PilT Protein Research Articles

Ectopic overexpression of MCPIP1 impairs adipogenesis by modulating microRNAs

Adipogenesis is a process of preadipocyte differentiation that requires action of numerous factors. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) possesses the N-terminus of the PilT protein (PilT N-terminus or PIN domain) that has RNase properties. This protein degrades transcripts coding for inflammation and differentiation – related proteins. Moreover, MCPIP1 is a broad suppressor of the miRNA biogenesis. We previously found that MCPIP1 degrades transcript encoding CCAAT/Enhancer Binding Protein Beta (C/EBPβ) and influences adipogenesis. Subsequently, we aimed to determine adipocyte miRNA expression profile in differentiating mouse preadipocytes, 3T3-L1, by overexpressing MCPIP1. Using Next-Generation Sequencing (NSG) we showed that MCPIP1 overexpression results in modulated levels of 58 miRNAs in adipocytes on day 2 of differentiation. Among them, 30 miRNAs showed significantly reduced levels and 28 showed increased levels in comparison to control. Approximately one third of the modulated miRNAs were not previously reported to be involved in adipocytes differentiation. Our analysis revealed that 24 down-regulated and 23 up-regulated miRNAs (at least 1.5-fold) influence 19 signaling pathways that are important for adipogenesis. Furthermore, reduced miRNA levels result in the up-regulation of their targets. By using luciferase reporter assay, we demonstrated that miR-32-5p and miR-9-3p directly target the 3′UTR region of Mapk8 and Tiam1, respectively. In addition, activation of MAP kinases pathway (JNK and p38), proposed as being regulated by down-regulated miRNAs, was higher in WTMCPIP1 than in D141NMCPIP1 or control 3T3-L1 adipocytes. Our results indicate a considerable impact of MCPIP1 on miRNAs levels and its significance in adipogenesis.

Type-IV Pilus deformation can explain retraction behavior.

Polymeric filament like type IV Pilus (TFP) can transfer forces in excess of 100 pN during their retraction before stalling, powering surface translocation(twitching). Single TFP level experiments have shown remarkable nonlinearity in the retraction behavior influenced by the external load as well as levels of PilT molecular motor protein. This includes reversal of motion near stall forces when the concentration of the PilT protein is loweblack significantly. In order to explain this behavior, we analyze the coupling of TFP elasticity and interfacial behavior with PilT kinetics. We model retraction as reaction controlled and elongation as transport controlled process. The reaction rates vary with TFP deformation which is modeled as a compound elastic body consisting of multiple helical strands under axial load. Elongation is controlled by monomer transport which suffer entrapment due to excess PilT in the cell periplasm. Our analysis shows excellent agreement with a host of experimental observations and we present a possible biophysical relevance of model parameters through a mechano-chemical stall force map.

Role of type IV pili in predation by Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus, as an obligate predator of Gram-negative bacteria, requires contact with the surface of a prey cell in order to initiate the life cycle. After attachment, the predator penetrates the prey cell outer membrane and enters the periplasmic space. Attack phase cells of B. bacteriovorus have polar Type IV pili that are required for predation. In other bacteria, these pili have the ability to extend and retract via the PilT protein. B. bacteriovorus has two pilT genes, pilT1 and pilT2, that have been implicated in the invasion process. Markerless in-frame deletion mutants were constructed in a prey-independent mutant to assess the role of PilT1 and PilT2 in the life cycle. When predation was assessed using liquid cocultures, all mutants produced bdelloplasts of Escherichia coli. These results demonstrated that PilT1 and PilT2 are not required for invasion of prey cells. Predation of the mutants on biofilms of E. coli was also assessed. Wild type B. bacteriovorus 109JA and the pilT1 mutant decreased the mass of the biofilm to 35.4% and 27.9% respectively. The pilT1pilT2 mutant was able to prey on the biofilm, albeit less efficiently with 50.2% of the biofilm remaining. The pilT2 mutant was unable to disrupt the biofilm, leaving 92.5% of the original biofilm after predation. The lack of PilT2 function may impede the ability of B. bacteriovorus to move in the extracellular polymeric matrix and find a prey cell. The role of Type IV pili in the life cycle of B. bacteriovorus is thus for initial recognition of and attachment to a prey cell in liquid cocultures, and possibly for movement within the matrix of a biofilm.

TFL, a hidden post-transcriptional modulator behind inflammation

Post-transcriptional modulation of inflammatory molecules is recently recognized for fine tuning of inflammatory initiation and maintenance. In these molecules, some families with three cytosines and a histidine type of zinc finger domain are gaining attention. Among them, MCP-1 induced protein (MCPIP), also known as regulatory RNase 1 (Regnase-1), encoded by the Zc3h12a gene is recently recognized as a novel player for the inflammatory response. Meanwhile, the Transformed follicular lymphoma ( TFL ) gene is identified as a putative tumor suppressor gene for lung carcinoma, as well as malignant lymphoma. TFL is a member of the Zc3h12 gene family; hence, it is named as Zc3h12d . It has Nedd4-binding protein 1 and bacterial YacP-like protein domains, which resemble the N-terminus of PilT protein, acts as a ribonuclease, and degrades several cytokines similar to Regnase-1. TFL encodes mainly the 58Kda protein, p58 TFL , which is expressed dominantly in lymphoid organs and is upregulated in activated T and B cells according to the inflammatory response. In contrast, MCPIP/Regnase-1 is expressed mainly in macrophages and for a short term in activated lymphocytes. TFL inhibits cell growth via upstream inhibition of the Rb signaling pathway like a tumor suppressor. Moreover, in contrast to other Zc3h12 family members, expression of TFL is increased slowly in activated lymphocytes, and inflammatory resolution is modulated, which is vital for tissue regeneration. Inflammatory dysregulation by TFL deficiencies brings about prolonged inflammation and may give rise to tumor susceptibility in lymphomagenesis. Therefore, TFL can be considered a novel regulatory marker of inflammation, as well as an indicator for tumor progression.

Cloning, expression, and functional analysis of molecular motor pilT and pilU genes of type IV pili in Acidithiobacillus ferrooxidans

PilT is a hexameric ATPase required for type IV pili (Tfp) retraction in gram-negative bacterium. Retraction of Tfp mediates intimate attachment and motility on inorganic solid surfaces. We investigated the cloning and expression of pilT and pilU genes of Acidithiobacillus ferrooxidans strains ATCC 23270, and the results indicate that PilT and PilU contain the canonical conserved AIRNLIRE and GMQTXXXXLXXL motifs that are the characteristic motifs of the PilT protein family; PilT and PilU also contain the canonical nucleotide-binding motifs, named with Walker A box (GxxGxGKT/S) and Walker B box (hhhhDE), respectively. The pilT and pilU genes were expressed to produce 37.1- and 42.0-kDa proteins, respectively, and co-transcribed induced by 10% mineral powder. However, ATPase activity of PilT was distinctly higher than those of PilU. These results indicated that the PilT protein was the real molecular motor of Tfp, while PilU could play a key role in the assembly, modification, and twitching motility of Tfp in A. ferrooxidans. However, PilT and PilU were nonetheless interrelated in the forming and function of the molecular motor of Tfp.

Loss of Meningococcal PilU Delays Microcolony Formation and Attenuates Virulence In Vivo

Neisseria meningitidis is a major cause of sepsis and bacterial meningitis worldwide. This bacterium expresses type IV pili (Tfp), which mediate important virulence traits such as the formation of bacterial aggregates, host cell adhesion, twitching motility, and DNA uptake. The meningococcal PilT protein is a hexameric ATPase that mediates pilus retraction. The PilU protein is produced from the pilT-pilU operon and shares a high degree of homology with PilT. The function of PilT in Tfp biology has been studied extensively, whereas the role of PilU remains poorly understood. Here we show that pilU mutants have delayed microcolony formation on host epithelial cells compared to the wild type, indicating that bacterium-bacterium interactions are affected. In normal human serum, the pilU mutant survived at a higher rate than that for wild-type bacteria. However, in a murine model of disease, mice infected with the pilT mutant demonstrated significantly reduced bacterial blood counts and survived at a higher rate than that for mice infected with the wild type. Infection of mice with the pilU mutant resulted in a trend of lower bacteremia, and still a significant increase in survival, than that of the wild type. In conclusion, these data suggest that PilU promotes timely microcolony formation and that both PilU and PilT are required for full bacterial virulence.

Functional Analysis of PilT from the Toxic Cyanobacterium Microcystis aeruginosa PCC 7806

The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 +/- 1.8 nmol P(i) min(-1) mg protein(-1), with a requirement for Mg(2+). Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed.

Pilus‐mediated adhesion of Neisseria meningitidis is negatively controlled by the pilus‐retraction machinery

The type IV pili (Tfp) of Neisseria meningitidis play an essential role in meningococcal virulence by mediating the initial interaction of bacteria with host cells. Tfp are also subject to retraction, which relies on the PilT protein. Among the other components of the Tfp machinery, PilC1, a pilus-associated protein, is important for Tfp biogenesis and adhesion. Adhesion of N. meningitidis to living epithelial cells was previously shown to rely on the upregulation of the pilC1 gene. On the other hand the lack of induction of pilC1 is believed to be responsible for the low adhesion of N. meningitidis onto fixed dead cells. Surprisingly, a pilT mutant, unable to retract its pili, has been shown to adhere very efficiently onto both living and fixed epithelial cells. To elucidate the mechanisms by which the pilus retraction machinery mediates meningococcal adhesion onto fixed cells, an analysis of gene expression levels in wild-type and pilT meningococci was performed using DNA microarrays. One of the upregulated genes in the pilT strain was pilC1. This result was confirmed using quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis. The transcription starting point responsible for the upregulation of pilC1 in a pilT background was shown to be different from those controlling the induction of pilC1 upon contact with living host cells. Subsequent work using a strain hyperproducing PilT confirmed that PilT downregulates the production of PilC1. Furthermore using a pilC1 allele under the control of IPTG, we demonstrated that the upregulation of pilC1 in a pilT background was responsible for the adhesive phenotype onto fixed dead cells. Taken together our results demonstrate that the pilus retraction machinery negatively controlled the adhesiveness of the Tfp via the expression of pilC1.

Aquifex aeolicus PilT, homologue of a surface motility protein, is a thermostable oligomeric NTPase.

Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a approximately equal 5- to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed.

The cyanobacterial PilT protein responsible for cell motility and transformation hydrolyzes ATP.

The unicellular cyanobacterium, Synechocystis sp. PCC 6803 is motile. A homologue of the PilT protein family, required for twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was found to be necessarily associated with cyanobacterial motility. The pilT1 (slr0161) mutant shows a pleotropic phenotype, defects in individual cell motility, and an increased number of long surface pili. Furthermore, the mutant loses its ability of natural competency. These findings demonstrate that PilT1 is essential for both cell motility and competency. Since the pilT gene contains a consensus ATP-binding motif (Walker boxes), the PilT protein is suggested for supplying energy for cell motility. The product of pilT1, overproduced in Escherichia coli and purified by Ni-affinity chromatography, hydrolyzes ATP in vitro.

Modification of type IV pilus-associated epithelial cell adherence and multicellular behavior by the PilU protein of Neisseria gonorrhoeae.

Expression of type IV pili (Tfp) correlates with the ability of Neisseria gonorrhoeae to colonize the human host, as well as with adherence to human epithelial tissue, twitching motility, competence for natural transformation, and autoagglutination. N. gonorrhoeae PilF (required for Tfp biogenesis) and PilT (required for twitching motility and transformation) share significant identities with members of a family of putative ATPases involved in membrane trafficking of macromolecules. An open reading frame downstream of the pilT locus encoding a 408-amino-acid protein with 33% identity with the gonococcal PilT protein and 45% identity with the PilU protein in Pseudomonas aeruginosa was characterized, and the corresponding gene was designated pilU. Unlike N. gonorrhoeae pilT mutants, pilU mutants express twitching motility and are competent for DNA transformation. However, loss-of-function mutations in pilU increased bacterial adherence to ME-180 human epithelial cells eightfold and disrupted in vitro Tfp-associated autoagglutination. Comparative alignment of N. gonorrhoeae PilU with other members of the TrbB-like family of traffic ATPases revealed a conserved carboxy-terminal domain unique to family members which are not essential for Tfp biogenesis but which specifically modify Tfp-associated phenotypes. Studies of the pilT-pilU locus by using Northern blotting, transcriptional fusions, and reverse transcription-PCR showed that the two genes encoding closely related proteins with dissimilar effects on Tfp phenotypes are transcribed from a single promoter.

Pilus retraction powers bacterial twitching motility.

Twitching and social gliding motility allow many gram negative bacteria to crawl along surfaces, and are implicated in a wide range of biological functions. Type IV pili (Tfp) are required for twitching and social gliding, but the mechanism by which these filaments promote motility has remained enigmatic. Here we use laser tweezers to show that Tfp forcefully retract. Neisseria gonorrhoeae cells that produce Tfp actively crawl on a glass surface and form adherent microcolonies. When laser tweezers are used to place and hold cells near a microcolony, retractile forces pull the cells toward the microcolony. In quantitative experiments, the Tfp of immobilized bacteria bind to latex beads and retract, pulling beads from the tweezers at forces that can exceed 80 pN. Episodes of retraction terminate with release or breakage of the Tfp tether. Both motility and retraction mediated by Tfp occur at about 1 microm s(-1) and require protein synthesis and function of the PilT protein. Our experiments establish that Tfp filaments retract, generate substantial force and directly mediate cell movement.

The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion.

The ability of Neisseria meningitidis (MC) to interact with cellular barriers is essential to its pathogenesis. With epithelial cells, this process has been modeled in two steps. The initial stage of localized adherence is mediated by bacterial pili. After this phase, MC disperse and lose piliation, thus leading to a diffuse adherence. At this stage, microvilli have disappeared, and MC interact intimately with cells and are, in places, located on pedestals of actin, thus realizing attaching and effacing (AE) lesions. The bacterial attributes responsible for these latter phenotypes remain unidentified. Considering that bacteria are nonpiliated at this stage, pili cannot be directly responsible for this effect. However, the initial phase of pilus-mediated localized adherence is required for the occurrence of diffuse adherence, loss of microvilli, and intimate attachment, because nonpiliated bacteria are not capable of such a cellular interaction. In this work, we engineered a mutation in the cytoplasmic nucleotide-binding protein PilT and showed that this mutation increased piliation and abolished the dispersal phase of bacterial clumps as well as the loss of piliation. Furthermore, no intimate attachment nor AE lesions were observed. On the other hand, PilT- MC remained adherent as piliated clumps at all times. Taken together these data demonstrate that the induction of diffuse adherence, intimate attachment, and AE lesions after pilus-mediated adhesion requires the cytoplasmic PilT protein.

Competence for natural transformation in Neisseria gonorrhoeae: a model system for studies of horizontal gene transfer.

A combined effort integrating studies of gonococcal Tfp biogenesis, the data made available from the gonococcal genome sequence project and applied molecular genetics have been used to identify the fibrillar filaments themselves, the PilT protein and the ComP protein as essential components for the DNA uptake phase of competence for natural transformation. Our ongoing studies are focused on identifying and understanding the complex interactions which exist between these essential constituents. These studies may be relevant not only to the early steps of genetic transformation but also to the two other venues for horizontal gene transfer based on recent findings. First, the thin pili of IncI1 conjugal plasmids required for liquid mating belong to the type IV family of pili (Yoshida et al., 1998). Secondly, type IV pili are required for lysogenic conversion of Vibrio cholerae by a filamentous phage encoding cholera toxin (Waldor and Mekalanos, 1996). How these highly conserved surface organelles contribute to such diverse forms of DNA translocation across membranes remains to be seen.

Role of BfpF, a member of the PilT family of putative nucleotide-binding proteins, in type IV pilus biogenesis and in interactions between enteropathogenic Escherichia coli and host cells.

Adherence of enteropathogenic Escherichia coli (EPEC) to epithelial cells is dependent on a type IV fimbria, termed the bundle-forming pilus (BFP). A cluster of 14 genes is required for expression of BFP. The eighth gene in the cluster, bfpF, encodes a putative nucleotide-binding protein which resembles the PilT protein of Pseudomonas aeruginosa. It has been proposed that PilT is required for the retraction of the P. aeruginosa pilus, which results in twitching motility. To test the role of BfpF in BFP function and EPEC pathogenesis, two different mutations were constructed in the bfpF gene, one in the cloned gene cluster in a laboratory E. coli strain and one in wild-type EPEC. Neither mutation affected prepilin synthesis, leader sequence processing, or pilus biogenesis. However, both mutations resulted in increased localized adherence. In addition, the EPEC bfpF mutant displayed increased aggregation. The EPEC bfpF mutant was not deficient in attaching and effacing activity or invasion capacity. These results suggest that BfpF decreases aggregation and adherence by EPEC but that subsequent steps in EPEC pathogenesis do not require this protein.

Identification, localization, and distribution of the PilT protein in Neisseria gonorrhoeae

A monoclonal antibody (MAb) directed against a highly conserved protein of Neisseria gonorrhoeae with a molecular size of 40 kDa was isolated and characterized. The protein antigen detected by this MAb was detected by enzyme-linked immunosorbent assay and immunoblotting in all strains of N. gonorrhoeae tested across a wide range of serovars. The 40-kDa protein was found to be expressed at relatively low levels and localized to both the cytosolic and cytoplasmic membrane fractions. Screening of a lambda gt11 expression library derived from gonococcal genomic DNA with the anti-40-kDa MAb and DNA sequence analysis suggested that the 40-kDa protein and the product of the gonococcal pilT gene were identical. Immunoblotting analysis of gonococcal mutants carrying defined mutations in the pilT gene confirmed that the 40-kDa protein was indeed PilT. The N-terminal sequence derived by microsequencing of the protein purified from gonococci led to the correction of the previously published pilT gene sequence. Sequencing of the pilT gene from three different strains revealed an extremely high degree of conservation at both the amino acid and DNA levels.