T4P Systems Research Articles

Probable Role of Type IV Pili of Aeromonas hydrophila in Human Pathogenicity.

Aeromonas hydrophila is a widely recognized broad-spectrum pathogen that primarily targets the gastrointestinal tract. Type IV pili (T4P) are proteinaceous nano-machines located on the bacterial cell surface, playing a crucial role in host colonization and infection. Regrettably, the T4P systems of A. hydrophila remain largely underexplored. A. hydrophila genomes with complete genome assembly and annotation reports up to 31 March 2023, were obtained from the NCBI Genome database or KEGG genome database, followed by a global search for T4P secretion system genes. Protein sequences of these manually curetted genes were used as secondary quarry for Synteny analysis. Protein-protein interaction analysis was performed by string analysis and in silico study of genomic islands. We identified 27 orthologs of type IV pili (T4P) nano-machine components in A. hydrophila. These orthologs are primarily distributed across three operons: pilABCD, pilMNOPQ, and pilVWXY. While the first two operons are commonly found in all experimental genomes, the presence of the pilVWXY operon, coding for 11 orthologs, is reported here for the first time in A. hydrophila. Notably, the complete pilVWXY operon is absent in nonvirulent strains. A genomic islands study between a nonvirulent and hypervirulent strain also confirms absence of most of the genes coded by pilVWXY in nonvirulent strain. Interestingly, among the 51 experimental genomes analyzed, the pilVWXY operon was completely absent in 10 strains, most of which are categorized as nonvirulent; Conclusions: The distribution of two major type IV pili (T4P) nano-machines, PilABCDMNOPQ and PilVWXY, is reported here for the first time in A. hydrophila. Additionally, this study suggests a potential role for the PilVWXY nano-machine in establishing human disease.

Characterizing BfpU, an Essential Protein in the Type IV Pilus of Enteropathogenic Escherichia coli

Type IV pili (T4Ps) are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. BfpU is an essential periplasmic protein of the T4P of Enteropathogenic Escherichia coli. Previous crosslinking and localization studies have shown that BfpU might interact with the outer membrane secretin BfpB, which is also essential for T4P biogenesis. To date, BfpU has no known homologs and its function is unknown. To purify BfpU, we performed affinity and size exclusion chromatography. Preliminary characterization of BfpU via thermal stability assay and circular dichroism suggests a melting temperature of 80 °C, and its secondary structure is comprised of ~40% anti‐parallel β‐sheet and ~45% random coil. The secondary structure profile is reminiscent of PilP, an essential periplasmic protein anchored in the membrane that interacts with secretins in other T4P systems. Additionally, random mutagenesis is in progress to assess essential residues of BfpU that may be involved in protein interactions within this T4P system. Single residue mutations from this mutagenesis screening will be used to confirm interaction sites within BfpU. The results of these studies will help clarify the role of BfpU in T4P biogenesis and its interactions with BfpB.

Motor-independent retraction of type IV pili is governed by an inherent property of the pilus filament

Type IV pili (T4P) are dynamic surface appendages that promote virulence, biofilm formation, horizontal gene transfer, and motility in diverse bacterial species. Pilus dynamic activity is best characterized in T4P that use distinct ATPase motors for pilus extension and retraction. Many T4P systems, however, lack a dedicated retraction motor, and the mechanism underlying this motor-independent retraction remains a mystery. Using the Vibrio cholerae competence pilus as a model system, we identify mutations in the major pilin gene that enhance motor-independent retraction. These mutants likely diminish pilin-pilin interactions within the filament to produce less-stable pili. One mutation adds a bulky residue to α1C, a universally conserved feature of T4P. We found that inserting a bulky residue into α1C of the retraction motor-dependent Acinetobacter baylyi competence T4P enhances motor-independent retraction. Conversely, removing bulky residues from α1C of the retraction motor-independent, V. cholerae toxin-coregulated T4P stabilizes the filament and diminishes pilus retraction. Furthermore, alignment of pilins from the broader type IV filament (T4F) family indicated that retraction motor-independent T4P, gram-positive Com pili, and type II secretion systems generally encode larger residues within α1C oriented toward the pilus core compared to retraction motor-dependent T4P. Together, our data demonstrate that motor-independent retraction relies, in part, on the inherent instability of the pilus filament, which may be a conserved feature of diverse T4Fs. This provides evidence for a long-standing yet previously untested model in which pili retract in the absence of a motor by spontaneous depolymerization.

Type IV pili are widespread among non-pathogenic Gram-positive gut bacteria with diverse carbohydrate utilization patterns.

Type IV pili (T4P) are bacterial surface-exposed appendages that have been extensively studied in Gram-negative pathogenic bacteria. Despite recent sequencing efforts, little is known regarding these structures in non-pathogenic anaerobic Gram-positive species, particularly commensals of the mammalian gut. Early studies revealed that T4P in two ruminal Gram-positive species are associated with growth on cellulose, suggesting possible associations of T4P with substrate utilization patterns. In the present study, genome sequences of 118 taxonomically diverse, mainly Gram-positive, bacterial strains isolated from anaerobic (gastrointestinal) environments, have been analysed. The genes likely to be associated with T4P biogenesis were analysed and grouped according to T4P genetic organization. In parallel, consortia of Carbohydrate Active enZYmes (CAZymes) were also analysed and used to predict carbohydrate utilization abilities of selected strains. The predictive power of this approach was additionally confirmed by experimental assessment of substrate-related growth patterns of selected strains. Our analysis revealed that T4P systems with diverse genetic organization are widespread among Gram-positive anaerobic non-pathogenic bacteria isolated from different environments, belonging to two phylogenetically distantly related phyla: Firmicutes and Actinobacteria.

Asymmetric distribution of type IV pili triggered by directional light in unicellular cyanobacteria

The type IV pili (T4P) system is a supermolecular machine observed in prokaryotes. Cells repeat the cycle of T4P extension, surface attachment, and retraction to drive twitching motility. Although the properties of T4P as a motor have been scrutinized with biophysics techniques, the mechanism of regulation remains unclear. Here we provided the framework of the T4P dynamics at the single-cell level in Synechocystis sp. PCC6803, which can recognize light direction. We demonstrated that the dynamics was detected by fluorescent beads under an optical microscope and controlled by blue light that induces negative phototaxis; extension and retraction of T4P was activated at the forward side of lateral illumination to move away from the light source. Additionally, we directly visualized each pilus by fluorescent labeling, allowing us to quantify their asymmetric distribution. Finally, quantitative analyses of cell tracking indicated that T4P was generated uniformly within 0.2 min after blue-light exposure, and within the next 1 min the activation became asymmetric along the light axis to achieve directional cell motility; this process was mediated by the photo-sensing protein, PixD. This sequential process provides clues toward a general regulation mechanism of T4P system, which might be essentially common between archaella and other secretion apparatuses.

The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.

Diversity and Evolution of Type IV pili Systems in Archaea.

Many surface structures in archaea including various types of pili and the archaellum (archaeal flagellum) are homologous to bacterial type IV pili systems (T4P). The T4P consist of multiple proteins, often with poorly conserved sequences, complicating their identification in sequenced genomes. Here we report a comprehensive census of T4P encoded in archaeal genomes using sensitive methods for protein sequence comparison. This analysis confidently identifies as T4P components about 5000 archaeal gene products, 56% of which are currently annotated as hypothetical in public databases. Combining results of this analysis with a comprehensive comparison of genomic neighborhoods of the T4P, we present models of organization of 10 most abundant variants of archaeal T4P. In addition to the differentiation between major and minor pilins, these models include extra components, such as S-layer proteins, adhesins and other membrane and intracellular proteins. For most of these systems, dedicated major pilin families are identified including numerous stand alone major pilin genes of the PilA family. Evidence is presented that secretion ATPases of the T4P and cognate TadC proteins can interact with different pilin sets. Modular evolution of T4P results in combinatorial variability of these systems. Potential regulatory or modulating proteins for the T4P are identified including KaiC family ATPases, vWA domain-containing proteins and the associated MoxR/GvpN ATPase, TFIIB homologs and multiple unrelated transcription regulators some of which are associated specific T4P. Phylogenomic analysis suggests that at least one T4P system was present in the last common ancestor of the extant archaea. Multiple cases of horizontal transfer and lineage-specific duplication of T4P loci were detected. Generally, the T4P of the archaeal TACK superphylum are more diverse and evolve notably faster than those of euryarchaea. The abundance and enormous diversity of T4P in hyperthermophilic archaea present a major enigma. Apparently, fundamental aspects of the biology of hyperthermophiles remain to be elucidated.

Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations.

Type IV pili (T4P) are dynamic protein filaments that mediate bacterial adhesion, biofilm formation, and twitching motility. The highly conserved PilMNOP proteins form an inner membrane alignment subcomplex required for function of the T4P system, though their exact roles are unclear. Three potential interaction interfaces for PilNO were identified: core-core, coiled coils (CC), and the transmembrane segments (TMSs). A high-confidence PilNO heterodimer model was used to select key residues for mutation, and the resulting effects on protein-protein interactions were examined both in a bacterial two-hybrid (BTH) system and in their native Pseudomonas aeruginosa context. Mutations in the oppositely charged CC regions or the TMS disrupted PilNO heterodimer formation in the BTH assay, while up to six combined mutations in the core failed to disrupt the interaction. When the mutations were introduced into the P. aeruginosa chromosome at the pilN or pilO locus, specific changes at each of the three interfaces--including core mutations that failed to disrupt interactions in the BTH system--abrogated surface piliation and/or impaired twitching motility. Unexpectedly, specific CC mutants were hyperpiliated but nonmotile, a hallmark of pilus retraction defects. These data suggest that PilNO participate in both the extension and retraction of T4P. Our findings support a model of multiple, precise interaction interfaces between PilNO; emphasize the importance of studying protein function in a minimally perturbed context and stoichiometry; and highlight potential target sites for development of small-molecule inhibitors of the T4P system. Pseudomonas aeruginosa is an opportunistic pathogen that uses type IV pili (T4P) for host attachment. The T4P machinery is composed of four cell envelope-spanning subcomplexes. PilN and PilO heterodimers are part of the alignment subcomplex and essential for T4P function. Three potential PilNO interaction interfaces (the core-core, coiled-coil, and transmembrane segment interfaces) were probed using site-directed mutagenesis followed by functional assays in an Escherichia coli two-hybrid system and in P. aeruginosa. Several mutations blocked T4P assembly and/or motility, including two that revealed a novel role for PilNO in pilus retraction, while other mutations affected extension dynamics. These critical PilNO interaction interfaces represent novel targets for small-molecule inhibitors with the potential to disrupt T4P function.

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the β-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.

Type IV Pili in Gram-Positive Bacteria

Type IV pili (T4P) are surface-exposed fibers that mediate many functions in bacteria, including locomotion, adherence to host cells, DNA uptake (competence), and protein secretion and that can act as nanowires carrying electric current. T4P are composed of a polymerized protein, pilin, and their assembly apparatuses share protein homologs with type II secretion systems in eubacteria and the flagella of archaea. T4P are found throughout Gram-negative bacterial families and have been studied most extensively in certain model Gram-negative species. Recently, it was discovered that T4P systems are also widespread among Gram-positive species, in particular the clostridia. Since Gram-positive and Gram-negative bacteria have many differences in cell wall architecture and other features, it is remarkable how similar the T4P core proteins are between these organisms, yet there are many key and interesting differences to be found as well. In this review, we compare the two T4P systems and identify and discuss the features they have in common and where they differ to provide a very broad-based view of T4P systems across all eubacterial species.

Type IV Pilus Proteins Form an Integrated Structure Extending from the Cytoplasm to the Outer Membrane

The bacterial type IV pilus (T4P) is the strongest biological motor known to date as its retraction can generate forces well over 100 pN. Myxococcus xanthus, a δ-proteobacterium, provides a good model for T4P investigations because its social (S) gliding motility is powered by T4P. In this study, the interactions among M. xanthus T4P proteins were investigated using genetics and the yeast two-hybrid (Y2H) system. Our genetic analysis suggests that there is an integrated T4P structure that crosses the inner membrane (IM), periplasm and the outer membrane (OM). Moreover, this structure exists in the absence of the pilus filament. A systematic Y2H survey provided evidence for direct interactions among IM and OM proteins exposed to the periplasm. For example, the IM lipoprotein PilP interacted with its cognate OM protein PilQ. In addition, interactions among T4P proteins from the thermophile Thermus thermophilus were investigated by Y2H. The results indicated similar protein-protein interactions in the T4P system of this non-proteobacterium despite significant sequence divergence between T4P proteins in T. thermophilus and M. xanthus. The observations here support the model of an integrated T4P structure in the absence of a pilus in diverse bacterial species.

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.

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.

Prime time for minor subunits of the type II secretion and type IV pilus systems

The type II secretion system (T2SS) exports folded proteins from the periplasms of Gram-negative bacteria. The type IV pilus system (T4PS) is a multifunctional machine used for adherence, motility and DNA transfer in bacteria and archaea. Partial sequence identity between the two systems suggests that they are related and might function via a similar mechanism, the dynamic assembly and disassembly of pseudopilus (T2SS) or pilus (T4PS) filaments. The major subunit in each system is thought to form the bulk of the (pseudo)pilus, while minor (low-abundance) subunits have proposed roles in assembly initiation, antagonism of disassembly, or modulation of (pseudo)pilus functional properties. In this issue, Cisneros et al. () extend their previous finding that pseudopilus assembly is primed by the minor pseudopilins, showing that the same proteins can initiate assembly of Escherichia coli T4P. Similarly, they show that the E. coli minor pilins prime the polymerization of T2S pseudopili, although unlike genuine pseudopili, the chimeric filaments did not support secretion. This work reinforces the notion of a common assembly mechanism for the T2S and T4P systems.

Structure of an Essential Type IV Pilus Biogenesis Protein Provides Insights into Pilus and Type II Secretion Systems

Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic Escherichia coli bundle-forming pilus is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner-membrane proteins BfpC and BfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β-domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein. Yet surprisingly, N-BfpC has far greater structural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components. Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE, and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems, imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that do not preclude component assembly.

Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.

Architecture of the Type II Secretion and Type IV Pilus Machineries

Motility and protein secretion are key processes contributing to bacterial virulence. A wealth of phylogenetic, biochemical and structural evidence support the hypothesis that the widely distributed type IV pilus (T4P) system, involved in twitching motility, and the type II secretion (T2S) system, involved in exoprotein release, are descended from a common progenitor. Both are composed of dedicated but dynamic assemblages, which have been proposed to function through alternate polymerization and depolymerization or degradation of pilin-like subunits. While ongoing studies aimed at understanding the details of assembly and function of these systems are leading to new insights, there are still large knowledge gaps with respect to several fundamental aspects of their biology, including the localization and stoichiometry of critical assembly components, and the nature of their interactions. This article highlights recent advances in understanding the architectures of the T4P and T2S systems, and the organization of their inner and outer membrane components. As structural data accumulates, it is becoming increasingly apparent that even components with little-to-no sequence similarity have similar folds, further supporting the idea that both systems function by a similar mechanism.

Roles of Putative Type II Secretion and Type IV Pilus Systems in the Virulence of Uropathogenic Escherichia coli

BackgroundType II secretion systems (T2SS) and the evolutionarily related type IV pili (T4P) are important virulence determinants in many Gram-negative bacterial pathogens. However, the roles of T2SS and T4P in the virulence of extraintestinal pathogenic Escherichia coli have not been determined.Methodology/Principal FindingsTo investigate the functions of putative T2SS and T4P gene clusters present in the model uropathogenic E. coli (UPEC) strains UTI89 and CFT073, we deleted the secretin gene present in each cluster. The secretin forms a channel in the outer membrane that is essential for the function of T2S and T4P systems. We compared the secretin deletion mutants with their wild type counterparts using tissue culture assays and the CBA/J mouse model of ascending urinary tract infection. No deficiencies were observed with any of the mutants in adherence, invasion or replication in human bladder or kidney cell lines, but UTI89 ΔhofQ and UTI89 ΔgspD exhibited approximately 2-fold defects in fluxing out of bladder epithelial cells. In the mouse infection model, each of the knockout mutants was able to establish successful infections in the bladder and kidneys by day one post-infection. However, UTI89 ΔhofQ and a CFT073 ΔhofQ ΔyheF double mutant both exhibited defects in colonizing the kidneys by day seven post-infection.Conclusions/SignificanceBased on our results, we propose that the putative T4P and T2S systems are virulence determinants of UPEC important for persistence in the urinary tract, particularly in renal tissues.

PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function inMyxococcus xanthus

Type IV pili (T4P) are dynamic surface structures that undergo cycles of extension and retraction. T4P dynamics center on the PilB and PilT proteins, which are members of the secretion ATPase superfamily of proteins. Here, we show that PilB and PilT of the T4P system in Myxococcus xanthus have ATPase activity in vitro. Using a structure-guided approach, we systematically mutagenized PilB and PilT to resolve whether both ATP binding and hydrolysis are important for PilB and PilT function in vivo. PilB as well as PilT ATPase activity was abolished in vitro by replacement of conserved residues in the Walker A and Walker B boxes that are involved in ATP binding and hydrolysis, respectively. PilB proteins containing mutant Walker A or Walker B boxes were nonfunctional in vivo and unable to support T4P extension. PilT proteins containing mutant Walker A or Walker B boxes were also nonfunctional in vivo and unable to support T4P retraction. These data provide genetic evidence that both ATP binding and hydrolysis by PilB are essential for T4P extension and that both ATP binding and hydrolysis by PilT are essential for T4P retraction. Thus, PilB and PilT are ATPases that act at distinct steps in the T4P extension/retraction cycle in vivo.

Weapons of mass retraction

Twitching motility is a unique form of bacterial propulsion on solid surfaces associated with cycles of extension, tethering and retraction of type IV pili (T4P). Although investigations over the last two decades in a number of species have identified the majority of the genes involved in this process, we are still learning how these pili are assembled and the mechanics by which bacteria use T4P to drag themselves from one place to another. Among the puzzles that remain to be solved is the mechanism by which hydrolysis of ATP is coupled to pilus assembly and disassembly, and how the cell envelope structure is modified to accommodate the passage of the pilus through the periplasm. Unravelling of these and other enigmas in the T4P system will not only teach us more about these important colonization and virulence factors, but also help us to understand related processes such as type II secretion, which relies on a set of proteins homologous to those in the T4P system, and bacterial conjugation, involving retractable pili belonging to the F-like subgroup of the type IV secretion family. This review focuses on recent discoveries relating to the assembly and function of T4P in generation of twitching motility.