Legionella pneumophila: from amoeba to macrophage metabolism

Abstract

Legionella pneumophila is an aquatic bacterium that has emerged as an accidental human pathogen. Within the aquatic environment, L. pneumophila has evolved virulence factors to survive predation by environmental amoebae. These virulence factors are hypothesised to allow the adaptation of the bacteria to replicate in human alveolar macrophages. During infection, L. pneumophila forms a replicative vacuole termed the Legionella-containing vacuole (LCV) that sustains the bacterial intracellular replication. Establishment of the LCV requires the Dot/Icm type IV secretion system (T4SS), that secretes over 330 bacterial proteins termed effectors into the infected host cell in order to manipulate host processes and facilitate bacterial replication. Despite their central role in LCV biogenesis, to date most effector proteins remain uncharacterised. Therefore, to aid in the characterisation of Dot/Icm effector proteins, in this study, we generated large genomic region mutants. To date, we have created nine genomic deletion mutants (A-I), as well as two multiple-region deletion mutants (FGHI and DFGHI) in L. pneumophila, resulting in the deletion of 68 effector genes and 138 non-effector genes.

These mutants were then used to identify the genomic regions important for bacterial replication in vitro and in vivo. Despite the loss of up to 42 effector-encoding genes, all mutants can replicate efficiently in THP1 macrophages. Meanwhile, in the protozoan host, at least six mutants showed a severe replication defect. Interestingly, in the mouse model, four mutants displayed an unexpected increase in bacterial burden, while one mutant showed a reduction in bacterial replication. Surprisingly, two of the mutants showing an increase in bacterial load in the mouse model were unable to replicate in Acanthamoeba castellanii. Together, these highlight the difference in requirements to survive in different hosts. This also suggests that the large effector repertoire of the Dot/Icm T4SS effectors likely evolved to enable an intracellular lifestyle in a diverse range of hosts.

Finally, the mutants were also used to identify a Dot/Icm effector protein that degraded host GAPDH mRNA. RNA sequencing of infected cells revealed that L. pneumophila downregulated multiple host glycolytic mRNAs which depended on a particular Dot/Icm effector. Taken together, this project has used mutants carrying large genomic deletions to identify genetic regions important for bacterial replication, as well as those manipulating host immune defence.