Lipid, Sugar and the Never-Ending Cycle

The Sun is rising up, and from the fifth-floor window Oranžový, Žlutá and Modrý are probably the first ones to see the beginning of a new day. It was a magnificent night; the millennial dance between the Sun, the Earth and the Moon took place again, culminating with the full Moon’s silvery light drizzle all over the city. But this story is about another cycle, a cycle that literally will make you sick. Not desirable indeed, yet fascinating nevertheless.

By now, I would assume that you have experienced gastrointestinal issues before. Here is a secret, we all have. Do I have your attention? Not alluring enough. Do not fray.

Oranžový, Žlutá and Modrý do not know anything about the digestive system, as they themselves do not require it. However, they know something about one of the culprits that may upset your digestive tract. They have been working with a deceptive looking organism. Like a caterpillar that morphs into a butterfly, this organism transforms its body from cyst to trophozoite, and vice versa. Its name has French and Czech origins, Giardia lamblia.

Cyst and trophozoite are true opposites. When it comes to disguise, cysts choose a minimalistic aesthetic, while trophozoites embrace an embellished appearance.

Cyst has an oval shape, a protector armor made up of a matrix of cyst wall filaments, and when you look at it, you can see a glimmer of shy smile.

On the other hand, trophozoite is the master of great first impression. It has a body shaped like a pear, eight flattering extensions called flagella, and two big nuclei that gives the appearance of looking right back at you.

While cyst prefers not to expend energy and can stay dormant in water for 1 to 3 months, trophozoite is an active swimmer and skillful ballet dancer; it can do several rounds of pirouette.

Trophozoite uses its swimming skill, and the aid of an adhesive disk to directly attach to the intestine wall. For trophozoite, this step is extremely crucial, literally life-or-death. If trophozoite fails to attach, it is discharged as the intestine is flushed. Outside of the intestine, trophozoite is really fragile, its lack of mitochondria makes the oxygen in the air poisonous for its survival. Thus, trophozoite’s secret is that it is an anaerobe, rather than aerobe like you and me.

Oranžový, Žlutá and Modrý recognize the importance of better understanding the transformation cycle that take place between trophozoite and cyst. In short, drinking contaminated water with cyst, in conjunction with exposure to the acidic environment of the stomach, prompts the cyst to transform into trophozoite. Trophozoite attaches, replicates, and feeds in the small intestine. Its presence is sensed and its declared “persona non grata”, which catalyze the all-well-known intestinal discomfort, diarrhea as wells as, malabsorption, and malnutrition.

“Trophozoite, lipids, sugars, cyst” Oranžový mumbled while viewing the sky.

It turns out trophozoite has one more trick up its sleeve, the encystation cycle; like metamorphosis, the encystation transforms the trophozoite to cyst. At some point, while trophozoite is attached to the intestinal wall, it makes the decision of traveling outside the body. This is a long and dangerous journey, but trophozoite knows exactly what and how to pack for the voyage. To start preparing for the transformation, trophozoite arrange tight ‘encystation-specific vesicles’ that carry components of the protector armor, ‘cyst wall protein’. This armor has to be transported and deposited on the outside of the plasma membrane, and slowly weave a shield.

What exactly makes trophozoite leave the intestinal wall and travel outside the body has remained in the dark.

Oranžový, Žlutá and Modrý have been researching the sphingolipid signaling in Giardia and have identified a key unique enzyme, the giardial glucosylceramide transferase-1 (gGlcT1). By manipulating the expression of this enzyme, they have been able to arrest or initiate the biogenesis of vesicles in the trophozoite. Vesicle biogenesis is a decisive step, drives the transformation from trophozoite to cyst, and most importantly it is essential for cyst viability and continuity of Giardia life cycle.

What does gGlcT1 do? gGlcT1 connects simple sugars to ceramide, leading to the conversion of ceramide to hexosylceramide; like wearing an outfit with a hat. Ceramide is a lipid and it is the core of sphingolipids. In the cells, ceramide is like the traffic light in a busy intersection, controlling traffic at metabolic junctions. However, Giardia has a truncated sphingolipid pathway, thus it does not produce ceramide. For Giardia the solution is to scavenge most nutrients from the host and keep few indispensable sphingolipids genes that can be stimulated and regulated at the right time. Talk about simplicity in a fancy way.

Oranžový, Žlutá and Modrý developed a research plan and were busy for several months trying to answer the question, is gGlcT1 activity indispensable for the encystation cycle?

First, they used a molecular approach to manipulate gGlcT1 protein expression and created a specific DNA construct carrying the gGlcT1 gene. Delivering this impermeable package to Giardia required literally shocking measures. An unexpected electrical pulse is applied to trophozoite, this opens small gaps in the cell membrane through which genetic material can enter the cytoplasm of the cell and then into the nuclei where the gene is expressed. If successful, trophozoite is persuaded to enter in the encystation cycle, and start the formation, packaging and transporting of vesicles towards the plasma membrane.

Second, they took advantage of a new blocking technology which prevented the protein factory to produce gGlcT1. This caused trophozoite to pause the encystation enterprise.

Third, like culinary artist, they spent some time perfecting the right soup for measuring gGlct1 transferase activity. They utilized a fluorescent probe (NBD-ceramide) to track the newly formed molecules, some luxurious sugars as substrates (UDP-glucose and UDP-galactose), and they combined in a pool of membranes as an enzyme source. To separate, visualize and quantify the hexosylceramides produced after the enzymatic reaction, they performed thin layer chromatography. Thin layer chromatography is like a race; samples are spotted at the bottom of the plate (the track), the plate is placed in a chamber with a specific solvent mix, and the molecules start running vertically. The polarity and structure of the molecules determines how fast the molecules will travel from the starting point.

Fourth, to complement their research, they incorporated the power of computer science to look for patterns within related proteins.

“It works!” Modrý shouted.

Giardia graciously transported NBD-ceramide, and converted it in two distinctive molecules, glucosylceramide and galactosylceramide. Modrý was pleased to announce that gGlcT1 activity was remarkably increased in the encysting stage in contrast to non-encysting stage where the products were not detected. This observation depicts gGlcT1 is functional and its activation was related to the encystation cycle.

Moreover, the appearance of unexpected galactosylceramide was a puzzle for everyone. Is galactosylceramide a product of gGlcT1? Is galactosylceramide a product of an unknown transferase? Is galactosylceramide required for the encystation process? To their knowledge Giardia would not have the means to create this molecule.

The answers came after a series of incubations, extractions, and partitions. It took everyone by surprise, gGlct1 was a promiscuous enzyme with the potential of creating glucosylceramide and galactosylceramide during the encystation cycle; and remarkably, gGlcT1 had strong preference for UDP-galactose as a substrate. Hence, both glucosylceramide and galactosylceramide may act as internal hibernation cues that trigger the production of ‘cyst wall protein’ to build the protector armor of cyst, while inducing a state of inactivity and metabolic depression. In the intestine, the cyst as a NASA spacecraft will be launched by a series of bowel movements, land in water, and stay there before the cycle starts again.

This discovery advances our knowledge of the encystation cycle, and put the sphingolipids as key signaling molecules to drive morphological transitions such as encystation in Giardia. Furthermore, this opens the possibility that the release of glucosylceramide and galactosylceramide by Giardia can be the cause of persistent inflammatory intestinal syndrome in patients. Thus, the sphingolipid pathway could expose new therapeutic strategies for the treatment of Giardia infections.

For Oranžový, Žlutá and Modrý, it is the end of spring, they had done a lot for the project, and as every year, they are getting ready to spend a day in the spa and wellness center for pipettes. When they get back, they will be ready to support you through all your experimental research.

This article is based on the following research paper:

Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation