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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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Validation of the inducible promoter system by β-galactosidase assay.Mycobacterium smegmatis mc2155 cultures harboring the plasmid pAZI 9018b were induced in triplicate with IPTG in a dose dependent manner (0,1,10,100,1000 μM). The β-galactosidase activity was monitored at O.D.410 in SpectraMax Plus384 spectrophotometer at 37°C after adding ONPG.
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pone-0005923-g003: Validation of the inducible promoter system by β-galactosidase assay.Mycobacterium smegmatis mc2155 cultures harboring the plasmid pAZI 9018b were induced in triplicate with IPTG in a dose dependent manner (0,1,10,100,1000 μM). The β-galactosidase activity was monitored at O.D.410 in SpectraMax Plus384 spectrophotometer at 37°C after adding ONPG.

Mentions: The β-galactosidase activity of M. smegmatis harboring the vector pAZI9018b was monitored. The assay revealed a linear increase in the enzyme activity (mO.D.410 nm/min) over a wide range of IPTG concentration (Figure 3). The upregulation of expression following induction was about 30 fold. The level of leaky expression was low as there was a fairly tight regulation at zero IPTG concentration. The results indicated that the constitutive promoter T150 produced enough repressor so as to block the transcription of the lacZ gene in the absence of any inducer. It was also observed with times of induction less than four hours the β-galactosidase activity reduced considerably and with induction time greater than 12 hours the fold induction reduced as there was an accumulation of leaky expression (data not shown). The results indicate that it took about a generation time (4-hours) in M. smegmatis for observing the optimized effect of the IPTG induction. It was also observed that various levels of IPTG did not have any effect on the growth rate of the host bacteria (data not shown).


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

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

Validation of the inducible promoter system by β-galactosidase assay.Mycobacterium smegmatis mc2155 cultures harboring the plasmid pAZI 9018b were induced in triplicate with IPTG in a dose dependent manner (0,1,10,100,1000 μM). The β-galactosidase activity was monitored at O.D.410 in SpectraMax Plus384 spectrophotometer at 37°C after adding ONPG.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005923-g003: Validation of the inducible promoter system by β-galactosidase assay.Mycobacterium smegmatis mc2155 cultures harboring the plasmid pAZI 9018b were induced in triplicate with IPTG in a dose dependent manner (0,1,10,100,1000 μM). The β-galactosidase activity was monitored at O.D.410 in SpectraMax Plus384 spectrophotometer at 37°C after adding ONPG.
Mentions: The β-galactosidase activity of M. smegmatis harboring the vector pAZI9018b was monitored. The assay revealed a linear increase in the enzyme activity (mO.D.410 nm/min) over a wide range of IPTG concentration (Figure 3). The upregulation of expression following induction was about 30 fold. The level of leaky expression was low as there was a fairly tight regulation at zero IPTG concentration. The results indicated that the constitutive promoter T150 produced enough repressor so as to block the transcription of the lacZ gene in the absence of any inducer. It was also observed with times of induction less than four hours the β-galactosidase activity reduced considerably and with induction time greater than 12 hours the fold induction reduced as there was an accumulation of leaky expression (data not shown). The results indicate that it took about a generation time (4-hours) in M. smegmatis for observing the optimized effect of the IPTG induction. It was also observed that various levels of IPTG did not have any effect on the growth rate of the host bacteria (data not shown).

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