Attachment Moieties Research Articles

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling.

Many life‐science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super‐resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of ‘linker’ compounds, that bridge biotarget, fluorophore and a functional moiety via well‐established labeling protocols. Linker molecules were synthesized via the Ugi four‐component reaction (Ugi‐4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation‐ and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon‐rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol‐maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self‐healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self‐healing linker–fluorophore conjugates and demonstrated their applications in super‐resolution imaging and single‐molecule spectroscopy.

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling

AbstractMany life‐science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super‐resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of ‘linker’ compounds, that bridge biotarget, fluorophore and a functional moiety via well‐established labeling protocols. Linker molecules were synthesized via the Ugi four‐component reaction (Ugi‐4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation‐ and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon‐rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol‐maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self‐healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self‐healing linker–fluorophore conjugates and demonstrated their applications in super‐resolution imaging and single‐molecule spectroscopy.

Glycosaminoglycan binding by arboviruses: a cautionary tale.

Arboviruses are medically important arthropod-borne viruses that cause a range of diseases in humans from febrile illness to arthritis, encephalitis and hemorrhagic fever. Given their transmission cycles, these viruses face the challenge of replicating in evolutionarily divergent organisms that can include ticks, flies, mosquitoes, birds, rodents, reptiles and primates. Furthermore, their cell attachment receptor utilization may be affected by the opposing needs for generating high and sustained serum viremia in vertebrates such that virus particles are efficiently collected during a hematophagous arthropod blood meal but they must also bind sufficiently to cellular structures on divergent organisms such that productive infection can be initiated and viremia generated. Sulfated polysaccharides of the glycosaminoglycan (GAG) groups, primarily heparan sulfate (HS), have been identified as cell attachment moieties for many arboviruses. Original identification of GAG binding as a phenotype of arboviruses appeared to involve this attribute arising solely as a consequence of adaptation of virus isolates to growth in cell culture. However, more recently, naturally circulating strains of at least one arbovirus, eastern equine encephalitis, have been shown to bind HS efficiently and the GAG binding phenotype continues to be associated with arbovirus infection in published studies. If GAGs are attachment receptors for many naturally circulating arboviruses, this could lead to development of broad-spectrum antiviral therapies through blocking of the virus-GAG interaction. This review summarizes the available data for GAG/HS binding as a phenotype of naturally circulating arbovirus strains emphasizing the importance of avoiding tissue culture amplification and artifactual phenotypes during their isolation.

Macro- and microporous polycaprolactone/duck’s feet collagen scaffold fabricated by combining facile phase separation and particulate leaching techniques to enhance osteogenesis for bone tissue engineering

Herein, a facile macro- and microporous polycaprolactone/duck’s feet collagen scaffold (PCL/DC) was fabricated and characterized to confirm its applicability in bone tissue engineering. A biomimetic scaffold for bone tissue engineering and regeneration for bone defects is an important element. PCL is a widely applied biomaterial for bone tissue engineering due to its biocompatibility and biodegradability. However, the high hydrophobicity and low cell attachment site properties of PCL lead to an insufficient microenvironment in designing a scaffold. Collagen is a nature-derived biomaterial that is widely used in tissue engineering and has excellent biocompatibility, mechanical properties, and cell attachment moieties. Among the resources from which collagen can be obtained, DC contains a high amount of collagen type I (COL1), is biocompatible, and is cost-effective. In this study, the scaffolds were fabricated by blending DC with PCL in various ratios and applied non-solvent-induced phase separation (NIPS) and thermal-induced phase separation (TIPS) (N-TIPS), solvent casting and particulate leaching (SCPL), and gas foaming method to fabricate macro- and microporous structure. The characterization of the fabricated scaffolds was carried out by morphological analysis, bioactivity test, physicochemical analysis, and mechanical test. In vitro study was carried out by viability test, morphology observation, and gene expression. The results showed that the incorporation of DC enhances the physicochemical and mechanical properties of the scaffolds. Also, a large amount of bone mimetic apatite was formed according to the DC content in the bioactivity test. The in vitro study showed that the PCL/DC scaffold is biocompatible and the existence of apatite and DC formed a favorable microenvironment for cell proliferation and differentiation. Overall, the novel porous PCL/DC scaffold can be a promising biomaterial model for bone tissue engineering and regeneration.

Biodistribution PET/CT Study of Hemoglobin-DFO-89Zr Complex in Healthy and Lung Tumor-Bearing Mice.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine β93 is the sole attachment moiety to the αβ-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

One-step enzymatic modification of RNA 3' termini using polymerase θ.

Site-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3′ terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3′ termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30–50 2′-deoxyribonucleotides to the 3′ terminus of RNA molecules of various lengths and sequences, and extends RNA 3′ termini with an assortment of 2′-deoxy and 2′,3′-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3′ terminal modification of RNA and potentially enzymatic synthesis of RNA.

Synthesis and characterization of a cobalt(II) tetrakis(3-fluorophenyl) porphyrin with a built-in 4-vinylphenyl surface attachment moiety

Metalloporphyrins serve important roles in biology and as components in emerging technological assemblies for energy conversion. In this report, we describe the synthesis and characterization of a novel cobalt(II) 5,10,15,20-tetrakis (3-fluorophenyl)porphyrin bearing a 4-vinylphenyl surface attachment group at a beta position on the macrocycle. Electrochemical measurements show the 3-fluorophenyl groups at the meso positions of the porphyrin perturb the reduction potentials of the complex to more positive values as compared to non-fluorinated analogs, thus allowing access to reduced cobalt porphyrin species at significantly less negative applied bias potentials. The complex, cobalt(II) 5,10,15,20-tetrakis(3-fluorophenyl)-2-(4-vinylphenyl)porphyrin, is abbreviated in this article as Gov-1 in honor of Govindjee for his pioneering investigations in the role of fluorine as a promoter of novel protein-substrate interactions and the inspirational role he continues to have in encouraging young investigators in the areas of natural and artificial photosynthesis.

Preferential Lentiviral Targeting of Astrocytes in the Central Nervous System

The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS) is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment.

Non-covalent functionalization of single-walled carbon nanotubes with modified polyethyleneimines for efficient gene delivery

Functionalized carbon nanotubes (CNTs) have been recently emerged as important class of vectors for delivery of DNA and other biomolecules into various cells. In this study, single-walled carbon nanotubes (SWNTs) were functionalized by non-covalent binding of hydrophobic moieties, which were covalently linked to polyethyleneimines (PEIs). PEIs of three molecular weights (25, 10 and 1.8kDa) were used. CNTs were functionalized with the PEI series either through phospholipid moiety (via a polyethyleneglycol linker) or through directly-attached long (18 carbons) or intermediate (10 carbons) hydrophobic alkyl moieties. All PEI-functionalized CNTs exhibited good stability and dispersibility in biological media. Visualizing of functionalized CNTs and lack of aggregation were confirmed by atomic force microscopy. The PEI derivatives bound to CNTs retained the ability to fully condense plasmid DNA at low N/P ratios and substantial buffering capacity in the endosomal pH range. PEI-functionalized CNTs exhibited increased transfection efficiency compared to underivatized PEIs up to 19-fold increase being observed in the functionalized CNT with the smallest PEI tested, the smallest hydrophobic attachment moiety tested and no linker. Also PEI-functionalized CNTs were effective gene delivery vectors in vivo following tail vein injection in mice with the largest expression occurring with the vector PEI-functionalized through a polyethyleneglycol linker.

The control of anchorage-dependent cell behavior within a hydrogel/microcarrier system in an osteogenic model

The use of injectable hydrogels for tissue engineering purposes such as bone regeneration has been hampered by the mass depletion of cells after encapsulation, due to the lack of a proper interface between hydrogel matrices and osteo-progenitor cells. Efforts to graft bioactive molecules as cell attachment moieties have achieved limited success. In this study, we devised a solution to promote cellular focal adhesion within hydrogels, and elicit the mechanism behind cellular survival/death therein. We found that the fulfillment of ligation between cellular integrins and extracellular ligands, instead of the expression of integrins per se, is essential to avoid apoptosis in gel-encapsulated anchorage-dependent cells (ADCs). Absence of such ligation brought about mass cell death in our osteogenic model with osteoblasts (as representative of ADCs) and failure of osteogenic commitment of mesenchymal stem cells (as representative of anchorage-dependent progenitors). We have designed a gel-based composite system that works as a suspension of injectable cell-laden microcarriers in hydrogel, as compared to the conventional cell-suspended hydrogels. Injectable microscopic anchors (microcarriers) not only provide platforms for cellular focal adhesion but also facilitate the cells to overcome gel enlacement and fully spread out into their natural morphology. Further in vitro and in vivo osteogenic investigations show the composites to be a competent potential injectable vehicle for the conveyance of ADCs and regenerations of bone and other tissues.

Replacement of Native Adenovirus Receptor-Binding Sites with a New Attachment Moiety Diminishes Hepatic Tropism and Enhances Bioavailability in Mice

The in vivo efficacy of adenoviral vectors (AdVs) in gene delivery strategies is hampered by the broad tissue tropism of the virus and its efficient binding to human erythrocytes. To circumvent these limitations, we developed a prototype AdV lacking native binding sites. We replaced the adenoviral fiber with a chimeric molecule consisting of the fiber tail domain, the reovirus sigma1 oligomerization domain, and a polyhistidine tag as model targeting moiety. We also abolished the integrin-binding motif in the penton base protein. The chimeric attachment molecule was efficiently incorporated onto AdV capsids, allowed efficient propagation of AdV without requirement for complementing fiber and conferred highly specific tropism to the AdV. Importantly, the targeted AdV exhibited markedly reduced tropism for liver cells. In comparison with control AdV with native tropism, the targeted AdV showed 1000-fold reduced transduction of HepG2 cells and 10,000-fold reduced transduction of mouse liver cells in freshly isolated liver slices. After intravenous inoculation of C57BL/6 mice, the targeted AdV exhibited delayed clearance in comparison with the native AdV, leaving approximately 10-fold greater levels in the blood 2 hr after inoculation. For all tissues analyzed, the targeted AdV displayed significantly reduced in vivo transduction in comparison with the native vector. Furthermore, in contrast to the native AdV, the targeted AdV did not bind human erythrocytes. Together, our findings suggest that the targeted AdV design described here provides a promising platform for systemic in vivo gene delivery.

Multidentate functionalized lubricant for ultralow head/disk spacing in a disk drive

As areal density in disk drive technology is marching towards the Tbit∕in.2 mark, the slider-to-disk spacing will be required to be within only a few nanometers. Coupled with very high slider velocities (>40m∕s), this leads to extremely high shear stresses on the nanometer-thin lubricant film at the disk surface. As a result, the lubricant film tends to exhibit local redistribution on a micrometer lateral level, decreasing the overall clearance and impacting the integrity of the interface. This paper describes a different approach to functionalized lubricant, where in addition to functional groups placed at the end of the chain, additional attachment moieties are introduced within the main polymer chain. The benefits of this approach is twofold: it increases adhesive interaction with the disk surface, therefore increasing the effective surface viscosity, while at the same time “tying” down the free backbone length for increased clearance. Fabrication, characterization, and performance data obtained on actual magnetic disks will be described.

Biomineralization in diatoms: Characterization of novel polyamines associated with silica

Pattern formation during silica biomineralization in diatoms appears to depend on long-chain polyamines as well as proteins covalently modified with polyamines (silaffins). Recently, the complete genome of the diatom Thalassiosira pseudonana has been sequenced making this species an attractive model organism for future studies on biomineralization. Mass- and NMR-spectroscopic analysis of the long-chain polyamines from this diatom species reveals the existence of a complex population with as yet unknown structural features. These include complex methylation patterns, different attachment moieties as well as the existence of quaternary ammonium functionalities.

Surface functionalization and grafting of heparin and/or RGD by an aqueous-based process to a poly(carbonate-urea)urethane cardiovascular graft for cellular engineering applications.

An aqueous-based process is reported for surface functionalization and grafting of anticoagulant and cell attachment moieties, such as heparin and/or arginine-glycine- aspartate (RGD) onto the lumenal surface of a prefabricated cardiovascular graft (5 mm i.d.) made of poly(carbonate- urea)urethane (MyoLink). It is a three-stage process, all aqueous: (1) hydroxylation using an azobis compound, particularly 2,2'-azobis(2-methylpropionamidine)dihydrochloride, which abstracts hydrogen via an electron transfer process from the polyurethane surface (strong oxygen purging); (2) grafting using the as-generated hydroxide groups to allow attachment of an acrylamide monomer using a conventional ceric ion technique (strong nitrogen purging); and (3) moiety attachment, preactivated with [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] in acidic solution. The technique was validated by attaching heparin and RGD/heparin to the MyoLink polymer. Following bonding, the graft segments were exposed to prolonged physiologic shear force in a flow circuit (10 h). The grafts first were analyzed by X-ray photoelectron spectroscopy (XPS) to determine the degree of attachment of the moieties and then by materials methods to assess whether any degradation of the graft material itself had occurred since polyurethanes with carbonate amorphous segments are readily susceptible to hydrolytic degradation following functionalization processes. XPS showed the moieties were present on the surface at a concentration of 10%. The S2p(3/2) states of sulfur indicated that there were high degrees of ionic covalent bonding, indicating high degrees of moiety bioactivity. Heparin was found to be present from the sulfur signal, namely NSO(3). RGD was found to be present from the nitrogen signal present at the binding energy of 399 eV. Macroscopic analysis and ESEM showed no signs of polyurethane degradation or small protuberances indicative of microgel formation. Quality control (QC) showed that the internal diameters and wall thicknesses of all the respective grafts postbonding remained within normal batch release limits (5 +/- 0.1mm, i.d.; 0.9 +/- 0.05 mm, wall thickness). Gel permeation chromatography (GPC) showed there were no statistical differences between the control, which was nonbonded (MN 45,300, MW 98,500, D 2.17) and all of the bonded samples, respectively (MN 41,800, MW 104,000, D 2.45). Radial tensile strength (RTS) analysis also showed that all of the respective samples postbonding (1.48N/mm) remained within batch release specifications (>1N/mm). A simple aqueous polymer surface functionalization and grafting technique has been developed for covalent bonding of anticoagulant and cell-attachment moieties onto poly(carbonate-urea)urethane(s) and has been validated by surface and materials analyses. The moieties were attached uniformly and were bioactive at a high surface density. No degradation in terms of a loss in mechanical properties was evident following bonding of the polyurethane.

Optimization of oligonucleotide-based DNA microarrays.

Oligonucleotide-based DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms. Here we report a systematic study of the sensitivity, specificity and dynamic range of microarray signals and their dependence on the labeling and hybridization conditions as well as on the length, concentration, attachment moiety and purity of the oligonucleotides. Both a controlled set of in vitro synthesized transcripts and RNAs from biological samples were used in these experiments. An algorithm is presented that allows the efficient selection of oligonucleotides able to discriminate a single nucleotide mismatch. Critical parameters for various applications are discussed based on statistical analysis of the results. These data will facilitate the design and standardization of custom-made microarrays applicable to gene expression profiling and sequencing analyses.

Microtextured Cell Culture Platforms: Biomimetic Substrates for the Growth of Cardiac Myocytes and Fibroblasts

In order to understand the role of tissue adaptation to altered physiological states, a more physiologically and dimensionally relevant in vitro model of cardiac myocyte organization has been developed. This new cell culture system, created by microfabrication technology, provides an environment in which cells can maintain a differentiated in vivo cell phenotype. By creating microtextured geometries of varied dimension, we are able to maximize the perpendicular surface area for myocyte attachment. These platforms provide a biocompatible surface with specific microarchitectures upon which cells exhibit enhanced cellular adhesion due to increased surface area, three-dimensional geometries, and bioacceptable attachment moieties.

Attachment role of gonococcal pili. Optimum conditions and quantitation of adherence of isolated pili to human cells in vitro.

Gonoccocal pili facilitate attachment of virulent Neisseria gonorrhoeae to human cells. To characterize this attachment function, purified gonococcal pili isolated from four strains possessing antigenically distinct pili were radiolabeled with 125I and used to measure the attachment of pili to various human cells in vitro. Human buccal and cervical-vaginal mucosal epithealial cells, fallopian tube mucosa, and sperm bound pili in greater numbers per micrometer2 of surface area (1--10) than fetal tonsil fibroblasts, HeLa M cells, erythrocytes, or polymorphonuclear leukocytes. This cell specificity of attachment suggests a greater density of membrane pili binding sites on cells similar or identical to cells from natural sites of infection. The pili binding sites were quantitated as 1 X 10(4) per cervical-vaginal squamous cell. Pili of all antigenic types attached equally to a given cell type, implying that the attachment moiety of each pilus was similar. Attachement of gonoccocal pili to human cells occurred quickly with saturation of presumed receptor sites within 20--60 min. Attachment was temperature dependent (37 degrees greater than 20 degrees greater than 4 degrees C), and pH dependent (3.5 less than 4.5 less than 5.5 less than 7.5). Attachment was inhibited by antibody to pili (homologous pili Ab greater than heterologous Ab). The extent of possible protection against gonococcal infection due to inhibition of pili-mediated attachment might prove limited as a result of the considerable antigenic heterogeneity among pili and the observation that blockage of pili attachment is maximal only with antibody to pili of the infecting strain.

Pili as a mediator of the attachment of gonococci to human erythrocytes.

Isolated pure gonococcal pili were found capable of producing direct agglutination of human erythrocytes. Four different strains of gonococci were compared, and hemagglutination was produced by isolated pili or piliated gonococci but not by nonpiliated gonococci of each strain. Pili from each of the four strains were antigenically distinguishable using antisera specific for pili to agglutinate piliated gonococci, form precipitin lines in Ouchterlony immunodiffusion, or inhibit hemagglutination caused by purified pili or piliated gonococci. However, these tests also demonstrate some shared antigenicity among pili. Shared antigens among the four pili types were quantitated at less than or equal to 2.5% by radioimmunoassay. Inhibition of hemagglutination was most marked with antiserum to the homologous pili type. Inhibition of hemagglutination by antiserum to heterologous pili suggested that shared antigens on pili from B and 2686 strains of gonococci are located near the erythrocyte attachment moiety of B strain pili and removed from the attachment moiety of 2686 strain pili. These results suggest that antigenic heterogeneity of pili will prove an important factor in any efforts to use pili as a vaccine for gonorrhea.