The Role of Lymphocyte Cytosolic Protein 1 in a Mycobacterium Marinum Infection

Abstract

Mycobacterium tuberculosis is an issue worldwide with multidrug‐resistant forms becoming prevalent due to the lack of effective treatments. The goal of this project is to identify if there are host genes that affect the disease outcome. We decided to study the influence of the Lymphocyte Cytosolic Protein 1 (LCP1) gene in zebrafish larvae infected with mycobacterium marinum. To understand the immune response in the fish we observed the bacterial burden in fish that had LCP1 (heterozygotes) and in fish that did not (knockout mutants). M. marinum was injected into the caudal vein of the zebrafish larvae and the fish were infected. Once the fish were fixed and euthanized, images of the bacterial infection were taken using a fluorescent microscope. The bacteria was tagged with tdTomato, a red fluorescent protein which allows for the bacteria to glow red under the microscope. The images were then analyzed, and the bacterial burden was quantified using ImageJ to count the number of glowing pixels. Following the imaging of the infected larvae, the fish were genotyped. The DNA from the larvae was extracted through a hotSHOT PCR protocol, then LCP1 Exon 2 was amplified through PCR amplification. The PCR product was then cut with the Bsl‐I enzyme to show where the gene was expressed, gel electrophoresis was then run to analyze where the Bsl‐I cut and identify whether the fish was a heterozygote or knockout mutant. We then matched the suitable images of the fish to the genotype and found that fish with LCP1 may have a higher rate of infection. Understanding why fish with LCP1 have a higher bacterial burden could provide an insight to the role LCP1 plays in the human body’s immune response. The future directions of this project are to observe more fish in order to replicate the findings and to test other phenotypes including granuloma size, macrophage motility, and extracellular growth in order to have a better understanding of LCP1's role in the disease outcome. Mycobacterium tuberculosis is an issue worldwide with multidrug‐resistant forms becoming prevalent.