Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Abstract

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.

Single-Nucleotide-Polymorphism Typing and Genetic Relationships of Salmonella enterica Serovar Typhi Isolates

“…Although most serovar Typhi isolates are monophasic for the expression of the flagellar antigen encoded by the fliC gene at the H1 locus, some Indonesian isolates have an additional z66 flagellar antigen, which was first described in 1981 (10) and was thought to be encoded by fljB at the H2 locus (19). The z66 flagellar antigen is now known to be encoded by a gene in the fljBA-like operon not located in the H2 locus (12). All the serovar Typhi isolates used in this study were typed for the presence of the z66 flagellar antigen gene by PCR using the primers described by Huang et al (12).…”

“…The z66 flagellar antigen is now known to be encoded by a gene in the fljBA-like operon not located in the H2 locus (12). All the serovar Typhi isolates used in this study were typed for the presence of the z66 flagellar antigen gene by PCR using the primers described by Huang et al (12). Among the 73 serovar Typhi isolates, 15 were known from serotyping results to express z66, and all 15 were confirmed to be positive for z66 by PCR (Table 1; data not shown).…”

PCR Method To Identify Salmonella enterica Serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella Isolates from the Blood of Patients with Clinical Enteric Fever

“…Strains positive for one of the three targets in the O grouping multiplex were then tested in the H multiplex. With the exception of some Indonesian strains that express H antigen j, all Salmonella serovar Typhi strains express antigen d (2,10,11,14,24,(28)(29)(30). Other Indonesian strains express "z66" antigen in addition to d or j.…”

Distribution Flagellin Gene Variants of Salmonella Typhi in Patients with Typhoid Fever in West Kutai, East Kalimantan, Indonesia

“…For z66Ind primer set designated Ind -F: 5' ATG TCG GAA ATC AAC CGT ATC T 3' and Ind-R:5' CAG GCC GTC AAC CTG AGA C 3' were selected for the specific amplification of a 597bp segment of the Ind gene. The PZ66-A and PZ66-B primers are located in the central region of the z66 gene that is largely deleted in the Ind gene and the primers Ind-F and Ind-R are located in the 5' and 3' portion of the Ind gene that shows homology with the z66 gene, but these primers are chosen such that the number of mismatches with this gene is too high to warrant efficient amplification of the z66 gene [11,12]. The Ind-specific PCR was performed with after an initial denaturation at 94°C for 2 min, for 35 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 1 min, followed by an extension step of 72°C for 5 min [11].…”