Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae

Abstract

The Vibrio cholerae genome contains a 5.4-kb pil gene cluster that resembles the Aeromonas hydrophila tap gene cluster and other type IV-A pilus assembly operons. The region consists of five complete open reading frames designated pilABCD and yacE, based on the nomenclature of related genes from Pseudomonas aeruginosa and Escherichia coli K-12. This cluster is present in both classical and El Tor biotypes, and the pilA and pilD genes are 100% conserved. The pilA gene encodes a putative type IV pilus subunit. However, deletion of pilA had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating that pilA does not encode the primary subunit of a pilus essential for these processes. The pilD gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences. Mutational analysis of the pilD gene showed that pilD is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice. Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences. Some pilD mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented in trans, indicating that the defect may not be directly due to a loss of pilD. Taken together, these data demonstrate the effectiveness of the V. cholerae genome project for rapid identification and characterization of potential virulence factors.

FIG. 1

Organization of six representative type IV pilus assembly operons. The bars at the top indicate the approximate locations and designations of DNA sequences obtained from the publicly available genomic database. Numbers in brackets indicate the percentages of amino acid identity of deduced protein sequences to the translated open reading frame of the appropriate V. cholerae gene. Percentages of identity were determined by a ClustalW alignment done with a MacVector software package. Sequences were assembled from GenBank and other public databases as follows: V. vulnificus (accession no. AF070934) (38), A. hydrophila (accession no. U20255) (39), P. aeruginosa (accession no. M32066 and M14849) (23, 35) (genomic data from www.pseudomonas.com), and E. coli (accession no. L28105, AE000409, and D26562) (4, 59), and N. gonorrhoeae (accession no. U32588 and X66144) (10, 43, 56).

FIG. 2

Genetic organization of pilD mutants and the complementing plasmid. Construction of strains and plasmids is detailed in Materials and Methods. In addition to the pilD deletion, KFV36 also has a deletion in pilA.

FIG. 3

Growth curves for pilD mutants. Overnight cultures were diluted 1:100 in 100 ml of LB broth with antibiotics and incubated with shaking at 37°C. At specific times, 1 ml of the cultures was removed and the optical density (A₆₀₀) was measured. Time zero is the time at which the cultures exited the stationary phase.

FIG. 4

Effect of pilD mutations on CT secretion. CT was measured as described in Materials and Methods. Strains carrying a pilD-complementing plasmid were grown in 0.05% arabinose to induce the expression of promoter P_BAD, as were N16961Sm and KFV18R, which contain plasmid pBAD18Cm-ToxT for constitutive overexpression of CT. For both classical (A) and El Tor (B) strains, enzyme-linked immunosorbent assay plates were read with a microtiter reader, and amounts (nanograms) of intracellular (hatched bars) and extracellular (solid bars) toxins were determined by comparison against a standard curve. Results are presented as a percentage of the total toxin produced. Averages and standard deviations were determined from duplicates in a single experiment that is representative of at least two experiments for each sample.

FIG. 5

Effect of pilD mutations on HAP secretion. Strains were heavily streaked onto 1% nonfat milk agar in divided glass plates and incubated at 37°C for 24 h. The agar contained 0.05% arabinose (ara) as indicated. Plates were photographed with a Bio-Rad Fluor-S MultiImager.

FIG. 6

Adherence of V. cholerae strains to HEp-2 cells. Assays were performed as described in Materials and Methods. KFV5R carrying either the pBAD18 vector or the pilD-complementing plasmid pKJF308 was grown in the presence of 0.05% arabinose to induce promoter P_BAD. Arabinose does not inhibit the adherence of wild-type O395 (data not shown). Results are presented as the percentage of input bacteria that adhered to HEp-2 cells after 1 h of incubation. Numbers given are averages from triplicate samples in a single assay and are representative of at least two experiments. Error bars represent standard deviations. (A) Adherence by O395 mutants of V. cholerae. (B) Complementation of KFV5R by pilD in trans.