The Worm's Exterior

From WormAtlas

In E. coli, synthetic biology has free reign to insert channels, receptors, and other proteins into the external membrane of the bacterium. The story in C. elegans is somewhat more complicated, as the nematode’s soft outer cells, the hypodermis, are protected by a layer of collagens and other proteins, called the cuticle.

Anatomically, the cuticle of C. elegans is divided into dorsal and ventral regions. In some developmental stages, including the adult worm, these regions are separated by longitudinal ridges called alae. The alae overlay the seam cells on the lateral side of the worm’s body, have different protein composition, and are distinct in appearance under an electron microscope. The cuticular structure is produced outside-first by cells underneath the cuticle, which insert additional layers of collagens during each developmental stage.

The cuticle itself is always covered with a layer of lipids and proteins called the epicuticle, some of which are glycosylated. These sugar moieties are diverse, and so complicate colonization by bacteria who thus need a significant number of different surface proteins to bind effectively and specifically, although there are some mutants (see section below) that disrupt collagen structure and may create a topology more friendly to retaining engineered bacteria, and some bacteria that bind despite this. Other known mutants (see section below) can affect the presence of various glycolipids and proteins in the epicuticle more directly. Similar glycosylation occurs inside the intestine, and is sometimes targeted by colonizing bacteria as well (see the article on the digestive system). The cuticle also bears holes and swellings where some neuronal cilia are exposed to the environment, and has a slightly negative electric charge that helps repel bacteria. The composition of the epicuticle changes during the dauer hibernation state and late larval development, but at adulthood returns to its earlier developmental form, suggesting it is well-maintained.

There are a number of bacteria known to be able to bind to C. elegans and other nematodes directly, however the majority are quite toxic to the worm and not very useful. A noteworthy exception to this is Microbacterium nematophilum, which attaches to the anal region of the cuticle as a method of hiding from its nematode predator, but is not particularly lethal to C. elegans. (M. nematophilum is, however, lethal to other nematodes in the Caenorhabditis genus.) However, the M. nematophilum genome is not well-studied and has not been sequenced. A lectin-binding assay may suggest a means to enable binding to the worm’s glycosylation.

Binding bacteria to the exterior of the nematode presents an exciting opportunity for bringing the benefits of nematodes to current synthetic biology work without requiring current prokaryotic solutions to be translated into eukaryotic genes. Such episymbiosis could permit a layer of “backwards compatibility” that would allow older projects to exploit the benefits of the worm's superior chemotaxis and powerful, precise sensory abilities for navigation without altering effector circuitry.

Embedding engineered proteins in the cuticle may also be possible. The cut family of genes codes for the cuticulins, a set of proteins that form structural polymers and complement the collagens. Importantly, they are well-understood as localized to specific regions of the cuticle, and not nearly as numerous, although their export mechanism is not yet well-understood. Removing the cuticle, however, is not practical, as it necessary to maintain hydrostatic pressure inside the worm. Punctures to it cause the worm to burst.

Cuticle Mutants

There are more than 170 collagen proteins that contribute to cuticular extracellular matrix. The majority of those that have been tested do not result in any distinctive phenotype, but members of the bli gene family such as bli-1 and bli-2 produce a ‘blistered’ appearance of particular interest without compromising normal development. (A number of cuticle mutants produce abnormal worm movement or shape; these have been listed under cuticular mutants as well.)

Epicuticle Mutants

The srf family of genes affects the presence and composition of the glycosylated proteins and lipids that surround the epicuticle. srf-1 is known to be necessary for the creation of an antigen that greatly reduces M. nematophilum’s ability to bind when it is absent. The bus family is similar, and contains many more genes that M. nematophilum requires in order to colonize C. elegans. It is probable that one of these can be exploited or over-expressed in order to make a more reliable binding mechanism. In pathogenic nematodes, these carbohydrates are also required for effective immune system evasion. (Medical projects utilizing C. elegans are, however, ill-advised.)

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