Cyclic diguanylate signaling proteins control intracellular growth of Legionella pneumophila

Abstract

Proteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle of Legionella pneumophila, the causative agent of Legionnaires' disease, has not previously been examined. The L. pneumophila genome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes as cdgS (cyclic diguanylate signaling) genes. Strains of L. pneumophila containing deletions of all individual cdgS genes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, several cdgS genes strongly decreased the ability of L. pneumophila to grow inside host cells. Expression of these cdgS genes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth in L. pneumophila. L. pneumophila strains overexpressing these cdgS genes were less cytotoxic to THP-1 macrophages than wild-type L. pneumophila but retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused by cdgS gene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of the cdgS genes in a Salmonella enterica serovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that several cdgS genes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability of L. pneumophila to grow in host cells.

FIG 1

Domain organization of predicted GGDEF/EAL/PilZ domain proteins in the Legionella pneumophila Philadelphia-1 genome. Graphic representation of the domain arrangement of Legionella pneumophila Philadelphia-1 GGDEF, EAL, or PilZ domain-containing proteins (domains are not drawn to scale). See the legend at the bottom of the figure for the annotations of the domains and other elements. The “Active domain” column indicates the signature motifs for the GGDEF and EAL conserved regions. The “I-site” column indicates the presence of the RXXD allosteric binding site; the conserved arginine (R) and aspartate (D) residues are shown in bold. The domain annotation was performed using the SMART web tool (70). DGC activity was determined using the S. Enteritidis ΔXII heterologous expression system as described in Results and in the legend to Fig. 3. The abilities of the different L. pneumophila strains overexpressing the indicated genes to grow in axenic growth medium are indicated in the AYE column, while the abilities to grow in a eukaryotic host are shown in the ICM column (“+++” represents WT-like levels; “−” represents no detectable growth).

FIG 3

Heterologous expression system for the in vivo detection of diguanylate cyclase activity. Phenotypes of L. pneumophila cdgS genes ectopically expressed in the background of the GGDEF-less strain Salmonella enterica serovar Enteritidis ΔXII. Each L. pneumophila cdgS gene was expressed in Salmonella strain ΔXII, and samples were spotted on LB plates containing calcofluor (upper left) or Congo red (upper right) or were spotted on motility agar (lower left). These three bioassays test for diguanylate cyclase activity. Cyclic di-GMP accumulation induces cellulose synthesis and appears as white on the LB calcofluor plates, promotes Congo red binding and appears red on Congo red plates, and decreases the swarm size on motility agar plates. The key at the lower right shows the identity of each sample, where DGC is a well-characterized C. crescentus protein that has strong diguanylate cyclase activity (DgcA). The control sample corresponds to the empty-vector plasmid.

FIG 2

Total intracellular cyclic diguanylate concentrations in whole-cell nucleotide extracts of L. pneumophila cdgS strains. L. pneumophila strains overexpressing (A) or lacking (B) cdgS genes were analyzed by reverse-phase HPLC, and intracellular levels of cyclic di-GMP were determined. The presented bar graphs in this figure represent a typical analysis profile. The y axis shows the amount of cyclic di-GMP in pmol extracted from 1 ml of culture at an OD₆₀₀ of 1.0.

FIG 4

Growth profiles of strains containing cdgS plasmids. (A) Growth in rich broth measured by OD₆₀₀. (B) Intracellular growth in THP-1 cells as measured by accumulation of GFP fluorescence. RFU, relative fluorescence units.

FIG 5

(A) Grazing activity of A. castellanii on strains overexpressing cdgS genes. A. castellanii monolayers were infected with different L. pneumophila cdgS-overexpressing strains at an MOI of 100. At each indicated time point, the extracellularly and intracellularly grown bacteria were pooled together, and aliquots were plated on a charcoal-yeast extract (CYE) plate. CFU were counted and calculated as the ratio of t_x/t₀. Error bars represent standard deviation (SD) values from three independent experiments. (B) Cytotoxicity of strains overexpressing cdgS genes for THP-1 cells. The MTT assay measures the number of viable macrophages following infection with L. pneumophila. Monolayers of THP-1 monocytes were infected with L. pneumophila cdgS-overexpressing strains for 6 days. The absorbance at 570 nm was measured and the CT₅₀ was calculated for each strain. THP-1 cell monolayers incubated with RPMI medium alone (null in graph) was used as a baseline for CT₅₀ calculations. THP-1 cell survival rate versus number of infecting CFU is plotted in this graph; error bars represent the SD of results from six independent replicates. CT₅₀ values are presented in the table. The dotA strain showed no cytotoxic effect in these experimental settings.

FIG 6

Dot/Icm-related activities of strains overexpressing cdgS genes. (A) Icm/Dot-dependent red blood cell (RBC) lysis by L. pneumophila overexpressing cdgS genes. (B) Detection of translocation of the LepA effector protein (LepA translocation is independent of the IcmS chaperone) and the SdeA effector protein (IcmS-dependent translocation). TEM-LepA or TEM-SdeA fusions were expressed in L. pneumophila, and the bacteria were used to infect THP-1 cell monolayers at an MOI of 40. The fluorescence intensity was measured, detecting emission wavelengths of 460 and 530 nm following excitation with UV at 405 nm. The ratios of the two emission intensities are plotted; error bars represent the SD of results from five independent infections.

FIG 7

Colocalization of LAMP-1 and L. pneumophila strains overexpressing cdgS genes. THP-1 cells were infected with GFP-labeled L. pneumophila cdgS-overexpressing strains, a JR32-negative control, and a dotA-positive control. Infected cells were fixed, and lysosomes were labeled using anti-LAMP-1 (α-LAMP-1) antibodies (red). (A) Representative confocal images demonstrating the locations of GFP-labeled WT (top panels) and dotA (bottom panels) bacteria and LAMP-1-labeled lysosomes. (B) Colocalization events were scored and calculated as ratios of LAMP-1 colocalized bacteria (yellow) to total bacterial number (yellow plus green) for all cdgS expressing strains. The graph represents average count of several fields. Error bars represent the 95% confidence interval of the mean. DIC, differential interface contrast.

FIG 8

Levels of flagellin in strains with different cdgS13 alleles. Whole-cell lysates of the indicated strains were loaded on a 12% SDS-PAGE gel and blotted on a polyvinylidene difluoride (PVDF) membrane. Western blot analysis using L. pneumophila α-FlaA polyclonal antibodies (Ab) was performed, and band intensities of three independent replicate blots were quantified and normalized to the level for the control sample using the ImageJ software package. Error bars represent standard deviation values from three independent blots. The results for a typical blot are presented under the corresponding strain name on the graph.