# Versatile regulation of effectors by novel orthologous regulators in the Legionella genus

## TL;DR

This study identifies new regulators in Legionella bacteria that control the expression of proteins used to infect host cells, showing how these regulators can act both locally and globally.

## Contribution

The discovery of LelB and LelC as novel regulators and the demonstration of LelB's versatile regulatory role across Legionella species.

## Key findings

- LelB and LelC regulate effector-encoding genes in L. pneumophila.
- LelB orthologs in some Legionella species regulate multiple effector genes.
- LelB can function as both a local and global regulator of effectors.

## Abstract

The intracellular pathogen Legionella pneumophila utilizes the Icm/Dot type IVB secretion system, which translocates >300 effector proteins into host cells. The expression of the effector-encoding genes (EEGs) is mediated by a combination of global and local transcriptional regulators. Here, we describe two novel LysR-type transcriptional regulators (LTTRs), LelB (lpg2138) and LelC (lpg1796), which function as local regulators of EEG in L. pneumophila. Bioinformatic analysis, expression assays, site-directed mutagenesis, and in vitro binding experiments led to the discovery that in L. pneumophila, LelB controls the expression of three EEGs, and LelC controls the expression of a single EEG. Moreover, we discovered a versatile regulator, LelB, that can serve either as a global or local regulator of EEGs. The LelB orthologs in the Legionella genus are divided into three clades, but only in two of them lelB is located adjacent to its target genes and functions as a local regulator. A genomic search using the validated LelB regulatory element identified multiple genes (between 19 and 36) harboring the LelB regulatory element in the five Legionella species belonging to the third clade. Examination of this collection of genes uncovered that most of them harbor common effector domains and other known effector features. Furthermore, experimental analysis of these genes from one of these species, L. longbeachae, indicated that LelB regulates their expression. Our results demonstrate that LelB functions as a local or global regulator of EEGs in different Legionella species, thus uncovering the remarkable versatility of effector regulation by LTTRs in the Legionella genus.

Legionella pneumophila translocates into host cells the largest number of effector proteins known in any pathogen. This cohort of proteins needs to be coordinated at the gene expression level to result in a successful infection. To date, the regulation of effector-encoding genes in L. pneumophila has been found to be mediated by global regulators that control the expression of multiple effectors and local regulators that control the expression of a single or few effectors. Here, we identified two novel regulators that function as local regulators of effectors in L. pneumophila. However, analysis of their orthologs across the Legionella genus revealed that one of them controls the expression of multiple genes in five Legionella species. The properties of the proteins encoded by these genes suggest that most of them function as effectors. Our data demonstrate a versatile regulator whose orthologs function as local or global regulators of EEGs in different Legionella species.