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PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence.

Gonzalo-Asensio J, Mostowy S, Harders-Westerveen J, Huygen K, Hernández-Pando R, Thole J, Behr M, Gicquel B, Martín C - PLoS ONE (2008)

Bottom Line: Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation.Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates.We focused this study to decipher the virulence networks regulated by PhoP.

View Article: PubMed Central - PubMed

Affiliation: Grupo de Genética de Micobacterias, Departamento de Microbiología, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, Spain.

ABSTRACT
Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation. Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates. We focused this study to decipher the virulence networks regulated by PhoP. A combined transcriptomic and proteomic analysis revealed that PhoP controls a variety of functions including: hypoxia response through DosR crosstalking, respiratory metabolism, secretion of the major T-cell antigen ESAT-6, stress response, synthesis of pathogenic lipids and the M. tuberculosis persistence through transcriptional regulation of the enzyme isocitrate lyase. We also demonstrate that the M. tuberculosis phoP mutant SO2 exhibits an antigenic capacity similar to that of the BCG vaccine. Finally, we provide evidence that the SO2 mutant persists better in mouse organs than BCG. Altogether, these findings indicate that PhoP orchestrates a variety of functions implicated in M. tuberculosis virulence and persistence, making phoP mutants promising vaccine candidates.

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Related in: MedlinePlus

Determination of sensitivity to the ICL inhibitor 3-NP.The wild type, phoP mutant and complemented strains were tested for their ability to grow in 7H9 medium supplemented with glucose or propionate as sole carbon sources in the presence of 3-NP. A change from blue to pink coloration is indicative of resazurin reduction and consequently it correlates with bacterial viability. No differences in 3-NP sensitivity were observed when the wild type, the phoP mutant or the complemented strains were grown in glucose as unique carbon source. The phoP mutant is less sensitive to 3-NP than the parental strain when grown in propionate as sole carbon supplier, indicating higher levels of ICL expression in the mutant. Complementation of the mutant with the phoPR operon renders bacteria as susceptible to 3-NP as the parental strain when propionate is the unique carbon source.
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pone-0003496-g005: Determination of sensitivity to the ICL inhibitor 3-NP.The wild type, phoP mutant and complemented strains were tested for their ability to grow in 7H9 medium supplemented with glucose or propionate as sole carbon sources in the presence of 3-NP. A change from blue to pink coloration is indicative of resazurin reduction and consequently it correlates with bacterial viability. No differences in 3-NP sensitivity were observed when the wild type, the phoP mutant or the complemented strains were grown in glucose as unique carbon source. The phoP mutant is less sensitive to 3-NP than the parental strain when grown in propionate as sole carbon supplier, indicating higher levels of ICL expression in the mutant. Complementation of the mutant with the phoPR operon renders bacteria as susceptible to 3-NP as the parental strain when propionate is the unique carbon source.

Mentions: In addition to hypoxia adaptation, the ability of M. tuberculosis to persist in the host depends largely on the capacity to utilise fatty acids during infection [3], [4]. Fatty acids are degraded to acetyl-CoA and propionyl-CoA subunits. The glyoxylate pathway is required for carbon anaplerosis of acetyl-CoA during starvation, while the methylcitrate cycle is required for propionyl-CoA metabolism and detoxification [40], [41]. Both pathways share the enzyme ICL [40]. Due to its role in the utilisation of intraphagosomal carbon sources, ICL has been shown to be required for persistence and virulence of M. tuberculosis in macrophage and mice [42]. Our results from the transcriptome and the proteome comparison consistently show as a novel finding that PhoP negatively regulates the expression of ICL as shown in Figure 1 and Figure 2. qRT-PCR studies showed that icl expression was higher in the phoP mutant than in the wild type strain (Figure 3). Unexpectedly, complementation did not restore expression of the icl gene to wild type levels. Consequently in vitro studies were performed to check the complementation of ICL expression. We tested the ability of the ICL inhibitor 3-nitropropionate (3-NP) [43] to block growth of the wild type and the phoP mutant in media containing either glucose or propionate as the sole carbon sources. No differences in 3-NP sensitivity were observed when the wild type or the phoP mutant strains were grown in glucose as carbon substrate. However, when bacteria were forced to induce icl expression to metabolise propionate as unique carbon source [40], the phoP mutant was less sensitive to 3-NP when compared to wild-type bacteria (Figure 5). In addition, complementation of the mutant with the phoPR operon restored 3-NP sensitivity (Figure 5). These results demonstrate that the phoP mutant is better pre-adapted than the parental strain to survive under environmental conditions which require icl expression, and this may possibly due to the higher ICL levels in this mutant.


PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence.

Gonzalo-Asensio J, Mostowy S, Harders-Westerveen J, Huygen K, Hernández-Pando R, Thole J, Behr M, Gicquel B, Martín C - PLoS ONE (2008)

Determination of sensitivity to the ICL inhibitor 3-NP.The wild type, phoP mutant and complemented strains were tested for their ability to grow in 7H9 medium supplemented with glucose or propionate as sole carbon sources in the presence of 3-NP. A change from blue to pink coloration is indicative of resazurin reduction and consequently it correlates with bacterial viability. No differences in 3-NP sensitivity were observed when the wild type, the phoP mutant or the complemented strains were grown in glucose as unique carbon source. The phoP mutant is less sensitive to 3-NP than the parental strain when grown in propionate as sole carbon supplier, indicating higher levels of ICL expression in the mutant. Complementation of the mutant with the phoPR operon renders bacteria as susceptible to 3-NP as the parental strain when propionate is the unique carbon source.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2566814&req=5

pone-0003496-g005: Determination of sensitivity to the ICL inhibitor 3-NP.The wild type, phoP mutant and complemented strains were tested for their ability to grow in 7H9 medium supplemented with glucose or propionate as sole carbon sources in the presence of 3-NP. A change from blue to pink coloration is indicative of resazurin reduction and consequently it correlates with bacterial viability. No differences in 3-NP sensitivity were observed when the wild type, the phoP mutant or the complemented strains were grown in glucose as unique carbon source. The phoP mutant is less sensitive to 3-NP than the parental strain when grown in propionate as sole carbon supplier, indicating higher levels of ICL expression in the mutant. Complementation of the mutant with the phoPR operon renders bacteria as susceptible to 3-NP as the parental strain when propionate is the unique carbon source.
Mentions: In addition to hypoxia adaptation, the ability of M. tuberculosis to persist in the host depends largely on the capacity to utilise fatty acids during infection [3], [4]. Fatty acids are degraded to acetyl-CoA and propionyl-CoA subunits. The glyoxylate pathway is required for carbon anaplerosis of acetyl-CoA during starvation, while the methylcitrate cycle is required for propionyl-CoA metabolism and detoxification [40], [41]. Both pathways share the enzyme ICL [40]. Due to its role in the utilisation of intraphagosomal carbon sources, ICL has been shown to be required for persistence and virulence of M. tuberculosis in macrophage and mice [42]. Our results from the transcriptome and the proteome comparison consistently show as a novel finding that PhoP negatively regulates the expression of ICL as shown in Figure 1 and Figure 2. qRT-PCR studies showed that icl expression was higher in the phoP mutant than in the wild type strain (Figure 3). Unexpectedly, complementation did not restore expression of the icl gene to wild type levels. Consequently in vitro studies were performed to check the complementation of ICL expression. We tested the ability of the ICL inhibitor 3-nitropropionate (3-NP) [43] to block growth of the wild type and the phoP mutant in media containing either glucose or propionate as the sole carbon sources. No differences in 3-NP sensitivity were observed when the wild type or the phoP mutant strains were grown in glucose as carbon substrate. However, when bacteria were forced to induce icl expression to metabolise propionate as unique carbon source [40], the phoP mutant was less sensitive to 3-NP when compared to wild-type bacteria (Figure 5). In addition, complementation of the mutant with the phoPR operon restored 3-NP sensitivity (Figure 5). These results demonstrate that the phoP mutant is better pre-adapted than the parental strain to survive under environmental conditions which require icl expression, and this may possibly due to the higher ICL levels in this mutant.

Bottom Line: Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation.Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates.We focused this study to decipher the virulence networks regulated by PhoP.

View Article: PubMed Central - PubMed

Affiliation: Grupo de Genética de Micobacterias, Departamento de Microbiología, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, Spain.

ABSTRACT
Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation. Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates. We focused this study to decipher the virulence networks regulated by PhoP. A combined transcriptomic and proteomic analysis revealed that PhoP controls a variety of functions including: hypoxia response through DosR crosstalking, respiratory metabolism, secretion of the major T-cell antigen ESAT-6, stress response, synthesis of pathogenic lipids and the M. tuberculosis persistence through transcriptional regulation of the enzyme isocitrate lyase. We also demonstrate that the M. tuberculosis phoP mutant SO2 exhibits an antigenic capacity similar to that of the BCG vaccine. Finally, we provide evidence that the SO2 mutant persists better in mouse organs than BCG. Altogether, these findings indicate that PhoP orchestrates a variety of functions implicated in M. tuberculosis virulence and persistence, making phoP mutants promising vaccine candidates.

Show MeSH
Related in: MedlinePlus