Dissertation/Thesis Abstract

Molecular Characterization of Thioester-Containing Proteins and Their Differential Gene Expression in Tissues of the Freshwater Snail, Biomphalaria glabrata
by Marquez, Joshua, M.S., New Mexico State University, 2020, 147; 27963435
Abstract (Summary)

Human schistosomiasis, also known as Bilharziasis, is a disease caused by trematode parasites of the genus Schistosoma, where each year approximately 200 million people need medical treatment worldwide. Avoidance of this disease is difficult in most of the populations affected, there is no vaccine available, and drugs that cure the disease do not prevent reinfection, therefore alternative approaches are warranted. The parasites in their asexual reproductive stage must infect an intermediate host, which are freshwater snails, and in their sexual reproductive adult stage must infect a vertebrate definitive host, which includes humans, to complete their life cycle. Since treatment and prevention methods have not worked in reducing the endemic nature of the disease, a multidisciplinary approach to reduce disease transmission includes the goal of understanding the immunobiology of the intermediate snail host. If the cycle can be stopped or reduced in the intermediate snail host, transmission to humans will be reduced.

There are snail strains with differences in susceptibility and resistance to schistosome infection, which is thought to be due to variability at the genetic level. Though, to what extent genetic variability affects schistosome resistance in different snail strains is not fully understood. Like many invertebrate organisms, snails lack an adaptive/acquired immune system, however, they do possess an innate immune system. Innate immune systems act in a relatively quick and non-specific manner against invading pathogens. Identification and removal of invading pathogens and their products is done by cell-mediated and humoral components, that is, protection provided by cells and secreted proteins. Several humoral components including molecules from the thioester-containing protein (TEP) superfamily have shown to be involved in immunological functions in many animals, from the most simple sponges and corals to humans. Most TEP superfamily members contain a characteristic (G)CGEQ thioester region (TER) in their protein sequence, which is a reactive site that tags or inactivates invading pathogens and their products. In some organisms, such as mosquitoes, TEPs have shown to be important in reducing their capacity as vectors in the transmission of diseases like malaria.

Thus, the main goal of this project is to identify, sequence, and compare TEP transcripts and their expression in susceptible (BB02) and resistant (BS90) Biomphalaria glabrata snail tissues. The working hypotheses are that B. glabrata snails possess a diverse array of TEPs, there is high sequence similarity between these TEP transcripts of the susceptible (BB02) and resistant (BS90) strains, and that these molecules will have differential expression in various snail tissues. This information provides insight into the evolutionary lineage of TEP sequences since conserved proteins like these and the genes that code for them undergo little evolutionary change over time. This signifies their importance in immunity not only in snails and other invertebrates but may expose important information about TEPs and other processes in humans as well. Furthermore, the presence and expression of TEPs may broaden our knowledge of the field of innate immune systems, and their possible interconnecting pathways that are not just part of immunity. It also provides further information available to scientists interested in these proteins that have studied them in other invertebrate and vertebrate organisms. Lastly, this information advances our insight into the TEP genes involved in the interconnected immune processes that render some snails susceptible and some resistant.

This study shows that there are 11 unique TEP superfamily genes expressed in B. glabrata snail strains. These include three members from the A2M group, three complement C3-like molecules, and five classical TEP-related subfamily proteins. Furthermore, the results show that there is high sequence identity in the transcripts between the susceptible and resistant snail strains, as well as high identities in the predicted amino acid sequences. In both snail strains, the characteristic TER was identified in six of the TEP sequences. However, two did not contain the TER, and three did not completely align with the representative (G)CGEQ residues, having one or more dissimilar amino acids in this protein motif. These results supported our hypothesis that both snail strains would contain a similar group of TEP superfamily members with subtle differences between strains. This suggests that these molecules may serve various functional roles and have possible differences in binding affinities between snails strains that render some TEPs more effective during innate immune responses.

In addition, tissue gene expression studies utilizing five different tissues associated with schistosome development (headfoot, kidney, stomach, digestive gland, and ovotestis) provides insight into what organ systems of the snail are involved in fighting infection. Furthermore, there was an interest in which snail strain produced more of the individual TEP transcripts (high expression level) within each tissue, relative to one another. The results in both snail strains showed constitutive expression (continuously making a transcript for the specific protein) in all snail tissues tested, tissue-specific expression, and varying expression levels of TEPs in different tissues. These results partially supported our hypothesis, since the TEPs in tissues had both similar and varying expression patterns, and the different snail strains did indeed have varying levels of TEP expression. This suggests that some tissues in one of the strains may have a more rapid response since there is greater availability of the specific TEP.

These findings are to be considered in the context of the limitations of this study. First, TEP transcripts reported here may be specific allelic forms from the individual snails from which the samples were extracted, and the possibility of other alleles will have to be tested using independent samples. Furthermore, the predicted amino acid sequences obtained from the TEP transcripts may not be the true form of the protein produced in vivo since post-transcriptional modifications are possible, such as alternative splicing. In addition, the expression studies presented are only preliminary, results are representative of only one biological replicate, and the reported transcript levels could vary within the snail’s population at different stages of development and physiological stresses encountered.

In conclusion, this study offers a novel understanding of the evolution and diversity of TEP molecules in metazoan animals. Furthermore, this study offers insight into their possible immunological role by investigating the expression of these genes in different snail tissues. A better understanding of the interactions that animals have with invading pathogens, and how genetic differences may confer varying resistance levels, such as the case with B. glabrata, could lead to a better understanding about the interactions that different snails in nature have with parasites. In turn, this may aid in the discovery and development of new methods to decrease the morbidity of human schistosomiasis.

Indexing (document details)
Advisor: Castillo, Maria G.
Commitee: Unguez, Graciela, Maio, William A.
School: New Mexico State University
Department: Biology
School Location: United States -- New Mexico
Source: MAI 81/12(E), Masters Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Immunology, Parasitology
Keywords: Biomphalaria glabrata, Differential gene expression, Innate immunity, Schistosoma mansoni, Schistosomiasis, Thioester-containing Proteins
Publication Number: 27963435
ISBN: 9781083529398
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