# Transcription factors DksA and PsrA are synergistic contributors to Legionella pneumophila virulence in Acanthamoeba castellanii protozoa

## TL;DR

This study shows that two bacterial transcription factors, DksA and PsrA, work together to help Legionella pneumophila survive and grow inside protozoa.

## Contribution

The study reveals a novel synergistic relationship between DksA and PsrA in regulating Legionella virulence traits.

## Key findings

- DksA1 mutation partially rescues growth defects when PsrA is expressed in trans.
- DksA and DksA1 can rescue ΔpsrA growth defects, but PsrA cannot rescue ΔdksA defects.
- Structural modeling suggests DksA1 impairs Mg2+ coordination in RNA polymerase, affecting transcription.

## Abstract

The environmental bacterium Legionella pneumophila, an intracellular parasite of free-living freshwater protozoa as well as an opportunistic human pathogen, has a biphasic lifestyle. The switch from the vegetative replicative form to the environmentally resilient transmissive phase form is governed by a complex stringent response-based regulatory network that includes RNA polymerase co-factor DksA. Here, we report that, through a dysfunctional DksA mutation (DksA1), a synergistic interplay was discovered between DksA and transcription regulator PsrA using the Acanthamoeba castellanii protozoan infection model. Surprisingly, in trans expression of PsrA partially rescued the growth defect of a dksA1 strain. Whilst in trans expression of DksA expectantly could fully rescue the growth defect of the dksA1 strain, it could also surprisingly rescue the growth defect of a ΔpsrA strain. Conversely, the severe intracellular growth defect of a ΔdksA strain could be rescued by in trans expression of DksA and DksA1, but not PsrA. In vitro phenotypic assays show that either DksA or DksA1 was required for extended culturability of bacterial cells, but normal cell morphology and pigmentation required DksA only. Comparative structural modelling predicts that the DksA1 mutation affects the coordination of Mg2+ into the active site of RNAP, compromising transcription efficiency. Taken together, we propose that PsrA transcriptionally assists DksA in the expression of select transmissive phase traits. Additionally, in vitro evidence suggests that the long-chain fatty acid metabolic response is mediated by PsrA together with DksA, inferring a novel regulatory link to the stringent response pathway.