The Temporal Expression of Global Regulator Protein CsrA Is Dually Regulated by ClpP During the Biphasic Life Cycle of Legionella pneumophila.

Zhen-Huang Ge,Qin-Sha Long,Yong-Jun Lu,Dong Shen,Pei-Bo Yuan,Xin Pan

doi:10.3389/fmicb.2019.02495

Abstract

Legionella pneumophila, an environmental bacterium that parasitizes protozoa, is the causative pathogen of Legionnaires' disease. L. pneumophila adopts a distinct biphasic life cycle that allows it to adapt to environmental conditions for survival, replication, and transmission. This cycle consists of a non-virulent replicative phase (RP) and a virulent transmissive phase (TP). Timely and fine-tuned expression of growth and virulence factors in a life cycle-dependent manner is crucial. Herein, we report evidence that CsrA, a key regulator of the switch between the RP and the TP, is dually regulated in a ClpP-dependent manner during the biphasic life cycle of L. pneumophila. First, we show that the protein level of CsrA is temporal during the life cycle and is degraded by ClpP during the TP. The ectopic expression of CsrA in a ΔclpP mutant, but not in the wild type, inhibits both the initiation of the RP in vitro and the invasiveness to Acanthamoeba castellanii, indicating that the ClpP-mediated proteolytic pathway regulates the CsrA protein level. We further show that the temporally expressed IHFB is the transcriptional inhibitor of csrA and is degraded via a ClpP-dependent manner during the RP. During the RP, the level of CsrA is increased by promoting the degradation of IHFB and reducing the degradation of the accumulated CsrA via a ClpP-dependent manner. During the TP, the level of CsrA is decreased by inhibiting the degradation of IHFB and promoting the degradation of the accumulated CsrA via a ClpP-dependent manner as well. In conclusion, our results show that the growth-stage-specific expression level of CsrA is dually regulated by ClpP-dependent proteolysis at both the transcription and protein levels during the biphasic life cycle of L. pneumophila.

Highlights

Legionella pneumophila is a Gram-negative intracellular bacterial pathogen that is the causative agent for most cases of Legionnaires’ disease (Fields et al, 2002; Newton et al, 2010; Guyard and Low, 2011)
We found that CsrA is required for bacterial cells in the transmissive phase (TP) to passage into the replicative phase (RP)
In order to assess the protein level of CsrA at different growth phases, we performed proteomic analysis of whole lysates obtained from cultures of L. pneumophila wild-type (WT) strain grown in liquid medium at indicated time points

Summary

Introduction

Legionella pneumophila is a Gram-negative intracellular bacterial pathogen that is the causative agent for most cases of Legionnaires’ disease (Fields et al, 2002; Newton et al, 2010; Guyard and Low, 2011). The bacteria differentiate into two forms—replicative and transmissive—undergoing physiological, morphogenetic, and metabolic changes (Molofsky and Swanson, 2004; Bruggemann et al, 2006). The bacteria enter exponential and post-exponential forms, requiring similar physiological, morphogenetic, and metabolic changes (Byrne and Swanson, 1998; Hammer and Swanson, 1999). The gene expression programs in replicative and transmissive bacteria in vivo are similar to that of exponential and post-exponential bacteria in vitro, respectively, suggesting that the biphasic life cycle is controlled globally by the bacterial growth phase and/or nutrient availability (Oliva et al, 2018). The exponential and post-exponential phase in broth cultures has been used to emulate the RP and TP of the L. pneumophila life cycle (Bruggemann et al, 2006)

Methods

Results

Conclusion