Cyclic AMP-Independent Control of Twitching Motility in Pseudomonas aeruginosa.

FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds-lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB-pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.IMPORTANCEPseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements-including the PilSR two-component system-to cell poles for optimal function.

The Type IVa Pilus Machinery Is Recruited to Sites of Future Cell Division.

Type IVa pili (T4aP) are ubiquitous microbial appendages used for adherence, twitching motility, DNA uptake, and electron transfer. Many of these functions depend on dynamic assembly and disassembly of the pilus by a megadalton-sized, cell envelope-spanning protein complex located at the poles of rod-shaped bacteria. How the T4aP assembly complex becomes integrated into the cell envelope in the absence of dedicated peptidoglycan (PG) hydrolases is unknown. After ruling out the potential involvement of housekeeping PG hydrolases in the installation of the T4aP machinery in Pseudomonas aeruginosa, we discovered that key components of inner (PilMNOP) and outer (PilQ) membrane subcomplexes are recruited to future sites of cell division. Midcell recruitment of a fluorescently tagged alignment subcomplex component, mCherry-PilO, depended on PilQ secretin monomers-specifically, their N-terminal PG-binding AMIN domains. PilP, which connects PilO to PilQ, was required for recruitment, while PilM, which is structurally similar to divisome component FtsA, was not. Recruitment preceded secretin oligomerization in the outer membrane, as loss of the PilQ pilotin PilF had no effect on localization. These results were confirmed in cells chemically blocked for cell division prior to outer membrane invagination. The hub protein FimV and a component of the polar organelle coordinator complex-PocA-were independently required for midcell recruitment of PilO and PilQ. Together, these data suggest an integrated, energy-efficient strategy for the targeting and preinstallation-rather than retrofitting-of the T4aP system into nascent poles, without the need for dedicated PG-remodeling enzymes. IMPORTANCE: The peptidoglycan (PG) layer of bacterial cell envelopes has limited porosity, representing a physical barrier to the insertion of large protein complexes involved in secretion and motility. Many systems include dedicated PG hydrolase components that create space for their insertion, but the ubiquitous type IVa pilus (T4aP) system lacks such an enzyme. Instead, we found that components of the T4aP system are recruited to future sites of cell division, where they could be incorporated into the cell envelope during the formation of new poles, eliminating the need for PG hydrolases. Targeting depends on the presence of septal PG-binding motifs in specific components, as removal of those motifs causes delocalization. This preinstallation strategy for the T4aP assembly system would ensure that both daughter cells are poised to extrude pili from new poles as soon as they separate from one another.

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.

UNLABELLED: FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE: FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Loss of membrane-bound lytic transglycosylases increases outer membrane permeability and ß-lactam sensitivity in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections. Its relatively impermeable outer membrane (OM) limits antibiotic entry, and a chromosomally encoded AmpC ß-lactamase inactivates ß-lactam antibiotics. AmpC expression is linked to peptidoglycan (PG) recycling, and soluble (sLT) or membrane-bound (mLT) lytic transglycosylases are responsible for generating the anhydromuropeptides that induce AmpC expression. Thus, inhibition of LT activity could reduce AmpC-mediated ß-lactam resistance in P. aeruginosa. Here, we characterized single and combination LT mutants. Strains lacking SltB1 or MltB had increased ß-lactam minimum inhibitory concentrations (MICs) compared to wild type, while only loss of Slt decreased MICs. An sltB1 mltB double mutant had elevated ß-lactam MICs compared to either the sltB1 or mltB single mutants (96 vs. 32 µg/mL cefotaxime), without changes to AmpC levels. Time-kill assays with ß-lactams suggested that increased MIC correlated with a slower rate of autolysis in the sltB1 mltB mutant - an antisuicide phenotype. Strains lacking multiple mLTs were more sensitive to ß-lactams and up to 16-fold more sensitive to vancomycin, normally incapable of crossing the OM. Multi-mLT mutants were also sensitive to bile salts and osmotic stress, and were hyperbiofilm formers, all phenotypes consistent with cell envelope compromise. Complementation with genes encoding inactive forms of the enzymes - or alternatively, overexpression of Braun's lipoprotein - reversed the mutants' cell envelope damage phenotypes, suggesting that mLTs help to stabilize the OM. We conclude that P. aeruginosa mLTs contribute physically to cell envelope stability, and that Slt is the preferred target for future development of LT inhibitors that could synergize with ß-lactams.

Structural and functional studies of the Pseudomonas aeruginosa minor pilin, PilE.

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilEΔ1-28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilXNm and PilVNm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilXNm and PilVNm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.

Biogenesis of Pseudomonas aeruginosa type IV pili and regulation of their function.

Type IV pili (T4P) are bacterial virulence factors involved in a wide variety of functions including deoxyribonucleic acid uptake, surface attachment, biofilm formation and twitching motility. While T4P are common surface appendages, the systems that assemble them and the regulation of their function differ between species. Pseudomonas aeruginosa, Neisseria spp. and Myxococcus xanthus are common model systems used to study T4P biology. This review focuses on recent advances in P. aeruginosa T4P structural biology, and the regulatory pathways controlling T4P biogenesis and function.