The nine ptl genes (A^I) are required for efficient secretion of pertussis toxin past the outer membrane. Mutations were made in ptlA^H by filling in unique restriction sites, generating in-frame deletions, or inserting a FLAG epitope tag. The mutations were cloned into a suicide shuttle plasmid containing the ptxptl operon and introduced into the adenylate cyclase locus of the chromosome of a Bordetella pertussis strain deleted for ptx. The wild-type ptxptl operon restored pertussis toxin expression and secretion. The ptl mutant constructs also restored expression of periplasmic pertussis toxin to the ptx deletion strain but the mutants had a statistically significant decrease in secretion of pertussis toxin of between 5-to 35-fold, suggesting all of the ptl genes must be intact for efficient pertussis toxin secretion. The mutations were also introduced into the adenylate cyclase locus of a wild-type ptxptl strain, resulting in a ptl diploid strain. The PtlC, PtlD, PtlE, PtlF, PtlG and PtlH mutants exerted dominance over the wild-type allele. ß

Pertussis toxin is a member of the AB 5 family of toxins and is composed of five subunits (S1 to S5) present in a 1:1:1:2:1 ratio. Secretion is a complex process. Each subunit has a secretion signal that mediates transport to the periplasm, where processing and assembly occur. Secretion of the assembled 105-kDa toxin past the outer membrane is mediated by the nine proteins encoded in the ptl operon. Previous studies have shown that S1, the catalytically active A subunit of pertussis toxin, is necessary for efficient secretion, suggesting that a domain on S1 may be required for interaction with the secretion apparatus. Previously, recombinant S1 from four different mutants (serine 54 to glycine, serine 55 to glycine, serine 56 to glycine, and arginine 57 to lysine) was shown to retain catalytic activity. We introduced these mutations into Bordetella pertussis and monitored pertussis toxin production and secretion. No pertussis toxin was detected in the serine 54-to-glycine mutant. The other S1 mutants produced periplasmic pertussis toxin, but little pertussis toxin secretion was observed. The arginine 57-to-lysine mutant had the most dramatic secretion defect. It produced wild-type levels of periplasmic pertussis toxin but secreted only 8% as much toxin as the wild-type strain. This phenotype was similar to that observed for strains with mutations in the ptl genes, suggesting that this region may have a role in pertussis toxin secretion.Pertussis toxin is a major virulence factor of Bordetella pertussis, the gram-negative bacterium that is the causative agent of whooping cough. It is a member of the AB 5 family of toxins, consisting of five subunits, S1, S2, S3, S4, and S5, present in a 1:1:1:2:1 ratio (18,20,27). S1 is the A or enzymatic subunit and catalyzes the ADP-ribosylation of G proteins in the target mammalian cell. Subunits S2 to S5 form the B pentamer, which delivers the S1 subunit to the target mammalian cell. Several of the mammalian cells targeted by pertussis toxin (including lymphocytes, macrophages, and neutrophils) are important effectors of the immune system, and toxin treatment compromises their ability to function, contributing to the severity of the disease (22,23,29).Pertussis toxin assembly and secretion is a complex process. Each subunit is synthesized with a signal peptide (20), which mediates secretion to the periplasm via the equivalent of the Sec-mediated secretion machinery of Escherichia coli. Folding and assembly of the subunits occurs in the periplasm, and the ptl (pertussis toxin liberation) operon is required for efficient secretion of assembled toxin past the outer membrane (7, 9, 31). The ptl secretion machinery is specific for pertussis toxin, since secretion of other known virulence factors occurs normally in ptl mutant strains (31, 32). The secretion machinery appears to discriminate between assembled and unassembled pertussis toxin, since only assembled toxin is efficiently released from the bacteria. Since secretion involves substrate recognition, the Ptl secretion machine...

A classic proinflammatory T helper cell type 1 (TH1) response directed against intracellular pathogens includes the cytokine osteopontin, which acts predominantly on macrophages, where it induces the secretion of interleukin (IL)-12 and suppresses the secretion of IL-10. As cell-mediated immune responses play an important role in the resistance to Lyme arthritis, a manifestation of infection by the extracellular pathogen Borrelia burgdorferi, we tested the hypothesis that osteopontin may be required to induce T(H)1 responses and inflammation. The role of osteopontin was tested in vivo and using ex vivo macrophages in B6129F3 mice susceptible to experimental Lyme arthritis. Mice of this genetic background and those fully backcrossed to C57BL/6, which lacked osteopontin expression (spp1-/-), were as susceptible to B. burgdorferi-induced arthritis as littermate controls. Furthermore, equal numbers of spirochetes, as measured by quantitative polymerase chain reaction of the B. burgdorferi gene recA in spp1-/- and B6129F3 wild-type littermates, suggested that susceptibility to infection was not dependent on this cytokine. Neither of the B6129F3 parental mouse strains lacked the ability to secrete osteopontin. spp1-/- mice and controls had immunoglobulin G2 titers, suggestive of a TH1 response. B. burgdorferi was able to directly stimulate the secretion of the proinflammatory cytokines IL-12 and tumor necrosis factor alpha from wild-type and spp1-/- macrophages alike. These results indicate that the usually critical role of osteopontin in the induction of cellular immune responses to intracellular pathogens was circumvented by the ability of the extracellular pathogen B. burgdorferi to induce macrophages directly to produce proinflammatory cytokines.

Pertussis toxin accumulates in the periplasm of Bordetella pertussis prior to secretion, and we examined its fate following treatment with antimicrobial agents. Both antibiotics that inhibit protein synthesis (erythromycin and chloramphenicol), transcription (rifampin), or cell wall biosynthesis (cefoperazone and piperacillin) and magnesium sulfate (which inhibits transcription of pertussis toxin, but not bacterial growth) did not prevent release of preformed toxin. In contrast, agents that affect bacterial membranes, such as polymyxin B, lidocaine, procaine, and ethanol, inhibited release of preformed pertussis toxin. These results suggest new protein synthesis is not required for pertussis toxin secretion, but a functional membrane complex is required.Pertussis toxin is a member of the AB 5 family of toxins, which includes cholera toxin, Escherichia coli heat-labile toxin, and Shiga toxin. It is a critical virulence factor for the gramnegative bacterium Bordetella pertussis, the causative agent of whooping cough (21,22,32). It is also the most complex bacterial toxin known, comprised of six subunits, called S1, S2, S3, S4, and S5 in a 1:1:1:2:1 ratio (14,17,27). S1 is the A component of pertussis toxin, the part of the toxin that mediates damage to host cells. S1 enzymatically attaches the ADP-ribose group from NAD onto mammalian GTP binding proteins, abolishing normal cellular regulation. S2 to S5 associate to form the B component, or B pentamer, which binds to the host cell and delivers S1 into the cytoplasm of target cells.This complex structure of pertussis toxin seems to necessitate an equally complex pathway for assembly and secretion from the bacterium. Each of the toxin subunits is synthesized with an N-terminal signal sequence, which mediates secretion to the periplasm, where the signal peptide is removed and the subunits fold and assemble into holotoxin (17). Finally, secretion of the assembled toxin past the outer membrane is mediated by the proteins encoded by the ptl operon (PtlA through PtlI) (7, 33). Interestingly, AB 5 toxins have only been found in gram-negative bacteria. A possible explanation is that without the compartment provided by the periplasm of the gram-negative bacterium, the six toxin subunits would be unable to efficiently associate and assemble. Gram-positive bacteria lack this compartment and might release predominantly nonfunctional (and possibly immunizing) subunits.While the periplasm may be needed for efficient assembly of the AB 5 toxins, it can still act as a barrier to toxin secretion, and strains producing AB 5 toxins tend to accumulate intracellular pools of toxin. A dose as small as 0.05 to 0.15 ng of pertussis toxin per ml can elicit a positive response in the Chinese hamster ovary (CHO) cell assay (9, 33), and we have observed that B. pertussis accumulates hundreds of nanograms of periplasmic pertussis toxin, a potentially significant dose.We were interested in examining what happened to this pool of toxin during antibiotic treatment.In some situations, antibiotic tr...

Arthritogenicity, as determined by joint swelling and synovial histology, was compared between or within two Borrelia genospecies that cause Lyme arthritis in humans. The spirochete burden in bladder tissue (a site of spirochete persistence) was documented by quantitative polymerase chain reaction, and immune responses were analyzed. In C3H/HeJ mice, three B. burgdorferi isolates and two of the three B. garinii isolates induced severe arthritis and swelling. Previous designation as invasive or noninvasive B. garinii, or RNA spacer type of B. burgdorferi did not determine arthritis severity induced by isolates. Compared with the other five isolates, the B. garinii PBi isolate induced significantly less arthritis, a lower humoral immune response, and persisted at a much lower level in bladder tissue. However, B. garinii PBi isolates induced similar Borrelia antigen-specific inflammatory T cell responses from the local draining lymph node. Thus, diverse B. burgdorferi and B. garinii isolates were highly arthritogenic in immune competent mice.