Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes

Abstract

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a wide range of acute and chronic infections. The transition to chronic infections is accompanied by physiological changes in the bacteria favoring formation of biofilm communities. Here we report the identification of LadS, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharides. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection by transcriptional and posttranscriptional mechanisms.

Fig. 1.

Characterization of P. aeruginosa ladS mutants. (A) Microtiter plate assay for biofilm formation. P. aeruginosa PAK, PAKΔpilA nonpiliated strain, and PAKΔpilA ladS::Tn5 mutant derivatives PAIH21, PAIC41, and PAIJ68 are shown. (B) Plastic tube assay for biofilm and pellicle formation. P. aeruginosa PAK, the respective isogenic ladS deletion mutant, and the PAK strain overexpressing the ladS gene are shown. (C) P. aeruginosa ladS mutant biofilm architecture. The gfp-tagged PAK and PAKΔladS strains were grown in a cell-flow system and observed with a confocal microscope. The large slide of the confocal images shows an overview of the biofilm structure, two transverse sections of the biofilm are shown to evaluate thickness and shape.

Fig. 2.

Expression of pel and exoS genes in the ladS mutant. (A) Expression of the pelA-lacZ fusion in either PAK or PAKΔladS strains. (B) Expression of the exoS-lacZ fusion in either PAK or PAKΔladS strains. Filled bars correspond to the activity obtained with the strains containing the vector pMP220, whereas open bars correspond to the activity obtained with the strains containing the pelA-lacZ fusion (A) or exoS-lacZ fusion (B).

Fig. 3.

Domain organization of LadS and RetS hybrid sensors. The transmitter domains are represented in black. This region contains the histidine kinase domain (HisKA) and the kinase domain (HATPase_C). The response regulator receiver domains (Response_reg) have been represented in light gray. The predicted transmembrane segments (TMs) are represented with white rectangle and allow anchoring of the sensor into the inner membrane. Each sensor possesses 8 TMs. The detector domains of LadS and RetS include the last 7 TMs (7TMR-DISM_7TM) and the putative signal-binding domain (7TMR-DISMED2), which is exposed to the periplasmic side of the cytoplasmic membrane. The positions of the Tn5 insertions found in the three original ladS mutants are indicated with a black triangle and the name of the corresponding mutant. The position of the first and last residue is indicated.

Fig. 4.

The activity of the rsmZ-lacZ fusion in various P. aeruginosa mutant backgrounds. Levels of β-galactosidase were measured in a culture of P. aeruginosa PAK, and the isogenic ladS, retS, gacS, and rsmA mutants. On the y axis, a break in scale has been introduced because of the high level of β-galactosidase activity obtained in the retS mutant.

Fig. 5.

Comparison between LadS-dependent and RetS-dependent gene regulation. The selected genes presented at least a 2-fold variation in the ladS mutant. (A) Genes that are up-regulated in a ladS mutant. (B) Genes that are down-regulated in a ladS mutant. The PA number and gene name, when annotated, is indicated on the left. Dark green and red genes are down- or up-regulated, respectively, by >5-fold. Genes shown in green and orange colors are down- or up-regulated, respectively, by >2-fold. Black genes do not reach the 2-fold cut-off. The expression ratio between PAK and an isogenic retS mutant were obtained from a previously published microarray analysis (11).

Fig. 6.

A model for the convergence of the signaling pathways during reciprocal regulation of virulence factors by LadS, RetS, and GacS through transcription of the small regulatory RNA RsmZ. The three sensors are anchored into the cytoplasmic membrane via their transmembrane domains. Unknown signals received by the input domains (7TMR-DISMED2 and HAMP) of the sensor kinases activate or repress the expression of genes specifying factors necessary for acute or chronic infection. The signaling cascade going through RetS and resulting in TTSS activation and biofilm repression is represented in blue. The signaling cascade going through LadS and resulting in TTSS repression and biofilm activation is represented in red. The small RNA RsmZ is represented by a curved line, which can form a complex with RsmA, resulting in biofilm formation and TTSS repression.