Antibacterial Agents and Release of Periplasmic Pertussis Toxin from
            <i>Bordetella pertussis</i>

Craig-Mylius, Kathleen A.; Weiss, Alison A.

doi:10.1128/aac.44.5.1383-1386.2000

Cited by 10 publications

(10 citation statements)

References 34 publications

(33 reference statements)

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“…B. pertussis strains containing a plasmid encoding GFP introduced by electroporation (22) were used, except where indicated. B. pertussis strains were grown on Bordet Gengou (BG) agar (BD Biosciences, Sparks, MD) or in Stainer-Scholte (SS) broth overlaid on BG agar (SS-BG), as described previously (30). All other strains were grown on Luria-Bertani agar plates.…”

Section: Bacterial Strains and Growth Conditionsmentioning

confidence: 99%

Bordetella pertussis Lipopolysaccharide Resists the Bactericidal Effects of Pulmonary Surfactant Protein A

Schaeffer¹,

McCormack

et al. 2004

The Journal of Immunology

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Surfactant protein A (SP-A) plays an important role in the innate immune defense of the respiratory tract. SP-A binds to lipid A of bacterial LPS, induces aggregation, destabilizes bacterial membranes, and promotes phagocytosis by neutrophils and macrophages. In this study, SP-A interaction with wild-type and mutant LPS of Bordetella pertussis, the causative agent of whooping cough, was examined. B. pertussis LPS has a branched core structure with a nonrepeating trisaccharide, rather than a long-chain repeating O-Ag. SP-A did not bind, aggregate, nor permeabilize wild-type B. pertussis. LPS mutants lacking even one of the sugars in the terminal trisaccharide were bound and aggregated by SP-A. SP-A enhanced phagocytosis by human monocytes of LPS mutants that were able to bind SP-A, but not wild-type bacteria. SP-A enhanced phagocytosis by human neutrophils of LPS-mutant strains, but only in the absence of functional adenylate cyclase toxin, a B. pertussis toxin that has been shown to depress neutrophil activity. We conclude that the LPS of wild-type B. pertussis shields the bacteria from SP-A-mediated clearance, possibly by sterically limiting access to the lipid A region.

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Section: Bacterial Strains and Growth Conditionsmentioning

confidence: 99%

Bordetella pertussis Lipopolysaccharide Resists the Bactericidal Effects of Pulmonary Surfactant Protein A

Schaeffer¹,

McCormack

et al. 2004

The Journal of Immunology

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“…To assay de novo production of pertussis toxin, strains were passed twice on BGA containing 40 mM MgSO 4 to modulate the bacteria and turn off transcription of the pertussis toxin operon (21), and bacteria from 24-h cultures were suspended in Stainer-Scholte broth to an optical density at 600 nm (OD 600 ) of 0.1. Six milliliters was plated on BGA plates containing appropriate antibiotics and incubated at 37°C for 30 h. The amounts of secreted and periplasmic pertussis toxin were determined as previously described (10)(11)(12). Briefly, the bacteria were harvested and pelleted by centrifugation, and the supernatants were filter sterilized for determination of secreted toxin.…”

Section: Methodsmentioning

confidence: 99%

“…For secretion assays, B. pertussis was grown in a thin layer of Stainer-Scholte broth on nutrient-rich BGA plates (10)(11)(12). Growth on this biphasic medium is reproducible and vigorous due to the large surface/volume ratio of the culture and the rich nutrient base of the semisolid agar.…”

Section: Methodsmentioning

confidence: 99%

DsbA and DsbC Are Required for Secretion of Pertussis Toxin by Bordetella pertussis

Stenson

Weiss

2002

Infect Immun

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The Dsb family of enzymes catalyzes disulfide bond formation in the gram-negative periplasm, which is required for folding and assembly of many secreted proteins. Pertussis toxin is arguably the most complex toxin known: it is assembled from six subunits encoded by five genes (for subunits S1 to S5), with 11 intramolecular disulfide bonds. To examine the role of the Dsb enzymes in assembly and secretion of pertussis toxin, we identified and mutated the Bordetella pertussis dsbA, dsbB, and dsbC homologues. Mutations in dsbA or dsbB resulted in decreased levels of S1 (the A subunit) and S2 (a B-subunit protein), demonstrating that DsbA and DsbB are required for toxin assembly. Mutations in dsbC did not impair assembly of periplasmic toxin but resulted in decreased toxin secretion, suggesting a defect in the formation of the Ptl secretion complex.Pertussis toxin is a major virulence factor of the gram-negative bacterium Bordetella pertussis, which is the causative agent of whooping cough (34,43). The toxin is a member of the AB 5 family of toxins, which includes Shiga toxin, cholera toxin, and Escherichia coli heat-labile toxin. It plays a crucial role in virulence by mediating ADP-ribosylation of host GTP-binding proteins (G i , G o , and G t ), thereby disrupting normal host cellular regulation (19,26). The systemic effects on the infected host include blocking of antimicrobial activity in a number of immune effector cells, resulting in a less effective immune response by the host (20, 42).Five structural toxin genes (S1 to S5) and the nine ptl (for pertussis toxin liberation) genes (ptlA to ptlI), encoding the secretion complex, are cotranscribed from a single operon (28, 45) that is positively regulated by the Bvg two-component regulatory system (22). S1 is the enzymatic A subunit of the toxin, while the B subunit or B pentamer binds mammalian cells and delivers the toxin into the mammalian cytoplasm (25,40). Pertussis toxin is a somewhat atypical AB 5 toxin. The B pentamer is not a homo-oligomer but rather consists of subunits S2, S3, S4, and S5, in a 1:1:2:1 ratio (30, 31, 39), which associate with the C terminus (2, 49) of the S1, or A, subunit. Each toxin subunit is translated with its own signal sequence (30, 31), and the subunits are secreted to the periplasm presumably via the Bordetella equivalent of the E. coli Sec machinery. Once in the periplasm, the subunits are assembled into the 105-kDa holotoxin that is recognized by the Ptl type IV secretion complex (10,15,35), which then moves the holotoxin through the outer membrane into the extracellular milieu (12,14,45). Additionally, it has been demonstrated that only the holotoxin is targeted for secretion, as expression of B subunit (S2 to S5) or A subunit (S1) in isolation did not result in extracellular secretion of toxin subunits (15). A region of the S1 subunit which might act as a recognition domain for this interaction of holotoxin with the Ptl secretion apparatus has been described (10).Interestingly, AB 5 toxins have been described only for gramn...

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“…Plasmids and bacterial strains used in this study are described in Table 1. B. pertussis strains expressing green fluorescent protein (GFP) were grown in Stainer Scholte (SS) broth on Bordet Gengou (BG) agar (Difco, Detroit, Mich.) plates for 24 h as described previously (11). Briefly, strains were suspended in 7 ml of SS broth containing the appropriate selective antibiotics to an optical density at 600 nm (OD 600 ) of approximately 0.1, overlaid on BG agar plates containing 15% defibrinated sheep's blood (Colorado Serum Company, Denver, Colo.) and appropriate antibiotics, and incubated at 37°C for 24 h.…”

Section: Methodsmentioning

confidence: 99%

Pertussis Toxin and Lipopolysaccharide Influence Phagocytosis ofBordetella pertussisby Human Monocytes

Schaeffer

Weiss

2001

Infect Immun

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The potential of human monocytes to mediate the clearance of Bordetella pertussis infection was examined. Bacteria expressing green fluorescent protein were incubated with adherent peripheral blood monocytes, and phagocytosis was quantified by using fluorescence microscopy. Monocytes internalized only a small percentage of the adherent bacteria. Surface-associated Bvg-regulated virulence factors, including adenylate cyclase toxin and filamentous hemagglutinin, did not affect attachment or phagocytosis. However, 1-h pretreatment with purified pertussis toxin inhibited the ability of monocytes to internalize wild-type bacteria. Mutations affecting the terminal trisaccharide of lipopolysaccharide resulted in reduced internalization without affecting adherence of bacteria to monocytes. Opsonization with human serum played only a modest role in promoting phagocytosis. The viability of internalized bacteria was determined by colony counts following treatment with polymyxin B and gentamicin. Less than 1% of internalized bacteria remained viable. These results suggest that pertussis toxin plays a role in the evasion of monocyte phagocytosis and that these cells represent a potential mediator of the clearance of B. pertussis infection.

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Antibacterial Agents and Release of Periplasmic Pertussis Toxin from Bordetella pertussis

Cited by 10 publications

References 34 publications

Bordetella pertussis Lipopolysaccharide Resists the Bactericidal Effects of Pulmonary Surfactant Protein A

Bordetella pertussis Lipopolysaccharide Resists the Bactericidal Effects of Pulmonary Surfactant Protein A

DsbA and DsbC Are Required for Secretion of Pertussis Toxin by Bordetella pertussis

Pertussis Toxin and Lipopolysaccharide Influence Phagocytosis ofBordetella pertussisby Human Monocytes

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