Abstract
Listeria monocytogenes (Lm) is a bacterial pathogen capable of causing meningitis, septicaemia and fetoplacental infections in the immunocompromised, elderly, and pregnant women. Disease caused by Lm (listeriosis) manifests as self-limiting gastroenteritis in immunocompetent individuals. Lm is a facultative intracellular pathogen that can survive extracellularly or adopt an intracellular lifecycle within a host cell. Lm has a family of structurally related proteins called ‘Internalin’ proteins, some of which are known virulence factors. Most strains of Lm have 25 Internalin proteins, with only five of these proteins (InlA, InlB, InlC, InlF, and InlP) being well-characterised in terms of biological function. Internalin proteins have a conserved leucine-rich repeat (LRR) region that adopts a horseshoe structure. In the case of the five well-characterised Internalin proteins, the concave surface mediates interaction with different human receptors. The majority of Internalin proteins have not been extensively studied.
The hypothesis that drove this project is that most or all Internalin proteins promote disease by interacting with human receptors. The overarching aim of this work was to identify human receptors of the three Internalin proteins Inl671, Inl369, and Inl2595. A yeast two-hybrid screen of a human cDNA library was previously performed by others in the Ireton laboratory to identify the human protein Ring1B as a potential receptor of Inl671. Riing1B is an E3 ubiquitin ligase that is known to regulate gene expression by mediating monoubiquitination of the histone 2A (H2A) protein. Western blotting and laser scanning confocal microscopy analysis was used to assess the effect of Listeria infection on Ring1B expression and activity. Although infection was correlated with decreased levels of Ring1B expression in some experiments, these effects were inconsistent among multiple experiments, making it difficult to conclude if Listeria affects Ring1B protein levels. Ring1B activity was assessed via Western blotting to detect monoubiquitinated H2AK119 (ubiquityl-H2AK119). However, difficulty in preserving H2A ubiquitination after solubilization of human cells prevented conclusions about whether Listeria infection affects monoubiquitination of H2A.
The Ireton laboratory previously performed a yeast two-hybrid screen to isolate two human proteins that potentially interact with the Internalin protein Inl369. However, the amino acid sequences of these human proteins were not characterized and their interaction with Inl369 was not validated through control experiments. In this Honours project, the interactions were validated by co-transforming, into a naïve strain of yeast, plasmids expressing Inl369 fused to the DNA binding domain of GAL4 and each of the two human proteins fused to the Gal4 transcription activation domain. Results showed that expression of yeast genes reporting interaction required both Inl671 and each of the two human proteins. It was therefore not due to promoter mutations in the reporter genes of the yeast strain where interaction was first detected. After validation of interaction, human cDNAs were sent for sequencing, leading to the identification of the human proteins centriolin and dynein axonemal assembly factor 11 (DNAAF11) as potential host receptors for Inl369. In addition to work with Inl671 and Inl369, the Internalin protein Inl2595 was studied for its suitability for yeast two-hybrid screens by determining if this protein lacked toxicity and the ability autoactivate gene expression in yeast. Unfortunately, Inl2595 constructs with the DNA binding domain of GAL4 fused to the N- or C-terminus of Inl2595 were found to autoactivate gene expression, indicating that they were not appropriate for yeast two-hybrid screening. The findings in this thesis suggest future work that may help assess the roles of Inl671 and Inl369 and their potential host receptors in Lm infection.