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W L Hynes

Publications and source records attributed to W L Hynes.

At least 19 recordsLinked to original sources
PubMed2005

A defensin-like gene expressed in the black-legged tick, Ixodes scapularis.

The black-legged tick Ixodes scapularis Linnaeus (Acari: Ixodidae) is an important vector of microbial pathogens. Knowledge of the tick's innate immune response, particularly defensin and other antimicrobial peptides, is important for understanding how microbes survive in this tick. A defensin gene (slnA) from I. scapularis was obtained by reverse transcription-polymerase chain reaction (RT-PCR) using mRNA extracted from tissues of female ticks. RT-PCR indicated the gene was expressed in the midgut, haemocytes, and fat-body, although no evidence of a peptide was found. Sequencing a cloned cDNA fragment revealed a 225 bp open reading frame encoding a 74 amino acid pre-prodefensin, including the putative 38 amino acid mature peptide. Similarity between the defensin amino acid sequences of I. scapularis and Dermacentor variabilis (Say) (Acari: Ixodidae) was 62.2% for the pre-prodefensin region; for the mature defensins from these two species the similarity was 78.9%, with the six cysteine residues being located in the same relative position. PCR amplification and sequencing of chromosomal DNA suggests that slnA, along with vsnA, the defensin gene from D. variabilis, does not contain any introns. This is in contrast to the defensins described for the soft tick, Ornithodoros moubata (sensu Walton) (Acari: Argasidae). The role of defensin in the innate immune response of I. scapularis following microbial invasions is discussed.

Amino Acid Sequence↗
PubMed2001

Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis.

Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1h post challenge. Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2kDa, was determined by MALDI-TOF mass spectrometry. N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K. A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70 degrees C. Although the tick defensin alone was not immediately effective against B. burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B. burgdorferi within 1h.

Amino Acid Sequence↗
PubMed2001

Contrasts in tick innate immune responses to Borrelia burgdorferi challenge: immunotolerance in Ixodes scapularis versus immunocompetence in Dermacentor variabilis (Acari: Ixodidae).

The blacklegged tick, Ixodes scapularis Say, transmits the Lyme disease spirochete Borrelia burgdorferi, whereas the American dog tick, Dermacentor variabilis (Say), is unable to transmit the bacterium. We compared the innate immune response of these ticks against spirochetes directly inoculated into the hemocoel cavity of ticks. In I. scapularis, some Borrelia were found associated with hemocytes, while numerous other spiral-shaped, intact bacteria remained free in the hemolymph. In contrast, in D. variabilis only remnants of the bacteria were evident in the hemolymph, indicating lysis; intact spirochetes were rare. Spirochetes were observed bound to or within the organs of both tick species, although many more spirochetes were found associated with the I. scapularis organs. The few spirochetes observed with the D. variabilis organs appeared to be dead because D. variabilis tissues rarely contained culturable bacteria, unlike I. scapularis tissues. When spirochetes were incubated with I. scapularis hemolymph plasma in vitro, bacterial survival and motility were not reduced. In contrast, incubation of spirochetes with D. variabilis hemolymph plasma resulted in > 50% of the spirochetes becoming nonmotile by 45 min. The differences in the responses of the two different tick species indicate that I. scapularis is immunotolerant when challenged with B. burgdorferi and dependent on a slow phagocytic response to clear Borrelia from the hemolymph. In contrast, D. variabilis is highly immunocompetent (i.e., innate immunity), using plasma borreliacidal factors and a rapid increase in phagocytic cells to clear the infection and limit tissue invasion.

Animals↗
PubMed2000

Enhancement of OspC expression by Borrelia burgdorferi in the presence of tick hemolymph.

Borrelia burgdorferi B31 cultured at 28 degrees C was used to assay the effects of tick hemolymph on outer surface protein C (OspC) surface expression. Using immunofluorescence, OspC surface expression was shown to increase in cultures treated with hemolymph from both Ixodes scapularis and Dermacentor variabilis, while control samples (non-hemolymph treated) showed no increase in the levels of OspC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed an increase in OspC from spirochetes exposed to tick hemolymph. In addition, a lower-molecular-mass protein band of unknown identity was also enhanced in the hemolymph-exposed spirochetes. The significance of these effects of tick hemolymph on B. burgdorferi outer surface protein expression is unknown.

Animals↗
PubMed2000

The extracellular hyaluronidase gene (hylA) of Streptococcus pyogenes.

Group A streptococci produce an extracellular hyaluronidase (hyaluronate lyase) which may be associated with the spread of the organism during infection. The gene for this hyaluronidase (hylA) encodes an 868 amino acid protein with a molecular size of 99636 Da. Cleavage of the proposed signal peptide results in an extracellular protein of 95941 Da. Comparison with other bacterial hyaluronidases indicates strong similarities to the genes from Streptococcus pneumoniae, Streptococcus agalactiae and Staphylococcus aureus. A region internal to the hylA gene was amplified from all 175 strains of Streptococcus pyogenes tested suggesting a widespread distribution of the gene.

DNA, Bacterial↗
PubMed2000

Hyaluronidases of Gram-positive bacteria.

Bacterial hyaluronidases, enzymes capable of breaking down hyaluronate, are produced by a number of pathogenic Gram-positive bacteria that initiate infections at the skin or mucosal surfaces. Since reports of the hyaluronidases first appeared, there have been numerous suggestions as to the role of the enzyme in the disease process. Unlike some of the other more well studied virulence factors, much of the information on the role of hyaluronidase is speculative, with little or no data to substantiate proposed roles. Over the last 5 years, a number of these enzymes from Gram-positive organisms have been cloned, and the nucleotide sequence determined. Phylogenetic analysis, using the deduced amino acid sequences of the Gram-positive hyaluronidases, suggests a relatedness among some of the enzymes. Molecular advances may lead to a more thorough understanding of the role of hyaluronidases in bacterial physiology and pathogenesis.

Gram-Positive Bacteria↗
PubMed2000

Response of the tick Dermacentor variabilis (Acari: Ixodidae) to hemocoelic inoculation of Borrelia burgdorferi (Spirochetales).

When Borrelia burgdorferi B31 low passage strain spirochetes were directly injected into the hemocoel of Dermacentor variabilis (Say) females, the bacteria were cleared from the hemocoel within < 24 h. Viable spirochetes were not found in hemolymph, salivary gland, or ovary tissues by subculturing or by IFA. The hemocyte population increased approximately 6 times within the first 6 h after inoculation compared with the uninoculated controls. In contrast, the soluble total hemolymph protein content decreased inversely with the increase in hemocytes. Borreliacidal activity was demonstrated with cell-free hemolymph from D. variabilis. In vitro antimicrobial assays using hemolymph from borrelia-challenged and nonchallenged ticks resulted in 72% spirochete reductions compared with only 11.5%, respectively, within 1 h. Additional evidence of induced antimicrobial hemolymph protein activity was demonstrated by SDS-PAGE, which revealed upregulation of a lysozyme-like peptide (approximately 15 kDa) (22% increase) and the induction of a approximately 5.8 kDa peptide in the B. burgdorferi-challenged ticks. In contrast with the nonvector borne Bacillus subtilis, D. variabilis presented a rapid and robust response to challenge with cultured B. burgdorferi spirochetes and lead to their early elimination. The role of the tick immune system, including possible differences between vector and nonvector ticks, in determining the success of invasive bacteria is discussed.

Animals↗
PubMed1999

Nucleotide sequence of the streptococcin A-FF22 lantibiotic regulon: model for production of the lantibiotic SA-FF22 by strains of Streptococcus pyogenes.

Streptococcin A-FF22 (SA-FF22) is a type AII linear lantibiotic produced by Streptococcus pyogenes strain FF22. Sequence analysis of an approximate 10 kb region of DNA showed it to contain nine open reading frames arranged in three operons responsible for regulation, biosynthesis and immunity of SA-FF22. This region is organized similarly to the Lactococcus lactis lacticin 481 region, however, unlike lacticin 481, a two-component regulatory system is essential for SA-FF22 production. Located immediately downstream of the scn region is a putative transposase gene, the presence of which supports earlier data that indicated a mobile nature to this region.

Amino Acid Sequence↗
PubMed1998

Control of bacterial infections in the hard tick Dermacentor variabilis (Acari: Ixodidae): evidence for the existence of antimicrobial proteins in tick hemolymph.

The ability of hard ticks to prevent infection by bacteria was investigated. During a 72-h period, virtually all Dermacentor variabilis females survived inoculation with Bacillus subtilis, Escherichia coli, and Staphyloccocus aureus but few survived infection with Pseudomonas aeruginosa. The hemocyte population increased to peak abundance at 48 h to approximately 6 times that of the uninfected controls. In contrast, the soluble hemolymph protein content decreased inversely as the hemocytes increased. D. variabilis hemolymph was found to be constitutively antimicrobial (i.e., hemolymph from bacteria-naive individuals inhibited bacterial growth). Infection with various bacterial species enhanced this innate capability. When hemolymph fractions separated by high-pressure liquid chromatography were tested for their ability to inhibit microbial growth, activity against the gram-positive bacterium, B. subtilis, was found in 2 polar fractions. Antimicrobial activity was lost when the fractions were incubated with protease. The least polar fraction contained 1 major protein, M(r) 14.5 kDa, that comigrated with human lysozyme. This protein, tentatively identified as tick lysozyme, was abundant in bacteria-naive ticks but increased greatly (43%) following challenge with B. subtilis. The identity of the other, more polar protein is unknown. Studies to characterize these antimicrobial proteins are planned.

Animals↗
PubMed1995

Analysis of a second bacteriophage hyaluronidase gene from Streptococcus pyogenes: evidence for a third hyaluronidase involved in extracellular enzymatic activity.

The hyaluronidase gene (hylP2) from a second group A streptococcal bacteriophage was isolated from ATCC T-type-22 hyaluronidase-producing strain 10403, a strain known to produce increased amounts of extracellular hyaluronidase. Sequence analysis of hylP2 and alignment with the previously described bacteriophage hyaluronidase gene (hylP) showed a high degree of similarity; however, hylP2 had deletions of regions specifying 34 amino acids. Twenty-eight of the deleted amino acids were in a region of HylP containing a series of collagen-like Gly-X-Y repeating units. By employing primers for both hylP and hylP2, PCR amplification resulted in fragments of appropriate sizes in 97% of the strains tested, with some strains producing two fragments, indicating the presence of at least two phages. When the hylP2 gene was introduced via a plasmid vector into a non-hyaluronidase-producing Streptococcus pyogenes strain, this strain was still unable to produce extracellular hyaluronidase, although intracellular hyaluronidase was present. These results, along with the absence of a typical N-terminal signal peptide, indicate that HylP2 is unable to be secreted into the extracellular milieu. Examination of more than 100 strains for production of hyaluronidase showed that only 23% of the strains produced extracellular hyaluronidase. One of these strains (strain 10403) contains a single bacteriophage hyaluronidase gene (hylP2) which, when inactivated by allelic replacement, still produces large amounts of extracellular hyaluronidase. These results suggest the presence of a different hyaluronidase gene encoding a protein that is actively secreted into the extracellular milieu.

Amino Acid Sequence↗
PubMed1993

Cloning of the gene encoding Streptococcin A-FF22, a novel lantibiotic produced by Streptococcus pyogenes, and determination of its nucleotide sequence.

Streptococcin A-FF22 (SA-FF22) is a lantibiotic produced by Streptococcus pyogenes FF22. The nucleotide sequence of the SA-FF22 structural gene (scnA) was determined and shown to encode a 51-amino-acid prepeptide. The proteolytic processing site of the SA-FF22 prepeptide differs from that which characterizes other type A lantibiotics.

Amino Acid Sequence↗
PubMed1992

PCR amplification of streptococcal DNA using crude cell lysates.

Gram-positive organisms such as streptococci and enterococci are often difficult to lyse. Obtaining DNA for procedures such as PCR amplification usually requires a large scale isolation for each strain under investigation. We describe a simple procedure for small volumes of whole cells, involving pretreatment with detergent and proteinase that allows for efficient release of DNA for PCR amplification. This procedure is fast, reproducible, can be used with a large number of samples, and has been successfully applied to a variety of streptococcal and enterococcal strains.

Base Sequence↗
PubMed1989

Sequence analysis and expression in Escherichia coli of the hyaluronidase gene of Streptococcus pyogenes bacteriophage H4489A.

The hyaluronidase gene (hylP) from Streptococcus pyogenes bacteriophage H4489A was previously cloned into Escherichia coli plasmid pUC8 as a 3.1-kilobase ThaI fragment. Southern hybridization experiments confirmed the origin of this fragment in bacteriophage H4489A before determination of the nucleotide sequence of the entire fragment. Two open reading frames (ORFs) were found, the first of which specified a 39,515-molecular-weight protein identified as the bacteriophage hyaluronidase. The second ORF encoded a 65,159-molecular-weight protein of unknown function. Putative transcription and translation control sequences for each ORF were identified by using a plasmid containing a promoterless chloramphenicol acetyltransferase gene. Controlled exclusive expression of the hylP gene via the T7 polymerase-promoter system in E. coli resulted in a 40,000-dalton protein, a result consistent with the coding capacity of the hylP gene.

Amino Acid Sequence↗
PubMed1987

Immunologic cross-reactivity of type A streptococcal exotoxin (erythrogenic toxin) and staphylococcal enterotoxins B and C1.

Immunologic cross-reactivity between Streptococcus pyogenes type A exotoxin (erythrogenic toxin) and Staphylococcus aureus enterotoxins B and C1 was demonstrated by Ouchterlony double diffusion, Western immunoblot, and immunodot analyses. Specific antiserum to type A streptococcal exotoxin reacted more strongly with staphylococcal enterotoxin B than with enterotoxin C1. The reactivity of type A streptococcal exotoxin with antiserum to staphylococcal enterotoxin B was greater than that of antiserum to enterotoxin C1. These results suggest that a conserved domain is present in the three exotoxins, which most likely originated from a common evolutionary ancestor.

Antibodies, Bacterial↗
PubMed1986

Proteinase-related broad-spectrum inhibitory activity among group-A streptococci.

Some 10% of group-A streptococci have inhibitory activity against all nine strains (eight of them streptococci) in a set of indicators in an inhibitor-production typing (P-typing) scheme. This activity was associated with the concurrent synthesis of cell-associated proteinase by the streptococcal strain. Inhibitor production was prevented either by incubation of the test strain in conditions inimical to proteinase production, e.g., at low temperature and alkaline pH, or by addition to the medium of substances, such as glucose, iodoacetic acid, lincomycin, Congo red or trypan blue, that had an anti-proteinase effect. Inhibitory activity was not detectable in liquid cultures, but freeze-thaw extracts of cultures of group-A streptococcus strain A1013 on Gibco Columbia Agar Base (Gibco Diagnostics, Madison, WI, USA) had some inhibitory activity. The inhibitor was concentrated and partially purified, and the active agent was shown to be a high-mol.-wt cationic protein which was bactericidal for various bacteria in the logarithmic growth phase, including the homologous producer strain.

Antibiosis↗