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Delineating bacteriostatic and bactericidal targets in mycobacteria using IPTG inducible antisense expression.

Kaur P, Agarwal S, Datta S - PLoS ONE (2009)

Bottom Line: By this method, we could differentiate between genes whose down regulation lead to bacteriostatic or bactericidal effect.The specificity of the antisense inhibition was conclusively shown in the case of auxotrophic gene ilvB.Additionally, under these conditions the expression of several genes in branched chain amino acid pathway was severely suppressed indicating targeted gene inactivation.

View Article: PubMed Central - PubMed

Affiliation: AstraZeneca India Pvt Ltd, Hebbal, Bangalore, India.

ABSTRACT
In order to identify novel high value antibacterial targets it is desirable to delineate whether the inactivation of the target enzyme will lead to bacterial death or stasis. This knowledge is particularly important in slow growing organisms, like mycobacteria, where most of the viable anti-tubercular agents are bactericidal. A bactericidal target can be identified through the conditional deletion or inactivation of the target gene at a relatively high cell number and subsequently following the time course of survival for the bacteria. A simple protocol to execute conditional inactivation of a gene is by antisense expression. We have developed a mycobacteria specific IPTG inducible vector system and monitored the effect of antisense inhibition of several known essential genes in mycobacteria by following their survival kinetics. By this method, we could differentiate between genes whose down regulation lead to bacteriostatic or bactericidal effect. Targets for standard anti-tubercular drugs like inhA for isoniazid, rpoB and C for rifampicin, and gyr A/B for flouroquinolones were shown to be bactericidal. In contrast targets like FtsZ behaved in a bacteriostatic manner. Induction of antisense expression in embB and ribosomal RNA genes, viz., rplJ and rpsL showed only a marginal growth inhibition. The specificity of the antisense inhibition was conclusively shown in the case of auxotrophic gene ilvB. The bactericidal activity following antisense expression of ilvB was completely reversed when the growth media was supplemented with the isoleucine, leucine, valine and pantothenate. Additionally, under these conditions the expression of several genes in branched chain amino acid pathway was severely suppressed indicating targeted gene inactivation.

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Survival kinetics of M. smegmatis (with M. tuberculosis gyrA-AS and gyrB –AS plasmids individually).Induction at 10 uM IPTG concentration. In the case of gyrA-AS r-M. smegmatis the cfu reduction is >3 log10 (bactericidal target), but gyrB-AS showed no cfu reduction as the orphan gyrB takes over once the other gyrB transcript is blocked.
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pone-0005923-g007: Survival kinetics of M. smegmatis (with M. tuberculosis gyrA-AS and gyrB –AS plasmids individually).Induction at 10 uM IPTG concentration. In the case of gyrA-AS r-M. smegmatis the cfu reduction is >3 log10 (bactericidal target), but gyrB-AS showed no cfu reduction as the orphan gyrB takes over once the other gyrB transcript is blocked.

Mentions: Full-length antisense genes of gyrA and gyrB were cloned into pAZI9018b and transformed into M. smegmatis and the survival kinetics following the antisense induction of the individual genes monitored. The results (Figure 7) indicate that while induction of antisense of gyrA resulted in more that 3-log10 reduction of the bacteria, the induction of antisense of gyrB was well tolerated and the bacteria grew normally. This data looked contradictory at the outset. However during the literature search it was seen that a gene designated as orphan gyrB was identified in M. smegmatis [19]. We speculated that while the wild type gyrB expression is reduced the orphan gyrB was capable in taking over the activity and thus preventing cell death. In order to prove this hypothesis we transformed the same constructs in M. tuberculosis and monitored the survival kinetics. The global analysis of M. tuberculosis genome did not indicate the presence of any additional putative gyrB; also the tranposon mutagens data indicated that both these genes were essential in M. tuberculosis [6]. The survival kinetics data (Figure 8) indicate that contrary to M. smegmatis, induction of antisense of both gyrA and gyrB in M. tuberculosis resulted in greater than 3 log10 reduction in cfu. Thus, validating our general hypothesis that both gyrA and gyrB were indeed bactericidal targets. Interestingly the kill kinetics of moxifloxacin even at its MBC of 1 ug/ml, is much rapid than the gyrase antisense (Figure 8). This probably is due to the fact that the drug not only inactivates gyrase, but also traps the enzyme DNA complex setting up a down stream complex cascade resulting in generation of oxidative stress leading to a faster cell death-a process that is probably not initiated in the simple downregulation or inhibition of gyrase.


Delineating bacteriostatic and bactericidal targets in mycobacteria using IPTG inducible antisense expression.

Kaur P, Agarwal S, Datta S - PLoS ONE (2009)

Survival kinetics of M. smegmatis (with M. tuberculosis gyrA-AS and gyrB –AS plasmids individually).Induction at 10 uM IPTG concentration. In the case of gyrA-AS r-M. smegmatis the cfu reduction is >3 log10 (bactericidal target), but gyrB-AS showed no cfu reduction as the orphan gyrB takes over once the other gyrB transcript is blocked.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691988&req=5

pone-0005923-g007: Survival kinetics of M. smegmatis (with M. tuberculosis gyrA-AS and gyrB –AS plasmids individually).Induction at 10 uM IPTG concentration. In the case of gyrA-AS r-M. smegmatis the cfu reduction is >3 log10 (bactericidal target), but gyrB-AS showed no cfu reduction as the orphan gyrB takes over once the other gyrB transcript is blocked.
Mentions: Full-length antisense genes of gyrA and gyrB were cloned into pAZI9018b and transformed into M. smegmatis and the survival kinetics following the antisense induction of the individual genes monitored. The results (Figure 7) indicate that while induction of antisense of gyrA resulted in more that 3-log10 reduction of the bacteria, the induction of antisense of gyrB was well tolerated and the bacteria grew normally. This data looked contradictory at the outset. However during the literature search it was seen that a gene designated as orphan gyrB was identified in M. smegmatis [19]. We speculated that while the wild type gyrB expression is reduced the orphan gyrB was capable in taking over the activity and thus preventing cell death. In order to prove this hypothesis we transformed the same constructs in M. tuberculosis and monitored the survival kinetics. The global analysis of M. tuberculosis genome did not indicate the presence of any additional putative gyrB; also the tranposon mutagens data indicated that both these genes were essential in M. tuberculosis [6]. The survival kinetics data (Figure 8) indicate that contrary to M. smegmatis, induction of antisense of both gyrA and gyrB in M. tuberculosis resulted in greater than 3 log10 reduction in cfu. Thus, validating our general hypothesis that both gyrA and gyrB were indeed bactericidal targets. Interestingly the kill kinetics of moxifloxacin even at its MBC of 1 ug/ml, is much rapid than the gyrase antisense (Figure 8). This probably is due to the fact that the drug not only inactivates gyrase, but also traps the enzyme DNA complex setting up a down stream complex cascade resulting in generation of oxidative stress leading to a faster cell death-a process that is probably not initiated in the simple downregulation or inhibition of gyrase.

Bottom Line: By this method, we could differentiate between genes whose down regulation lead to bacteriostatic or bactericidal effect.The specificity of the antisense inhibition was conclusively shown in the case of auxotrophic gene ilvB.Additionally, under these conditions the expression of several genes in branched chain amino acid pathway was severely suppressed indicating targeted gene inactivation.

View Article: PubMed Central - PubMed

Affiliation: AstraZeneca India Pvt Ltd, Hebbal, Bangalore, India.

ABSTRACT
In order to identify novel high value antibacterial targets it is desirable to delineate whether the inactivation of the target enzyme will lead to bacterial death or stasis. This knowledge is particularly important in slow growing organisms, like mycobacteria, where most of the viable anti-tubercular agents are bactericidal. A bactericidal target can be identified through the conditional deletion or inactivation of the target gene at a relatively high cell number and subsequently following the time course of survival for the bacteria. A simple protocol to execute conditional inactivation of a gene is by antisense expression. We have developed a mycobacteria specific IPTG inducible vector system and monitored the effect of antisense inhibition of several known essential genes in mycobacteria by following their survival kinetics. By this method, we could differentiate between genes whose down regulation lead to bacteriostatic or bactericidal effect. Targets for standard anti-tubercular drugs like inhA for isoniazid, rpoB and C for rifampicin, and gyr A/B for flouroquinolones were shown to be bactericidal. In contrast targets like FtsZ behaved in a bacteriostatic manner. Induction of antisense expression in embB and ribosomal RNA genes, viz., rplJ and rpsL showed only a marginal growth inhibition. The specificity of the antisense inhibition was conclusively shown in the case of auxotrophic gene ilvB. The bactericidal activity following antisense expression of ilvB was completely reversed when the growth media was supplemented with the isoleucine, leucine, valine and pantothenate. Additionally, under these conditions the expression of several genes in branched chain amino acid pathway was severely suppressed indicating targeted gene inactivation.

Show MeSH
Related in: MedlinePlus