Amoebae as a host for Legionella replication
AffiliationMicrobiology & Immunology
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-08-05. This item is currently available to University of Melbourne staff and students only, login required.
© 2020 Pengfei Li
Legionella pneumophila (L. pneumophila) is a gram-negative bacterium found ubiquitously in natural water sources where it replicates within amoebae, such as Acanthamoeba castellanii (A. castellanii). The evolution in amoebal hosts allows L. pneumophila to ‘accidentally’ infect human lungs after inhalation of aerosols containing L. pneumophila. In both human macrophages and amoebae, L. pneumophila replicates intracellularly within a vacuole known as the Legionella containing vacuole (LCV) that avoids fusion with the endocytic pathway. Fundamental to this process is the translocation of over 330 proteins by L. pneumophila into host cells by the Dot/Icm type 4 secretion system. To date, only a subset of Dot/Icm effector proteins has been characterised. These proteins are involved in manipulating host cellular processes such as ER vesicle recruitment, post-translational modifications and host cell survival. The majority of L. pneumophila effector proteins remain uncharacterised due to functional redundancy of effector proteins and complex effector protein regulation. In addition, despite the ecological and evolutionary significance of the intra-A. castellanii stage of the L. pneumophila life cycle, very little is known about this host-pathogen interaction. In this study, we constructed in-frame markerless mutants in L. pneumophila 130b according to genomic regions enriched for putative Dot/Icm effector genes and revealed host-specific genomic regions that are required for optimal L. pneumophila replication in A. castellanii. Specifically, by identifying a putative glutamate transporter necessary for replication in A. castellanii, but not in macrophages, this study suggests an aspect of L. pneumophila metabolism adaptation in different hosts. A. castellanii presents two life stages: active trophozoites and dormant cysts. Trophozoites act as reservoirs for L. pneumophila and are hijacked by L. pneumophila for bacterial replication. Cysts are resistant to anti-microbial treatments and were previously thought to facilitate L. pneumophila persistence and dissemination. However, in this study, we showed that the infection of A. castellanii with L. pneumophila led to inhibition of the transition from trophozoites to cysts in a Dot/Icm dependent manner. This inhibition partially required the gene letA, which encodes the regulator of two-component system LetAS. Pathogenic amoeba cysts prose a human health problem. This study provides a biological model for anti-cyst research and provides clues into critical encystment pathways. L. pneumophila can interfere with a broad range of host cellular pathways to establish the replication niche. Here, we investigate A. castellanii transcriptional response with L. pneumophila infection by RNA sequencing analysis. As a result, A. castellanii genes associated with mitosis, DNA replication and cell wall synthesis were significantly downregulated at late infection phase, which might contribute to the inhibition of A. castellanii proliferation and encystment. Compared with transcriptional profile in macrophage, L. pneumophila displayed different regulation on host eEF1a expression and genes associated with ATP production. Furthermore, a gene encoding sirtuin family protein, Sir6f, was upregulated during L. pneumophila infection, and silencing of sir6f led to decreased bacterial replication, suggesting that Sir6f is a host factor that facilitates L. pneumophila replication in A. castellanii. Evolution of L. pneumophila in amoebal hosts is hypothesized to allow its adaptations to survive in macrophages. Overall, this study highlights a number of strategies utilised by L. pneumophila during A. castellanii infection, including host-specific virulence factors, inhibition of A. castellanii encystment, affect host transcriptome and hijack of host factors. These microbial strategies will add valuable insights and provide potential targets regarding the development of new mechanisms for L. pneumophila control and prevention.
KeywordsLegionella pneumophila; Acanthamoeba castellanii; Host-pathogen interaction; Intracellular replication; Encystment; Transcriptional rewiring
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