Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa

Abstract

Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.

Keywords: mechanotransduction; surface sensing; type IV pili; virulence.

Fig. 1.

Surface contact regulates the PaQa operon. P. aeruginosa was transformed with a plasmid encoding a yfp reporter fused to the promoter sequence of PaQa. A cfp reporter fused to the rpoD promoter served as an internal control. (A) Cells growing on solid agarose (Right) have higher YFP fluorescence compared with cells growing in liquid medium (Left), whereas CFP fluorescence only slightly decreased. (Scale bar, 1 μm.) (B) The fluorescence level of cell populations grown on various hydrogels for 3 h was enhanced relative to liquid growth. All fluorescence values are normalized to the levels measured in liquid. Error bars represent 95% confidence intervals (n = 4, each measurement is the average from more than 100 cells).

Fig. 2.

The Chp chemosensory system and TFP extension/retraction mediate surface sensing. (A) The relative fluorescence intensity of the PaQa YFP reporter (normalized to rpoD-mKate) in WT versus Chp system mutants was compared during growth in liquid media or when associated with a 1% agarose hydrogel. Cells lacking PilJ or the histidine kinase ChpA failed to activate the PaQa reporter upon surface contact. Error bars represent 95% confidence intervals (n = 4). (B) The fluorescence intensity of PaQa YFP-expressing bacteria growing on an agarose pad was determined as they transition from a liquid culture. (C) The fluorescence intensity ratio PaQa-YFP:rpoD-mKate was measured for each cell in the colony grown on 1% agarose. Shown is the average ratio per cell as a function of time. WT colonies demonstrate a strong increase in PaQa-YFP, whereas mutants lacking TFP fail to activate PaQa-YFP. Solid lines represent the mean fluorescence over multiple colonies (n = 12), and shaded regions correspond to 95% confidence intervals. (Inset) The rate of increase in florescence (dF/dt) reveals a strong pulse in PaQa promoter activity that is absent in the pilA mutant and diminished in strains with defective TFP extension (pilB) and retraction (pilTU).

Fig. 3.

The activity of PaQa upon surface contact depends on hydrogel mechanical properties. The relative fluorescence intensity of the PaQa YFP reporter (normalized to RpoD-mKate) per cell is averaged for each colony and reported as a function of time. Dashed lines represent the mean over multiple colonies (n = 12) and the shaded regions represent 95% confidence intervals. (Inset) The rate of change of fluorescence F. Higher agarose concentration yields hydrogels with smaller pore size, thus increasing interaction between TFP and the substrate.

Fig. 4.

Proposed molecular model for surface sensing mediated by type IV pilus. (A) The pilus extends and retracts by assembly and disassembly, respectively, of PilA monomers into a polymer. During growth in liquid, TFP do not attach, therefore TFP are not actuated upon retraction (i). Upon contact with a solid surface, attachment and retraction exerts tension on TFP leading to an uncharacterized modification (ii). This modulates the interaction between the periplasmic domain of the MCP chemosensory protein PilJ with PilA and transduces the signal to the cytoplasm: PilJ activates the CheA homolog ChpA complexed with the CheW homolog PilI, stimulating CyaB activity and cAMP production. cAMP thus activates the transcription factor Vfr, increasing transcription of a wide range of genes including virulence factors. Simultaneously, the Chp chemosensory system stimulates TFP extension/retraction via PilG and PilH response regulators, leading to a positive feedback of twitching and surface sensing upon contact. (B) A model for the initiation of Vfr-dependent transcription on abiotic surfaces. (i) Single planktonic cells possess background TFP activity as they swim in the bulk of the fluid. (ii) Upon substrate contact, extended TFP attach onto the surface. Retraction rapidly generates tensile load on the fiber. (iii) Mechanotransduction activates the Chp chemosensory system, leading to an increase in TFP extension and retraction frequency, and induction of cAMP production. (iv) With increased twitching motility and cAMP levels, cells simultaneously explore the substrate and activate Vfr-dependent genes, including type II and III secretion systems, potentially leading to acute infection of a host. Vfr also activates the quorum sensing system thereby stimulating the secretion of autoinducers.