FimL regulates cAMP synthesis in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa, a ubiquitous bacteria found in diverse ecological niches, is an important cause of acute infections in immunocompromised individuals and chronic infections in patients with Cystic Fibrosis. One signaling molecule required for the coordinate regulation of virulence factors associated with acute infections is 3', 5'-cyclic adenosine monophosphate, (cAMP), which binds to and activates a catabolite repressor homolog, Vfr. Vfr controls the transcription of many virulence factors, including those associated with Type IV pili (TFP), the Type III secretion system (T3SS), the Type II secretion system, flagellar-mediated motility, and quorum sensing systems. We previously identified FimL, a protein with histidine phosphotransfer-like domains, as a regulator of Vfr-dependent processes, including TFP-dependent motility and T3SS function. In this study, we carried out genetic and physiologic studies to further define the mechanism of action of FimL. Through a genetic screen designed to identify suppressors of FimL, we found a putative cAMP-specific phosphodiesterase (CpdA), suggesting that FimL regulates cAMP levels. Inactivation of CpdA increases cAMP levels and restores TFP-dependent motility and T3SS function to fimL mutants, consistent with in vivo phosphodiesterase activity. By constructing combinations of double and triple mutants in the two adenylate cyclase genes (cyaA and cyaB), fimL, and cpdA, we show that ΔfimL mutants resemble ΔcyaB mutants in TM defects, decreased T3SS transcription, and decreased cAMP levels. Similar to some of the virulence factors that they regulate, we demonstrate that CyaB and FimL are polarly localized. These results reveal new complexities in the regulation of diverse virulence pathways associated with acute P. aeruginosa infections.

Figure 1. cAMP levels are decreased in fimL mutants.

Intracellular cAMP levels were measured in the presence (2 mM) or absence of calcium in (A) PAO1, (B) PAO1ΔcpdA, or (C) PA103. Gene names denote in-frame deletions. PAO1ΔcpdA(comp) fimL-FL denotes complementation of PAO1ΔcpdAΔfimL with fimL-3X-FLAG by gene replacement in the fimL locus. PAO1ΔcpdAΔcyaB (CTX-cyaB) denotes complementation of PAO1ΔcpdAΔcyaB with cyaB at the CTX phage attachment site. Note the scale differences. Shown are mean results of triplicate samples from 3 experiments. Error bars indicate SD (for cpdA in (A) +/−4.7 in the presence of Ca²⁺ and +/−3.6 in the absence of Ca²⁺). (***) P<0.001 compared to the wild type strain grown in the absence of calcium.

Figure 2. Loss of fimL does not affect cyaB transcription or protein levels.

(A) β-galactosidase activity was measured in the presence or absence of calcium in PAO1 or PAO1ΔfimL. All strains carry a lacZ transcriptional reporter integrated at the CTX site without a promoter (P₀-lacZ) or with the cyaB promoter (PcyaB-lacZ). Error bars indicate SD of the average rate from 14 data points taken from two experiments. (B) Immunoblot of PAO1 or PAO1ΔfimL in which the wild type cyaB gene has been replaced with cyaB-His. Lysates were prepared from bacteria grown in the presence or absence of Ca²⁺ and probed with anti-His antibody (upper panel) or anti-PilA antibody (lower panel) as a loading control.

Figure 3. FimL, CyaB, and Vfr are required for exoT transcription and TM.

All strains harbor the PexoT-lacZ transcriptional reporter fusion integrated at the CTX site as a readout for transcription of the T3SS. β-galactosidase activity was measured in the presence or absence of calcium. Gene names denote in-frame deletions in PAO1 (A and C) or PA103 (B and D). (A and B) Shown are mean of 12 data points from triplicate samples from 2 or 3 experiments. (C and D). Shown is the mean diameter from a minimum of 5 colonies from 2 or 3 experiments. Error bars denote SD. (***) indicates P<0.001 compared to the wild type strain grown in the absence of Ca²⁺.

Figure 4. Ectopic expression of fimL decreases cAMP.

cAMP levels were measured in (A) PAO1ΔcpdA or (B) PA103 with p (empty vector) or pfimL-FL (vector with fimL-3X-FLAG). Shown are mean values of 3 experimental repetitions performed in triplicate. Error bars indicate SD. (***) P<0.001 compared to the respective strains containing vector only and grown in the absence of Ca²⁺.

Figure 5. Ectopic expression of fimL inhibits exoT transcription.

All strains harbor the PexoT-lacZ transcriptional reporter fusion integrated at the CTX site as a readout for transcription of the T3SS and either include p (vector only) or pfimL-FL (vector with fimL-3X-FLAG). β-galactosidase activity was measured in the presence or absence of Ca²⁺ in the indicated mutants in (A) PAO1 or (B) PA103. Shown is the mean of 12 data points from 3 or 4 experiments. Error bars indicate SD. (***) P<0.001 compared to the respective strain containing vector only grown in the absence of Ca²⁺.

Figure 6. FimL and CyaB are polarly localized.

Images of (A) log-phase or (B) stationary phase grown PAO1 fimL-GFP in which fimL-gfp replaced the native fimL gene. Scale bar is approximately 10 µm. (C) PAO1 GFP with pGFP (stationary phase). (D) PAO1 ΔfimL + pfimL-GFP without arabinose induction (log phase). (E) PAO1 + pfimL-GFP with arabinose induction (stationary phase). (F) PAO1 ΔcyaB + pcyaB-GFP with arabinose induction (log phase).

Figure 7. FimL may function to link the Chp system and CyaB to regulate cAMP levels in P. aeruginosa.

FimL may interact with ChpA and/or CyaB to affect cAMP biosynthesis activity directly or indirectly. The Chp chemosensory system is thought to regulate pilus extension and retraction via two-component signaling. An input signal to the methyl-accepting chemotaxis protein PilJ induces autophosphorylation of the hybrid histidine kinase ChpA. PilG and PilH are putative response regulators that accept phosphoryl groups from phosphorylated ChpA and regulate pilus function. CpdA is a phosphodiesterase that degrades cAMP. cAMP is an allosteric regulator of Vfr which regulates multiple virulence pathways. OM and IM refer to the outer membrane and inner membrane respectively.