Binding Of Pili Research Articles

Cloning and Expression of Pigeon-Derived Escherichia coli Type 1 Pilus Clusters and Analysis of Amino Acid Sequence Characteristics of Functional Proteins.

Type 1 pili, as an important virulence factor of E. coli, has certain homology between APEC and UPEC, but the homology degree is not clear enough. This study aims to compare the homology between them. The recombinant bacteria were constructed by homologous recombination. The pili were observed by TEM, and the hemagglutination characteristics were determined by MHSA. The complete gene sequence was determined by sequencing, and the amino acid sequences of the functional proteins of type 1 pili of APEC and UPEC were compared. TEM showed that they could express pili, which were slender, straight, and dense. Stable-pUC-fimBH has MHSA but stable-pUC-fimBG does not. The amino acid sequence similarity of FimB of NJ05 and UPEC was 98.8%, FimE was 99.4%, and the similarity between them was 51.5%. Compared with UPEC's type 1 pili FimC and FimD sequences, the similarity was 99.52% and 87.8%, respectively. The amino acid sequence of FimA of NJ05 was 89-96%, similar to UPEC, and the N-terminal and C-terminal amino acid sequences were exactly the same. The gene sequence and amino acid sequence similarity of FimH between them were both above 99%. The similarity of the pilus binding domain of FimH was 52.8%, but only 27.6% in the receptor binding domain. A few of the same amino acid residues were found in the corresponding regions of FimA, FimF, FimG, and FimH. The type 1 pili of APEC and UPEC come from the same origin, which is helpful to further reveal the pathogenic mechanism of E. coli infection in the poultry respiratory tract.

Structural mechanisms of Tad pilus assembly and its interaction with an RNA virus.

Caulobacter crescentus Tad (tight adherence) pili, part of the type IV pili family, are crucial for mechanosensing, surface adherence, bacteriophage (phage) adsorption, and cell-cycle regulation. Unlike other type IV pilins, Tad pilins lack the typical globular β sheet domain responsible for pilus assembly and phage binding. The mechanisms of Tad pilus assembly and its interaction with phage ΦCb5 have been elusive. Using cryo-electron microscopy, we unveiled the Tad pilus assembly mechanism, featuring a unique network of hydrogen bonds at its core. We then identified the Tad pilus binding to the ΦCb5 maturation protein (Mat) through its β region. Notably, the amino terminus of ΦCb5 Mat is exposed outside the capsid and phage/pilus interface, enabling the attachment of fluorescent and affinity tags. These engineered ΦCb5 virions can be efficiently assembled and purified in Escherichia coli, maintaining infectivity against C. crescentus, which presents promising applications, including RNA delivery and phage display.

Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study.

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.

Comparative transcriptome analysis revealed the role and mechanism of a FeoC-like LuxR-type regulator in intracellular survival of Aeromonas hydrophila

A. hydrophila is one of the most important pathogens in the aquaculture industry, and its drug-resistant strains continue to emerge due to the irregular use of drugs. Previous studies found A. hydrophila could survive in fish macrophages, so it was speculated that A. hydrophila may hide in host macrophages to escape the killing of drugs and other antibacterial factors. The mechanism of A. hydrophila survival in fish macrophages is worthy of further study. In this study, the expression of a FeoC-like LuxR-type regulator was stable silenced by shRNA to construct the silencing strain feoC-RNAi. And the intracellular survival rate of feoC-RNAi decreased by about 67.5% compared with that of the wild-type strain. Comprehensive transcriptome analysis suggested that this FeoC-like LuxR-type regulator may regulate at least 1286 genes. According to GO and KEGG analysis of these 1286 DEGs, it can be speculated that this regulator may affect A. hydrophila intracellular survival by regulating bacterial metal ion metabolism, chemotaxis, type IV pilus biogenesis and binding, then further regulate the bacterial intracellular survival. Further comparison on biological phenotype of A. hydrophila B11 and feoC-RNAi revealed this FeoC-like LuxR-type regulator could regulate the expression of genes related to iron and magnesium metabolism and greatly affect bacterial growth under the limited magnesium concentration, but has no effect on bacterial growth under the limited iron concentration. And inhibited expression of this FeoC-like LuxR-type regulator led to a decrease in chemotactic ability of A. hydrophila by about 60%, disappearance of pilus, and a decrease in adhesion by about 50%. So a conclusion could be made that this FeoC-like LuxR-type regulator might play a vital role in regulating the uptake of magnesium and the homeostasis of magnesium and iron, and chemotaxis, type IV pilus biogenesis and adhesion of A. hydrophila, then further protect bacteria from the microbicidal actions and keep survival in host macrophages.

DMBT1 inhibition of Pseudomonas aeruginosa twitching motility involves its N-glycosylation and cannot be conferred by the Scavenger Receptor Cysteine-Rich bacteria-binding peptide domain

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1–220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

Type IV pili: dynamics, biophysics and functional consequences.

The surfaces of many bacteria are decorated with long, exquisitely thin appendages called type IV pili (T4P), dynamic filaments that are rapidly polymerized and depolymerized from a pool of pilin subunits. Cycles of pilus extension, binding and retraction enable T4P to perform a phenomenally diverse array of functions, including twitching motility, DNA uptake and microcolony formation. On the basis of recent developments, a comprehensive understanding is emerging of the molecular architecture of the T4P machinery and the filament it builds, providing mechanistic insights into the assembly and retraction processes. Combined microbiological and biophysical approaches have revealed how T4P dynamics influence self-organization of bacteria, how bacteria respond to external stimuli to regulate T4P activity for directed movement, and the role of T4P retraction in surface sensing. In this Review, we discuss the T4P machine architecture and filament structure and present current molecular models for T4P dynamics, with a particular focus on recent insights into T4P retraction. We also discuss the functional consequences of T4P dynamics, which have important implications for bacterial lifestyle and pathogenesis.

Differential Regulation of Escherichia coli fim Genes following Binding to Mannose Receptors.

Regulation of the uropathogenic Escherichia coli (UPEC) fimB and fimE genes was examined following type 1 pili binding to mannose-coated Sepharose beads. Within 25 min after mannose attachment, fimE expression dropped eightfold, whereas fimB transcription increased about two- to fourfold. Because both fim genes encode site-specific recombinases that affect the position of the fimS element containing the fimA promoter, the positioning of fimS was also examined. The fimS element changed to slightly more Phase-OFF in bacteria mixed with plain beads, whereas UPEC cells interacting with mannose-coated beads had significantly less Phase-OFF orientation of fimS under pH 7 conditions. On the other hand, Phase-OFF oriented fimS increased fourfold when UPEC cells were mixed with plain beads in a pH 5.5 environment. Positioning of fimS was also affected by fimH mutations, demonstrating that the FimH ligand binding to its receptor facilitates the changes. Moreover, enzyme immunoassays showed that UPEC cells had greater type 1 pili expression when mixed with mannose-coated beads versus plain beads. These results indicate that, after type 1 pilus binding to tethered mannose receptors, the physiology of the E. coli cells changes to maintain the expression of type 1 pili even when awash in an acidic environment.

Mucosal fluid glycoprotein DMBT1 suppresses twitching motility and virulence of the opportunistic pathogen Pseudomonas aeruginosa.

It is generally thought that mucosal fluids protect underlying epithelial surfaces against opportunistic infection via their antimicrobial activity. However, our published data show that human tear fluid can protect against the major opportunistic pathogen Pseudomonas aeruginosa independently of bacteriostatic activity. Here, we explored the mechanisms for tear protection, focusing on impacts of tear fluid on bacterial virulence factor expression. Results showed that tear fluid suppressed twitching motility, a type of surface-associated movement conferred by pili. Previously, we showed that twitching is critical for P. aeruginosa traversal of corneal epithelia, exit from epithelial cells after internalization, and corneal virulence. Inhibition of twitching by tear fluid was dose-dependent with dilutions to 6.25% retaining activity. Purified lactoferrin, lysozyme, and contrived tears containing these, and many other, tear components lacked the activity. Systematic protein fractionation, mass spectrometry, and immunoprecipitation identified the glycoprotein DMBT1 (Deleted in Malignant Brain Tumors 1) in tear fluid as required. DMBT1 purified from human saliva also inhibited twitching, as well as P. aeruginosa traversal of human corneal epithelial cells in vitro, and reduced disease pathology in a murine model of corneal infection. DMBT1 did not affect PilA expression, nor bacterial intracellular cyclicAMP levels, and suppressed twitching motility of P. aeruginosa chemotaxis mutants (chpB, pilK), and an adenylate cyclase mutant (cyaB). However, dot-immunoblot assays showed purified DMBT1 binding of pili extracted from PAO1 suggesting that twitching inhibition may involve a direct interaction with pili. The latter could affect extension or retraction of pili, their interactions with biotic or abiotic surfaces, or cause their aggregation. Together, the data suggest that DMBT1 inhibition of twitching motility contributes to the mechanisms by which mucosal fluids protect against P. aeruginosa infection. This study also advances our understanding of how mucosal fluids protect against infection, and suggests directions for novel biocompatible strategies to protect our surface epithelia against a major opportunistic pathogen.

Binding of CFA/I Pili of Enterotoxigenic Escherichia coli to Asialo-GM1 Is Mediated by the Minor Pilin CfaE.

CFA/I pili are representatives of a large family of related pili that mediate the adherence of enterotoxigenic Escherichia coli to intestinal epithelial cells. They are assembled via the alternate chaperone-usher pathway and consist of two subunits, CfaB, which makes up the pilus shaft and a single pilus tip-associated subunit, CfaE. The current model of pilus-mediated adherence proposes that CFA/I has two distinct binding activities; the CfaE subunit is responsible for binding to receptors of unknown structure on erythrocyte and intestinal epithelial cell surfaces, while CfaB binds to various glycosphingolipids, including asialo-GM1. In this report, we present two independent lines of evidence that, contrary to the existing model, CfaB does not bind to asialo-GM1 independently of CfaE. Neither purified CfaB subunits nor CfaB assembled into pili bind to asialo-GM1. Instead, we demonstrate that binding activity toward asialo-GM1 resides in CfaE and this is essential for pilus binding to Caco-2 intestinal epithelial cells. We conclude that the binding activities of CFA/I pili for asialo-GM1, erythrocytes, and intestinal cells are inseparable, require the same amino acid residues in CfaE, and therefore depend on the same or very similar binding mechanisms.

Phenotypical analysis of the Lactobacillus rhamnosus GG fimbrial spaFED operon: surface expression and functional characterization of recombinant SpaFED pili in Lactococcus lactis.

A noticeable genomic feature of many piliated Gram-positive bacterial species is the presence of more than one pilus-encoding operon. Paradigmatically, the gut-adapted Lactobacillus rhamnosus GG strain contains two different fimbrial operons in its genome. However, whereas one of these operons (called spaCBA) is encoding for the functionally mucus-/collagen-binding SpaCBA pilus, for the other operon (called spaFED) any native expression of the SpaFED-called pili is still the subject of some uncertainty. Irrespective of such considerations, we decided it would be of relevance or interest to decipher the gross structure of this pilus type, and as well assess its functional capabilities for cellular adhesion and immunostimulation. For this, and by following the approach we had used previously to explicate the immuno-properties of SpaCBA pili, we constructed nisin-inducible expression clones producing either wild-type or SpaF pilin-deleted surface-assembled L. rhamnosus GG SpaFED pili on Lactococcus lactis cells. Using these piliated lactococcal constructs, we found that the pilin-polymerized architecture of a recombinant-produced SpaFED pilus coincides with sequence-based functional predictions of the related pilins, and in fact is prototypical of those other sortase-dependent pilus-like structures thus far characterized for piliated Gram-positive bacteria. Moreover, we confirmed that among the different pilin subunits encompassing spaFED operon-encoded pili, the SpaF pilin is a main adhesion determinant, and when present in the assembled structure can mediate pilus binding to mucus, certain extracellular matrix proteins, and different gut epithelial cell lines. However, somewhat unexpectedly, when recombinant SpaFED pili are surface-attached, we found that they could not potentiate the existing lactococcal cell-induced immune responses so elicited from intestinal- and immune-related cells, but rather instead, they could dampen them. Accordingly, we have now provided the first phenotypical description of a spaFED pilus operon, and with that furthered the functional understanding of surface piliation for a particular gut-commensalic genre of piliated Gram-positive bacteria.

Structure and Assembly of CFA/I pili from Enterotoxigenic Escherichia Coli That Cause Traveler's Diarrhea

Enterotoxigenic Escherichia coli (ETEC) bacteria that cause traveler's diarrhea utilize pili to initiate infection via pilus binding to epithelial cells in the small intestine. According to the World Health Organization, ETEC cause the largest number of recorded community-acquired cases of childhood diarrhea in the developing world, and are the most common cause of Traveler's diarrhea. Through a multi-disciplinary approach that includes x-ray crystallography, electron microscopy, site-directed mutagenesis, and genetic sequence analysis we elucidate the structure and assembly of CFA/I pili expressed on ETEC. We show that the distinction between Class I pili from the chaperone/usher pathway (e.g., P-pili from uropathogenic bacteria) and Class 5 pili from the alternate chaperone pathway (e.g., CFA/I pili), which was based on the lack of genetic sequence homology, does not correlate with any major structural or functional differences between these classes of pili. Pilin subunits transit the outer membrane through an usher that can accommodate single subunits, but not the assembled helical filament. We identify a proline residue in the major pilin, CfaB, that appears to isomerize from the trans to the cis conformation, producing the conformational change required for assembly of the mature pilus filament comprising about 1,000 subunits. Lastly, analysis of genetic variability among clinical strains representative of the eight discrete Class 5 fimbrial subtypes, in combination with structural data, show that each bacterial strain presents a distinct outer surface of CfaB, while the interior and protein-protein interface residues are more highly conserved. These data suggest that protein surface variability facilitates evasion of the immune system by ETEC.

Binding of Neisseria meningitidis Pili to Berry Polyphenolic Fractions

Blocking bacterial adhesion to host surfaces provides novel potential to control infections. The present study was directed to binding and inhibitory activity of different fresh berries and berry and fruit juices against Neisseria meningitidis . Berries and juices were fractionated according to their molecular size into three fractions. A microtiter well assay for binding of N. meningitidis pili to berry and juice fractions was constructed. In addition, adhesion inhibition to human epithelial cells (HEC-1B) was tested. The active fractions were then subfractionated by employing solid-phase extraction. Subfractions were characterized by RP-HPLC-DAD, and the pili binding was evaluated by using microtiter well binding assay. Binding and inhibitory activity were detected to bilberry, cranberry, lingonberry, and crowberry fractions, which contained anthocyanins or a mixture of proanthocyanidins and flavonols. Thus, the findings identify several previously unknown binding and inhibitory activities and may suggest Vaccinium berries and crowberry as promising sources against meningococcal adherence.

Human and Bovine Milk Oligosaccharides Inhibit Neisseria meningitidis Pili Attachment In Vitro

Milk oligosaccharides have been shown to interfere with adhesion of many pathogens to host mucosal surfaces. Characterization of the adhesion mechanisms of the bacteria to host cell surface is needed to develop novel functional food, infant formulas, and anti-infective drugs. Adhesion of Neisseria meningitidis, a human specific pathogen causing meningitis and septicemia, is not completely understood but is mediated by type IV pili. Here, we developed a microtiter well pili binding assay to investigate the binding activities of N. meningitidis isolated type IV pili to different glycoproteins. Pili binding activities to bovine thyroglobulin and human salivary agglutinin but not to chicken ovalbumin were present. Inhibition of these binding activities was demonstrated by fractionated human or bovine milk oligosaccharides. The binding of neisserial pili to bovine thyroglobulin was most effective and was clearly inhibited by human milk neutral or bovine milk acidic oligosaccharides.

The mechanism of bacterial infection by filamentous phages involves molecular interactions between TolA and phage protein 3 domains.

The early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized.

A co-operative interaction between Neisseria gonorrhoeae and complement receptor 3 mediates infection of primary cervical epithelial cells.

Little is known about the pathogenesis of gonococcal infection within the lower female genital tract. We recently described the distribution of complement receptor 3 (CR3) on epithelia of the female genital tract. Our studies further indicate that CR3-mediated endocytosis serves as a primary mechanism by which N. gonorrhoeae elicits membrane ruffling and cellular invasion of primary, human, cervical epithelial cells. We have extended these studies to describe the nature of the gonococcus-CR3 interaction. Western Blot analysis demonstrated production of alternative pathway complement components by ecto- and endocervical cells which allows C3b deposition on gonococci and its rapid conversion to iC3b. Anti-iC3b and -factor I antibodies significantly inhibited adherence and invasion of primary cervical cells, suggesting that iC3b covalently bound to the gonococcus serves as a primary ligand for CR3 adherence. However, gonococcal porin and pili also bound to the I-domain of CR3 in a non-opsonic manner. Binding of porin and pili to CR3 were required for adherence to and invasion of cervical epithelia. Collectively, these data suggest that gonococcal adherence to CR3 occurs in a co-operative manner, which requires gonococcal iC3b-opsonization, porin and pilus. In conjunction, these molecules facilitate targeting to and successful infection of the cervical epithelium.

CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae.

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.

Binding of pili from uropathogenic Escherichia coli to membranes secreted by human colonocytes and enterocytes.

PapG adhesins mediate the binding of uropathogenic Escherichia coli. Although receptors for these adhesins have not been demonstrated in intestinal epithelia, the colonic microflora includes strains of uropathogenic E. coli. We now report that surfactant-like particles secreted by the human intestine contain receptors for PapG adhesins and may provide an intestinal habitat for uropathogenic bacteria.

Binding of the pili of Pseudomonas aeruginosa to a low-molecular-weight mucin and neutral cystatin of human submandibular-sublingual saliva.

In previous experiments, we have shown that [125I]pili of Pseudomonas aeruginosa exhibited specific binding to a low-molecular-weight mucin (MG2) of human submandibular-sublingual saliva (HSMSL; Reddy MS, Levine MJ, Paranchych W. Crit Rev Oral Biol Med 4:315-323, 1993). In the present study, I have utilized unlabeled pili and immunostaining to identify the receptor molecules in HSMSL. In addition to MG2, pili also bound to neutral cystatin (CsnSN). Binding of unlabeled pili to MG2 and CsnSN could be abolished by treatment of HSMSL with trypsin to hydrolyze the peptide moieties or N-acetylation to neutralize the positive charges of the lysine residues. Reductive methylation of HSMSL, which modifies the lysine residues to methyl lysines while retaining the positive charges, did not affect the binding of pili to either MG2 or CsnSN. Further, pili also exhibited binding to a recombinant MG2 peptide (aa 1-86). Collectively, the data suggested that a protein-to-protein interaction via electrostatic forces mediates the binding of the pili to MG2 and CsnSN. Iodination of pili, which modifies tyrosine-24 and/or -27 residues to O-iodotyrosine(s), abolished its binding to CsnSN but not to MG2. These results suggested that the conformation of pili also plays a role in interaction with CsnSN. Conformational change(s) of pili induced by iodination also made it susceptible to hydrolysis with trypsin.

Effect of lipopolysaccharide mutations on recipient ability of Salmonella typhimurium for incompatibility group H plasmids.

Previous investigations of the incompatibility group F, P, and I plasmid systems revealed the important role of the outer membrane components in the conjugal transfer of these plasmids. We have observed variability in transfer frequency of three incompatibility group H plasmids (IncHI1 plasmid R27, IncHI2 plasmid R478, and a Tn7 derivative of R27, pDT2454) upon transfer into various Salmonella typhimurium lipopolysaccharide (LPS) mutants derived from a common parental strain, SL1027. Recipients with truncated outer core via the rfaF LPS mutation increased the transfer frequency of the IncH plasmids by up to a factor of 10(3). Mutations which resulted in the truncation of the residues following 3-deoxy-D-manno-octulosonic acid, such as the rfaE and rfaD mutations, decreased the transfer frequency to undetectable levels. Addition of phosphorylethanolamine, a component of wild-type LPS, to the media decreased the frequency of transfer of R27 into wild-type and rfaF LPS mutant recipients tested. Reversing the direction of transfer, by mating LPS mutant donors with wild-type recipients, did not affect the frequency of transfer compared to the standard matings of wild-type donor with LPS mutant recipient. These findings demonstrate that conjugation interactions affected by LPS mutation are not specific for the recipient cell. Our results suggest that LPS mutation does not affect conjugation via altered pilus binding but affects some later steps in the conjugative process, and alteration of transfer frequency by O-phosphorylethanolamine and LPS truncation is due to charge-related interactions between the donor and recipient cell.

Structure-Function Analysis of the Adherence-Binding Domain on the Pilin of Pseudomonas aeruginosa Strains PAK and KB7

The pili of Pseudomonas aeruginosa mediate bacterial binding to human epithelial cell surfaces. We have previously shown that a 17-residue synthetic peptide, KCTSDQDEQFIPKGCSK, corresponding to the C-terminal sequence of the PAK pilin protein (residues 128-144) contains the adherence binding domain. Another pilin strain, KB7, has been cloned and sequenced [Paranchych et al. (1990) in Pseudomonas Biotransformations, Pathogenesis and Evolving Biotechnology, pp 343-351, American Society for Microbiology, Washington, DC]. The C-terminal 17-residue sequence of the KB7 pilin is SCATTVDAKFRPNGCTD, which is semiconserved as compared to the PAK sequence. In this study, the interactions between the A549 human lung carcinoma cells and the two P. aeruginosa pilin strains were elucidated using a single alanine replacement analysis on the C-terminal 17-residue synthetic peptide of the pilins. The ability of these peptide analogs to inhibit the binding of the biotinylated PAK pili to A549 cells was assessed. Six PAK amino acid side chains (Ser131, Gln136, Ile138, Pro139, Gly141, and Lys144) and nine KB7 side chains (Ala130, Thr131, Thr132, Val133, Asp134, Ala135, Lys136, Arg138, and Pro139) were found to be important in mediating the pilus adhesin binding to A549 cells. In addition, a flexible peptide analog with both cysteine residues replaced by alanine failed to inhibit the binding of PAK pili to A549 cells. This suggests that the interactions between the pilin ligand and the A549 cell surface receptors are dependent on the conformation mediated by the disulfide bridge (Cys129 and Cys142). The residues considered to contribute to bacterial adherence are referred to as the "adhesintope". Four PAK and three KB7 side chains were located in a structurally more rigid region of the disulfide-bridged peptide as revealed by two-dimensional NMR studies [McInnes et al. (1993) Biochemistry 32, 13432-13440]. The structural aspects of the pilin-receptor interactions related to the mapped adhesintope sequences are discussed. The dissimilarities between the PAK and KB7 adhesintopes may suggest that compensatory mutations could occur among different pilin strains so as to allow the pilin adhesins to interact with the same receptor.