Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli - PubMed (original) (raw)
Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli
C Eslava et al. Infect Immun. 1998 Jul.
Abstract
Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a beta-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenic E. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects.
Figures
FIG. 1
Map of the cloned insert of pJPN201. The fimbrial subunit of the AAF/II antigen is encoded on the far right end of the fragment (17). At the left end of the insert is the pet gene, followed in opposite orientation by the astA gene embedded within an IS-like element and then by four other IS-homologous sequences (see text). Subclones used for sequencing and expression of pet are indicated. Restriction sites introduced by PCR are indicated by asterisks.
FIG. 2
Alignment of the predicted Pet protein with its closest homologs, EspP (accession no. X97542), EspC (U69128), and SepA (Z48219). Shaded residues represent identity. Coordinates are shown for Pet only. The asterisk at residue 53 indicates the first amino acid of the mature Pet protein. The arrowhead at residue 1018 indicates the position of cleavage of the β domain. The serine protease motif is in boldface.
FIG. 2
Alignment of the predicted Pet protein with its closest homologs, EspP (accession no. X97542), EspC (U69128), and SepA (Z48219). Shaded residues represent identity. Coordinates are shown for Pet only. The asterisk at residue 53 indicates the first amino acid of the mature Pet protein. The arrowhead at residue 1018 indicates the position of cleavage of the β domain. The serine protease motif is in boldface.
FIG. 3
(a) SDS-PAGE analysis of clone pCEFN1, encoding the complete pet gene. Lanes: A, Triton X-100-insoluble fraction of HB101(pCEFN1); B, Triton X-100-insoluble fraction of HB101 harboring the cloning vector pSPORT1; C, supernatant of HB101(pCEFN1); D, supernatant of HB101(pSPORT1). The arrow at 104 kDa represents the Pet passenger domain (the mature form of the protein); the arrow at ca. 30 kDa represents the β domain inserted into the bacterial outer membrane. (b) Western immunoblot of bacterial supernatants reacted with anti-Pet antiserum. Lanes: A, periplasmic fraction of HB101(pCEFN1); B, periplasmic fraction of HB101(pJPN205) harboring the C-terminal deletion mutant of the pet gene; C, cytoplasmic fraction of HB101(pCEFN1); D, cytoplasmic fraction of HB101(pJPN205); E, supernatant of HB101(pCEFN1); F, supernatant of HB101(pJPN205). Arrows denote expected sizes of mature Pet and the expected truncated species produced by pJPN205. Smaller species reacting with antibodies in lanes B and E most likely represent breakdown products of the mature toxin.
FIG. 4
Enterotoxic activity of the Pet protein derived from pCEFN1. Supernatants from overnight cultures were size fractionated (>50 kDa), and 5 μg of protein was added per Ussing chamber into which was mounted full-thickness rat jejunal tissue. The supernatants of HB101(pCEFN1) and HB101(pSPORT1) are illustrated in lanes C and D, respectively of Fig. 3a. Data points represent the means of at least three experiments; error bars represent standard errors of the means. The insert of pCEFN1 generates significant rises in PD and Isc compared with negative controls (P < 0.05 by Student’s t test).
Similar articles
- In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli.
Navarro-García F, Eslava C, Villaseca JM, López-Revilla R, Czeczulin JR, Srinivas S, Nataro JP, Cravioto A. Navarro-García F, et al. Infect Immun. 1998 Jul;66(7):3149-54. doi: 10.1128/IAI.66.7.3149-3154.1998. Infect Immun. 1998. PMID: 9632579 Free PMC article. - The serine protease motif of EspC from enteropathogenic Escherichia coli produces epithelial damage by a mechanism different from that of Pet toxin from enteroaggregative E. coli.
Navarro-García F, Canizalez-Roman A, Sui BQ, Nataro JP, Azamar Y. Navarro-García F, et al. Infect Immun. 2004 Jun;72(6):3609-21. doi: 10.1128/IAI.72.6.3609-3621.2004. Infect Immun. 2004. PMID: 15155671 Free PMC article. - Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells.
Navarro-García F, Canizalez-Roman A, Luna J, Sears C, Nataro JP. Navarro-García F, et al. Infect Immun. 2001 Feb;69(2):1053-60. doi: 10.1128/IAI.69.2.1053-1060.2001. Infect Immun. 2001. PMID: 11160002 Free PMC article. - Enteroaggregative Escherichia coli plasmid-encoded toxin.
Navarro-Garcia F. Navarro-Garcia F. Future Microbiol. 2010 Jul;5(7):1005-13. doi: 10.2217/fmb.10.69. Future Microbiol. 2010. PMID: 20632801 Review. - Autotransporters and virulence of enteroaggregative E. coli.
Navarro-Garcia F, Elias WP. Navarro-Garcia F, et al. Gut Microbes. 2011 Jan-Feb;2(1):13-24. doi: 10.4161/gmic.2.1.14933. Gut Microbes. 2011. PMID: 21637014 Review.
Cited by
- Genomic Dissection of an Enteroaggregative Escherichia coli Strain Isolated from Bacteremia Reveals Insights into Its Hybrid Pathogenic Potential.
Del Carpio AMG, Freire CA, Andrade FB, Piazza RMF, Silva RM, Carvalho E, Elias WP. Del Carpio AMG, et al. Int J Mol Sci. 2024 Aug 26;25(17):9238. doi: 10.3390/ijms25179238. Int J Mol Sci. 2024. PMID: 39273188 Free PMC article. - Plasmid-encoded toxin of Escherichia coli cleaves complement system proteins and inhibits complement-mediated lysis in vitro.
Correa GB, Freire CA, Dibo M, Huerta-Cantillo J, Navarro-Garcia F, Barbosa AS, Elias WP, Moraes CTP. Correa GB, et al. Front Cell Infect Microbiol. 2024 Feb 2;14:1327241. doi: 10.3389/fcimb.2024.1327241. eCollection 2024. Front Cell Infect Microbiol. 2024. PMID: 38371299 Free PMC article. - A Mini-Review of Enteroaggregative Escherichia coli with a Specific Target on the Virulence Factors Controlled by the AggR Master Regulator.
Izquierdo-Vega JA, Castillo-Juarez RJ, Sánchez-Gutiérrez M, Ares MA, De La Cruz MA. Izquierdo-Vega JA, et al. Pol J Microbiol. 2023 Dec 16;72(4):347-354. doi: 10.33073/pjm-2023-037. eCollection 2023 Dec 1. Pol J Microbiol. 2023. PMID: 37875068 Free PMC article. Review. - The Diversity of Escherichia coli Pathotypes and Vaccination Strategies against This Versatile Bacterial Pathogen.
Pokharel P, Dhakal S, Dozois CM. Pokharel P, et al. Microorganisms. 2023 Jan 30;11(2):344. doi: 10.3390/microorganisms11020344. Microorganisms. 2023. PMID: 36838308 Free PMC article. Review. - Novel organisation and regulation of the pic promoter from enteroaggregative and uropathogenic Escherichia coli.
Alhammadi MM, Godfrey RE, Ingram JO, Singh G, Bathurst CL, Busby SJW, Browning DF. Alhammadi MM, et al. Virulence. 2022 Dec;13(1):1393-1406. doi: 10.1080/21505594.2022.2111754. Virulence. 2022. PMID: 35971774 Free PMC article.
References
- Alting-Mees M A, Sorge J A, Short J M. pBluescriptII: multifunctional cloning and mapping vectors. Methods Enzymol. 1992;216:483–495. - PubMed
- Ausubel F M, Brent R, Kingston R E, Moore D D, Smith J A, Seidman J G, Struhl K, editors. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons; 1989.
- Bachovchin W W, Plaut A G, Flentke G R, Lynch M, Kettner C A. Inhibition of IgA1 proteinases from Neisseria gonorrhoeae and Haemophilus influenzae by peptide prolyl boronic acids. J Biol Chem. 1990;265:3738–3743. - PubMed
- Bardwell J C, McGovern K, Beckwith J. Identification of a protein required for disulfide bond formation in vivo. Cell. 1991;67:581–589. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources