Crash landing of Vibrio cholerae by MSHA pili-assisted braking and anchoring in a viscoelastic environment.

Mannose-sensitive hemagglutinin (MSHA) pili and flagellum are critical for the surface attachment of Vibrio cholerae, the first step of V. cholerae colonization on host surfaces. However, the cell landing mechanism remains largely unknown, particularly in viscoelastic environments such as the mucus layers of intestines. Here, combining the cysteine-substitution-based labeling method with single-cell tracking techniques, we quantitatively characterized the landing of V. cholerae by directly observing both pili and flagellum of cells in a viscoelastic non-Newtonian solution consisting of 2% Luria-Bertani and 1% methylcellulose (LB+MC). The results show that MSHA pili are evenly distributed along the cell length and can stick to surfaces at any point along the filament. With such properties, MSHA pili are observed to act as a brake and anchor during cell landing which includes three phases: running, lingering, and attaching. Importantly, loss of MSHA pili results in a more dramatic increase in mean path length in LB+MC than in 2% LB only or in 20% Ficoll solutions, indicating that the role of MSHA pili during cell landing is more apparent in viscoelastic non-Newtonian fluids than viscous Newtonian ones. Our work provides a detailed picture of the landing dynamics of V. cholerae under viscoelastic conditions, which can provide insights into ways to better control V. cholerae infections in a real mucus-like environment.

Minor pilins are involved in motility and natural competence in the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibers that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Three further gene products with similarity to PilX-like minor pilins have a function in flocculation of Synechocystis. The results of our study indicate that different minor pilins facilitate distinct pilus functions. Further, our microarray analysis demonstrated that the transcription levels of the minor pilin genes change in response to surface contact. A total of 122 genes were determined to have altered transcription between planktonic and surface growth, including several plasmid genes which are involved exopolysaccharide synthesis and the formation of bloom-like aggregates.

PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion.

Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly ubiquitous in prokaryotes. They are predominantly polymers of one major pilin but also contain minor pilins whose functions are often poorly defined and likely to be diverse. Here, we show that the minor pilin PilB from the T4P of Streptococcus sanguinis displays an unusual bimodular three-dimensional structure with a bulky von Willebrand factor A-like (vWA) module "grafted" onto a small pilin module via a short loop. Structural modeling suggests that PilB is only compatible with a localization at the tip of T4P. By performing a detailed functional analysis, we found that 1) the vWA module contains a canonical metal ion-dependent adhesion site, preferentially binding Mg2+ and Mn2+, 2) abolishing metal binding has no impact on the structure of PilB or piliation, 3) metal binding is important for S. sanguinis T4P-mediated twitching motility and adhesion to eukaryotic cells, and 4) the vWA module shows an intrinsic binding ability to several host proteins. These findings reveal an elegant yet simple evolutionary tinkering strategy to increase T4P functional versatility by grafting a functional module onto a pilin for presentation by the filaments. This strategy appears to have been extensively used by bacteria, in which modular pilins are widespread and exhibit an astonishing variety of architectures.

Fresh Extension of Vibrio cholerae Competence Type IV Pili Predisposes Them for Motor-Independent Retraction.

Bacteria utilize dynamic appendages, called type IV pili (T4P), to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can occur with the aid of an ATPase motor or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remain poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization. IMPORTANCE Extracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions; however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.

Collective Dynamics of Model Pili-Based Twitcher-Mode Bacilliforms.

Pseudomonas aeruginosa, like many bacilliforms, are not limited only to swimming motility but rather possess many motility strategies. In particular, twitching-mode motility employs hair-like pili to transverse moist surfaces with a jittery irregular crawl. Twitching motility plays a critical role in redistributing cells on surfaces prior to and during colony formation. We combine molecular dynamics and rule-based simulations to study twitching-mode motility of model bacilliforms and show that there is a critical surface coverage fraction at which collective effects arise. Our simulations demonstrate dynamic clustering of twitcher-type bacteria with polydomains of local alignment that exhibit spontaneous correlated motions, similar to rafts in many bacterial communities.

The impact of the ancillary pilus-1 protein RrgA of Streptococcus pneumoniae on colonization and disease.

The Gram-positive bacterium Streptococcus pneumoniae, the pneumococcus, is an important commensal resident of the human nasopharynx. Carriage is usually asymptomatic, however, S. pneumoniae can become invasive and spread from the upper respiratory tract to the lungs causing pneumonia, and to other organs to cause severe diseases such as bacteremia and meningitis. Several pneumococcal proteins important for its disease-causing capability have been described and many are expressed on the bacterial surface. The surface located pneumococcal type-1 pilus has been associated with virulence and the inflammatory response, and it is present in 20%-30% of clinical isolates. Its tip protein RrgA has been shown to be a major adhesin to human cells and to promote invasion through the blood-brain barrier. In this review we discuss recent findings of the impact of RrgA on bacterial colonization of the upper respiratory tract and on pneumococcal virulence, and use epidemiological data and genome-mining to suggest trade-off mechanisms potentially explaining the rather low prevalence of pilus-1 expressing pneumococci in humans.

Structure and function of minor pilins of type IV pili.

Type IV pili are versatile and highly flexible fibers formed on the surface of many Gram-negative and Gram-positive bacteria. Virulence and infection rate of several pathogenic bacteria, such as Neisseria meningitidis and Pseudomonas aeruginosa, are strongly dependent on the presence of pili as they facilitate the adhesion of the bacteria to the host cell. Disruption of the interactions between the pili and the host cells by targeting proteins involved in this interaction could, therefore, be a treatment strategy. A type IV pilus is primarily composed of multiple copies of protein subunits called major pilins. Additional proteins, called minor pilins, are present in lower abundance, but are essential for the assembly of the pilus or for its specific functions. One class of minor pilins is required to initiate the formation of pili, and may form a complex similar to that identified in the related type II secretion system. Other, species-specific minor pilins in the type IV pilus system have been shown to promote additional functions such as DNA binding, aggregation and adherence. Here, we will review the structure and the function of the minor pilins from type IV pili.

Role of CpxR in Biofilm Development: Expression of Key Fimbrial, O-Antigen and Virulence Operons of Salmonella Enteritidis.

Salmonella Enteritidis is a non-typhoidal serovar of great public health significance worldwide. The RpoE sigma factor and CpxRA two-component system are the major regulators of the extracytoplasmic stress response. In this study, we found that the CpxR has highly significant, but opposite effects on the auto-aggregation and swarming motility of S. Enteritidis. Auto-aggregation was negatively affected in the ∆cpxR mutant, whereas the same mutant significantly out-performed its wild-type counterpart with respect to swarming motility, indicating that the CpxR plays a role in biofilm-associated phenotypes. Indeed, biofilm-related assays showed that the CpxR is of critical importance in biofilm development under both static (microtiter plate) and dynamic (flow cell) media flow conditions. In contrast, the RpoE sigma factor showed no significant role in biofilm development under dynamic conditions. Transcriptomic analysis revealed that the cpxR mutation negatively affected the constitutive expression of the operons critical for biosynthesis of O-antigen and adherence, but positively affected the expression of virulence genes critical for Salmonella-mediated endocytosis. Conversely, CpxR induced the expression of curli csgAB and fimbrial stdAC operons only during biofilm development and flagellar motAB and fliL operons exclusively during the planktonic phase, indicating a responsive biofilm-associated loop of the CpxR regulator.

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili.

Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.

Adhesive mechanism of different Salmonella fimbrial adhesins.

Salmonellosis is a serious threat to human and animal health. Salmonella adhesion to the host cell is an initial and most crucial step in the pathogenesis of salmonellosis. Many factors are involved in the adhesion process of Salmonella infection. Fimbriae are one of the most important factors in the adhesion of Salmonella. The Salmonella fimbriae are assembled in three types of assembly pathways: chaperon-usher, nucleation-precipitation, and type IV fimbriae. These assembly pathways lead to multiple types of fimbriae. Salmonella fimbriae bind to host cell receptors to initiate adhesion. So far, many receptors have been identified, such as Toll-like receptors. However, several receptors that may be involved in the adhesive mechanism of Salmonella fimbriae are still un-identified. This review aimed to summarize the types of Salmonella fimbriae produced by different assembly pathways and their role in adhesion. It also enlisted previously discovered receptors involved in adhesion. This review might help readers to develop a comprehensive understanding of Salmonella fimbriae, their role in adhesion, and recently developed strategies to counter Salmonella infection.

The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase.

Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.

Long polar fimbriae contribute to pathogenic Escherichia coli infection to host cells.

Long polar fimbria (LPF) is one of the few fimbrial adhesins of enterohemorrhagic Escherichia coli (E. coli) O157:H7 associated with colonization on host intestine, and both two types of LPF (including LPF1 and LPF2) play essential roles during the bacterial infection process. Though the fimbriae had been well studied in intestinal pathogenic E. coli strains, new evidences from our research revealed that it might be the key virulence for bovine mastitis pathogenic E. coli (MPEC) as well. This article summarizes the current knowledge on the LPF in E. coli, focusing on its genetic characteristics, prevalence, expression regulation, and adherence mechanism in different pathotypes of E. coli strains.

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity.

Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 was proposed to be a mechanosensory component that-in conjunction with minor pilins-triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins/PilY1 and virulence. pilW, pilX, and pilY1 mutants had reduced virulence towards Caenorhabditis elegans relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the AlgR regulon and delaying C. elegans killing. Reporter assays confirmed that FimS-AlgR were required for increased expression of the minor pilin operon upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via a phosphomimetic mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants required FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR that suppresses expression of acute-phase virulence factors and delays killing. This mechanism could contribute to adaptation of P. aeruginosa in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors-such as alginate-that are characteristic of such infections.

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion.

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable fibronectin-binding, collagen-binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT-6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain-of-function mutants we show that, unlike other GAS pili, the FCT-6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT-6 pili.

Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology.

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable.

Motility and adhesion through type IV pili in Gram-positive bacteria.

Type IV pili are hair-like bacterial surface appendages that play a role in diverse processes such as cellular adhesion, colonization, twitching motility, biofilm formation, and horizontal gene transfer. These extracellular fibers are composed exclusively or primarily of many copies of one or more pilin proteins, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the pilus core. Type IV pili have been characterized extensively in Gram-negative bacteria, and recent advances in high-throughput genomic sequencing have revealed that they are also widespread in Gram-positive bacteria. Here, we review the current state of knowledge of type IV pilus systems in Gram-positive bacterial species and discuss them in the broader context of eubacterial type IV pili.

Role of Mfa5 in Expression of Mfa1 Fimbriae in Porphyromonas gingivalis.

Fimbriae are protein-based filamentous appendages that protrude from the bacterial cell surface and facilitate host adhesion. Two types of fimbriae, FimA and Mfa1, of the periodontal pathogen Porphyromonas gingivalis are responsible for adherence to other bacteria and to host cells in the oral cavity. Both fimbrial forms are composed of 5 proteins, but there is limited information about their polymerization mechanisms. Here, the authors evaluated the function of Mfa5, one of the Mfa1 fimbrial accessory proteins. Using mfa5 gene disruption and complementation studies, the authors revealed that Mfa5 affects the incorporation of other accessory proteins, Mfa3 and Mfa4, into fibers and the expression of fimbriae on the cell surface. Mfa5 is predicted to have a C-terminal domain (CTD) that uses the type IX secretion system (T9SS), which is limited to this organism and related Bacteroidetes species, for translocation across the outer membrane. To determine the relationship between the putative Mfa5 CTD and the T9SS, mutants were constructed with in-frame deletion of the CTD and deletion of porU, a C-terminal signal peptidase linked to T9SS-mediated secretion. The ∆CTD-expressing strain presented a similar phenotype to the mfa5 disruption mutant with reduced expression of fimbriae lacking all accessory proteins. The ∆porU mutants and the ∆CTD-expressing strain showed intracellular accumulation of Mfa5. These results indicate that Mfa5 function requires T9SS-mediated translocation across the outer membrane, which is dependent on the CTD, and subsequent incorporation into fibers. These findings suggest the presence of a novel polymerization mechanism of the P. gingivalis fimbriae.

How Bacteria Use Type IV Pili Machinery on Surfaces.

The bacterial type IV pilus (T4P) is a versatile molecular machine with a broad range of functions. Recent advances revealed that the molecular components and the biophysical properties of the machine are well conserved among phylogenetically distant bacterial species. However, its functions are diverse, and include adhesion, motility, and horizontal gene transfer. This review focusses on the role of T4P in surface motility and bacterial interactions. Different species have evolved distinct mechanisms for intracellular coordination of multiple pili and of pili with other motility machines, ranging from physical coordination to biochemical clocks. Coordinated behavior between multiple bacteria on a surface is achieved by active manipulation of surfaces and modulation of pilus-pilus interactions. An emerging picture is that the T4P actively senses and responds to environmental conditions.

Mfa4, an Accessory Protein of Mfa1 Fimbriae, Modulates Fimbrial Biogenesis, Cell Auto-Aggregation, and Biofilm Formation in Porphyromonas gingivalis.

Porphyromonas gingivalis, a gram-negative obligate anaerobic bacterium, is considered to be a key pathogen in periodontal disease. The bacterium expresses Mfa1 fimbriae, which are composed of polymers of Mfa1. The minor accessory components Mfa3, Mfa4, and Mfa5 are incorporated into these fimbriae. In this study, we characterized Mfa4 using genetically modified strains. Deficiency in the mfa4 gene decreased, but did not eliminate, expression of Mfa1 fimbriae. However, Mfa3 and Mfa5 were not incorporated because of defects in posttranslational processing and leakage into the culture supernatant, respectively. Furthermore, the mfa4-deficient mutant had an increased tendency to auto-aggregate and form biofilms, reminiscent of a mutant completely lacking Mfa1. Notably, complementation of mfa4 restored expression of structurally intact and functional Mfa1 fimbriae. Taken together, these results indicate that the accessory proteins Mfa3, Mfa4, and Mfa5 are necessary for assembly of Mfa1 fimbriae and regulation of auto-aggregation and biofilm formation of P. gingivalis. In addition, we found that Mfa3 and Mfa4 are processed to maturity by the same RgpA/B protease that processes Mfa1 subunits prior to polymerization.

Peptide-Mediated PEGylation of Polysulfone Reduces Protein Adsorption and Leukocyte Activation.

The exposure of blood to bioincompatible materials used for dialysis triggers leukocyte activation and protein adsorption. We describe a single-step, postmanufacturing method for surface modification to create biomaterials used in medical devices and dialysis with altered surface characteristics. Peptides derived from the receptor-binding domain of the type IV pilin of Pseudomonas aeruginosa were synthesized using L and D-amino acids to generate L-K122-4, enantiomer D-K122-4, and D-retroinverso RI-K122-4 peptides. L-K122-4, D-K122-4, and RI-K122-4 peptides, but not control peptides, bound durably to the surfaces of materials used in medical devices and dialysis including silicone and polysulfone. D-K122-4 enantiomeric peptides were protease resistant on polysulfone and could remain bound to the surface for up to 28 days. To demonstrate that K122-4 peptides could be used to modify material surfaces, D-K122-4 peptide was conjugated to polyethylene glycol (D-K122-4-PEG) and applied to polysulfone. When compared with untreated material, D-K122-4-PEG reduced the surface adsorption of albumin or immunoglobulin G to polysulfone. In coincubation experiments, although uncoated polysulfone induced pro-interleukin-1ß cytokine expression in leukocytes, cellular activation was prevented when leukocytes were incubated with D-K122-4-PEG-modified polysulfone. These data demonstrate the proof of principle that K122-4 peptides can be applied to modify the surface characteristics of materials used for dialysis.