Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis

The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory. A devR::kan mutant of M. tuberculosis was constructed by allelic exchange. The devR mutant strain showed reduced cell-to-cell adherence in comparison to the parental strain in laboratory culture media. This phenotype was reversed on complementation with a wild-type copy of devR. The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells. The expression of DevR was not modulated upon entry of M. tuberculosis into human monocytes. However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain. Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M. tuberculosis within human monocytes in vitro. The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M. tuberculosis within tissues.     

The devR gene product is characteristic of receivers of two-component regulatory systems and is essential for heterocyst development in the filamentous cyanobacterium Nostoc sp. strain ATCC 29133.

Strain UCD 311 is a transposon-induced mutant of Nostoc sp. strain ATC C 29133 that is unable to fix nitrogen in air but does so under anoxic conditions and is able to establish a functional symbiotic association with the hornwort Anthoceros punctatus. These properties of strain UCD 311 are consistent with previous observations that protection against oxygen inactivation of nitrogenase is physiologically provided within A. punctatus tissue. Upon deprivation of combined nitrogen, strain UCD 311 clearly differentiates heterocysts and contains typical heterocyst-specific glycolipids; it also makes apparently normal akinetes upon phosphate starvation. Sequence analysis adjacent to the point of the transposon insertion revealed an open reading frame designated devR. Southern analysis established that similar sequences are present in other heterocyst-forming cyanobacteria. devR putatively encodes a protein of 135 amino acids with high similarity to the receiver domains of response regulator proteins characteristics of two-component regulatory systems. On the basis of its size and the absence of other functional domains, DevR is most similar to CheY and Spo0F. Reconstruction of the mutation with an interposon vector confirmed that the transposition event was responsible for the mutant phenotype. The presence of wild-type devR on a plasmid in strain UCD 311 restored the ability to fix nitrogen in air. While devR was not essential for differentiation of akinetes, its presence in trans in Nostoc sp. strain ATCC 29133 stimulated their formation to above normal levels in aging medium. On the basis of RNA analysis, devR is constitutively expressed with respect to the nitrogen source for growth. The devR gene product is essential to the development of mature heterocysts and may be involved in a sensory pathway that is not directly responsive to cellular nitrogen status.     

Biochemical and Genetic Evidence for Participation of DevR in a Phosphorelay Signal Transduction Pathway Essential for Heterocyst Maturation in Nostoc punctiforme ATCC 29133

In a test of the hypothesis that DevR is a response regulator protein that functions in a phosphorelay signal transduction system involved in heterocyst development in Nostoc punctiforme ATCC 29133, purified affinity-tagged DevR was shown to be phosphorylated in vitro by the noncognate sensor kinase EnvZ. Site-directed mutagenesis was used to generate N. punctiforme mutants with single amino acid substitutions at the putative phosphorylation site of DevR. These mutants exhibited a Fox- phenotype like the original devR insertion mutant UCD 311, consistent with a phosphotransferase role for DevR.     

Large Mg(2+)-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae

Mycobacteria adapt to a decrease in oxygen tension by entry into a non-replicative persistent phase. It was shown earlier that the two-component system, DevR-DevS, was induced in Mycobacterium tuberculosis and Mycobacterium bovis BCG cultures during hypoxia, suggesting that it may play a regulatory role in their adaptation to oxygen limitation. The presence of a homologous genetic system in Mycobacterium smegmatis was predicted by scanning its unfinished genome sequence with devR and devS genes of M. tuberculosis. Rv3134c, which is cotranscribed with devR-devS in M. tuberculosis, was also present in M. smegmatis at a similar location upstream from devR. The expression of all three genes was induced at the RNA and protein levels in M. smegmatis cultures grown under microaerobic and anaerobic conditions. The M. smegmatis genome also contained the hspX gene, encoding chaperone alpha-crystallin, Acr, that was induced during hypoxia. The similarity in sequences and hypoxia-responsive behaviour of devR-devS, Rv3134c and hspX genes in M. smegmatis and M. tuberculosis suggests that the molecular mechanisms involved in the dormancy response are likely conserved in these two species. M. smegmatis could therefore serve as a useful model for the delineation of the hypoxia response in general and DevR-DevS regulated pathways in particular.     

Co-Expression of DevR and DevRN-Aph Proteins Is Associated with Hypoxic Adaptation Defect and Virulence Attenuation of Mycobacterium tuberculosis

Background

The DevR response regulator is implicated in both hypoxic adaptation and virulence of Mycobacterium tuberculosis (M. tb). DevR regulon genes are powerfully induced in vivo implicating them in bacterial adaptation to host control strategies. A better understanding of DevR function will illumine the way for new strategies to control and treat tuberculosis.

Methodology/Principal Findings

Towards this objective, we used a combination of genetic, microbiological, biochemical, cell biological tools and a guinea pig virulence assay to compare the hypoxic adaptation and virulence properties of two novel M. tb strains, namely, a devR disruption mutant, Mut1, that expresses C-terminal truncated N-terminal domain of DevR (DevR_NTD) as a fusion protein with AphI (DevR_N-Kan), and its complemented strain, Comp1, that expresses intact DevR along with DevR_N-Kan. Comp1 bacteria exhibit a defect in DevR-mediated phosphosignalling, hypoxic induction of HspX and also hypoxic survival. In addition, we find that Comp1 is attenuated in virulence in guinea pigs and shows decreased infectivity of THP-1 cells. While Mut1 bacilli are also defective in hypoxic adaptation and early growth in spleen, they exhibit an overall virulence comparable to that of wild-type bacteria.

Conclusions/Significance

The hypoxic defect of Comp1 is associated to a defect in DevR expression level. The demonstrated repression of DevR function by DevR_N-Kan suggests that such a knockdown approach could be useful for evaluating the activity of DevRS and other two-component signaling pathways. Further investigation is necessary to elucidate the mechanism underlying Comp1 attenuation.     

Global role for ClpP-containing proteases in stationary-phase adaptation of Escherichia coli

To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the clpA, clpX, and clpP protease mutants of Escherichia coli were subjected to proteome analysis during growth and during carbon starvation. For most of the growth-phase-regulated proteins detected on our gels, the clpA, clpX, or clpP mutant failed to mount the growth-phase regulation found in the wild type. For example, in the clpP and clpA mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells. On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase. The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC). These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism. From our data we conclude that the levels of most major growth-phase-regulated proteins in E. coli are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins. Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type. Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.     

Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12

Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, sigma(S) (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). We now report that a second global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of an rpoS strain had acquired a GASP mutation in lrp. This GASP allele, lrp-1141, encodes a mutant protein lacking the critical glycine in the turn of the helix-turn-helix DNA-binding domain. The lrp-1141 allele behaves as a null mutation when in single copy and is dominant negative when overexpressed. Hence, the mutant protein appears to retain stability and the ability to dimerize but lacks DNA-binding activity. We also demonstrated that a lrp null allele generated by a transposon insertion has a fitness gain identical to that of the lrp-1141 allele, verifying that cells lacking Lrp activity have a competitive advantage during prolonged starvation. Finally, we tested by genetic analysis the hypothesis that the lrp-1141 GASP mutation confers a fitness gain by enhancing amino acid catabolism during carbon starvation. We found that while amino acid catabolism may play a role, it is not necessary for the lrp GASP phenotype, and hence the lrp GASP phenotype is due to more global physiological changes.     

Function of the cytochrome bc1-aa3 branch of the respiratory network in mycobacteria and network adaptation occurring in response to its disruption

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc1 complex and terminating in the aa3-type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc1 complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O2-limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd-type oxidase were revealed by expression profiling of the bc1-deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc1-aa3 pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.     

Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae.

Starvation of cells of the yeast Saccharomyces cerevisiae causes cessation of proliferation and acquisition of characteristic physiological properties. The stationary-phase state that results represents a unique developmental state, as shown by a novel conditional phenotype (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987): mutant cells cannot proliferate at the restrictive temperature when stimulated to reenter the mitotic cell cycle from stationary phase but are unaffected and continue proliferation indefinitely if transferred to the restrictive temperature during exponential growth. We have exploited this reentry mutant phenotype to demonstrate that the same stationary-phase state is generated by nitrogen, sulfur, or carbon starvation and by the cdc25-1 mutation, which conditionally impairs the cyclic AMP-mediated signal transduction pathway. We also show that heat shock, a treatment that elicits physiological perturbations associated with stationary phase, does not cause cells to enter stationary phase. The physiological properties associated with stationary phase therefore do not result from residence in stationary phase but from the stress conditions that bring about stationary phase.     

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr(128) protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationary-phase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the rpsL gene promoter (which heads the Mycobacterium smegmatis operon encoding the tuf gene encoding EF-Tu) or the rrnA promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so rpsL expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing rpsL at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.     

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in stationary-phase cultures of Bacillus subtilis.

Glutamine phosphoribosylpyrophosphate amidotransferase (ATase) activity is rapidly inactivated in stationary-phase cells of Bacillus subtilis. The inactivation of APase requires both the cessation of rapid cell growth and the presence of oxygen. ATase is inactivated in two protease-deficient mutant strains at a rate similar to that seen in the wild type, and is stable in anaerobic cell-free extracts of the parent strain. These results suggest that the inactivation of ATase is not the result of general proteolysis. The inactivation of ATase in stationary-phase cultures can be inhibited by oxygen starvation. This oxygen requirement does not reflect a dependence on the generation of metabolic energy, but appears to be a direct requirement for molecular oxygen. ATase synthesis is repressed by the addition of adenosine, and is inactivated only after the cessation of exponential growth. Addition of chloramphenicol or rifampin to exponential- and stationary-phase cells does not inhibit ATase inactivation, suggesting that protein or ribonucleic acid synthesis is not required for inactivation. ATase is inactivated at the end of exponential growth in cells that have exhausted a required amino acid.