Mutations in the S1 Subunit of Pertussis Toxin That Affect Secretion

Abstract

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...

Temporal Expression of Pertussis Toxin and Ptl Secretion Proteins byBordetella pertussis

“…The C-terminal portion of S1 has been proposed to mediate association with the membrane (12); however, the C-terminal portion of S1 is required for association with the B pentamer (20), and assembly into holotoxin would promote disassociation of S1 from the membrane. In previous studies workers have identified a domain on S1 (amino acids 55 to 57), distal from the C terminus, which is required for secretion of pertussis toxin (9). This region is thought to mediate recognition of pertussis toxin by the Ptl secretion complex; however, it is currently not known how the Ptl secretion system can discriminate among the secretion substrate, assembled pertussis toxin, and unassembled subunits.…”

Bordetella pertussis risA , but Not risS , Is Required for Maximal Expression of Bvg-Repressed Genes

“…A second homologous recombination resulting in the excision of pSS2141 DNA was selected for by plating transconjugates on media containing streptomycin. Isolates were screened for the ⌬risA mutant by PCR using the risAfor and risArev primer pair and template prepared from bacterial colonies (13). The in-frame ⌬risA mutant was confirmed in one clone, BP⌬OR, which was selected for further analysis.…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed as previously described (13). Proteins were resolved using 15% (37.5:1) acrylamide/bis-acrylamide separating gels.…”

Reduced Glutathione Is Required for Pertussis Toxin Secretion by Bordetella pertussis

“…S1 modifies mammalian G-proteins, which play a critical role in cellular signaling, and disruption of signaling by these proteins impairs development of the immune response to B. pertussis. Unlike the other AB 5 toxins where the five Bsubunits are identical, pertussis toxin has four different subunits (encoded by the ptxS2 to -S5 genes) in the B-pentamer.…”

“…Each of the five subunits possesses a secretion signal peptide, which directs secretion to the periplasm (18,20). In the periplasm, the signal peptide is removed and the subunits fold and assemble (5,9,10,21,26). Assembled pertussis toxin is released from the periplasm by the Ptl secretion complex, a member of the type IV secretion systems (7,8,10,21,29).…”