Biomedical subjects

B Helen Jost

Publications and source records attributed to B Helen Jost.

24 records · Page 2Linked to original sources

PubMed2002

Identification and role in virulence of putative iron acquisition genes from Corynebacterium pseudotuberculosis.

Four genes, fagA, B, C and D, encoding products with 32-47% identity to proteins involved in bacterial iron uptake systems, were identified immediately downstream of the Corynebacterium pseudotuberculosis phospholipase D gene. beta-Galactosidase assays on a C. pseudotuberculosis strain carrying a fagA-lacZ fusion indicated that the putative fagABC operon was poorly expressed in iron-rich media. However, similar experiments in iron-limited media resulted in an approximately three-fold increase in beta-galactosidase activity, suggesting that this operon is regulated by iron in vitro. Although no defect in iron utilization could be determined for a C. pseudotuberculosis fagB(C) mutant in vitro, this mutant showed reduced virulence compared to wild-type in a goat model of caseous lymphadenitis. Thus, expression of the fag genes in the host appears to contribute to virulence.

Animals↗

PubMed2002

The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity.

The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of Arcanobacterium pyogenes, has a variant undecapeptide sequence (EATGLAWDPWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P(499)), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P(499) is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 0.1% activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO.C(492)) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C(492), which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.

Actinomycetaceae↗

PubMed2002

Widespread distribution of a tet W determinant among tetracycline-resistant isolates of the animal pathogen Arcanobacterium pyogenes.

Tetracycline resistance is common among isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes. The tetracycline resistance determinant cloned from two bovine isolates of A. pyogenes was highly similar at the DNA level (92% identity) to the tet(W) gene, encoding a ribosomal protection tetracycline resistance protein, from the rumen bacterium Butyrivibrio fibrisolvens. The tet(W) gene was found in all 20 tetracycline-resistant isolates tested, indicating that it is a widely distributed determinant of tetracycline resistance in this organism. In 25% of tetracycline-resistant isolates, the tet(W) gene was associated with a mob gene, encoding a functional mobilization protein, and an origin of transfer, suggesting that the determinant may be transferable to other bacteria. In fact, low-frequency transfer of tet(W) was detected from mob+ A. pyogenes isolates to a tetracycline-sensitive A. pyogenes recipient. The mobile nature of this determinant and the presence of A. pyogenes in the gastrointestinal tract of cattle and pigs suggest that A. pyogenes may have inherited this determinant within the gastrointestinal tracts of these animals.

Actinomycetaceae↗

PubMed2002

Immunomagnetic separation (IMS)-fluorescent antibody detection and IMS-PCR detection of seeded Cryptosporidium parvum oocysts in natural waters and their limitations.

Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.

Animals↗

PubMed2002

Identification of a second Arcanobacterium pyogenes neuraminidase and involvement of neuraminidase activity in host cell adhesion.

Arcanobacterium pyogenes, a common inhabitant of the upper respiratory and urogenital tracts of economically important animals, such as cattle and swine, is also an opportunistic pathogen associated with suppurative infections in these animals. A. pyogenes expresses neuraminidase activity encoded by the nanH gene, and previously, construction of a nanH mutant of A. pyogenes BBR1 indicated that a second neuraminidase is present in this strain. A 5,112-bp gene, nanP, was cloned and sequenced, and this gene conferred neuraminidase activity on an Escherichia coli host strain. The predicted 186.8-kDa NanP protein exhibited similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. As expected, insertional inactivation of the nanP gene in A. pyogenes BBR1 resulted in a mutant with reduced neuraminidase activity. However, insertional inactivation of the nanP gene in the nanH mutant strain resulted in a complete lack of neuraminidase activity. Like NanH, NanP was localized to the A. pyogenes cell wall. However, unlike the nanH gene, which was present in 100% of the strains examined, nanP was present in only 64.2% of the isolates (n = 53). A. pyogenes adheres to HeLa cells, and a nanP mutant displayed a wild-type adhesion phenotype with these cells. In contrast, the ability of a nanH nanP double mutant to bind to HeLa cells was reduced by 53%. The wild-type adhesion phenotype was restored by providing nanP in trans. These data indicate that the neuraminidases of A. pyogenes play a role in adhesion of this organism to host epithelial cells.

Actinomycetaceae↗

PubMed2002

Evaluation of a test for Clostridium difficile toxins A and B for the diagnosis of neonatal swine enteritis.

A commercially available 1-hour enzyme immunoassay (EIA) for detecting the presence of Clostridium difficile toxins A and B was evaluated for use in diagnosis of C. difficile infections in neonatal swine. This test was compared with a tissue culture cytotoxicity assay, which is considered to be the reference standard for the detection of C. difficile toxins. Twenty-seven samples of colonic contents and 23 fecal samples were collected from freshly euthanized neonatal swine with a history of scours. Of the 50 specimens tested, 20 were positive by the EIA test and tissue culture and 24 were negative by both tests, for an overall correlation of 88%. The sensitivity and specificity of the EIA were 91% and 86%, respectively, and the positive and negative predictive values were 84% and 86%, respectively. The EIA test is considered suitable as an aid for the diagnosis of C. difficile enteritis because of the high correlation between EIA results and those of the tissue culture cytotoxicity assay.

Animals↗