Infection and Immunity
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A 38-Kilobase Pathogenicity Island Specific for<i>Mycobacterium avium</i>subsp.<i>paratuberculosis</i>Encodes Cell Surface Proteins Expressed in the Host ABSTRACT We have used representational difference analysis to identify a novelMycobacterium avium subsp.paratuberculosis -specific ABC transporter operon (mpt ), which comprises six open reading frames designatedmptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that thempt operon is flanked on one end by afep cluster encoding proteins involved in the uptake of Fe3+ and on the other end by asid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kbM. avium subsp.paratuberculosis -specific locus flanked by an insertion sequence similar to IS1110 . Expression studies using Western blot analyses showed that MptC is present in the envelope fraction ofM. avium subsp.paratuberculosis . The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening ofMycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes anM. avium subsp.paratuberculosis pathogenicity island.
Infection and Immunity - Tập 72 Số 3 - Trang 1265-1274 - 2004
<i>Mycobacterium avium</i> Invades the Intestinal Mucosa Primarily by Interacting with Enterocytes ABSTRACT
Previous studies have demonstrated that
Mycobacterium avium
can invade intestinal epithelial cells both in vitro and in vivo. When given to mice orally,
M. avium
preferentially interacts with the intestinal mucosa at the terminal ileum. We evaluated the mechanism(s) of
M. avium
binding and invasion of the intestinal mucosa using three different systems: (i) electron microscopy following administration of
M. avium
into an intestinal loop in mice, (ii) quantitative comparison of the bacterial load in Peyer's patch areas of the terminal ileum versus areas that do not contain Peyer's patches, and (iii) investigation of the ability of
M. avium
to cause disseminated infection following oral administration using B-cell-deficient mice, lacking Peyer's patches, in comparison with C57BL/6 black mice. By all approaches,
M. avium
was found to invade the intestinal mucosa by interacting primarily with enterocytes and not with M cells.
Infection and Immunity - Tập 69 Số 3 - Trang 1515-1520 - 2001
Relationship between virulence of Mycobacterium avium strains and induction of tumor necrosis factor alpha production in infected mice and in in vitro-cultured mouse macrophages We studied the ability of two Mycobacterium avium strains with different virulences to induce tumor necrosis factor alpha (TNF) synthesis by mouse resident peritoneal macrophages (RPM phi) in vitro in an experiment to look for a possible correlation between virulence and this TNF-inducing capacity. The low-virulence strain, 1983, induced significantly higher production of TNF by RPM phi than did the high-virulence strain, ATCC 25291. TNF neutralization during culture of infected RPM phi resulted in enhancement of growth of strain 1983 and had no effect on growth of strain ATCC 25291; TNF treatment of strain ATCC 25291-infected macrophages had no effect on mycobacterial growth. The extent of M. avium growth and the amount of TNF synthesis were independent of the presence of contaminating T cells or NK cells in the macrophage monolayers. Intraperitoneal administration of anti-TNF monoclonal antibodies to BALB/c mice infected intravenously with M. avium 1983 abrogated the elimination of the bacteria in the liver and caused a slight increase in bacterial growth in the spleen. Neutralization of TNF led to a minor increase in the proliferation of M. avium ATCC 25291 in the liver and spleen of BALB/c mice late in infection. Anti-TNF treatment did not affect the growth of the two M. avium strains in BALB/c.Bcgr (C.D2) mice, suggesting that restriction of M. avium strains to induce TNF production by macrophages may limit their ability to proliferate both in vitro and in vivo.
Infection and Immunity - Tập 63 Số 10 - Trang 3759-3764 - 1995
Purification and Properties of Staphylococcal Delta Hemolysin
Large amounts (200 mg per liter of culture supernatant fluid) of highly purified staphylococcal soluble delta hemolysin were obtained by adsorption to and selective elution from hydroxyapatite followed by exhaustive dialysis against water, concentration by polyvinylpyrrolidone or polyethylene glycol 20,000 dialysis, and a final water dialysis. No carbohydrate, phosphorus, or inactive 280-nm absorbing material was detected in the preparation; however, analysis by density gradient centrifugation, gel filtration, analytical ultracentrifugation, carboxymethyl cellulose chromatography, polyacrylamide disc gel electrophoresis, isoelectric focusing, and electron microscopy revealed that the lysin was molecularly heterogeneous. The preparation contained an acidic fibrous lysin (
S
20,w
of 11.9) and a basic lysin component composed of a population of granular aggregates of various sizes, with a maximum
S
20,w
of approximately 4.9. No other staphylococcal products were detected in the preparation. The lysin was active against erythrocytes from many animal species and acted synergistically with staphylococcal beta hemolysin against sheep erythrocytes. It was soluble in chloroform-methanol (2:1), was inactivated by various phospholipids, normal sera, and proteolytic enzymes, but was partially resistant to heat inactivation. Activity was not affected by Ca
2+
, Mg
2+
, citrate, ethylenediaminetetraacetic acid, or cysteine. The lysin preparation also disrupted bacterial protoplasts and spheroplasts, erythrocyte membranes, lysosomes, and lipid spherules, was growth-inhibitory for certain bacteria, and clarified egg yolk-agar. Large amounts produced dermonecrosis in rabbits and guinea pigs. The minimum lethal intravenous dose for mice and guinea pigs was approximately 110 and 30 mg/kg, respectively.
Infection and Immunity - Tập 3 Số 3 - Trang 449-465 - 1971
Cellular location of a Treponema denticola chymotrypsinlike protease and importance of the protease in migration through the basement membrane A number of immunological methods were used to localize a cell-associated Treponema denticola chymotrypsinlike protease. Indirect immunofluorescence staining, immunogold labeling, and an enzyme-linked immunosorbent assay all indicated that the protease was attached to the outside of the cell envelope. The invasive capability of T. denticola was evaluated by following the degradation of a reconstituted basement membrane material (Matrigel) and the release of spirochetes from the gel. Under conditions where the chymotrypsinlike activity was increased, more spirochetes migrated from the gel. Protease inhibitors strongly reduced the number of cells that moved out of the gel. The purified chymotrypsinlike protease degraded the basement membrane components type IV collagen, laminin, and fibronectin. The study suggests that the T. denticola chymotrypsinlike protease may play an important role in the invasion and destruction of basement membrane.
Infection and Immunity - Tập 58 Số 2 - Trang 347-351 - 1990
Characterization of <i>Porphyromonas gingivalis</i> -Induced Degradation of Epithelial Cell Junctional Complexes ABSTRACT
Porphyromonas gingivalis
is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that
P. gingivalis
has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (β1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of
P. gingivalis
on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to
P. gingivalis
(>10
9
bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and β1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral
P. gingivalis
. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to
P. gingivalis
. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and β1-integrin in lysates derived from MDCK cells exposed to
P. gingivalis
. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by
P. gingivalis
, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that
P. gingivalis
is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of
P. gingivalis
to initiate epithelial barrier destruction.
Infection and Immunity - Tập 68 Số 3 - Trang 1441-1449 - 2000
Proteases of Treponema denticola outer sheath and extracellular vesicles Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath- and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 x 10(4) M(-1) min-1. Inhibition of the enzyme by phenylboronic acid was rapid (< 1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases.
Infection and Immunity - Tập 63 Số 10 - Trang 3973-3979 - 1995
Cytopathic Effects of the Major Surface Protein and the Chymotrypsinlike Protease of<i>Treponema denticola</i> ABSTRACT Prominent antigens ofTreponema denticola have been suggested to be mediators of the cytopathic effects typically seen in periodontal disease. In the present study of theT. denticola major surface protein (Msp) and the surface-expressed chymotrypsinlike protease complex (CTLP), we characterized the ability of these proteins to adhere to and lyse epithelial cells. Msp and CTLP were closely associated in spirochete outer membranes. Purified Msp, both native and recombinant, and CTLP bound to glutaraldehyde-fixed periodontal ligament epithelial cells. Adherence of Msp was partially blocked by specific antibodies. Adherence of CTLP was partially blocked by serine protease inhibitors and was further inhibited by specific antibodies. Both native Msp and CTLP were cytotoxic toward periodontal ligament epithelial cells, and their cytotoxicity was inhibited by the same treatments that inhibited adherence. Msp, but not CTLP, lysed erythrocytes. Msp complex (partially purified outer membranes free of protease activity) was cytotoxic toward a variety of different cell types. Pore-forming activities of recombinant Msp in black lipid model membrane assays and in HeLa cell membranes were similar to those reported for the native protein, supporting the hypothesis that Msp cytotoxicity was due to its pore-forming activity.
Infection and Immunity - Tập 66 Số 5 - Trang 1869-1877 - 1998
Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells The effects of Treponema denticola and its outer membrane-bound chymotrypsin-like proteinase on periodontal ligament epithelial cell cultures at different stages of maturity were studied. In sparse cultures with migrating epithelial cells, large intracellular vacuoles were formed rapidly following exposure to live T. denticola. Treponemes showing structural damage were seen occasionally inside membrane-bound vesicles. Intensive membrane blebbing occurred in infected cells and continued for up to 48 h before the cell died. Blebbing could also be induced by a purified chymotrypsin-like proteinase of T. denticola. Cortical actin and alpha-actinin of the bacterium-treated cells showed disorganization, and pericellular fibronectin was degraded by both whole T. denticola and the isolated proteinase. Epithelial cells with well-formed lateral cell contacts appeared to be more resistant to the effects of T. denticola than migrating isolated cells. In multilayer epithelial cultures, adhesion of T. denticola and membrane blebbing were observed infrequently. There was no evidence of invasion of T. denticola into epithelial multilayers. However, immunogold electron microscopy showed rapid transport of T. denticola chymotrypsin-like proteinase into newly formed large intracellular vacuoles within the epithelial layers. These vacuoles were lined by membranes studded with ribosomes. T. denticola-treated epithelial multilayers had loose cell contacts, collapsed intercellular spaces, and increased permeability. Through its capacity to cause these unique cytopathic effects, the chymotrypsin-like proteinase of T. denticola has the potential to contribute to the initiation of periodontal disease.
Infection and Immunity - Tập 63 Số 9 - Trang 3401-3410 - 1995
Complementation of a <i>Treponema denticola flgE</i> Mutant with a Novel Coumermycin A1-Resistant <i>T. denticola</i> Shuttle Vector System ABSTRACT
By using the mutated
gyrB
gene from a spontaneous coumermycin A1-resistant
Treponema denticola
, an
Escherichia coli-T. denticola
shuttle vector that renders
T. denticola
resistant to coumermycin was constructed. The complete
T. denticola flgE
gene was cloned into the shuttle vector pKMCou, and the vector was transformed into the
T. denticola
ATCC 33520
flgE
erythromycin-resistant knockout mutant HL210. The coumermycin-resistant transformants were motile and restored FlgE activity. This complementation system should prove useful in studying the virulence factors of
T. denticola
and uncultivable pathogenic spirochetes.
Infection and Immunity - Tập 70 Số 4 - Trang 2233-2237 - 2002
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