A screen for compounds that inhibit replication of Vibrio cholerae chromosome II
The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens, such as Vibrio cholerae, the cause of cholera. All organisms in this family have two chromosomes, and replication of the smaller one depends on rctB, a gene that is restricted to the Vibrionaceae. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new more ..
BioActive Compounds: 351
The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens, such as Vibrio cholerae, the cause of cholera. All organisms in this family have two chromosomes, and replication of the smaller one depends on rctB, a gene that is restricted to the Vibrionaceae. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. This high throughput cell-based screen was performed to identify small molecule inhibitors of RctB. We identified a compound that blocked growth of an E. coli strain bearing an rctB-dependent plasmid but did not influence growth of E. coli lacking this plasmid. This compound, designated vibrepin, had potent cidal activity against V. cholerae and inhibited the growth of all vibrio species tested. Vibrepin blocked RctB oriCII unwinding, apparently by promoting formation of large non-functional RctB complexes. Although vibrepin also appears to have targets other than RctB, our findings suggest that RctB is an attractive target for generation of novel antibiotics that only block growth of vibrios. Vibrio-specific agents, unlike antibiotics currently used in clinical practice, will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms.
The day before screening, E. coli strain YBA685 was grown overnight (~24 h) in Luria-Bertani (LB) broth with 50 microg/mL kanamycin at 37 degrees C with shaking.
On the day of screening, the overnight culture of YBA685 was inoculated at a 1:500 dilution into LB broth containing 50 microg/mL kanamycin; the fresh culture was then grown at 37 degrees C until reaching an OD600 of ~0.1. 30 microl aliquots of the culture were transferred into 384-well plates. Experimental compounds were then pin-transferred to the plates at 100 nL per well. Wells in column 23 served as the negative controls. Column 24 was filled with the positive control (mach1 in 50 microg/mL kanamycin). Final assay well volume was 30 uL.
After the plates were incubated at 37 degrees C for 3 hours, the OD600 of the wells was measured.
The mean and standard deviation of OD600 values from the experimental wells in each replicate plate were obtained, and z-scores for each well were calculated. Activity scores for each replicate well were calculated by dividing the z-score by 5, multiplying by 100 and then applying the inverse sign. Activity scores < 0 were assigned a value of 0; activity scores > 100 were assigned a value of 100. The average of the replicate activity scores was used as the final activity score for each well. Wells with activity scores >= 40 and z-scores < -2.0 in both replicates were considered active.
Data Table (Concise)