Effects of crp2-1 knockout to crp2-2 and crp3 expression : Innate immune response to pneumococcal infection in zebrafish

Abstract

Streptococcus pneumoniae is the leading cause of pneumonia, causing severe risk to those with impaired immunity, such as children and the elderly. It is estimated that this bacterium causes the annual death of 300 000 children under five years old globally. The globular, gram-positive pneumococcus can be found inhabiting large percentages of population, with mainly non- symptomatic carriers. To better understand the immunological characteristics and mechanisms underlying pneumococcal infection and immunity, various model organisms such as zebrafish have been utilized. Zebrafish are becoming increasingly more common model organisms in immunological studies. Their immune system comprises of both innate and adaptive immunity, with components and mechanisms similar or analogous to human immunity. Their adaptive immunity does not develop until a month post fertilization, allowing for the study of solely innate immunity within the first weeks. An important part of the innate immune response against pathogens, such as pneumococcus, are C-reactive proteins (CRP). They are evolutionarily conserved proteins of the pentraxin family, capable of recognizing pathogens and activating necessary immunological responses such as phagocytosis. Compared to humans, the zebrafish Crps showcase a variety of gene isoforms, splice variants, and monomeric as well as trimeric protein structures.

The earlier forward genetic screen and whole genome transcriptome analysis of zebrafish suggested that Crps, especially Crp2-1 has an important role in the acute phase response in S. pneumoniae infection. The experiment detailed in this thesis studied the expression of three Crp isoforms in two-day-old zebrafish larvae subjected to Streptococcus pneumoniae infection for 68 hours. Utilizing clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR- associated protein 9 (Cas9) mutagenesis, a zebrafish line with dysfunctional crp2-1 gene was created. The expression of three Crps were compared between this homozygote line to zebrafish with functional Crps. The aim of this experiment was to better characterize the expression of genetically similar Crps to crp2-1 during infection. The data gathered implied that these genes, especially crp3, could have overlapping functions in pneumococcal infection. Further studies verified the assumption, suggesting that these genes have a conserved function in anti- pneumococcal immunity. The ability of zebrafish with crp2-1 knockout to induce crp3 expression implies of a compensatory mechanism capable of targeting expression in relevant genes, when the primary gene provides inadequate response.

In addition to qPCR results regarding the expression of genes crp2-1, crp2-2, and crp3 between the two lines of zebrafish, this thesis describes the suitability of zebrafish as a model organism for immunological research and explores the diverse characteristics of CRPs. Finer detailing of the mechanisms described in this thesis could lead to novel applications in immunology, with improved diagnostics and better treatment for patients with pneumococcal infection.