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Title: Interactions of legionella pneumophila with amoeba and human hosts : cellular and molecular mechanisms
Authors: Mou, Qianqian
Degree: Ph.D.
Issue Date: 2019
Abstract: Background: Legionella pneumophila is a gram-negative bacterial species that has a wide distribution in both natural and artificial fresh water systems. Amoebas in biofilm in the water environment play a crucial role in the survival of L. pneumophila. Once ingested by an amoeba, Legionella multiply inside the amoeba instead of dying. Hence, the amoeba host facilitates the intracellular replication and spread of L. pneumophila. The organism is then released when the amoeba host ruptures. Inhalation by humans of aerosols contaminated with L. pneumophila causes Legionnaires' disease, a community-acquired pneumonia that has been reported worldwide in association with immunocompromised individuals. The virulence of the pathogen in humans is mainly due to its resistance to macrophages. The survival of L. pneumophila within various hosts hinges on its successful evasion of the hosts' conserved phagocytic killing pathways. The organism possesses a repertoire of effector proteins that manipulate the host cell signaling and metabolic pathways. However, whether similar L. pneumophila virulence factors are expressed during intracellular replication in amoebas and macrophages remains unknown. The underlying mechanisms that lead to the outcomes of the two host types after L. pneumophila infection are also poorly understood. Evidence has shown that L. pneumophila grown in the environmental host Acanthamoeba castellanii were more virulent and more readily infected human monocytes and macrophages. To understand the interactions between L. pneumophila and amoebas, a detailed analysis of gene expression must be performed in both the organism and the host. Aims: The main studies involved in this thesis include a detailed comparison of the intracellular bacterial replication and the virulence gene expression levels of L. pneumophila and host cell death during intracellular growth in A. castellanii and monocyte THP-1. To determine precisely the interaction between L. pneumophila and its natural host Acanthamoeba, gfp-transfected L. pneumophila was used to infect A. castellanii, fluorescence-activated cell sorting was used to isolate the L. pneumophila- infected Acanthamoeba, and dual transcriptome profiling was performed for both the bacterial pathogen and the amoeba host. Key findings: For intracellular growth of L. pneumophila in different hosts, the findings show that the growth of L. pneumophila in THP-1 cells caused no apparent increase in the bacterial count during the first 24 h after infection and an increase in bacterial cells of less than 10-fold from T36 to T48. In contrast, the growth of L. pneumophila in A. castellanii led to a 10-fold increase in the L. pneumophila count during the first 24 h after infection, and the bacterial count remained stable from T24 to T48. The results show that L. pneumophila replicated more rapidly in Acanthamoeba than in THP-1 cells. The two hosts showed different expression patterns of L. pneumophila virulence genes during intracellular growth. When L. pneumophila was grown in THP-1, flaA involved in the induction of pyroptosis was downregulated, whereas sdhA involved indirectly in the inhibition of host cell death was upregulated during the infection. The vipD and sidF genes involved in the induction and suppression of apoptosis showed no obvious change. In contrast, when L. pneumophila was grown in Acanthamoeba, flaA and vipD were upregulated during the late phases of infection, whereas sdhA and sidF were downregulated during the late phases. The gene expression patterns of L. pneumophila suggest that, during intracellular growth, cell death was suppressed in THP-1 but induced in Acanthamoeba.
In our investigation of host cell death, L. pneumophila induced the expression of caspase-1 but not caspase-3 or cell death in THP-1 at T48. The findings show that active caspase-1-associated pyroptosis could be more involved in THP-1 death than caspase-3- associated apoptosis. Notably, very small differences in active caspase production and cell death percentages between L. pneumophila-infected and uninfected THP-1 were seen, which suggests that L. pneumophila infection had little effect on THP-1 death. In contrast, L. pneumophila caused a high rate of cell death in A. castellanii from T12 to T48. Microscopic studies showed that the infected A. castellanii generated large numbers of rounded-up cells and cysts during the late stages. Furthermore, the metacaspase-1 responsible for encystation was upregulated in Acanthamoeba during the later stages of Legionella infection; this finding differs from the reports of other research groups. This study showed that A. castellanii cell death-mediated lytic release and A. castellanii encystment-mediated nonlytic release could co-occur in L. pneumophila-infected A. castellanii. To elucidate the interactions between L. pneumophila and Acanthamoeba, A. castellanii infected with gfp-transfected L. pneumophila for 48 h was enriched by fluorescence-activated cell sorting for dual transcriptome analysis. The results show that the transcriptome of L. pneumophila genes was dominated by the upregulation of genes involved in flagellar activity, bacterial protein synthesis, and amino acid metabolism. However, the transcriptome of L. pneumophila-infected A. castellanii was dominated by the downregulation of genes involved in protein synthesis and amino acid metabolism. In addition, it was observed that the pvc genes in L. pneumophila were significantly upregulated during intracellular growth. The putative functions of the pvc genes were involved in the amino acid metabolism. The pvcA-knockout and pvcB-knockout L. pneumophila released from A. castellanii showed lower replication in THP-1 when compared with wild-type L. pneumophila. Replication in Acanthamoeba triggered the expression of L. pneumophila pvc genes, which could regulate the L. pneumophila virulence that further affects bacterial pathogenesis in human macrophages. Conclusions: The studies performed as part of this thesis demonstrated that Legionella pneumophila better adapted to and more readily induced cell death to facilitate bacterial release in Acanthamoeba castellanii. In contrast, THP-1 preferentially induced pyroptosis during Legionella infection, which led to host cell death and limited bacterial growth. Dual transcriptomes of both the pathogen and amoeba host indicated that during the late stages of intracellular growth, flagellar assembly and energy metabolism were activated in L. pneumophila, whereas metabolic activities were inhibited in the Acanthamoeba host.
Subjects: Hong Kong Polytechnic University -- Dissertations
Legionella pneumophila
Legionnaires' disease -- Molecular aspects
Pages: xxiv. 239 pages : color illustrations
Appears in Collections:Thesis

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