[Show abstract][Hide abstract] ABSTRACT: Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin
from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF
family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA
in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9,
and each was cleaved at a single site in either the tRNAPro14 D-loop or within the tRNALys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural
determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for
recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that
also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role
for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial
Preview · Article · Jan 2016 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Toxin-antitoxin (TA) systems are implicated in the downregulation of bacterial cell growth associated with stress survival and latent tuberculosis infection, yet the activities and intracellular targets of these TA toxins are largely uncharacterized. Here, we use a specialized RNA-seq approach to identify targets of a Mycobacterium tuberculosis VapC TA toxin, VapC-mt4 (also known as VapC4), which have eluded detection using conventional approaches. Distinct from the one other characterized VapC toxin in M. tuberculosis that cuts 23S rRNA at the sarcin-ricin loop, VapC-mt4 selectively targets three of the 45 M. tuberculosis tRNAs (tRNA(Ala2), tRNA(Ser26) and tRNA(Ser24)) for cleavage at, or adjacent to, their anticodons, resulting in the generation of tRNA halves. While tRNA cleavage is sometimes enlisted as a bacterial host defense mechanism, VapC-mt4 instead alters specific tRNAs to inhibit translation and modulate growth. This stress-linked activity of VapC-mt4 mirrors basic features of eukaryotic tRNases that also generate tRNA halves and inhibit translation in response to stress.
No preview · Article · Jul 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium tuberculosis genome encodes five putative "alternative" ribosomal proteins whose expression is repressed at high Zn(2+) concentration. Each alternative protein has a primary homolog that is predicted to bind Zn(2+) . We hypothesized that zinc triggers a switch between these paired homologous proteins and therefore chose one of these pairs, S18-1/S18-2, to study mechanisms of the predicted competition for their incorporation into ribosomes. As predicted, our data show that Zn(2+) -depletion causes accumulation of both S18-2 mRNA and protein. In contrast, S18-1 mRNA levels are unchanged to slightly elevated under Zn(2+) -limited conditions. However the amount of S18-1 protein is markedly decreased. We further demonstrate that both S18 proteins interact with ribosomal protein S6, a committed step in ribosome biogenesis. Zn(2+) is absolutely required for the S18-1/S6 interaction, while it is dispensable for S18-2/S6 dimer formation. These data suggest a model in which the S18-1 is the dominant ribosome constituent in high zinc conditions, e.g. inside of phagosomes, but that it can be replaced by S18-2 when zinc is deficient, e.g. in the extracellular milieu. Consequently, Zn(2+) -depletion may serve as a signal for building alternative ribosomes when M. tuberculosis is released from macrophages, to allow survival in the extracellular environment.
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No preview · Article · Apr 2015 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.
Full-text · Article · Sep 2014 · The Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: To persist and cause disease in the host, Mycobacterium tuberculosis must adapt to its environment during infection. Adaptations include changes in nutrient utilization and alterations in growth
rate. M. tuberculosis Rv1422 is a conserved gene of unknown function that was found in a genetic screen to interact with the mce4 cholesterol uptake locus. The Rv1422 protein is phosphorylated by the M. tuberculosis Ser/Thr kinases PknA and PknB, which regulate cell growth and cell wall synthesis. Bacillus subtilis strains lacking the Rv1422 homologue yvcK grow poorly on several carbon sources, and yvcK is required for proper localization of peptidoglycan synthesis. Here we show that Mycobacterium smegmatis and M. tuberculosis strains lacking Rv1422 have growth defects in minimal medium containing limiting amounts of several different carbon sources.
These strains also have morphological abnormalities, including shortened and bulging cells, suggesting a cell wall defect.
In both mycobacterial species, the Rv1422 protein localizes uniquely to the growing cell pole, the site of peptidoglycan synthesis
in mycobacteria. An M. tuberculosis ΔRv1422 strain is markedly attenuated for virulence in a mouse infection model, where it elicits decreased inflammation in
the lungs and shows impaired bacterial persistence. These findings led us to name this gene cuvA (carbon utilization and virulence protein A) and to suggest a model in which deletion of cuvA leads to changes in nutrient uptake and/or metabolism that affect cell wall structure, morphology, and virulence. Its role
in virulence suggests that CuvA may be a useful target for novel inhibitors of M. tuberculosis during infection.
Full-text · Article · Jul 2014 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium tuberculosis genome contains an unusually high number of toxin-antitoxin modules, some of which have been suggested to play a role in the establishment and maintenance of latent tuberculosis. Nine of these toxin-antitoxin loci belong to the mazEF family, encoding the intracellular toxin MazF and its antitoxin inhibitor MazE. Nearly every MazF ortholog recognizes a unique three- or five-base RNA sequence and cleaves mRNA. As a result, these toxins selectively target a subset of the transcriptome for degradation and are known as "mRNA interferases." Here we demonstrate that a MazF family member from M. tuberculosis, MazF-mt6, has an additional role-inhibiting translation through targeted cleavage of 23S rRNA in the evolutionarily conserved helix/loop 70. We first determined that MazF-mt6 cleaves mRNA at (5)(')UU↓CCU(3') sequences. We then discovered that MazF-mt6 also cleaves M. tuberculosis 23S rRNA at a single UUCCU in the ribosomal A site that contacts tRNA and ribosome recycling factor. To gain further mechanistic insight, we demonstrated that MazF-mt6-mediated cleavage of rRNA can inhibit protein synthesis in the absence of mRNA cleavage. Finally, consistent with the position of 23S rRNA cleavage, MazF-mt6 destabilized 50S-30S ribosomal subunit association. Collectively, these results show that MazF toxins do not universally act as mRNA interferases, because MazF-mt6 inhibits protein synthesis by cleaving 23S rRNA in the ribosome active center.
Full-text · Article · May 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.
[Show abstract][Hide abstract] ABSTRACT: We report a remarkably good outcome in a 14-month-old boy with early clinical diagnosis and aggressive empirical treatment of neural larva migrans caused by the raccoon roundworm Baylisascaris procyonis. He presented with fever, meningismus, lethargy, irritability and asymmetric spastic extremity weakness. Early findings of marked blood and cerebrospinal fluid eosinophilia and of diffuse white matter signal abnormality in the brain and spinal cord on MRI suggested a parasitic encephalomyelitis. Rapid presumptive treatment with albendazole and high-dose steroids halted progression of clinical signs. The diagnosis was confirmed by 2 sequential enzyme-linked immunosorbent assay studies positive for B procyonis serum immunoglobulin G and by Western blot. Field examination with soil sampling yielded infective Baylisascaris eggs. Repeat MRI 3 months later showed atrophy and diffuse, chronic white matter abnormalities, discordant with the marked clinical improvement in this interval. At 10 months, residual neurologic deficits included subtle paraparesis and moderate language delay. This case is the first in which spinal involvement in human Baylisascaris infection was clinically suspected and confirmed by neuroimaging. Importantly, early diagnosis and aggressive treatment of Baylisascaris meningo-encephalitis and myelitis with albendazole and high-dose steroids likely contributed to the good outcome in this patient, in contrast with previous reports.
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA.
Preview · Article · Feb 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Due to the inexorable invasion of our hospitals and communities by drug-resistant bacteria, there is a pressing need for novel antibacterial agents. Here we report the development of a sensitive and robust but low-tech and inexpensive high-throughput metabolic screen for novel antibiotics. This screen is based on a colorimetric assay of pH that identifies inhibitors of bacterial sugar fermentation. After validation of the method, we screened over 39,000 crude extracts derived from organisms that grow in the diverse ecosystems of Costa Rica and identified 49 with reproducible antibacterial effects. An extract from an endophytic fungus was further characterized, and this led to the discovery of three novel natural products. One of these, which we named mirandamycin, has broad-spectrum antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, methicillin-resistant Staphylococcus aureus, and Mycobacterium tuberculosis. This demonstrates the power of simple high throughput screens for rapid identification of new antibacterial agents from environmental samples.
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.
[Show abstract][Hide abstract] ABSTRACT: While protein kinases are key components in multiple cellular processes, efficient identification of cognate in vivo substrates remains challenging. Here we describe a powerful method to screen potential substrates of protein kinases by partial transfer of proteins from a 2D-PAGE gel to a Western blot membrane. This approach allowed precise pinpointing of candidate substrate spots in the 2D gel, and identifying physiological substrates of protein kinases in Mycobacterium tuberculosis.
No preview · Article · Jun 2011 · The Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins.
Preview · Article · Apr 2010 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The ability to ectopically control gene expression is a fundamental tool for the study of bacterial physiology and pathogenesis. While many efficient inducible expression systems are available for Gram-negative bacteria, few are useful in phylogenetically distant organisms, such as mycobacteria. We have adapted a highly-inducible regulon of Rhodococcus rhodochrous to artificially regulate gene expression in both rapidly-growing environmental mycobacteria and slow-growing pathogens, such as Mycobacterium tuberculosis. We demonstrate that this artificial regulatory circuit behaves as a bistable switch, which can be manipulated regardless of growth phase in vitro, and during intracellular growth in macrophages. High-level overexpression is also possible, facilitating biochemical and structural studies of mycobacterial proteins produced in their native host.
Preview · Article · Oct 2008 · Tuberculosis (Edinburgh, Scotland)
[Show abstract][Hide abstract] ABSTRACT: SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in Mycobacterium tuberculosis, and this sigma factor is required for virulence in animal models of infection. SigH is negatively regulated by RshA, its cognate anti-sigma factor, which functions as a stress sensor and redox switch. While RshA provides a direct mechanism for sensing stress and activating transcription, bacteria use several types of signal transduction systems to sense the external environment. M. tuberculosis encodes several serine-threonine protein kinase signaling molecules, 2 of which, PknA and PknB, are essential and have been shown to regulate cell morphology and cell wall synthesis. In this work, we demonstrate that SigH and RshA are phosphorylated in vitro and in vivo by PknB. We show that phosphorylation of RshA, but not SigH, interferes with the interaction of these 2 proteins in vitro. Consistent with this finding, negative regulation of SigH activity by RshA in vivo is partially relieved in strains in which pknB is over-expressed, resulting in increased resistance to oxidative stress. These findings demonstrate an interaction between the signaling pathways mediated by PknB and the stress response regulon controlled by SigH. The intersection of these apparently discrete regulatory systems provides a mechanism by which limited activation of the SigH-dependent stress response in M. tuberculosis can be achieved. Coordination of the PknB and SigH regulatory pathways through phosphorylation of RshA may lead to adaptive responses that are important in the pathogenesis of M. tuberculosis infection.
Preview · Article · Oct 2008 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7) and determined cleavage specificities for MazF-mt1 and MazF-mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF-mt3 cleaves RNA at UU CCU or CU CCU and MazF-mt7 at U CGCU ( indicates the cleavage site). As pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF-mt3 and MazF-mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF-mt3 and MazF-mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression through differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection.
Full-text · Article · Jun 2008 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes, and the products of two of these, PknA and PknB, are key components of a signal transduction pathway that regulates cell division and/or morphology. Previously, we have shown that one substrate of these kinases is Wag31, a homologue of the cell division protein DivIVA that is present, but not known to be phosphorylated, in other Gram-positive bacteria. Here, we investigate the localization and function of Wag31 and its phosphorylation. We demonstrate that Wag31 is localized to the cell poles. We further show that wag31 is an essential gene and that depletion of its product causes a dramatic morphological change in which one end of the cell becomes round rather than rod-shaped. This abnormal morphology appears to be caused by a defect in polar peptidoglycan synthesis. Finally, expression of M. tuberculosis wag31 in the wag31 conditional mutant of Mycobacterium smegmatis altered the growth rate in a manner that depended on the phospho-acceptor residue encoded by the allele being expressed. Taken together, these results indicate that Wag31 regulates cell shape and cell wall synthesis in M. tuberculosis through a molecular mechanism by which the activity of Wag31 can be modulated in response to environmental signals.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterial SigE and SigH both initiate transcription from the sigB promoter, suggesting that they recognize similar sequences. Through mutational and primer extension analyses, we determined
that SigE and SigH recognize nearly identical promoters, with differences at the 3′ end of the −35 element distinguishing
between SigE- and SigH-dependent promoters.
Preview · Article · Apr 2008 · Journal of bacteriology