J Vet Sci.  2010 Dec;11(4):315-319. 10.4142/jvs.2010.11.4.315.

Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains

• Kobayashi
• Gaziri LC
• Vidotto MC

• KMID: 1072180
• DOI: http://doi.org/10.4142/jvs.2010.11.4.315

Abstract

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh(s)) and a 33 kDa beta-domain (Tsh(beta)). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh(s) (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh(beta) (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37degrees C or 42degrees C. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26degrees C, 37degrees C, or 42degrees C, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37degrees C in mucin agar, and it was not detected when the bacteria were grown at 26degrees C in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.

Keyword

avian Escherichia coli; functional activities; temperature-sensitive hemagglutinin (Tsh); virulence factor

MeSH Terms

Adhesins, Escherichia coli/*metabolism
Brazil
Escherichia coli/*metabolism
*Gene Expression Regulation, Bacterial
Hemagglutination
Mucins/metabolism
Protein Transport
Recombinant Proteins/isolation & purification/metabolism

Figure

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  Fig. 1 Western blot of fractions from APEC 13 grown in different media and at different temperatures. (A) Supernatant fractions; Lane 1: control, recombinant E. coli BL 21 Star (DE3) strain/Tsh; Lane 2: LB medium, 26℃; Lane 3: CFA, 26℃; Lane 4: CFA, 37℃; Lane 5: CFA, 42℃; Lane 6: mucin, 26℃; Lane 7: mucin, 37℃; Lane 8: mucin, 42℃. (B) Pellet fractions; Lane 1: E. coli BL 21 Star (DE3) strain/Tsh; Lane 2: LB, 26℃; Lane 3: LB, 42℃; Lane 4: CFA, 26℃; Lane 5: CFA, 37℃; Lane 6: CFA, 42℃; Lane 7: mucin, 26℃; Lane 8: mucin, 37℃; Lane 9: mucin, 42℃. The positions of molecular mass markers (kDa) are shown at the left. APEC: avian pathogenic E. coli, LB: Luria-Bertani, CFA: colonization factor antigen.

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  Fig. 2 The mucinolytic activity of fractions from APEC 13 grown at 37℃ tested in a medium containing 1.5% agarose. (A) Supernatant of E. coli HB101 (as a negative control); (B) Supernatant of recombinant E. coli BL21/Tsh at 37℃ (+++).

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