Stabilization of the Pertussis Toxin Secretion Apparatus by the C Terminus of PtlD

ABSTRACT Pertussis toxin (PT) is secreted from Bordetella pertussis by a type IV secretion system, known as the Ptl transporter, that comprises nine different proteins, PtlA to PtlI. In this study, we found that PtlD is required for the stability of three Ptl proteins, PtlE, PtlF, and PtlH. A region limited to the C-terminal 72 amino acids of PtlD (amino acids 392 to 463) was sufficient for maintaining the stability of PtlE, PtlF, and PtlH, although this region was not sufficient to support secretion of the toxin. Further analysis demonstrated that a stretch of 10 amino acids at the C-terminal end of PtlD (amino acids 425 to 434) contributes to transporter stability.

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Analysis of Relative Levels of Production of Pertussis Toxin Subunits and Ptl Proteins in Bordetella pertussis

Infection and Immunity ◽

10.1128/iai.72.4.2057-2066.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2057-2066 ◽

Cited By ~ 5

Author(s):

Anissa M. Cheung ◽

Karen M. Farizo ◽

Drusilla L. Burns

Keyword(s):

Alkaline Phosphatase ◽

Pertussis Toxin ◽

Alkaline Phosphatase Activity ◽

Bordetella Pertussis ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Type Iv ◽

Multiple Copies ◽

Relationship Of

ABSTRACT Pertussis toxin is transported across the outer membrane of Bordetella pertussis by the type IV secretion system known as the Ptl transporter, which is composed of nine different proteins. In order to determine the relative levels of production of pertussis toxin subunits and Ptl proteins in B. pertussis, we constructed translational fusions of the gene for alkaline phosphatase, phoA, with various ptx and ptl genes. Comparison of the alkaline phosphatase activity of strains containing ptx′- or ptl′-phoA fusions indicated that pertussis toxin subunits are produced at higher levels than Ptl proteins, which are encoded by genes located toward the 3′ end of the ptx-ptl operon. We also engineered strains of B. pertussis by introducing multiple copies of the ptl genes or subsets of these genes and then examined the ability of each of these strains to secrete pertussis toxin. From these studies, we determined that certain Ptl proteins appear to be limiting in the secretion of pertussis toxin from the bacteria. These results represent an important first step in assessing the stoichiometric relationship of pertussis toxin and its transporter within the bacterial cell.

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Agrobacterium-Mediated T-DNA Transfer and Integration by Minimal VirD2 Consisting of the Relaxase Domain and a Type IV Secretion System Translocation Signal

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-11-1356 ◽

2009 ◽

Vol 22 (11) ◽

pp. 1356-1365 ◽

Cited By ~ 28

Author(s):

Maartje van Kregten ◽

Beatrice I. Lindhout ◽

Paul J. J. Hooykaas ◽

Bert J. van der Zaal

Keyword(s):

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Terminal Part ◽

Type Iv ◽

C Terminus ◽

Localization Signal ◽

Translocation Domain ◽

First Time

The VirD2 protein of Agrobacterium tumefaciens is essential for processing and transport of the T-DNA. It has at least three functional domains: a relaxase domain at the N terminus, a bipartite nuclear localization signal (NLS), and a sequence called ω at the C terminus. We confirm here that deletions of the C-terminal part of VirD2 led to lack of transfer of T-DNA but, for the first time, we report that virulence is restored when these truncations are supplemented at the C terminus by a short translocation signal from the VirF protein. The lack of virulence of C-terminal deletions suggests that the C-terminal part contains all or part of the translocation signal of VirD2. Using a novel series of mutant VirD2 proteins, the C-terminal half of VirD2 was further investigated. We demonstrate that the C-terminal 40 amino acids of VirD2, which include the NLS and ω, contain all or part of the translocation domain necessary for transport of VirD2 into plant cells, while another element is present in the middle of the protein. The finding that a type IV secretion system transport signal at the C terminus of VirD2 is necessary for virulence provides evidence for the role of VirD2 as a pilot protein driving translocation of the T-strand.

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The PtlE Protein of Bordetella pertussis Has Peptidoglycanase Activity Required for Ptl-Mediated Pertussis Toxin Secretion

Journal of Bacteriology ◽

10.1128/jb.184.11.2863-2869.2002 ◽

2002 ◽

Vol 184 (11) ◽

pp. 2863-2869 ◽

Cited By ~ 27

Author(s):

Amy A. Rambow-Larsen ◽

Alison A. Weiss

Keyword(s):

Fusion Protein ◽

Pertussis Toxin ◽

Bordetella Pertussis ◽

Active Site ◽

Type Iv Secretion ◽

Active Site Residues ◽

Outer Membranes ◽

Type Iv ◽

Toxin Secretion ◽

Peptidoglycan Layer

ABSTRACT Pertussis toxin of Bordetella pertussis is secreted by a type IV secretion system comprised of the products of the nine ptl (pertussis toxin liberation) genes. These proteins are believed to form a complex spanning both the inner and outer membranes and passing through the peptidoglycan layer. Peptidoglycan acts as a barrier for transport through the periplasm of large folded molecules. Assembled pertussis toxin and the secretion component proteins PtlC through PtlH are too large to diffuse through intact peptidoglycan. Therefore, we hypothesized that the Ptl system contains a peptidoglycanase activity. The PtlE protein was found to exhibit a sequence match to the active site of glycohydrolase enzymes. An N-terminally polyhistidine-tagged PtlE fusion protein, constructed and expressed in Escherichia coli and in B. pertussis, exhibited peptidoglycanase activity on activity gels. A fusion protein with alanine substitutions at the putative active site residues (aspartic acid at position 53 and glutamic acid at position 62) lacked peptidoglycanase activity. B. pertussis strains with the amino acid substitutions were deficient for pertussis toxin secretion. Based on these results, we concluded that PtlE is a peptidoglycanase responsible for the local removal or rearrangement of the peptidoglycan layer during Ptl secretion complex assembly.

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