Deciphering the possible role of ctxB7 allele on higher production of cholera toxin by Haitian variant Vibrio cholerae O1

Abstract

Cholera continues to be an important public health concern in developing countries where proper hygiene and sanitation are compromised. This severe diarrheal disease is caused by the Gram-negative pathogen Vibrio cholerae belonging to serogroups O1 and O139. Cholera toxin (CT) is the prime virulence factor and is directly responsible for the disease manifestation. The ctxB gene encodes cholera toxin B subunit (CTB) whereas the A subunit (CTA) is the product of ctxA gene. Enzymatic action of CT depends on binding of B pentamers to the lipid-based receptor ganglioside G M1 . In recent years, emergence of V. cholerae Haitian variant strains with ctxB7 allele and their rapid spread throughout the globe has been linked to various cholera outbreaks in Africa and Asia. These strains produce classical type (WT) CTB except for an additional mutation in the signal sequence region where an asparagine (N) residue replaces a histidine (H) at the 20 th amino acid position (H20N) of CTB precursor (pre-CTB). Here we report that Haitian variant V. cholerae O1 strains isolated in Kolkata produced higher amount of CT compared to contemporary O1 El Tor variant strains under in vitro virulence inducing conditions. We observed that the ctxB7 allele, itself plays a pivotal role in higher CT production. Based on our in silico analysis, we hypothesized that higher accumulation of toxin subunits from ctxB7 allele might be attributed to the structural alteration at the CTB signal peptide region of pre-H20N CTB. Overall, this study provides plausible explanation regarding the hypertoxigenic phenotype of the Haitian variant strains which have spread globally, possibly through positive selection for increased pathogenic traits.

Elucidating the correlation between the number of TTTTGAT heptamer repeats and cholera toxin promoter activity in Vibrio cholerae O1 pandemic strains

“…Therefore, six pandemic strains of V. cholerae with different numbers of TTTTGAT heptad repeats within P ctxAB were tested to determine CT production in the presence and absence of HCO 3 - . As earlier reported (20), El Tor variant strain MCM168 and the Haitian variant IDH03595 (17) demonstrated heightened CT production compared to the prototype El Tor strain N16961, although the amount of CT produced varied among the strains. This enhanced CT production by the Haitian variant strain could also be attributed to the signature ctxB7 allele, which results in a histidine to asparagine substitution (H20N) in the CT B signal sequence (17).…”

“…As earlier reported (20), El Tor variant strain MCM168 and the Haitian variant IDH03595 (17) demonstrated heightened CT production compared to the prototype El Tor strain N16961, although the amount of CT produced varied among the strains. This enhanced CT production by the Haitian variant strain could also be attributed to the signature ctxB7 allele, which results in a histidine to asparagine substitution (H20N) in the CT B signal sequence (17). Strikingly, MCM168, which has the minimal number of the TTTTGAT heptamer sequence within P ctxAB , produced the highest amount of CT in vitro even when HCO 3 - was not included in the AKI medium.…”

“…Furthermore, MCM168 produced 2.6-fold heightened CT over both of the classical strains O395 and 569B ( P value 0.03). IDH03595, a Haitian variant strain isolated from Kolkata with 5 TTTTGAT perfect heptad repeats in P ctxAB region (17), produced CT comparable (1827.3 ng/ml/O.D.600nm) to that secreted by 569B (1406.1 ng/ml/O.D.600nm), 1.5 times higher than O395 ( P value 0.02), and 19.7 fold larger amounts of CT than H218 ( P value 0.001). IDH03595 demonstrated a more than 4-fold increase in the amount of CT produced in comparison with N16961 (415.3 ng/ml/O.D.600nm, P value 0.002), whereas O395 and 569B secreted 2.7 times and 1.5-fold higher CT than N16961 ( P value 0.0001 and 0.02, respectively).…”

“…Derivatives of E. coli K37 strain JW419 transformed with recombinant plasmids carrying P ctxAB fusion constructs were grown at 37° C overnight, and diluted 1:100 in fresh LB medium supplemented with 1 mM IPTG and were allowed to grow at 30° C for 3 hours. β-Galactosidase activity was measured in Miller Units (MU) as described previously (17). A minimum of three individual experiments for each strain were performed, and mean values were plotted to with error bars signifying standard deviations for each strain.…”

Dissecting serotype-specific contributions to live oral cholera vaccine efficacy

“…This isolate has since been used as the predominant human challenge strain, including the most recently reported volunteer challenge OCV trial 45 . Recent 7PET evolution over the last three decades has led to strains with altered virulence traits, and potentially immunogenicity [61][62][63][64] . Our data suggest that currently circulating late 7PET V. cholerae, which have never been used as challenge strains, may interact differently with OCV-induced immunity than early 7PET and 6 th pandemic isolates, and thus should be considered for use not only as next-generation live OCVs, but as challenge strains in future human volunteer studies.…”