic Variation in Giardia Lamblia."Cellular Microbiology (2009)

Prucca, César G., Ileana Slavin, Rodrigo Quiroga, Eliana V. Elías, Fernando D. Rivero, Alicia Saura, Pedro G. Carranza, and Hugo D. Luján. "Antigenic Variation in Giardia Lamblia Is Regulated by RNA Interference." Nature 456.7223 (2008): 750-54.

Rivero, Fernando D., Alicia Saura, Cesar G. Prucca, Pedro G. Carranza, Alessandro Torri, and Hugo D. Lujan. "Disruption of Antigenic Variation Is Crucial for Effective Parasite Vaccine." Nature Medicine 16.5 (2010): 551-57.

Photo credits:

http://post-mitotic.tumblr.com/post/47534689780. TEM of Giardia on the microvilli of small intestine

http://www.nature.com/nrmicro/journal/v8/n6/images/nrmicro2317-f3.jpg schematic of VSP structure and regulation

http://www.pathobio.sdu.edu.cn/sdjsc/webteaching/Course/webteach/Protozoan/Giardia-20lamblia/GiardiaTroph(1).jpg Giardia is a teardrop-shaped organism with a smile on its face.