A conservative amino acid mutation in the master regulator FleQ renders Pseudomonas aeruginosa aflagellate

Abstract

Flagellar-based motility plays a critical role in Pseudomonas aeruginosa pathogenesis, influencing both the establishment of bacterial infection and the host's response to the pathogen. Nonetheless, aflagellate clinical strains are often isolated from acutely and chronically infected patients and include the virulent laboratory strain PA103. We determined that PA103's aflagellate phenotype is the result of a single amino acid change (G240V) in the master flagellar regulator, FleQ. This mutation, which lies just outside the Walker B box of FleQ, abrogates the ability of FleQ to positively regulate flagellar gene expression. Reversal of this seemingly conservative amino acid substitution is sufficient to restore swimming motility to PA103, despite the presence of mutations in other flagellar genes of PA103. We also investigated the consequences of restoring flagellar assembly on PA103 virulence. Although a negative correlation between flagellar assembly and Type 3 secretion system (T3SS) expression has been reported previously, we did not observe downregulation of T3SS expression or function in Fla+ PA103. Restoration of flagellar assembly did, however, amplify IL-1 signals measured during murine pulmonary infection and was associated with increased bacterial clearance. These experiments suggest that loss of flagellar motility may primarily benefit PA103 by attenuating pathogen recognition and clearance during acute infection.

Figure 1. Expression of FleQ_PAO1 is sufficient for PA103 to swim.

Swimming zone diameters were measured 16% LB agar plates. Bars represent mean ± S.D. of three replicates. VC: vector control.

Figure 2. FleQ is expressed in both PAO1 and PA103.

Whole cell lysates of PAO1 and PA103 strains (normalized to total protein) were separated by SDS-PAGE and immunoblotted with anti-FleQ antiserum.

Figure 3. Reversion of the Valine240 mutation to Glycine in FleQ_PA103 restores FleQ function.

(A) Swimming zones were measured for PA103 carrying pMQ72 (VC), or plasmids encoding FleQ_PAO1, FleQ_PA103, or the “corrected” allele FleQ(V240G)_PA103 16 hr after inoculating 0.3% LB agar plates containing gentamicin and 0.2% arabinose. All genes are expressed from an arabinose inducible promoter (P_ara). Bars represent mean ± SD of three replicates. (B) The same set of plasmids was transformed into PA14 fleQ::Tn. Swimming was measured on 0.3% LB agar containing gentamicin and 0.2% arabinose. Bars represent mean ± SD of three replicates.

Figure 4. PA103 assembles flagella upon expression of corrected FleQ(V240G)_PA103.

Western blots of surface-associated (upper panel) and total flagellin (middle panel) were prepared from bacteria grown in LB without (uninduced) or with (induced) 0.2% arabinose. Samples were normalized to total protein (as measured in the cell pellet) prior to SDS-PAGE and Western blotting. Whole cell pellets were also probed with anti-Hfq antiserum to confirm equal loading (lower panel). Lanes 1-4 are PA103 carrying (1) empty pMQ72 plasmid, (2) FleQ_PAO1, (3) FleQ_PA103 and (4) FleQ(V240G)_PA103. Lane 5: PAO1 carrying an empty pMQ72 plasmid. Note difference in electrophoretic mobility between a-type flagellin of PA103 and b-type flagellin of PAO1.

Figure 5. T3SS expression by PA103 is not altered when flagellar assembly is restored.

(A) PA103 carrying lux transcriptional reporters for the exoU, exoS or exoT promoters, or a promoterless control, was transformed with P_ara-FleQ(V240G)_PA103 (Fla⁺) or empty vector (Fla^-). RLU (luminescence/OD₆₀₀) was measured hourly (0–4 h) after diluting bacteria into T3SS inducing media (MinS+NTA). Bars represent the mean ± SD of triplicate samples, and the experiment was independently reproduced three times. (B) Western blot of secreted T3SS effectors. Culture supernatants of PA103 carrying pMQ72 (VC), FleQ_PAO1, FleQ_PA103 or FleQ(V240G)_PA103 were prepared from bacteria grown in MinS+NTA with (induced) or without (uninduced) 0.2% arabinose. Each lane was loaded with supernatant equivalent to 15 µg total protein, as measured in the cell pellet. ExoU, ExoT, PopB and PopD were detected by Western blotting with polyclonal antiserum. Cell pellets were immunoblotted with anti-Hfq antiserum to confirm equal loading. (C) Cytotoxicity toward HeLa cells as measured by LDH release. HeLa cells were infected with bacteria (MOI = 10) in triplicate and sampled at 1 h (white bars) and 2 h (grey bars) post-infection. Values are normalized to cytotoxicity caused by PA103 at each time point. The assay was repeated three times, and bars show the mean ± S.D. of an assay, which is representative of three independent experiments.

Figure 6. Flagellated bacteria are cleared more efficiently in mouse lung.

C57BL/6 mice were intranasally infected with P. aeruginosa strain PA103 or fla ⁺ PA103, then sacrificed 6 hpi. (A) Lung bacterial burden expressed as number of bacteria recovered per lung (output) divided by measured inoculum administered (input). Each symbol represents an individual animal; the line indicates the geometric mean for each group. (B) Total cells, (C) neutrophils and (D) macrophages were counted in BAL. Each symbol represents an individual animal; lines indicate medians. Log-transformed data were analyzed using Mann-Whitney test (**, p<0.01).

Figure 7. Fla⁺ PA103 elicits more IL-1 cytokines than aflagellate (wild-type) PA103.

BAL fluid was collected 6 hpi, and IL-1α (A) and IL-1β (B) were measured by sandwich ELISA. Bars indicate mean ± S.E.M. for groups of mice (n = 6-7); these were compared by t-test (*, p<0.05; **, p<0.01).