Hanjeong Harvey

PilM/N/O/P Proteins Form an Inner Membrane Complex That Affects the Stability of the Pseudomonas aeruginosa Type IV Pilus Secretin

The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. With the exception of the outer membrane secretin, a multimer of PilQ subunits, the specific functions of the products encoded by this gene cluster are poorly characterized. Orthologous proteins in the related bacterial type II secretion system have been shown to interact to form an inner membrane complex required for protein secretion. In this study, we provide evidence that the PilM/N/O/P proteins form a functionally equivalent type IVa pilus complex. Using Pseudomonas aeruginosa as model organism, we found that all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane. Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. Furthermore, complementation studies revealed that the stoichiometry of the components is important for the correct formation of a stable complex in vivo. We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa, as PilQ stability is negatively affected in its absence. Finally, we show that, in the absence of the pilin subunit, the levels of membrane-bound components of the inner membrane complex are negatively regulated by the PilR/S two-component system, suggesting a role for PilR/S in sensing the piliation status of the cell.

Modification of Pseudomonas aeruginosa Pa5196 Type IV Pilins at Multiple Sites with d-Araf by a Novel GT-C Family Arabinosyltransferase, TfpW

Pseudomonas aeruginosa Pa5196 produces type IV pilins modified with unusual alpha1,5-linked d-arabinofuranose (alpha1,5-D-Araf) glycans, identical to those in the lipoarabinomannan and arabinogalactan cell wall polymers from Mycobacterium spp. In this work, we identify a second strain of P. aeruginosa, PA7, capable of expressing arabinosylated pilins and use a combination of site-directed mutagenesis, electrospray ionization mass spectrometry (MS), and electron transfer dissociation MS to identify the exact sites and extent of pilin modification in strain Pa5196. Unlike previously characterized type IV pilins that are glycosylated at a single position, those from strain Pa5196 were modified at multiple sites, with modifications of alphabeta-loop residues Thr64 and Thr66 being important for normal pilus assembly. Trisaccharides of alpha1,5-D-Araf were the principal modifications at Thr64 and Thr66, with additional mono- and disaccharides identified on Ser residues within the antiparallel beta sheet region of the pilin. TfpW was hypothesized to encode the pilin glycosyltransferase based on its genetic linkage to the pilin, weak similarity to membrane-bound GT-C family glycosyltransferases (which include the Mycobacterium arabinosyltransferases EmbA/B/C), and the presence of characteristic motifs. Loss of TfpW or mutation of key residues within the signature GT-C glycosyltransferase motif completely abrogated pilin glycosylation, confirming its involvement in this process. A Pa5196 pilA mutant complemented with other Pseudomonas pilins containing potential sites of modification expressed nonglycosylated pilins, showing that TfpWs pilin substrate specificity is restricted. TfpW is the prototype of a new type IV pilin posttranslational modification system and the first reported gram-negative member of the GT-C glycosyltransferase family.

The Peptidoglycan-Binding Protein FimV Promotes Assembly of the Pseudomonas aeruginosa Type IV Pilus Secretin

The Pseudomonas aeruginosa inner membrane protein FimV is among several proteins of unknown function required for type IV pilus-mediated twitching motility, arising from extension and retraction of pili from their site of assembly in the inner membrane. The pili transit the periplasm and peptidoglycan (PG) layer, ultimately exiting the cell through the PilQ secretin. Although fimV mutants are nonmotile, they are susceptible to killing by pilus-specific bacteriophage, a hallmark of retractable surface pili. Here we show that levels of recoverable surface pili were markedly decreased in fimV pilT retraction-deficient mutants compared with levels in the pilT control, demonstrating that FimV acts at the level of pilus assembly. Levels of inner membrane assembly subcomplex proteins PilM/N/O/P were decreased in fimV mutants, but supplementation of these components in trans did not restore pilus assembly or motility. Loss of FimV dramatically reduced the levels of the PilQ secretin multimer through which pili exit the cell, in part due to decreased levels of PilQ monomers, while PilF pilotin levels were unchanged. Expression of pilQ in trans in the wild type or fimV mutants increased total PilQ monomer levels but did not alter secretin multimer levels or motility. PG pulldown assays showed that the N terminus of FimV bound PG in a LysM motif-dependent manner, and a mutant with an in-frame chromosomal deletion of the LysM motif had reduced motility, secretin levels, and surface piliation. Together, our data show that FimVs role in pilus assembly is to promote secretin formation and that this function depends upon its PG-binding domain.

Pseudomonas aeruginosa Minor Pilins Prime Type IVa Pilus Assembly and Promote Surface Display of the PilY1 Adhesin

Background: Type IV pili (T4P) are virulence factors composed of major and minor pilins. Results: Minor pilins prime pilus assembly and traffic anti-retraction protein PilY1 to the surface; FimU and GspH are structurally similar. Conclusion: Minor pilins are essential for pilus assembly and function. Significance: This work expands the structural and functional similarities between the T4P and T2S systems. Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility.

Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility

PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.

Functional mapping of PilF and PilQ in the Pseudomonas aeruginosa type IV pilus system.

Pseudomonas aeruginosa uses type IV pili (T4P) to interact with the environment and as key virulence factors when acting as an opportunistic pathogen. Assembly of the outer membrane PilQ secretin channel through which the pili are extruded is essential for pilus biogenesis. The P. aeruginosa T4P pilotin, PilF, is required for PilQ outer membrane localization and assembly into secretins and contains six tetratricopeptide (TPR) protein-protein interaction motifs, suggesting that the two proteins interact. In this study, we found that the first four TPR motifs of PilF are sufficient for PilQ outer membrane targeting, oligomerization, and function. Guided by our structure of PilF, site-directed mutagenesis of the protein surface revealed that a hydrophobic groove on the first TPR is required for PilF-mediated PilQ assembly. Deletion of individual domains within PilQ suggests that the N0, KH-like, or secretin domain, but not the C-terminus, interacts with PilF. Purified PilQ was found to pull down PilF from Pseudomonas cell lysates. Together, these data allow us to propose a model for PilF function in the T4P system. PilF interacts directly or indirectly with the PilQ monomer after translocation of both proteins through the inner membrane and acts as a co-chaperone with the Lol system to facilitate transit across the periplasm to the outer membrane. The mechanism of PilQ insertion and assembly, which appears to be independent of the Bam system, remains to be determined.

Pseudomonas aeruginosa d-Arabinofuranose Biosynthetic Pathway and Its Role in Type IV Pilus Assembly

Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked d-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-d-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to d-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilAIV produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae.

Nanoscale Pulling of Type IV Pili Reveals Their Flexibility and Adhesion to Surfaces over Extended Lengths of the Pili

Type IV pili (T4P) are very thin protein filaments that extend from and retract into bacterial cells, allowing them to interact with and colonize a broad array of chemically diverse surfaces. The physical aspects that allow T4P to mediate adherence to many different surfaces remain unclear. Atomic force microscopy (AFM) nanoscale pulling experiments were used to measure the mechanical properties of T4P of a mutant strain of Pseudomonas aeruginosa PAO1 unable to retract its T4P. After adhering bacteria to the end of an AFM cantilever and approaching surfaces of mica, gold, or polystyrene, we observed adhesion of the T4P to all of the surfaces. Pulling of single and multiple T4P on retraction of the cantilever from the surfaces could be described using the worm-like chain (WLC) model. Distinct peaks in the measured distributions of the best-fit values of the persistence length Lp on two different surfaces provide strong evidence for close-packed bundling of very flexible T4P. In addition, we observed force plateaus indicating that adhesion of the T4P to both hydrophilic and hydrophobic surfaces occurs along extended lengths of the T4P. These data shed new light, to our knowledge, on T4P flexibility and support a low-affinity, high-avidity adhesion mechanism that mediates bacteria-surface interactions.

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

ABSTRACT 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. 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.

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

Background: Type IVa pilus (T4aP) assembly is primed by minor pilins. Results: Non-core subunit PilE interacts with core minor pilins and is incorporated into pili; PilE is structurally similar to Neisseria PilX and PilV. Conclusion: PilE connects the priming complex and the major pilin. Significance: This function may be broadly conserved for non-core minor components in T4aP. 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.