Vaccine errors.
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Haemophilus influenzae↗
Biomedical subjects
C F Marrs
Publications and source records attributed to C F Marrs.
Comparison of hemagglutinating pili of type b and nontypeable Haemophilus influenzae.
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Bacterial Adhesion↗
Comparison of the alpha-pilins of Haemophilus influenzae type b strains Eagan (p+), M43 (p+), and 770235bof+.
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Amino Acid Sequence↗
Identification of type 4 pili in Kingella denitrificans.
Kingella denitrificans is an occasionally pathogenic member of the family Neisseriaceae and is a member of the normal respiratory flora. Electron microscopy, colony morphology types, DNA transformation patterns, and immunoblots suggest that K. denitrificans and K. kingae have type 4 pili. This was confirmed by N-terminal amino acid sequencing for K. denitrificans.
Amino Acid Sequence↗
The type 4 pilin of Moraxella nonliquefaciens exhibits unique similarities with the pilins of Neisseria gonorrhoeae and Dichelobacter (Bacteroides) nodosus.
Moraxella nonliquefaciens is a bacterium which is part of the normal flora of the human upper respiratory tract and is an occasional cause of disease. Using a previously cloned type 4 pilin gene (tfpQ) from Moraxella bovis as a hybridization probe, we have cloned an 826 bp Sau3 AI fragment which contains an M. nonliquefaciens type 4 pilin gene (tfpA) from strain NCTC 7784. The pilin gene is expressed in Escherichia coli. We have examined NCTC 7784 and nine other M. nonliquefaciens strains by genomic Southern hybridization using tfpA as a probe, and they all appeared to have more than one pilin gene. While the predicted amino acid sequence of the M. nonliquefaciens tfpA pilin has conserved regions as compared to pilins of M. bovis and M. lacunata, it also shows similarities to both the type 4 pilin of Neisseria gonorrhoeae and the type 4 pilin of Dichelobacter nodosus (formerly Bacteroides nodosus).
Amino Acid Sequence↗
Shared antigenicity and immunogenicity of type 4 pilins expressed by Pseudomonas aeruginosa, Moraxella bovis, Neisseria gonorrhoaea, Dichelobacter nodosus, and Vibrio cholerae.
Immunoblotting with polyclonal rabbit antibodies raised against pilins expressed by Pseudomonas aeruginosa, Moraxella bovis, Neisseria gonorrhoeae, Dichelobacter nodosus, and Vibrio cholerae was used to demonstrate that these polypeptides display conserved antigenic and, in most cases, immunogenic determinants. These determinants appear to be localized to the highly homologous amino-terminal domains (residues 1 to 25).
Animals↗
Comparison and analysis of the nucleotide sequences of pilin genes from Haemophilus influenzae type b strains Eagan and M43.
Previous studies have demonstrated antigenic differences among the pili expressed by various strains of Haemophilus influenzae type b (Hib). In order to understand the molecular basis for these differences, the structural gene for pilin was cloned from Hib strain Eagan (p+) and the nucleotide sequence was compared to those of strains M43 (p+) and 770235 b0f+, which had been previously determined. The pilin gene of Hib strain Eagan (p+) had a 648-bp open reading frame that encoded a 20-amino-acid leader sequence followed by the 196 amino acids found in mature pilin. The translated sequence was three amino acids larger than pilins of strains M43 (p+) and 770235 b0f+ and was 78% identical and 95% homologous when conservative amino acid substitutions were considered. Differences between the amino acid sequences were not localized to any one region but rather were distributed throughout the proteins. Comparison of protein hydrophilicity profiles showed several hydrophilic regions with sequences that were conserved between strain Eagan (p+) and pilins of other Hib strains, and these regions represent potentially conserved antigenic domains. Southern blot analyses using an intragenic probe from the pilin gene of strain Eagan (p+) showed that the pilin gene was conserved among all type b and nontypeable strains of H. influenzae examined, and only a single copy was present in these strains. Homologous genes were not present in the phylogenetically related species Pasteurella multocida, Pasteurella haemolytica, and Actinobacillus pleuropneumoniae. These data indicate that the pilin gene was highly conserved among different strains of H. influenzae and that small differences in the pilin amino acid sequences account for the observed antigenic differences of assembled pili from these strains.
Amino Acid Sequence↗
Interesting sequence differences between the pilin gene inversion regions of Moraxella lacunata ATCC 17956 and Moraxella bovis Epp63.
The bacterium Moraxella lacunata is a causative agent of human conjunctivitis and keratitis. We have previously reported construction of plasmid pMxL1, which includes a 5.9-kb fragment on which the pilin gene inversion region of M. lacunata resides. The inversion region of pMxL1 was shown to invert when pMxL1 was in an Escherichia coli host cell. In this report, we present Western immunoblot analysis using Moraxella bovis Epp63 anti-I and anti-Q pilin sera which demonstrate that pMxL1 makes pilin only when in orientation 1. The sequence of the pMxL1 plasmid containing the invertible region contains a perfect tandem repeat of 19 bp in the orientation 1 nonexpressed pilin gene at the middle of the recombination junction site. This 19-bp insert causes a frameshift and disrupts the pilin gene. The predicted amino acid sequence of this nonfunctional pilin gene (with the 19-bp repeat subtracted) bears closest resemblance to M. bovis Epp63 Q pilin sequence, although the other (functional) M. lacunata pilin encoded by pMxL1 shows slightly higher homology to Q pilin. Comparison of the pMxL1 sequence with that of the M. bovis Epp63 sequence shows two other particularly interesting differences. One is a 15-bp sequence addition found in pMxL1 at the 60-bp region previously reported as a possible M. bovis recombinational enhancer. The second is an AT deletion in pMxL1 compared with Epp63 within an open reading frame (tfpB) which results in the pMxL1 tfpB open reading frame being one-third shorter than in Epp63. The DNA sequences in these three altered regions from the M. lacunata strain from which pMxL1 was derived were amplified by polymerase chain reaction and sequenced. The parent strain was found to contain the differences seen in pMxL1. Comparison of the M.bovis and M. lacunata pilin gene amino acid sequences is also presented.
Amino Acid Sequence↗
Pili (fimbriae) of Branhamella species.
PURPOSE: Pili (fimbriae) have frequently been found to be involved in the attachment of bacteria to mucosal epithelial cells, an important initial step in the disease process. The purpose of this study was to determine if Branhamella catarrhalis expresses type 4 pili. MATERIALS AND METHODS: Piliated B. catarrhalis phenotypic characteristics of colony morphology, agar corrosion, twitching motility, competence for deoxyribonucleic acid (DNA) transformation, autoagglutination, and pellical formation were observed. DNA was isolated from Branhamella spp. and used in genomic Southern hybridizations with a Moraxella bovis pilin gene as a probe. Electron microscopy of negatively stained bacteria was carried out to visualize pili. RESULTS: B. catarrhalis has several (but not all) of the phenotypic characteristics that are related to the presence of type 4 (MePhe) pili in closely related Moraxella spp., including competence for DNA transformation, autoagglutination, pellicle formation, colony morphology, and pitting of agar. The one phenotype we have not found that is generally characteristic of type 4 piliated bacteria is twitching motility. Genomic Southern hybridization analysis using a cloned M. bovis Q pilin gene as a probe reveals DNA homologous to the Q pilin gene in B. catarrhalis, Branhamella ovis, Branhamella caviae, and Branhamella cuniculi. Examination of B. catarrhalis strain ATCC25240 by electron microscopy reveals two different kinds of pili. One kind appears similar to other type 4 pili, whereas a second class is short pili extending outward from all portions of the bacteria. CONCLUSION: Phenotypic, electron-microscopic, and hybridization data are all consistent with type 4 pili being present on some B. catarrhalis strains.
Fimbriae, Bacterial↗
Kinetics and regulation of transcription of bacteriophage Mu.
Mu transcription was analyzed by hybridization of [3H]uridine pulse-labeled RNA from heat-induced Mu lysogens to Mu DNA restriction fragments on nitrocellulose blots. Based on their time of appearance and dependence on Mu functions, we have defined three classes of transcripts: early, middle, and late. Replication-defective prophages containing A or B amber mutations or a deletion of the beta (right) end produced only early RNA derived from the left-most 8 to 10 kb of the Mu genome. A replication-proficient C amber mutant exhibited similar early transcription but at later times also produced middle transcripts from a region including C, which encodes the activator of late transcription. The C mutant did not produce late transcripts from the right-most 26 kb of the Mu genome encoding genes involved in phage morphogenesis and release. These results indicate that Mu DNA replication is required for efficient expression of middle RNA, which is itself required for expression of late transcripts. Amber mutations in essential genes other than A, B, and C had no significant effect on transcription except for polarity of one E mutation. Uninduced Mu c+ and Mu cts prophages produced very low levels of Mu-specific RNA derived from several regions including the c (immunity) gene and the region between genes B and C.
Bacteriophage mu↗
Cloning, expression, and sequence analysis of the Haemophilus influenzae type b strain M43p+ pilin gene.
By using antiserum against Haemophilus influenzae type b (Hib) strain M43p+ denatured pilin, we screened a genomic library of Hib strain M43p+ and identified a clone that expressed pilin, but not assembled pili, on its surface. Southern blot analysis revealed the presence of one structural gene, which was also present in strain M42p-, a nonpiliated variant. Five exonuclease III deletion mutants, two of which had deletions that extended into the structural gene and failed to express pilin, were used to obtain the nucleotide sequence of the structural gene. The amino acid sequence of the open reading frame agrees with 38 of 40 amino acids from the published sequence of purified Hib M43p+ pilin. The pilin gene coded for a mature protein of 193 amino acids, with a calculated molecular mass of 21,101 daltons. Comparison of the Hib M43p+ pilin amino acid sequence with those of pilins of other bacteria revealed strong conservation of amino- and carboxy-terminal regions in M43p+ and Escherichia coli F17, type 1C, and several members of the P pili family, as well as Klebsiella pneumoniae type 3 MR/K, Bordetella pertussis serotype 2, and Serratia marcescens US46 fimbriae.
Amino Acid Sequence↗
Sequence analysis of the inversion region containing the pilin genes of Moraxella bovis.
Moraxella bovis EPP63 is able to produce two antigenically distinct pili called Q and I pili (previously called beta and alpha pili). Hybridization studies have shown that the transition between the types is due to inversion of a 2.1-kilobase segment of chromosomal DNA. We present the sequence of a 4.1-kilobase region of cloned DNA spanning the entire inversion region in orientation 1 (Q pilin expressed). Comparison of this sequence with the sequence of the polymerase chain reaction-amplified genomic DNA from orientation 2 (I pilin expressed) allows the site-specific region of recombination to be localized to a 26-base-pair region in which sequence similarity to the left inverted repeat of the Salmonella typhimurium hin system was previously noted. In addition, 50% sequence similarity was seen in a 60-base-pair segment of our sequence to the recombinational enhancer of bacteriophage P1, an inversion system related to the hin system of S. typhimurium. Finally, two open reading frames representing potential genes were identified.
Amino Acid Sequence↗
Morphogenetic expression of Moraxella bovis fimbriae (pili) in Pseudomonas aeruginosa.
Type 4 fimbriae (pili) are found in a wide variety of gram-negative bacteria and are composed of small structural subunits which share significant sequence homology among different species, especially at their amino-terminal ends. Previous studies demonstrating morphogenetic expression of Bacteroides nodosus fimbriae from cloned subunit genes in Pseudomonas aeruginosa suggested that there is a common mechanism for type 4 fimbriae assembly and that the structural subunits are interchangeable (J. S. Mattick et al., J. Bacteriol. 169:33-41, 1987). Here we have examined the expression of Moraxella bovis fimbrial subunits in P. aeruginosa. M. bovis subunits were assembled into extracellular fimbriae in this host, in some cases as a homopolymer but in others as a mosaic with the indigenous subunit, indicating structural equivalence. This result contrasts with other studies in which recombinant P. aeruginosa expressing different subunits produced fimbriae composed almost exclusively of one subunit or the other (T. C. Elleman and J. E. Peterson, Mol. Microbiol. 1:377-380, 1987). Both observations can be explained by reversibility of subunit-subunit interactions at the site of assembly, with the forward equilibrium favoring chain extension between compatible subunits.
DNA, Recombinant↗
Identification, cloning, and sequencing of piv, a new gene involved in inverting the pilin genes of Moraxella lacunata.
Moraxella lacunata is a bacterium that is a causative agent of human conjunctivitis and keratitis. We have previously cloned the Q and I pilin (formerly called beta and alpha pilin) genes of Moraxella bovis and determined that an inversion of 2 kilobases (kb) of DNA determines which pilin gene is expressed. Using an M. bovis pilin gene as a hybridization probe to screen a lambda ZAP library of M. lacunata DNA, we have isolated a clone that not only contains the entire type 4 pilin gene inversion region of M. lacunata but inverts the 2-kb region on a plasmid subclone (pMxL1) in Escherichia coli. Deletion derivatives of pMxL1 yielded some plasmids that still had the entire inversion region but were phase locked into one or the other of the two potential orientations. Similarly, insertions of a 2-kb streptomycin-resistant element (omega) within some regions outside of the inversion also resulted in phase-locked plasmids. These deletions and insertions thus localize a probable invertase necessary for the inversion event. The region was sequenced, and an open reading frame with over 98% DNA sequence homology to an open reading frame that we previously found in M. bovis and called ORF2 appeared to be a strong candidate for the invertase. This conclusion was confirmed when a plasmid containing the M. bovis ORF2 supplied, in trans, the inversion function missing from one of the M. lacunata phase-locked inversion mutants. We have named these putative invertase genes piv(ml) (pilin inversion of M. lacunata) and piv(mb) (pilin inversion of M. bovis). Despite previously noted sequence similarities between the M. bovis sites of inversion and those of the Hin family of invertible segments and a 60-base-pair region within the inversion with 50% sequence similarity to the cin recombinational enhancer, there is no significant sequence similarity of the Piv invertases to the Hin family of invertases.
Amino Acid Sequence↗
Purification, characterization, and pathogenicity of Moraxella bovis pili.
Pilins composed of the alpha or beta pilins of Moraxella bovis strain Epp63 were purified, subjected to chemical or enzymatic cleavage, and the resulting fragments sequenced by automated Edman degradation. alpha Pilin was found to be a 155-amino-acid polypeptide with a single intramolecular disulfide bridge. The beta pilin amino acid sequence substantiated the previously reported structure derived from the beta pilin gene DNA sequence, and indicated that the alpha and beta pilins of this strain are approximately 70% homologous. DNA hybridization studies of genomic DNA from the alpha- and beta-piliated variants of strain Epp63 indicated that the expression of the two pilin types was governed by an oscillating mechanism of chromosomal rearrangement. The alpha and beta pili were evaluated serologically and found to exhibit approximately 50% shared antigenicity, indicating that regions of conserved and heterologous sequence specify both type-specific and crossreacting epitopes. The pathogenicity of the alpha- and beta-piliated variants was studied by ocular inoculation of calves eyes; beta-piliated organisms were significantly more infectious than alpha-piliated organisms, indicating that beta pili confer, or are associated with, a relative advantage during the first stages of ocular infection. Preliminary analysis of other M. bovis strains suggests that each strain produces two types of pilin, and that this property may be characteristic of the species.
Amino Acid Sequence↗
Pilin-gene phase variation of Moraxella bovis is caused by an inversion of the pilin genes.
Moraxella bovis Epp63 can express either of two different pilin proteins, called alpha and beta. We have previously cloned and sequenced the beta-pilin gene and now report that DNAs isolated from bacteria expressing alpha pilin have hybridization patterns consistently different from those of bacteria expressing beta pilin. The phase variation between alpha- and beta-pilin gene expression appears to be associated with an inversion of about 2 kilobases of DNA, whose endpoints occur within the coding region of the expressed pilin gene. Comparisons of the beta-pilin gene sequence with those of well-studied bacterial inversion systems revealed a stretch of 58% sequence similarity (21 of 36 base pairs) between the left inverted repeat of the Salmonella typhimurium flagellar hin control region and the amino-terminal portion of the beta-pilin gene.
Bacterial Outer Membrane Proteins↗
Cloning and sequencing of a Moraxella bovis pilin gene.
Moraxella bovis pili have been shown to play a major role in both infectivity and protective immunity of bovine infectious keratoconjunctivitis. Sonicated M. bovis DNA from the piliated strain EPP63 was inserted into the vector lambda gt11 with EcoRI linkers. Recombinant phage were screened with an oligonucleotide probe based on the amino-terminal portion of the DNA sequence of a Neisseria gonorrhoeae pilin gene. Two candidate phages produced a protein that comigrated with EPP63 beta pilin in sodium dodecyl sulfate-polyacrylamide gels and bound anti-pilus antisera. The 1.9-kilobase insert from one of these, lambda gt11M182, was subcloned in both orientations into pBR322, forming the plasmids pMxB7 and pMxB9, both of which produced beta pilin, as did pMxB12, a HindIII deletion derivative of pMxB7. In HB101(pMxB12), the M. bovis pilin protein was shown to be primarily localized in the inner membrane. The entire 939-base-pair insert of pMxB12 was sequenced, revealing a ribosome binding site just upstream of the coding region and an AT-rich region further upstream containing some potential RNA polymerase recognition sites. The translation of the sequence predicts a six-amino-acid leader sequence preceding the phenylalanine that begins the mature protein. Codon usage analysis of the M. bovis beta pilin gene revealed greater use of the CUA codon for leucine than usual for a well-expressed Escherichia coli gene. Comparisons of the M. bovis EPP63 beta pilin protein sequence with other pilin gene sequences are presented.
Amino Acid Sequence↗