Multi-Dot/Icm effector mutants reveal distinct host responses in Legionella infected macrophages

Legionella pneumophila is the causative agent of Legionnaires' disease, an accidental and severe form of pneumonia. The replication of L. pneumophila within alveolar macrophages requires the Dot/Icm type IV secretion system that translocates more than 330 bacterial effector proteins into the infected host cell. Many Dot/Icm effectors possess eukaryotic-like domains and manipulate host processes by functional mimicry. However, only a minority of effectors have been fully characterized. Redundancy among effector protein activities and host targets has complicated functional characterization of individual effector proteins. To explore the collective impact of Dot/Icm effectors on host cell processes, we created a library of 14 L. pneumophila genomic mutant strains lacking effector-rich regions (a total of 85 effector genes deleted) and used the multi-deletion mutants to interrogate host-pathogen interactions. While none of the mutants exhibited a defect in intracellular replication in THP-1 macrophages, several were attenuated in amoebae, including a mutant lacking a predicted amino acid transporter. In THP-1 cells, we observed that wild-type L. pneumophila 130b triggered a strong inflammatory and cellular stress transcriptional response in macrophages, whereas six mutants displayed reduced interferon signalling and one mutant displayed a marked decrease in the global expression of host heat shock response (HSR) genes. Through the creation of further sub-deletions, we found that the Dot/Icm effector kinase LegK4 was responsible for induction of the host HSR via the canonical transcription factor heat shock factor 1 (HSF1). Infection of THP-1 macrophages deficient in HSF1 revealed a potential role for the host HSR in the early stages of L. pneumophila infection. Together, these results reinforce the utility of multi-gene deletions to identify Dot/Icm effector phenotypes and the importance of the host HSR to L. pneumophila infection.