Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

Abstract

g Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/ Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over-and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole. C yclic-di-GMP (c-di-GMP) is a nearly ubiquitous bacterial second messenger involved in signaling pathways that control lifestyle transitions, among which are the transition from the motile to the sessile state and, for pathogenic bacteria, the transition from pathogenic to environmental lifestyles or from acute/planktonic infections to chronic/biofilm infections (1-4). c-di-GMP is produced from GTP by diguanylate cyclases (DGCs), is degraded by phosphodiesterases (PDEs), and acts through various effector proteins or RNA riboswitches (5-8). The catalytic site of DGCs is associated with the GGDEF domain, while PDE activity involves either the EAL or the HD-GYP domains. Genes encoding these proteins have been identified in all major bacterial phyla and are particularly expanded in gammaproteobacteria, with a median of 22 genes per genome (9). Consistently, the genomes of gammaproteobacterium Legionella pneumophila strains encode from 22 to 24 GGDEF/EAL proteins (10, 11). Few are specific to each strain, but most of them are highly conserved in L. pneumophila strains (11). c-di-GMP seems to be strongly involved in Legionella cell signaling, since the 22 GGDEF/EAL proteins represent 13.5% of proteins contain...

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

“…The observation that 1 to 3% of the inoculum entered into the macrophages is similar to past observations made with L. pneumophila and this type of gentamicin protection assay (53). To confirm our results, we utilized a trypan blue quenching method that was recently adapted to assess L. pneumophila entry into host cells (54)(55)(56). In this case, we infected the BMD macrophages with GFP-expressing bacteria at an MOI equal to 50, and after treatment with trypan blue to quench residual extracellular bacteria, the level of trypan blue-resistant fluorescence remaining associated with the monolayer (i.e., intracellular bacteria) was assessed using a fluorescence microplate reader.…”

“…Entry was measured as the percentage of intracellular CFU recovered from the monolayers compared to the number of CFU in the inoculum. As a second means of monitoring bacterial entry, we utilized a trypan blue quenching-based entry assay which has been described previously for L. pneumophila (54)(55)(56). To that end, BMD macrophages (5 ϫ 10 4 ) or differentiated U937 cells (1 ϫ 10 5 ) were seeded into black, clear-bottomed, 96-well microtiter plates (Falcon), and bacteria suspended in RPMI medium lacking phenol red (in order to reduce background fluorescence) were added at an MOI of 50 to six replicate wells.…”

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

“…e medium was supplemented with chloramphenicol (Cm) (5 μg ml −1 ) when necessary. In particular, all bio lm experiments were performed with Lens wild-type and mutant strains transformed with pJCL2968 vector ('pCOMP−') (Allombert et al 2014), or derivatives of pJCL2968 vector, carrying a Cm resistance gene ('pCOMP+'). Bio lms were formed as described previously in SBB-Cm medium in 24-well microplates (BD Falcon, France) at 32.5°C for nine days.…”

“…e inactivation fragments consisted of a selection marker (kanamycin (km)-resistance cassette) anked by the 5 and 3 sequences of the targeted genes. For complementation experiments, wild type alleles were cloned in pJCL2968 vector carrying a Cm resistance cassette (Allombert et al 2014). All the strains used in this study were subsequently transformed either by the empty vector pJCL2968, or by the complementation vector.…”

“…Genes encoding GGDEF/EAL proteins were inactivated by allelic exchange between an inactivation fragment carried by a suicide plasmid (pAV695) and the wild type chromosome (Allombert et al 2014). e inactivation fragments consisted of a selection marker (kanamycin (km)-resistance cassette) anked by the 5 and 3 sequences of the targeted genes.…”