Publication Date

8-2023

Advisor(s) - Committee Chair

Simran Banga, Rodney King, Ajay Srivastava

Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

The crucial virulence factor of accidental human pathogen Legionella pneumophila during the course of Legionnaire disease is the over 300 effector proteins secreted from its Dot/Icm secretion system. Eukaryotic host cells usually elicit an arsenal of immune responses against invading L. pneumophila. Nonetheless, the bacteria unexpectedly subvert these defense mechanisms to survive and proliferate unhindered in the host. Although some effector proteins have been proposed to play a significant role in this host-pathogen interaction, many still need to be characterized. The LneB and MavA proteins are examples of those effectors that need characterization. Thus, this study aimed to investigate the structural and functional characteristics of LneB and MavA proteins using several bioinformatics predictive pipelines and transcriptomics data supplemented experimentally through cell-based and biochemical assays to support the prediction.

The LneB protein was predicted to have histone acetylation activity (HAT) based on bioinformatics analysis. To investigate the HAT activity of LneB in vitro, the protein was ectopically expressed in the Escherichia coli BL21 strain and purified using nickel ion chromatography. The HAT activity assay was carried out on the purified LneB protein and on the nuclear extracts from LneB-GFP transfected 293T cells. Transcriptomics analysis shows that the LneB protein differentially induces upregulation of early growth factor and dehydrogenase (DHRS2) compared to the GFP control.

There was no significant difference between the in vitro HAT activity of LneB protein and the elution buffer (p-value = 0.1137, t-value = 5.537). In vivo, HAT activity was significantly reduced in cells transfected with LneB protein compared to the GFP control (p-value = 0.0025, t-value = 20.08). The HAT activity is not significantly different at a MOI of 10 or 100 when infected cells (Dot/Icm mutant and wild-type L. pneumophila) are compared to uninfected U937 cells (p-value = 0.8969 and 0.5384, respectively). However, the HAT activity in cells infected with an L. pneumophila Dot/Icm mutant at MOI of 100 was significantly lower than in cells that were not infected (p-value = 0.0236). This result suggests that the effector protein from the wild type plays a significant role in acetylating histone protein in the host. Further investigation is required to understand the HAT activity of LneB and other roles the protein could play in the host.

Our bioinformatics analysis suggested that the MavA protein possesses Ras-GEF domains and potentially binds to GTP. The protein is predicted to possess two coiled-coil domains and also interact with GTP, Ras and actin. The transcriptomic data from cells expressing MavA protein showed significant upregulation of sixteen genes, which are involve in steroid hormone metabolic processes, endocytic recycling, cilia movement among others. The sortilin receptor protein was the only repressed gene in the cell when compared to a GFP protein control. Connecting the bioinformatics finding and the review of literature, we suggested that the MavA protein could be involved in the biological process in the cell such as internalization of L. pneumophila, creation of Legionella-containing vacuoles in host cells through endosomal remodeling or cytoskeletal reorganization.

Disciplines

Biology | Life Sciences

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