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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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FtsZ antisense expression in mycobacteria.Microscopic (1000x) observation of M. tuberculosis FtsZ antisense construct in M. smegmatis mc2155: M. smegmatis mc2155 transformed with Mtu FtsZ antisense (AS) construct. The colonies appeared after 8 days (vs.control in 3 days) & showed poor growth in broth (inhibited). Culture concentrated to normalize the cell number (vs. WT) for induction with IPTG. Panel A. M. smegmatis mc2155 host (WT) and Panel B. M. smegmatis mc2155 with Mtu FtsZ antisense gene, both induced for 48 hrs. with 1 mM IPTG. The induced cultures of FtsZ antisense clone (Panel B) showed filamentation with elongated cells (no septation), as observed under phase contrast conditions in 1000X. Micrographs were taken using AxioVision Rel. 4.5 software.
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pone-0005923-g004: FtsZ antisense expression in mycobacteria.Microscopic (1000x) observation of M. tuberculosis FtsZ antisense construct in M. smegmatis mc2155: M. smegmatis mc2155 transformed with Mtu FtsZ antisense (AS) construct. The colonies appeared after 8 days (vs.control in 3 days) & showed poor growth in broth (inhibited). Culture concentrated to normalize the cell number (vs. WT) for induction with IPTG. Panel A. M. smegmatis mc2155 host (WT) and Panel B. M. smegmatis mc2155 with Mtu FtsZ antisense gene, both induced for 48 hrs. with 1 mM IPTG. The induced cultures of FtsZ antisense clone (Panel B) showed filamentation with elongated cells (no septation), as observed under phase contrast conditions in 1000X. Micrographs were taken using AxioVision Rel. 4.5 software.

Mentions: FtsZ protein forms a ring at the site of bacterial cell division. Over or under production of this protein causes an imbalance in the cell division and results in cell lysis or filamentation respectively [16], [17]. We down-regulated FtsZ gene by cloning it in antisense orientation replacing the LacZ in pAZI9018b. Though it was the M. tuberculosis FtsZ gene that had been cloned in antisense orientation, it could down regulate the M. smegmatis FtsZ expression, as there was about 92% sequence identity at the nucleotide level. Compared to the wild type strain, the induced antisense FtsZ strain showed a high degree of filamentation (Figure 4). This observation is in accordance to the earlier observation that deficiency of FtsZ causes filamentation in mycobacteria [18], proving that the antisense mRNA was capable of inhibiting the expression of the genomic driven FtsZ sense gene. In order to delineate whether the downstream effect of a target downregulation is bacteriostatic or bactericidal one has to define a mycobacterial benchmark. This is illustrated in the generic mycobacterial survival kinetics (Figure 5) where various zones have been defined. Though, the standard 99.9% kill is defined as bactericidal for antibiotics, the slow growth of M. tuberculosis brings in an element of time dependence while evaluating cidality and stasis. From the survival kinetics of FtsZ antisense strain it was seen that the target behaved in a bacteriostatic fashion. There was a meagre drop of 1.5 log10 in cfu at an inducer concentration of 10 µM IPTG (Figure 6). There was no further reduction in cfu at higher IPTG concentration.


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

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

FtsZ antisense expression in mycobacteria.Microscopic (1000x) observation of M. tuberculosis FtsZ antisense construct in M. smegmatis mc2155: M. smegmatis mc2155 transformed with Mtu FtsZ antisense (AS) construct. The colonies appeared after 8 days (vs.control in 3 days) & showed poor growth in broth (inhibited). Culture concentrated to normalize the cell number (vs. WT) for induction with IPTG. Panel A. M. smegmatis mc2155 host (WT) and Panel B. M. smegmatis mc2155 with Mtu FtsZ antisense gene, both induced for 48 hrs. with 1 mM IPTG. The induced cultures of FtsZ antisense clone (Panel B) showed filamentation with elongated cells (no septation), as observed under phase contrast conditions in 1000X. Micrographs were taken using AxioVision Rel. 4.5 software.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005923-g004: FtsZ antisense expression in mycobacteria.Microscopic (1000x) observation of M. tuberculosis FtsZ antisense construct in M. smegmatis mc2155: M. smegmatis mc2155 transformed with Mtu FtsZ antisense (AS) construct. The colonies appeared after 8 days (vs.control in 3 days) & showed poor growth in broth (inhibited). Culture concentrated to normalize the cell number (vs. WT) for induction with IPTG. Panel A. M. smegmatis mc2155 host (WT) and Panel B. M. smegmatis mc2155 with Mtu FtsZ antisense gene, both induced for 48 hrs. with 1 mM IPTG. The induced cultures of FtsZ antisense clone (Panel B) showed filamentation with elongated cells (no septation), as observed under phase contrast conditions in 1000X. Micrographs were taken using AxioVision Rel. 4.5 software.
Mentions: FtsZ protein forms a ring at the site of bacterial cell division. Over or under production of this protein causes an imbalance in the cell division and results in cell lysis or filamentation respectively [16], [17]. We down-regulated FtsZ gene by cloning it in antisense orientation replacing the LacZ in pAZI9018b. Though it was the M. tuberculosis FtsZ gene that had been cloned in antisense orientation, it could down regulate the M. smegmatis FtsZ expression, as there was about 92% sequence identity at the nucleotide level. Compared to the wild type strain, the induced antisense FtsZ strain showed a high degree of filamentation (Figure 4). This observation is in accordance to the earlier observation that deficiency of FtsZ causes filamentation in mycobacteria [18], proving that the antisense mRNA was capable of inhibiting the expression of the genomic driven FtsZ sense gene. In order to delineate whether the downstream effect of a target downregulation is bacteriostatic or bactericidal one has to define a mycobacterial benchmark. This is illustrated in the generic mycobacterial survival kinetics (Figure 5) where various zones have been defined. Though, the standard 99.9% kill is defined as bactericidal for antibiotics, the slow growth of M. tuberculosis brings in an element of time dependence while evaluating cidality and stasis. From the survival kinetics of FtsZ antisense strain it was seen that the target behaved in a bacteriostatic fashion. There was a meagre drop of 1.5 log10 in cfu at an inducer concentration of 10 µM IPTG (Figure 6). There was no further reduction in cfu at higher IPTG concentration.

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