Secretion of Pertussis Toxin from Bordetella pertussis

Production and secretion of pertussis toxin (PT) is essential for the virulence of Bordetella pertussis. Due to the large oligomeric structure of PT, transport of the toxin across bacterial membrane barriers represents a significant hurdle that the bacteria must overcome in order to maintain pathogenicity. During the secretion process, PT undergoes a two-step transport process. The first step involves transport of the individual polypeptide chains of PT across the inner membrane utilizing a generalized secretion pathway, most likely the bacterial Sec system. The second step involves the use of a specialized apparatus to transport the toxin across the outer membrane of the bacterial cell. This apparatus, which has been termed the Ptl transporter and which is unique to the PT secretion pathway, is a member of the type IV family of bacterial transporters. Here, the current understanding of the PT secretion process is reviewed including a description of the Ptl proteins that assemble to form the transporter, the general structure of type IV transporters, the known similarities and differences between canonical type IV substrate transport and Ptl-mediated transport of PT, as well as the known sequence of events in the assembly and secretion of PT.

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Importance of Holotoxin Assembly in Ptl-Mediated Secretion of Pertussis Toxin from Bordetella pertussis

Infection and Immunity ◽

10.1128/iai.68.7.4049-4054.2000 ◽

2000 ◽

Vol 68 (7) ◽

pp. 4049-4054 ◽

Cited By ~ 39

Author(s):

Karen M. Farizo ◽

Theresa Huang ◽

Drusilla L. Burns

Keyword(s):

Pertussis Toxin ◽

Bordetella Pertussis ◽

Structural Components ◽

Type Iv ◽

System A

ABSTRACT We examined the structural components of pertussis toxin that are required for efficient export from Bordetella pertussis via the Ptl system, a member of the type IV family of macromolecular transporters. First, we constructed a strain of B. pertussis that contains a functional Ptl system but does not produce pertussis toxin. Plasmids which express either the S1 subunit or the B oligomer were then introduced into this strain. We found that the B oligomer of the toxin is not secreted in the absence of the S1 subunit. Conversely, the S1 subunit is also not secreted by a Ptl-mediated mechanism in the absence of the B oligomer. Thus, an assembled holotoxin is required for Ptl-mediated export of pertussis toxin from B. pertussis.

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

Journal of Bacteriology ◽

10.1128/jb.01106-08 ◽

2008 ◽

Vol 190 (21) ◽

pp. 7285-7290 ◽

Cited By ~ 7

Author(s):

Anita Verma ◽

Anissa M. Cheung ◽

Drusilla L. Burns

Keyword(s):

Amino Acids ◽

Pertussis Toxin ◽

Bordetella Pertussis ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Type Iv ◽

C Terminus ◽

Toxin Secretion ◽

The Stability

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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Temporal Expression of Pertussis Toxin and Ptl Secretion Proteins by Bordetella pertussis

Journal of Bacteriology ◽

10.1128/jb.186.1.43-50.2004 ◽

2004 ◽

Vol 186 (1) ◽

pp. 43-50 ◽

Cited By ~ 16

Author(s):

Amy A. Rambow-Larsen ◽

Alison A. Weiss

Keyword(s):

Pertussis Toxin ◽

Time Course ◽

Growth Cycle ◽

Lag Phase ◽

Logarithmic Growth Phase ◽

Type Iv ◽

Constant Rate ◽

Toxin Secretion ◽

The Individual ◽

Logarithmic Growth

ABSTRACT Pertussis toxin is an AB5 toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting.

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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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Requirements for Assembly of PtlH with the Pertussis Toxin Transporter Apparatus of Bordetella pertussis

Infection and Immunity ◽

10.1128/iai.00008-07 ◽

2007 ◽

Vol 75 (5) ◽

pp. 2297-2306 ◽

Cited By ~ 14

Author(s):

Anita Verma ◽

Drusilla L. Burns

Keyword(s):

Pertussis Toxin ◽

Bordetella Pertussis ◽

Wild Type ◽

Inner Membrane ◽

Type Iv ◽

Pt Complex ◽

Specific Subset ◽

Tight Association ◽

Altered Form ◽

Membrane Spanning

ABSTRACT PtlH is an essential component of the Ptl system, the type IV transporter responsible for secretion of pertussis toxin (PT) across the outer membrane of Bordetella pertussis. The nine Ptl proteins are believed to interact to form a membrane-spanning apparatus through which the toxin is secreted. In this study, we monitored the subcellular localization of PtlH in strains of B. pertussis lacking PT, lacking other Ptl proteins, or from which ATP has been depleted in order to gain insight into the requirements for assembly of PtlH with the remainder of the Ptl transporter complex that is thought to be tightly embedded in the membrane. We found that PtlH is exclusively localized to the inner membrane fraction of the cell in a wild-type strain of B. pertussis. In contrast, PtlH localized to both the cytoplasmic and inner membrane fractions of a mutant strain of B. pertussis that does not produce PT. In comparison to how it localized in wild-type strains of B. pertussis, PtlH exhibited aberrant localization in strains lacking PtlD, PtlE, PtlF, and PtlG. We also found that localization of PtlH was perturbed in B. pertussis strains that were treated with carbonyl cyanide m-chlorophenylhydrazone and sodium arsenate, which are capable of depleting cellular ATP levels, and in strains of B. pertussis that produce an altered form of PtlH that lacks ATPase activity. When taken together, these results indicate that tight association of PtlH with the membrane, likely through interactions with components of the transporter-PT complex, requires the toxin substrate, a specific subset of the Ptl proteins, and ATP. Based on these data, a model for the assembly of the Ptl transporter-PT complex is presented.

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Faculty Opinions recommendation of Definition of a bacterial type IV secretion pathway for a DNA substrate.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019206.216470 ◽

2004 ◽

Author(s):

Tracy Palmer

Keyword(s):

Type Iv Secretion ◽

Secretion Pathway ◽

Type Iv ◽

Definition Of ◽

Dna Substrate ◽

Bacterial Type

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Gender Determination by Linear Dimension of Permanent Canine: An Odontometric Analysis

Janaki Medical College Journal of Medical Science ◽

10.3126/jmcjms.v2i1.11392 ◽

2014 ◽

Vol 2 (1) ◽

pp. 23-27

Author(s):

B Sharma ◽

N Balaji ◽

MK Sumathi

Keyword(s):

Sexual Dimorphism ◽

Sex Determination ◽

Linear Dimension ◽

Medical Sciences ◽

Medical College ◽

Type Iv ◽

Racial Background ◽

Significant Difference ◽

Natural Change ◽

The Individual

Background and objectives: Identification, an aspect of forensic anthropology, is the recognition of an individual based on the physical characteristics unique to the individual. Among the four main attributes i.e. gender, age, stature and ethnic or racial background of an individual’s biological identity, sex determination is usually the first step in the human identification process. Teeth can be used as a means of sex determination as teeth are resistant to post-mortem degradation and survive deliberate, accidental or natural change. This study was carried out with an objective to determine the sexual dimorphism of maxillary and mandibular canine by linear tooth diameter for permanent dentition in Moradabad population. Material and Methods: A total number of 40 subjects (20 Males and 20 Females) were included in this study. After obtaining an informed written consent, alginate impression was taken with help of perforated impression trays and study models were prepared with type IV dental stone. Linear (MD, BL, Crown Height) were taken with digital vernier caliper. Results: It was observed that males’ shows more mean linear crown diameter as compared to females. Also, the mesiodistal and buccolingual measurement shows statistically significant difference for all canines, being higher for males than females. Conclusion: The present study has expressed sexual dimorphism of permanent canine using Student’s test and indicate that linear dimension of maxillary canine can be used for sexual diamorphism with accuracy along with other accepted procedure for sex determination. DOI: http://dx.doi.org/10.3126/jmcjms.v2i1.11392 Janaki Medical College Journal of Medical Sciences (2014) Vol. 2 (1): 23-27

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