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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

Quantification of gene expression by qRT-PCR.Relative expression levels of the dosR, nuoB, lipF, pks3 and icl genes. The relative quantity (RQ) for each gene in the phoP mutant and the complemented strain were calculated with respect to the gene expression levels in the wild type strain. The expression levels of each gene in each strain were normalized to the levels of sigA mRNA. Primers and probe sequences for the aforementioned genes as well as for the endogenous control sigA are listed in Table S3.
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pone-0003496-g003: Quantification of gene expression by qRT-PCR.Relative expression levels of the dosR, nuoB, lipF, pks3 and icl genes. The relative quantity (RQ) for each gene in the phoP mutant and the complemented strain were calculated with respect to the gene expression levels in the wild type strain. The expression levels of each gene in each strain were normalized to the levels of sigA mRNA. Primers and probe sequences for the aforementioned genes as well as for the endogenous control sigA are listed in Table S3.

Mentions: Under the initial hypoxic conditions within macrophages, M. tuberculosis enters a dormant state characterized by the induction of the so called dormancy regulon which includes approximately 50 genes [5], 18 under the control of 2CS DosRST [19]–[21]. In this work we show that part of the DosR regulon, including the dosRS genes themselves, is under the control of PhoP as indicated as initial hypoxic response in Figure 1. In addition, alpha crystallin - a latency antigen which also belongs to the DosR regulon [5] - also appears downregulated in the phoP mutant in our proteome comparison (Table S2). Altogether, these observations indicate that PhoP might regulate the dormancy regulon through crosstalking with DosR. To really confirm that dosR is under the control of PhoP, we carried out qRT-PCR analyses. Our results demonstrate that dosR expression is reduced in the phoP mutant with respect to the wild type strain and, complementation of the phoP mutant with the phoPR operon restores dosR expression to wild type levels (Figure 3). The DosRS 2CS was initially discovered for being higher expressed in the virulent M. tuberculosis H37Rv than in its avirulent counterpart H37Ra [22], [23]. Hence, DosRS was initially named DevRS, an acronym for differentially expressed in virulent strain. Here, we demonstrate by qRT-PCR that dosR is downregulated in H37Ra with respect to H37Rv (Figure S1). This is probably a consequence of a Ser219Leu mutation in PhoP from H37Ra. On the other hand, it has been recently demonstrated that following the initial adaptation to hypoxia through the DosR regulon, M. tuberculosis initiates an EHR [7]. Interestingly, we show for the first time that PhoP also regulates a subset of genes from the EHR as indicated as enduring hypoxic response in Figure 1. In sum, these findings suggest that PhoP serves as a link between the early and enduring hypoxia responses in M. tuberculosis.


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)

Quantification of gene expression by qRT-PCR.Relative expression levels of the dosR, nuoB, lipF, pks3 and icl genes. The relative quantity (RQ) for each gene in the phoP mutant and the complemented strain were calculated with respect to the gene expression levels in the wild type strain. The expression levels of each gene in each strain were normalized to the levels of sigA mRNA. Primers and probe sequences for the aforementioned genes as well as for the endogenous control sigA are listed in Table S3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003496-g003: Quantification of gene expression by qRT-PCR.Relative expression levels of the dosR, nuoB, lipF, pks3 and icl genes. The relative quantity (RQ) for each gene in the phoP mutant and the complemented strain were calculated with respect to the gene expression levels in the wild type strain. The expression levels of each gene in each strain were normalized to the levels of sigA mRNA. Primers and probe sequences for the aforementioned genes as well as for the endogenous control sigA are listed in Table S3.
Mentions: Under the initial hypoxic conditions within macrophages, M. tuberculosis enters a dormant state characterized by the induction of the so called dormancy regulon which includes approximately 50 genes [5], 18 under the control of 2CS DosRST [19]–[21]. In this work we show that part of the DosR regulon, including the dosRS genes themselves, is under the control of PhoP as indicated as initial hypoxic response in Figure 1. In addition, alpha crystallin - a latency antigen which also belongs to the DosR regulon [5] - also appears downregulated in the phoP mutant in our proteome comparison (Table S2). Altogether, these observations indicate that PhoP might regulate the dormancy regulon through crosstalking with DosR. To really confirm that dosR is under the control of PhoP, we carried out qRT-PCR analyses. Our results demonstrate that dosR expression is reduced in the phoP mutant with respect to the wild type strain and, complementation of the phoP mutant with the phoPR operon restores dosR expression to wild type levels (Figure 3). The DosRS 2CS was initially discovered for being higher expressed in the virulent M. tuberculosis H37Rv than in its avirulent counterpart H37Ra [22], [23]. Hence, DosRS was initially named DevRS, an acronym for differentially expressed in virulent strain. Here, we demonstrate by qRT-PCR that dosR is downregulated in H37Ra with respect to H37Rv (Figure S1). This is probably a consequence of a Ser219Leu mutation in PhoP from H37Ra. On the other hand, it has been recently demonstrated that following the initial adaptation to hypoxia through the DosR regulon, M. tuberculosis initiates an EHR [7]. Interestingly, we show for the first time that PhoP also regulates a subset of genes from the EHR as indicated as enduring hypoxic response in Figure 1. In sum, these findings suggest that PhoP serves as a link between the early and enduring hypoxia responses in M. tuberculosis.

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