View source for P.69 Pertactin Structure and Function

==Introduction==
Pertactin is a virulence toxin of ''Bordetella parapertussis'' and close relatives, such as ''Bordetella pertussis''. It is an outer surface membrane protein involved in the binding of ''B. parapertussis'' to host cells, which aids the bacteria in infection of host cells with whooping cough. Many of the conserved regions in this protein, such as its passenger and autotransporter domains, contribute directly to the overall virulence and pathogenicity of these organisms. 

Autotransporters make up the largest protein family in Gram-negative bacteria. They are usually comprised of a C-terminal beta-barrel-shaped transporter domain anchored in the outer membrane and an N-terminal passenger domain that crosses the outer membrane through the beta barrel (Figure 1a). The autotransporter is considered a virulence factor with the passenger domain contributing to the virulence of the pathogen. This N-terminal domain is similar in structure between different species, but the functions vary greatly. However, the C-terminal beta-barrel domain is a highly conserved structure for transport across the membrane but can vary greatly in the sequence. Many factors including biogenesis, use of accessory proteins, and fate of the beta-barrel translocator are not well known.

Seeing that the structure of the passenger domain is similar between various bacteria, it was found that about 97% of them contain an extended right handed beta-helical structure. Specifically, pertactin has a passenger domain with a 16-turn parallel β-helix with a V-shaped cross-section and a hydrophobic core. Each turn contains approximately 25 residues, which make up three beta strands that are linked by loops. It is predicted that the alpha-helical passenger domain traverses the hydrophilic pore of the transporter domain after the transporter domain is inserted into the outer membrane <ref name="BEN">Benz, I., & Schmidt, M. (n.d.). Structures and functions of autotransporter proteins in microbial pathogens. International Journal of Medical Microbiology, 461-468</ref>.

[[Image:autotransporter.jpg]]

'''Figure 1a'''. The Organization of an Autotransporter. The passenger domain and transporter domain are cleaved from the signal sequences and released into the periplasmic space where the C-terminal translocator forms in the outer membrane and transports the N-terminal through the pore. 


==2D image==

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==3D image==

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==Structure and Protein Adhesion Properties==

Pertactin is a single unit protein consisting of a 16-stranded parallel beta-helix with a v-shaped cross section.  There are two conserved domains within Pertactin, an auto transporter domain located at the N-terminus and a passenger domain located at the C-terminus <ref name= "CON">"Conserved Protein Domain Family." CDD: Conserved Domain Database. NCBI, n.d. Web. 12 Nov. 2015.</ref>.

Pathogens such as ''Bordetella pertussis'' and ''Bordetella parapertussis'' utilize virulence factors to adhere to target cells, which contribute to the overall pathogenicity of the organism. Pertactin shares homology with other proteins that are known to aid in cell-cell adhesion, emphasizing the importance of structure and how it relates to function. P.69 pertactin has been shown to adhere to mammalian cells, and has several features that contribute to this functionality <ref name="EMS">Emsley, P., Charles, I., Fairweather, N., & Isaacs, N. (1996). Structure of ''Bordetella pertussis'' virulence factor P.69 pertactin. Nature, 90-92.</ref>.

One of these features is the <scene name='71/716564/Arg_gly_asp/1'>Arg-Gly-Asp (RGD) tripeptide motif</scene> that allows for protein-protein interactions <ref name="EMS" />. This motif has been found in several proteins, and has been shown to support cell adhesion in most cases. A subset of cell-surface proteins, called integrins, act as receptors for cell adhesion molecules. These integrins recognize the RGD motif within their ligands, and allow for cell-substratum and cell-cell interactions <ref>D'Souza, S. E., Ginsberg, M. H., & Plow, E. F. (1991). Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends In Biochemical Sciences, 16(7), 246-250.</ref>.

Additionally, P.69 contains two <scene name='71/716564/Proline/3'>proline-rich regions</scene> which are thought to provide important binding sites, and are characteristic of proteins exhibiting binding capabilities <ref name="EMS" />. Proline is a very unusual amino acid, and its structure limits the possible conformations it can adopt. The rigidity of these structures allow for reliable binding sites in many different proteins especially when chains of proline are bound to each other. These regions are typically non-specific, and allow for rapid binding. This is advantageous due to the wide range of ligands that can be bound, increasing the versatility of the proteins that utilize these regions <ref>Williamson, M. P. (1994). The structure and function of proline-rich regions in proteins. Biochemical Journal, 297(Pt 2), 249–260.</ref>.

The linear form of pertactin that protrudes from the surface of ''B. pertussis'' also has a high surface area that could be well suited for targeting mammalian cells <ref name="EMS" />. Studies have shown that adhesive area strongly affects integrin binding and adhesion strength. The positioning of binding regions also affects adhesion strength, making the combination of these two factors particularly important for proteins that serve this function <ref>Gallant, N. D., Michael, K. E., & García, A. J. (2005). Cell Adhesion Strengthening: Contributions of Adhesive Area, Integrin Binding, and Focal Adhesion Assembly. Molecular Biology of the Cell, 16(9), 4329–4340.</ref>.
== Function==

The closely related organisms ''B. pertussis, B. parapertussis, and B. bronchiseptica'' all produce slightly different forms of pertactin, which differ in the passenger domain (P.69, P.70, P.68 respectively). Specifically, the difference in size is due to the number of internal  repeats of (GGXXP) in a proline rich region within the passenger domain. These different forms of pertactin all share two RGD motifs that are thought to be relevant to cell binding. One is found in the passenger domain and one in the transporter domain. There is further evidence that pertactin functions in adhesion, however, the data is contradictory. ''In vitro'' adhesion assays show that pertactin allows for adhesion to general cell lines such as CHO and HeLa cells, however, no evidence for was found to convey that pertactin aids in adhesion of bacterial cells to either bronchial or laryngeal cells. Furthermore, no receptor has been identified for pertactin <ref name= "HEN">Henderson, I., & Nataro, J. (2001). Virulence Functions of Autotransporter Proteins. Infection and Immunity, 1231-1243.</ref>. This data supports the conflict of whether or not the function of pertactin is adhesion to host cells. 

In ''B. bronchiseptica'', pertactin seemed to be involved in the cytotoxicity of mononuclear phagocytic cells. A possible explanation is that pertactin promoted stable adhesion of the bacterium to the phagocyte. Researchers also found that pertactin is produced ''in vitro'' at an intermediate time in ''B. pertussis'' growth: after another virulence factor, FHA, but before pertussis toxin. This occurrence supports the idea that pertactin is involved in a closer adhesion to mammalian cells but prior to toxin release <ref name="HEN" />. If pertactin truly is an adhesion molecule, it would be a huge contribution to the overall pathogenesis of the species. 

Regardless if pertactin functions as an adhesion molecule, it is very biologically relevant. Pertactin would make for a good vaccine candidate. Manipulation of the passenger domain can be performed so that a gene for a certain protein from a different species can be inserted in the passenger domain thus replacing the wild type passenger domain <ref name="BEN" />. This would make for a very good vaccine candidate. Not only would the translocator of ''B. pertussis'' can be recognized as an antigen by the host as well as the heterologous protein inserted in the passenger domain. This could be the basis for a heterologous vaccine (combating two species).

The last step of secretion in autotransporters is C→ N terminal threading of the passenger domain through the outer membrane-spanning portion of the protein. After this step, the original structure of pertactin is formed. Interestingly, this translocation process does not depend on the consumption of ATP nor the presence of a proton gradient <ref>Junker, M., Schuster, C. C., McDonnell, A. V., Sorg, K. A., Finn, M. C., Berger, B., & Clark, P. L. (2006). Pertactin β-helix folding mechanism suggests common themes for the secretion and folding of autotransporter proteins. Proceedings of the National Academy of Sciences of the United States of America, 103(13), 4918–4923.</ref>.

== Relevance==

P.69 has recently been shown to be an agglutinogen, an antigen that produces agglutinin which causes particles to coagulate <ref>Charles, I. G., Dougan, G., Pickard, D., Chatfield, S., Smith, M., Novotny, P., … Fairweather, N. F. (1989). Molecular cloning and characterization of protective outer membrane protein P.69 from ''Bordetella pertussis''. Proceedings of the National Academy of Sciences of the United States of America, 86(10), 3554–3558.</ref>. Due to agglutinogen properties as well as the ability to kill ''B. pertussis'', P.69 has the potential for use in an acellular vaccine as an antigen for whooping cough <ref>Gotto, J. W., Eckhardt, T., Reilly, P. A., Scott, J. V., Cowell, J. L., Metcalf, T. N., … Siegel, M. (1993). Biochemical and immunological properties of two forms of pertactin, the 69,000-molecular-weight outer membrane protein of ''Bordetella pertussis''. Infection and Immunity, 61(5), 2211–2215.</ref>. Specifically, region 1 of pertactin has been found to be responsible for immunity properties due to its polymorphic attributes <ref>King A, Berbers G, van Oirschot H, Hoogerhout P, Knipping K, Mooi F (2001). Microbiology 147(11):2885-2895 doi:10.1099/00221287-147-11-2885.
</ref>.

==Pertactin vs Pertussis Toxin: Virulence Factors==

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Pertactin and {{:Pertussis_Toxin_ATP-Complex}} are both virulence factors that contribute to respiratory tract infection and whooping cough. Both are responsible for binding the foreign bacterial cell to the host organism’s cells. Despite similar functions, Pertactin and Pertussis toxin have very different structures. Pertussis toxin is a virulence factor only produced by ''Bordetella pertussis''. It is known to cause systemic symptoms of pertussis disease, such as leukocytosis and histamine sensitivity. It has also, recently, been discovered to promote respiratory infection by inhibiting and modulating host cell immune responses. Pertussis toxin promotes infection by acting as a soluble factor, which attacks resident cells of the trachea and lungs, such as macrophages<ref name= "Car">Carbonetti, Nicholas H. “Pertussis Toxin and Adenylate Cyclase Toxin: Key Virulence Factors of Bordetella Pertussis and Cell Biology Tools.” Future microbiology 5 (2010): 455–469. PMC. Web. 17 Nov. 2015.</ref>. Pertactin is also a virulence factor known to contribute to whooping cough. Pertactin is found in ''Bordetella pampertussis'', and a 91.3% homologous protein is found in ''Bordetella pertussis'', the species which produces Pertussis toxin. An amino acid alignment between the two strains reveals that the proteins differ in the number of repeated sequences. ''B. pampertussis'' has a series of approximately twenty more amino acids beginning at the 580th position ([[Media:Prn6.png| Figure 6]]). Similarly to Pertussis toxin, Pertactin is involved in binding the bacterial cell to respiratory host cells, however, less is known about the specific cells Pertactin binds to in order to promote infection.<ref name="CON" />

Despite similar functions, Pertussis toxin and Pertactin have very different structures.  As mentioned previously, Pertactin’s structure consists of a 16-stranded parallel beta-helix with a v-shaped cross section, while Pertussis toxin consists of five subunits.<ref name="CON" />BLAST results of Pertactin revealed that two domains, autotransporter and PL1_Passenger_AT, are conserved. The autotransporter domain is located at the C-terminus of Pertactin and functions to transport the passenger domain, located at the N-terminus of Pertactin, in to the host cell, where the two domains are typically cleaved.<ref name="CON" /> This process is responsible for the bacterial cell binding to the host cell.  While Pertactin is a single polypeptide chain, Pertussis toxin consists of five different subunits: S1 makes up subunit A and subunit B is a pentameric ring made of S2, S3, two S4 and S5. BLAST results revealed that a single domain is contained within subunits 1, 4 and 5: Pertussis_S1 superfamily, Pertussis_S4 superfamily and Pertussis_S5 superfamily. Subunits 2 and 3 contain an ATP superfamily and Pertussis_S2S3 Superfamily, which represent the N-terminal domain of aerolysin and pertussis toxin and the C-terminal domain, respectively.  The individual structures may be responsible for Pertussis toxin’s ability to recognize receptors on numerous cell types<ref name="Car" />.

As a whole, Pertactin and Pertussis toxin are structurally very different. However, amino acid alignments between Pertactin and each of the five subunits of Pertussis toxin reveals that various sections of Pertactin are the same or highly similar to individual subunits of Pertussis toxin ([[Media:Prn1.png | Figure 1]], [[Media:Prn2.png | Figure 2]], [[Media:Prn3.png | Figure 3]], [[Media:Prn4.png | Figure 4]], [[Media:Prn5.png | Figure 5]]). BLAST results also revealed that subunits 4 and 5’s structures consist of an OB fold and a closed or partly open beta barrel.<ref name="CON" /> Similar amino acid sequences, as well as similarities between Pertactin’s beta barrel structure and the beta barrel structures of subunits 4 and 5 may be how Pertactin and Pertussis toxin have similar functions.

Supplemental Information:
#REDIRECT [[1dab]]

== References ==
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