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Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro.

Hingley-Wilson SM, Lougheed KE, Ferguson K, Leiva S, Williams HD - Tuberculosis (Edinb) (2010)

Bottom Line: However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines.Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb.The possibility remains that these USPs are functionally redundant in Mtb.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
Mycobacterium tuberculosis has 10 universal stress proteins, whose function is unknown. However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines. Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb. Knock-out mutants of individual usp genes encoding the USPs Rv1996, Rv2005c, Rv2026c and Rv2028c were generated and their growth and survival under hypoxic and other stress conditions examined. Although the majority of usp genes are highly induced in hypoxic conditions, mutation did not affect the long term survival of Mtb under these conditions, or in response to a range of stress conditions chosen to represent the environmental onslaughts experienced by the bacillus during an infection, nor during infection of mouse and human - derived macrophage cell lines. The possibility remains that these USPs are functionally redundant in Mtb.

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hspX promoter activity in M. tuberculosis H37Rv and ΔRv2026c. The plasmid pMH108 carrying the hspX promoter region upstream of the firefly luciferase genes was transformed into Mtb H37Rv and ΔRv2026c. The promoter activity was measured in cultures grown under oxygen-sufficient conditions to mid-exponential phase and stationary phase and in cultures grown to hypoxic stationary phase. Relative light units (RLU) were determined and normalised using the OD of the cultures. The hspX promoter was seen to be active in both the wildtype and the ΔRv2026c mutants strain. Error bars represent the standard deviations of 3 independent cultures.
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fig5: hspX promoter activity in M. tuberculosis H37Rv and ΔRv2026c. The plasmid pMH108 carrying the hspX promoter region upstream of the firefly luciferase genes was transformed into Mtb H37Rv and ΔRv2026c. The promoter activity was measured in cultures grown under oxygen-sufficient conditions to mid-exponential phase and stationary phase and in cultures grown to hypoxic stationary phase. Relative light units (RLU) were determined and normalised using the OD of the cultures. The hspX promoter was seen to be active in both the wildtype and the ΔRv2026c mutants strain. Error bars represent the standard deviations of 3 independent cultures.

Mentions: Rv2026c is not a part of the DosR regulon, and its expression is not induced by hypoxia or NO, and while it is expressed during starvation in PBS (Table 1), the ΔRv2026c mutant showed a comparable survival defect to the wildtype under these conditions (data not shown). RT-PCR experiments showed that Rv2026c was transcribed during exponential phase, normoxic and hypoxic stationary phase and as expected mutation of dosR did not affect expression levels (not shown). Rv2026c is adjacent to dosT (Rv2027c) (Figure 2C), encoding the DosT histidine kinase partner of DosR, and its apparent constitutive expression led us to consider whether this USP might be involved in DosR mediated induction of genes. Both the sensor kinases DosT and DosS activate the response regulator DosR, resulting in induction of the DosR regulon, with the evidence being that DosT preferentially responds to hypoxia,52 and a dosT mutant was only able to induce DosR regulon expression to 40–45% of normal levels (Roberts et al., 2004). The possible role of Rv2026c in the DosR pathway was tested using a luciferase reporter assay with a plasmid construct containing the hspX promoter region upstream of firefly luciferase gene.20 The marked induction of hspX expression under hypoxic conditions occurred in wildtype H37Rv, as expected, and the ΔRv2026c mutant showed no difference in hspX expression indicating that Rv2026c is not involved in the expression of DosR-regulated genes (Figure 5).


Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro.

Hingley-Wilson SM, Lougheed KE, Ferguson K, Leiva S, Williams HD - Tuberculosis (Edinb) (2010)

hspX promoter activity in M. tuberculosis H37Rv and ΔRv2026c. The plasmid pMH108 carrying the hspX promoter region upstream of the firefly luciferase genes was transformed into Mtb H37Rv and ΔRv2026c. The promoter activity was measured in cultures grown under oxygen-sufficient conditions to mid-exponential phase and stationary phase and in cultures grown to hypoxic stationary phase. Relative light units (RLU) were determined and normalised using the OD of the cultures. The hspX promoter was seen to be active in both the wildtype and the ΔRv2026c mutants strain. Error bars represent the standard deviations of 3 independent cultures.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2914252&req=5

fig5: hspX promoter activity in M. tuberculosis H37Rv and ΔRv2026c. The plasmid pMH108 carrying the hspX promoter region upstream of the firefly luciferase genes was transformed into Mtb H37Rv and ΔRv2026c. The promoter activity was measured in cultures grown under oxygen-sufficient conditions to mid-exponential phase and stationary phase and in cultures grown to hypoxic stationary phase. Relative light units (RLU) were determined and normalised using the OD of the cultures. The hspX promoter was seen to be active in both the wildtype and the ΔRv2026c mutants strain. Error bars represent the standard deviations of 3 independent cultures.
Mentions: Rv2026c is not a part of the DosR regulon, and its expression is not induced by hypoxia or NO, and while it is expressed during starvation in PBS (Table 1), the ΔRv2026c mutant showed a comparable survival defect to the wildtype under these conditions (data not shown). RT-PCR experiments showed that Rv2026c was transcribed during exponential phase, normoxic and hypoxic stationary phase and as expected mutation of dosR did not affect expression levels (not shown). Rv2026c is adjacent to dosT (Rv2027c) (Figure 2C), encoding the DosT histidine kinase partner of DosR, and its apparent constitutive expression led us to consider whether this USP might be involved in DosR mediated induction of genes. Both the sensor kinases DosT and DosS activate the response regulator DosR, resulting in induction of the DosR regulon, with the evidence being that DosT preferentially responds to hypoxia,52 and a dosT mutant was only able to induce DosR regulon expression to 40–45% of normal levels (Roberts et al., 2004). The possible role of Rv2026c in the DosR pathway was tested using a luciferase reporter assay with a plasmid construct containing the hspX promoter region upstream of firefly luciferase gene.20 The marked induction of hspX expression under hypoxic conditions occurred in wildtype H37Rv, as expected, and the ΔRv2026c mutant showed no difference in hspX expression indicating that Rv2026c is not involved in the expression of DosR-regulated genes (Figure 5).

Bottom Line: However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines.Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb.The possibility remains that these USPs are functionally redundant in Mtb.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
Mycobacterium tuberculosis has 10 universal stress proteins, whose function is unknown. However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines. Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb. Knock-out mutants of individual usp genes encoding the USPs Rv1996, Rv2005c, Rv2026c and Rv2028c were generated and their growth and survival under hypoxic and other stress conditions examined. Although the majority of usp genes are highly induced in hypoxic conditions, mutation did not affect the long term survival of Mtb under these conditions, or in response to a range of stress conditions chosen to represent the environmental onslaughts experienced by the bacillus during an infection, nor during infection of mouse and human - derived macrophage cell lines. The possibility remains that these USPs are functionally redundant in Mtb.

Show MeSH
Related in: MedlinePlus