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The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro.

Kana BD, Gordhan BG, Downing KJ, Sung N, Vostroktunova G, Machowski EE, Tsenova L, Young M, Kaprelyants A, Kaplan G, Mizrahi V - Mol. Microbiol. (2008)

Bottom Line: The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB.Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection.In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

View Article: PubMed Central - PubMed

Affiliation: MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, School of Pathology, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg 2000, South Africa.

ABSTRACT
Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

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Growth and survival of multiple rpf-like mutants in human monocytes and in B6D2/F1 mice.A. Human monocytes were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and growth was monitored over 4 days. Experiments were conducted with cells isolated from two healthy donors.B. Growth and survival of quadruple mutants in mouse lungs. B6D2/F1 mice were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and the bacillary loads in the lungs of the infected animals were determined by cfu assessment over a period of 240 days. Each point represents the mean of three mice per group and the error bars denote the standard deviations. The lung bacillary loads that differ significantly from those of the wild-type control are denoted by an asterisk above the relevant data point (P < 0.0001 for all points except ΔACBD at 240 days, where P = 0.0024).C. Effect of complementation of ΔACBE with rpfC, rpfD and rpfE genes on growth in mouse lungs. B6D2/F1 mice were infected with H37Rv (♦), ΔACBE (▴) or ΔACBE + CDE (□) and lung bacillary loads were assessed over 77 days. The individual bacillary loads or the mean of two or three infected mice per group are shown.
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fig03: Growth and survival of multiple rpf-like mutants in human monocytes and in B6D2/F1 mice.A. Human monocytes were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and growth was monitored over 4 days. Experiments were conducted with cells isolated from two healthy donors.B. Growth and survival of quadruple mutants in mouse lungs. B6D2/F1 mice were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and the bacillary loads in the lungs of the infected animals were determined by cfu assessment over a period of 240 days. Each point represents the mean of three mice per group and the error bars denote the standard deviations. The lung bacillary loads that differ significantly from those of the wild-type control are denoted by an asterisk above the relevant data point (P < 0.0001 for all points except ΔACBD at 240 days, where P = 0.0024).C. Effect of complementation of ΔACBE with rpfC, rpfD and rpfE genes on growth in mouse lungs. B6D2/F1 mice were infected with H37Rv (♦), ΔACBE (▴) or ΔACBE + CDE (□) and lung bacillary loads were assessed over 77 days. The individual bacillary loads or the mean of two or three infected mice per group are shown.

Mentions: To assess the effect of rpf-like gene loss on intracellular growth of M. tuberculosis, human peripheral blood mononuclear cells (PBMCs) were infected with ΔACBD, ΔACBE or H37Rv and growth monitored over 4 days. Both quadruple mutants grew similarly to the wild-type strain in this model (Fig. 3A). Moreover, both strains retained their respective colony-forming abilities following passage in monocytes: normal for ΔACBD and retarded in the case of ΔACBE (data not shown).


The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro.

Kana BD, Gordhan BG, Downing KJ, Sung N, Vostroktunova G, Machowski EE, Tsenova L, Young M, Kaprelyants A, Kaplan G, Mizrahi V - Mol. Microbiol. (2008)

Growth and survival of multiple rpf-like mutants in human monocytes and in B6D2/F1 mice.A. Human monocytes were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and growth was monitored over 4 days. Experiments were conducted with cells isolated from two healthy donors.B. Growth and survival of quadruple mutants in mouse lungs. B6D2/F1 mice were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and the bacillary loads in the lungs of the infected animals were determined by cfu assessment over a period of 240 days. Each point represents the mean of three mice per group and the error bars denote the standard deviations. The lung bacillary loads that differ significantly from those of the wild-type control are denoted by an asterisk above the relevant data point (P < 0.0001 for all points except ΔACBD at 240 days, where P = 0.0024).C. Effect of complementation of ΔACBE with rpfC, rpfD and rpfE genes on growth in mouse lungs. B6D2/F1 mice were infected with H37Rv (♦), ΔACBE (▴) or ΔACBE + CDE (□) and lung bacillary loads were assessed over 77 days. The individual bacillary loads or the mean of two or three infected mice per group are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229633&req=5

fig03: Growth and survival of multiple rpf-like mutants in human monocytes and in B6D2/F1 mice.A. Human monocytes were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and growth was monitored over 4 days. Experiments were conducted with cells isolated from two healthy donors.B. Growth and survival of quadruple mutants in mouse lungs. B6D2/F1 mice were infected with H37Rv (♦), ΔACBD (▪) or ΔACBE (▴) and the bacillary loads in the lungs of the infected animals were determined by cfu assessment over a period of 240 days. Each point represents the mean of three mice per group and the error bars denote the standard deviations. The lung bacillary loads that differ significantly from those of the wild-type control are denoted by an asterisk above the relevant data point (P < 0.0001 for all points except ΔACBD at 240 days, where P = 0.0024).C. Effect of complementation of ΔACBE with rpfC, rpfD and rpfE genes on growth in mouse lungs. B6D2/F1 mice were infected with H37Rv (♦), ΔACBE (▴) or ΔACBE + CDE (□) and lung bacillary loads were assessed over 77 days. The individual bacillary loads or the mean of two or three infected mice per group are shown.
Mentions: To assess the effect of rpf-like gene loss on intracellular growth of M. tuberculosis, human peripheral blood mononuclear cells (PBMCs) were infected with ΔACBD, ΔACBE or H37Rv and growth monitored over 4 days. Both quadruple mutants grew similarly to the wild-type strain in this model (Fig. 3A). Moreover, both strains retained their respective colony-forming abilities following passage in monocytes: normal for ΔACBD and retarded in the case of ΔACBE (data not shown).

Bottom Line: The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB.Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection.In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

View Article: PubMed Central - PubMed

Affiliation: MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, School of Pathology, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg 2000, South Africa.

ABSTRACT
Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

Show MeSH
Related in: MedlinePlus