Identification and characterization of Dot/Icm-independent determinants of Legionella pneumophila replication in mammalian host cells
Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology
First Advisor
Roy, Craig
Abstract
Legionella pneumophila is a natural parasite of freshwater amoebae that becomes an accidental pathogen of humans upon inhalation by a susceptible individual. Once L. pneumophila is taken up by a phagocytic cell, it uses its main virulence factor, the Dot/Icm type IV secretion system, to deliver over 300 different bacterial proteins to the host cytosol. Together, these proteins protect the phagosome from lysosomal fusion and manipulate the host cell to create a niche for bacterial replication known as the Legionella-containing vacuole. While many of the steps leading to the formation of this compartment have been studied in depth, much remains unknown about the requirements for L. pneumophila to survive and replicate in its vacuole. To further our understanding of how L. pneumophila is able to replicate in mammalian hosts, I used transposon insertion sequencing to define bacterial genes and functions that are required for fitness in primary murine macrophages and a mouse model of disease. I additionally determined which L. pneumophila genes are required for growth on laboratory media, showing that these requirements are distinct from those during infection. Focusing on genes that were crucial to intracellular but not axenic replication revealed a cluster of seven genes, lpg2928-lpg2922, that contributed to L. pneumophila infection. The genes lpg2925-lpg2922 are predicted to encode an ATP-binding cassette transporter similar to other bacterial efflux pumps, which are capable of translocating substrates with diverse properties. Infection assays confirmed that this transporter, now called Lit (Legionella infectivity transporter), is important for L. pneumophila infection of bone marrow-derived macrophages, mice, alveolar macrophage-like cells, and human monocyte-like cells. Expression of the Lit transporter increased early in infection of macrophages and may be regulated in response to ambient arginine levels, possibly through the action of the arginine repressor ArgR. The Lit transporter is homologous, by sequence and predicted structure, to an unusual class of efflux pump typified by MacAB-TolC, which spans both bacterial membranes but transports substrates from the periplasm to the extracellular environment. Its ability to prevent accumulation of a common efflux pump substrate, ethidium bromide, provided experimental support for bioinformatic predictions. Expression studies indicated that the transporter may be co-transcribed with one or more of the genes upstream, which encode a dimethyladenosine transferase, a hypothetical protein with potential helicase/relaxase activity, and a diadenosine tetraphosphatase. The exact functional relationship between the Lit transporter and Lpg2928-Lpg2926 is unclear, but RNA-sequencing data revealed that genes that were differentially expressed in a Lit-deficient mutant were similarly affected in an Lpg2927-deficient mutant. Deletion of lpg2927 also led to an increase in expression of genes downstream. While the substrate of the Lit transporter remains unknown, we have ruled out substrates of related pumps and other commonly effluxed molecules. We hypothesize that the Lit transporter supports L. pneumophila intracellular replication by removal of a toxic molecule, secretion of a signaling molecule, release of a resource-scavenging molecule, or export of a ‘public good’ that modulates the vacuolar environment to benefit the bacteria. Overall, this study adds to our understanding of the mechanisms supporting L. pneumophila replication in a mammalian host, and contributes a new member to a fascinatingly versatile class of transporter.
Recommended Citation
Moss, Caitlin Elizabeth, "Identification and characterization of Dot/Icm-independent determinants of Legionella pneumophila replication in mammalian host cells" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1249.
https://elischolar.library.yale.edu/gsas_dissertations/1249
