Characterization of vibrio cholerae O1 antigen as the bacteriophage K139 receptor and identification of IS1004 insertions aborting O1 antigen biosynthesis

Abstract

Bacteriophage K139 was recently characterized as a temperate phage of O1 Vibrio cholerae. In this study we have determined the phage adsorption site on the bacterial cell surface. Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor. In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139. Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide. By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection. Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfb region). Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004 into the rfb gene cluster. Treating one wbeW::IS1004 serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance. Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation. This strain had a significant reduction in its ability to colonize the mouse small intestine.

FIG. 1

(A) Inactivation of bacteriophage K139.cm9 with purified LPS derived from V. cholerae O1 classical Inaba strain 569B (Sigma). The inhibition data represent the means ± the standard errors of three independent experiments. (B) SDS-PAGE pattern of purified LPS after silver staining. Strains used for LPS preparation for lanes were as follows: 1, 569B; 2, O395; 3, O395R-1; 4, MAK757; 5, P27459; 6, AI1838; 7, MO10; 8, Salmonella serovar Typhimurium. The inactivation of phage K139.cm9 was determined in plaque inhibition assays and is indicated as a percentage (mean values of at least two independent experiments); the first and second values in each set were obtained using 10 and 100 μg of purified LPS per ml, respectively.

FIG. 2

(A) Analysis of LPS from parent and mutant strains. Lanes a to e represent LPS patterns of phage-resistant P27459 mutants and lane f represents the LPS pattern of the clinical isolate CO966. LPS was prepared from strains P27459 (wt), P27res30 (a), P27res118 (b), P27res29 (c), P27res144 (d), P27res108 (e), and CO966 (f). The inactivation of phage K139.cm9 was determined in plaque inhibition assays and is indicated as a percentage; the first and second values in each set were obtained using 10 and 100 μg of purified LPS per ml, respectively. n.d., not done. (B) Western blot analysis using a polyclonal antiserum against O1 LPS (O1 common; Difco) corresponding to the samples from panel A. The molecular size standard is indicated in kilodaltons according to the Kaleidoscope polypeptide standard (Bio-Rad).

FIG. 3

(A) The rfb region of V. cholerae O1. The representation is based on the sequences submitted to GenBank (see text) and of Stroeher et al. (45) and Bik et al. (4). The transcriptional directions are indicated by arrows. Probes A to C, used for Southern hybridization, are indicated by horizontal lines. The scales are indicated in kilobases. A, AvaI; S, SacI; O1 to O6, primers used to amplify the probes. (B) Southern blot analysis of HpaII-digested chromosomal DNA with IS1004- and wbeW-specific DNA probes. Lanes 1, P27459; lanes 2, P27res30; lanes 3, P27res30(pACYC177); lanes 4, P27res30(pJNwbeW); lanes 5, P27res30rev. The additional band that hybridizes with the wbeW probe in P27res30(pJNwbeW) (lane 4) is due to the digestion of the complementing plasmid containing wbeW. (C) LPS pattern after SDS-PAGE and silver staining (lanes contain samples from the strains listed for panel B. (D) Southern blot analysis of HpaII-digested chromosomal DNA with IS1004- and manB-specific DNA probes. Lanes 6, MAK757; lanes 7, MAKres3. (E) DNA sequences at sites of IS1004 insertions. PCR fragments amplified by primers O5 and O6 out of strains P27459 (P27), P27res30, and P27res30rev were sequenced with sequencing primer wbeWseq, and P27res30 was sequenced additionally with primer IS1004seq (panel A). Sequence analysis of PCR fragments generated with primers manB1 and manB2 out of strains MAK757 and MAKres3 was performed with manBseq and IS1004seq (MAKres3 only) primers (panel A). Sequences for the other IS1004 insertions have been retrieved from the literature or GenBank. Possible duplicated AT base pairs at the insertion site are underlined.

FIG. 4

Immunogold detection of O1 antigen. V. cholerae O1 Ogawa strains were stained with anti-Ogawa O1 antiserum and anti-rabbit immunoglobulin-gold conjugate and visualized as electron-dense particles in electron micrographs. (A) The O antigen is present over the whole cell surface of strain MAK757, including the LPS-sheathed flagellum. (B) Strain CO966 possesses only a small number of O antigen-containing LPS molecules. (C) No O1 antigen could be detected in the mutant MAKres3 (manB::IS1004). These results confirmed the LPS analysis by SDS-PAGE and silver staining (Fig. 2A). Bars indicate 0.5 μm.