A Novel Legionella Genomic Island Encodes a Copper-Responsive Regulatory System and a Single Icm/Dot Effector Protein Transcriptionally Activated by Copper

Abstract

The intracellular pathogen Legionella pneumophila utilizes the Icm/Dot type IV secretion system to translocate >300 effector proteins into host cells during infection. The regulation of some of these effector-encoding genes was previously shown to be coordinated by several global regulators, including three two-component systems (TCSs) found in all the Legionella species examined. Here, we describe the first Legionella genomic island encoding a single Icm/Dot effector and a dedicated TCS, which regulates its expression. This genomic island, which we named Lci, undergoes horizontal gene transfer in the Legionella genus, and the TCS encoded from this island (LciRS) is homologous to TCSs that control the expression of various metal resistance systems found in other bacteria. We found that the L. pneumophila sensor histidine kinase LciS is specifically activated by copper via a unique, small periplasmic sensing domain. Upon activation by LciS, the response regulator LciR directly binds to a conserved regulatory element and activates the expression of the adjacently located lciE effector-encoding gene. Thus, LciR represents the first local regulator of effectors identified in L. pneumophila. Moreover, we found that the expression of the lciRS operon is repressed by the Fis1 and Fis3 regulators, leading to Fis-mediated effects on copper induction of LciE and silencing of the expression of this genomic island in the absence of copper. This island represents a novel type of effector regulation in Legionella, shedding new light on the ways by which the Legionella pathogenesis system evolves its effector repertoire and expands its activating signals. IMPORTANCE Legionella pneumophila is an intracellular human pathogen that utilizes amoebae as its environmental host. The adaptation of L. pneumophila to the intracellular environment requires coordination of expression of its multicomponent pathogenesis system, which is composed of a secretion system and effector proteins. However, the regulatory factors controlling the expression of this pathogenesis system are only partially uncovered. Here, we discovered a novel regulatory system that is activated by copper and controls the expression of a single effector protein. The genes encoding both the regulatory system and the effector protein are located on a genomic island that undergoes horizontal gene transfer within the Legionella genus. This regulator-effector genomic island represents the first reported case of local regulation of effectors in Legionella. The discovery of this regulatory mechanism is an important step forward in the understanding of how the regulatory network of effectors functions and evolves in the Legionella genus.

A horizontally acquired Legionella genomic island encoding a LuxR type regulator and effector proteins displays variation in gene content and regulation

“…The expression levels of regK3 and legK3 were relatively low in wild‐type L. pneumophila (Figures 2b and ), and genes acquired by HGT have been previously shown to be repressed by nucleoid‐associated proteins (NAPs) (Navarre et al., 2006) such as Fis in L. pneumophila (Linsky et al., 2020). Therefore, we examined the regK3 , legK3 , and cegK3 regulatory regions for potential Fis regulatory elements (TG‐N 13 ‐C) (Cho et al., 2008; Hengen et al., 1997), and identified four potential Fis sites.…”

“…This regulatory cascade includes the LetAS TCS, which constitutes the LetS SHK; the LetA RR, which harbors a LuxR type HTH; the two small RNAs (sRNAs) RsmY and RsmZ; and the posttranscriptional repressor CsrA (Gal‐Mor & Segal, 2003b; Hammer et al., 2002; Nevo et al., 2014; Rasis & Segal, 2009; Sahr et al., 2009, 2017). The fourth regulatory system includes the Fis1 and Fis3 repressors, which repress the expression of more than 20 EEGs including EEGs acquired by horizontal gene transfer (HGT) (Linsky et al., 2020; Zusman et al., 2014). Fis proteins function as nucleoid‐associated proteins (NAPs) and are direct regulators of gene expression (Dillon & Dorman, 2010).…”

“…The LciRS TCS, constitutes the LciS SHK and the LciR RR, which, similarly to CpxR and PmrA, belongs to the wHTH type regulators. LciR was found to regulate the expression of a single EEG, lciE , in response to elevated copper concentrations (Linsky et al., 2020). The lciRSE genes form a genomic island acquired by HGT and the expression of this island, which is present only in several Legionella genomes, is repressed by Fis (Linsky et al., 2020).…”

REC protein family expansion by the emergence of a new signaling pathway

“…Domain swapping in general is an important biological phenomenon that can impact all domains of life (Forslund et al, 2019). Complicating our interpretation of bacterial evolution, HGT is known to mobilize genes or entire gene clusters that can be subsequently integrated into foreign genomes (Bellieny-Rabelo et al, 2020;Linsky et al, 2020;Liu et al, 2017;Price et al, 2008;Treangen and Rocha, 2011;Wu et al, 2011). The narrative of HGT becomes even more entwined when we consider domain swapping.…”

The Two-Component System RstA/RstB Regulates Expression of Multiple Efflux Pumps and Influences Anaerobic Nitrate Respiration in Pseudomonas fluorescens

“…Two-component systems (TCSs), which are ubiquitously distributed in prokaryotic genomes, play a pivotal role in bacterial sensing and responding to changes in their environment ( 1 ). A TCS comprises a sensor kinase and a response regulator, which are usually encoded adjacently in genomes ( 2 , 3 ). A typical sensor kinase is composed of an N-terminal sensor domain facing the extracellular or periplasmic space and a C-terminal histidine kinase domain located in the cytosol ( 4 ).…”