Secretion of environmental TcpF proteins by the pathogenic strain. (A) Coomassie brilliant blue staining of the culture supernatant (CS) and periplasmic (P) fractions revealed that the pathogenic strain mediates the secretion of the environmental TcpF proteins. (B) In the absence of TcpC, the TCP apparatus secretin, these proteins are no longer detected in the culture supernatant, indicating that this secretion is dependent on the TCP apparatus.

Clustal W alignment of SCE4 environmental TcpF, which is 66% identical to pathogenic TcpF, reveals regions that are divergent between these proteins. TcpF derivatives that were found to be defective in colonization (Fig. 2) are represented by the designations of the plasmids encoding them, given above the alignment at the location of the 5-amino-acid insertion. Residues in the SCE environmental TcpF protein that are identical (*), conserved (:), or semiconserved (.) with respect to the pathogenic TcpF protein are indicated below the sequence.

Lack of HtrA restores the stability of protein derivatives that are defective in extracellular secretion. The TcpF protein derivatives that were degraded into products A and B, as shown in Fig. 3, were analyzed in the presence and absence of HtrA by Western blotting of the periplasmic (P) and culture supernatant (CS) fractions with anti-TcpF polyclonal antibodies. Although HtrA restored these protein derivatives to a stable full-length product in the periplasm, they still could not be detected in the culture supernatant.

Alignment of the pathogenic TcpF protein with divergent environmental TcpF proteins. Clustal W alignment reveals residues that are identical (*), conserved (:), and semiconserved (.) between the pathogenic TcpF protein and environmental TcpF proteins. Plasmids encoding linker scan protein derivatives that were found to influence secretion are annotated above the alignment with respect to the location of the 5-amino-acid insertion. The level of identity between each environmental TcpF protein and pathogenic TcpF, as reported by Mukhopadhyay et al. in 2001 (26), is given on the left. (Adapted from reference 26 with permission.)

Summary of TcpF secretion and colonization determinants. The shaded box represents TcpF after translocation through the Sec apparatus (mature form of the protein). TcpF derivatives generated by linker scanning mutagenesis are represented by the designations of the plasmids encoding them, given at the approximate location of the 5-amino-acid insertion. Open boxes represent regions found to be important for extracellular secretion. Striped boxes represent TcpF derivatives that are stable in the culture supernatant and are still able to mediate colonization. Filled boxes represent regions found to be important in mediating colonization.

Summary of the locations of tcpF mutations. (A) Amino acid sequence of mature TcpF. Shaded letters represent the Sec-dependent signal sequence. The locations of the linker scanning mutations are indicated above the TcpF sequence. Internal deletions are underlined and labeled below the TcpF sequence. (B) Alteration of each TcpF derivative. Plasmids carrying each tcpF linker scan (LS) mutation are listed, and the insertions are given according to the nomenclature described in Materials and Methods. Chromosomal internal deletions of tcpF are indicated by numbers 1 through 7, and the amino acids (aa) deleted are shown.

Western blot analysis of TcpF linker scan derivatives and the mutant with internal deletion 1 for extracellular secretion. Western blotting with anti-TcpF polyclonal antibodies was performed on the culture supernatant (CS), periplasmic (P), and outer membrane (OM) fractions. (A) TcpF derivative fractions were loaded according to the location of the 5-amino-acid insertion, from the N terminus to the C terminus. Full-length TcpF derivatives constructed by linker scanning mutagenesis were detected in either the periplasm or the outer membrane fraction, or both, but were not detected in the culture supernatant. (B) The mutant with internal deletion 1 was detected in the periplasm but not in the outer membrane or the culture supernatant.

Environmental TcpF proteins cannot mediate colonization in the infant mouse cholera model. The competitive index was determined for each environmental TcpF variant by competing each strain against the reference strain, SJK9, expressing wild-type TcpF. Each circle represents a mouse, and each horizontal line marks the average competitive index of the five or six mice tested for one strain. The positive control is the competition between SJK9 and SJK7, both expressing wild-type TcpF. The negative control is the competition between SJK9, expressing wild type TcpF, and SJK71, harboring the empty vector. The competitive index was determined for each mouse, and Student's t test was used to determine which strains were significantly defective in colonization compared to the positive control. Environmental TcpF proteins with various levels of divergence from pathogenic TcpF were found to be significantly defective in colonization compared to the positive control, as indicated by asterisks (P < 0.016).

Analysis of TcpF derivatives in colonization. TcpF derivatives constructed by linker scanning mutagenesis that were detected in the culture supernatant were analyzed for their functions in colonization. (A) Stable TcpF derivatives in the culture supernatant were loaded onto the protein gel according to the location of the 5-amino-acid insertion in TcpF, from the N terminus to the C terminus, and were detected by Western blotting using anti-TcpF polyclonal antibodies. Plasmids encoding the TcpF derivatives are given above the Western blot, followed by the alteration of TcpF in brackets. wt, wild type. (B) The competitive index was determined for each TcpF derivative by competing each strain in the infant mouse model against the reference strain, SJK9, expressing wild-type TcpF. The competitive index of each TcpF derivative was graphed from the N terminus to the C terminus according to the location of the 5-amino-acid insertion. Each circle represents a mouse, and each horizontal line represents the average competitive index for the five or six mice tested for one strain. The positive (+) control is the competition between SJK9 and SJK7, both expressing wild-type TcpF. The negative (−) control is the competition between SJK9, expressing wild-type TcpF, and SJK71, harboring the empty vector. Student's t test was used to determine which of the strains were significantly defective in colonization compared to the positive control. TcpF derivatives with 5-amino-acid insertions that mapped toward the C terminus were found to be significantly defective in colonization, as indicated by asterisks (P < 0.01).