Evaluation of cytokine profiles related to Mycobacterium tuberculosis latent antigens using a whole-blood assay in the Philippines.

Introduction: There is no useful method to discriminate between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB). This study aimed to investigate the potential of cytokine profiles to discriminate between LTBI and active PTB using whole-blood stimulation with Mycobacterium tuberculosis (MTB) antigens, including latency-associated antigens. Materials and methods: Patients with active PTB, household contacts of active PTB patients and community exposure subjects were recruited in Manila, the Philippines. Peripheral blood was collected from the participants and used for whole-blood stimulation (WBS) with either the early secretory antigenic target and the 10-kDa culture filtrate protein (ESAT-6/CFP-10), Rv3879c or latency-associated MTB antigens, including mycobacterial DNA-binding protein 1 (MDP-1), α-crystallin (Acr) and heparin-binding hemagglutinin (HBHA). Multiple cytokine concentrations were analyzed using the Bio-Plex™ multiplex cytokine assay. Results: A total of 78 participants consisting of 15 active PTB patients, 48 household contacts and 15 community exposure subjects were eligible. The MDP-1-specific IFN-γ level in the active PTB group was significantly lower than that in the household contact group (p < 0.001) and the community exposure group (p < 0.001). The Acr-specific TNF-α and IL-10 levels in the active PTB group were significantly higher than those in the household contact (TNF-α; p = 0.001, IL-10; p = 0.001) and community exposure (TNF-α; p < 0.001, IL-10; p = 0.01) groups. However, there was no significant difference in the ESAT-6/CFP-10-specific IFN-γ levels among the groups. Conclusion: The patterns of cytokine profiles induced by latency-associated MTB antigens using WBS have the potential to discriminate between LTBI and active PTB. In particular, combinations of IFN-γ and MDP-1, TNF-α and Acr, and IL-10 and Acr are promising. This study provides the first demonstration of the utility of MDP-1-specific cytokine responses in WBS.

Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals' blood cells in combination with CpG-DNA.

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

Genetic engineering employing MPB70 and its promoter enables efficient secretion and expression of foreign antigen in bacillus Calmette Guérin (BCG) Tokyo.

Vaccination is an important factor in public health. The recombinant bacillus Calmette Guérin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA-sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze-drying process. The freeze-dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs.

Dynamic action of an intrinsically disordered protein in DNA compaction that induces mycobacterial dormancy.

Mycobacteria are the major human pathogens with the capacity to become dormant persisters. Mycobacterial DNA-binding protein 1 (MDP1), an abundant histone-like protein in dormant mycobacteria, induces dormancy phenotypes, e.g. chromosome compaction and growth suppression. For these functions, the polycationic intrinsically disordered region (IDR) is essential. However, the disordered property of IDR stands in the way of clarifying the molecular mechanism. Here we clarified the molecular and structural mechanism of DNA compaction by MDP1. Using high-speed atomic force microscopy, we observed that monomeric MDP1 bundles two adjacent DNA duplexes side-by-side via IDR. Combined with coarse-grained molecular dynamics simulation, we revealed the novel dynamic DNA cross-linking model of MDP1 in which a stretched IDR cross-links two DNA duplexes like double-sided tape. IDR is able to hijack HU function, resulting in the induction of strong mycobacterial growth arrest. This IDR-mediated reversible DNA cross-linking is a reasonable model for MDP1 suppression of the genomic function in the resuscitable non-replicating dormant mycobacteria.

Limited proteolysis of mycobacterial DNA-binding protein 1 with an extended, lysine-rich, intrinsically disordered region to unveil posttranslational modifications.

The basic, intrinsically disordered regions of eukaryotic histones and their bacterial counterparts are presumed to act as signaling hubs to regulate the compaction of chromosomes or nucleoids and various DNA processes such as gene expression, recombination, and DNA replication. Posttranslational modifications (PTMs) on these regions are pivotal in regulating chromosomal or nucleoid compaction and DNA processes. However, the low sequence complexity and the presence of short lysine-rich repeats in the regions have hindered the accurate determination of types and locations of PTMs using conventional proteomic procedures. We described a limited proteolysis protocol using trypsin to analyze PTMs on mycobacterial DNA-binding protein 1 (MDP1), a nucleoid-associated protein in mycobacterial species that possesses an extended, lysine-rich, intrinsically disordered region in its C-terminal domain. This limited proteolysis approach successfully revealed significant methylation on many lysine residues in the C-terminal domain of MDP1 purified from Mycobacterium tuberculosis, which was lacking in the corresponding region of recombinant MDP1 expressed in Escherichia coli.

Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression.

Survival of the live attenuated Bacillus Calmette-Guérin (BCG) vaccine amidst harsh host environments is key for BCG effectiveness as it allows continuous immune response induction and protection against tuberculosis. Mycobacterial DNA binding protein 1 (MDP1), a nucleoid associated protein, is essential in BCG. However, there is limited knowledge on the extent of MDP1 gene regulation and how this influences BCG survival. Here, we demonstrate that MDP1 conditional knockdown (cKD) BCG grows slower than vector control in vitro, and dies faster upon exposure to antibiotics (bedaquiline) and oxidative stress (H2O2 and menadione). MDP1-cKD BCG also exhibited low infectivity and survival in THP-1 macrophages and mice indicating possible susceptibility to host mediated stress. Consequently, low in vivo survival resulted in reduced cytokine (IFN-gamma and TNF-alpha) production by splenocytes. Temporal transcriptome profiling showed more upregulated (81-240) than downregulated (5-175) genes in response to MDP1 suppression. Pathway analysis showed suppression of biosynthetic pathways that coincide with low in vitro growth. Notable was the deferential expression of genes involved in stress response (sigI), maintenance of DNA integrity (mutT1), REDOX balance (WhiB3), and host interactions (PE/PE_PGRS). Thus, this study shows MDP1's importance in BCG survival and highlights MDP1-dependent gene regulation suggesting its role in growth and stress adaptation.

Antibodies against native proteins of Mycobacterium tuberculosis can detect pulmonary tuberculosis patients.

Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn't distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734-0.890, p < 0.0001) and 0.821 (95% CI 0.752-0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB's POCT by antibody-based diagnosis.

Virulence of Mycobacterium intracellulare clinical strains in a mouse model of lung infection - role of neutrophilic inflammation in disease severity.

BACKGROUND: Mycobacterium intracellulare is a major etiological agent of Mycobacterium avium-intracellulare pulmonary disease (MAC-PD). However, the characteristics of the virulence of M. intracellulare and the in vivo chemotherapeutic efficacy remain unclear. In this study, we examined the virulence of nine M. intracellulare strains with different clinical phenotypes and genotypes in C57BL/6 mice. RESULTS: We classified three types of virulence phenotypes (high, intermediate, and low) based on the kinetics of the bacterial load, histological lung inflammation, and neutrophilic infiltration. High virulence strains showed more severe neutrophilic infiltration in the lungs than intermediate and low virulence strains, with 6.27-fold and 11.0-fold differences of the average percentage of neutrophils in bronchoalveolar lavage fluid, respectively. In particular, the high virulence strain M.i.198 showed the highest mortality in mice, which corresponded to the rapid progression of clinical disease. In mice infected with the drug-sensitive high virulence strain M019, clarithromycin-containing chemotherapy showed the highest efficacy. Monotherapy with rifampicin exacerbated lung inflammation with increased lymphocytic and neutrophilic infiltration into the lungs. CONCLUSIONS: The virulence phenotypes of clinical strains of M. intracellulare were diverse, with high virulence strains being associated with neutrophilic infiltration and disease progression in infected mice. These high virulence strains were proposed as a useful subject for in vivo chemotherapeutic experiments.

Lysocin E Targeting Menaquinone in the Membrane of Mycobacterium tuberculosis Is a Promising Lead Compound for Antituberculosis Drugs.

Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria. Lysocin E is an antibiotic that showed antibacterial activity against Staphylococcus aureus by binding to its menaquinone (commonly known as vitamin K2). Unlike S. aureus, menaquinone is essential in both growing and dormant Mycobacterium tuberculosis. This study aims to evaluate the antituberculosis activities of lysocin E and decipher its mode of action. We show that lysocin E has high in vitro activity against both drug-susceptible and drug-resistant Mycobacterium tuberculosis var. tuberculosis and dormant mycobacteria. Lysocin E is likely bound to menaquinone, causing M. tuberculosis membrane disruption, inhibition of oxygen consumption, and ATP synthesis. Thus, we have concluded that the high antituberculosis activity of lysocin E is attributable to its synergistic effects of membrane disruption and respiratory inhibition. The efficacy of lysocin E against intracellular M. tuberculosis in macrophages was lower than its potent activity against M. tuberculosis in culture medium, probably due to its low ability to penetrate cells, but its efficacy in mice was still superior to that of streptomycin. Our findings indicate that lysocin E is a promising lead compound for the development of a new tuberculosis drug that cures drug-resistant and latent tuberculosis in a shorter period.

Monitoring IgG against Mycobacterium tuberculosis proteins in an Asian elephant cured of tuberculosis that developed from long-term latency.

Tuberculosis (TB) is fatal in elephants, hence protecting elephants from TB is key not only in the conservation of this endangered animal, but also to prevent TB transmission from elephants to humans. Most human TB cases arise from long-term asymptomatic infections. Significant diagnostic challenges remain in the detection of both infection and disease development from latency in elephants due to their huge bodies. In this study, we assessed cryopreserved sera collected for over 16 years, from the first Japanese treatment case of elephant TB. Semi-quantification of IgG levels to 11 proteins showed high detection levels of 3 proteins, namely ESAT6/CFP10, MPB83 and Ag85B. The level of IgG specific to these 3 antigens was measured longitudinally, revealing high and stable ESAT6/CFP10 IgG levels regardless of onset or treatment. Ag85B-specifc IgG levels were largely responsive to onset or treatment, while those of MPB83 showed intermediate responses. These results suggest that ESAT6/CFP10 is immunodominant in both asymptomatic and symptomatic phases, making it useful in the detection of infection. On the other hand, Ag85B has the potential to be a marker for the prediction of disease onset and in the evaluation of treatment effectiveness in elephants.

Identification and functional analysis of a new type of Z,E-mixed prenyl reductase from mycobacteria.

Isoprenoids with reduced Z,E-mixed prenyl groups are found in various organisms. To date, only polyprenol reductases (PR-Dol) involved in dolichol biosynthesis have been identified as enzymes capable of reducing Z,E-mixed prenyl groups. Although C35 -isoprenoids with reduced Z,E-mixed prenyl groups are found in mycobacteria, Z,E-mixed heptaprenyl reductase (HepR) remains unidentified. In the present study, the identification and functional analysis of HepR was performed. No PR-Dol homolog gene was detected in the genome of Mycolicibacterium vanbaalenii. However, a homolog of geranylgeranyl reductase (GGR), which reacts with an all-E prenyl group as a substrate, was encoded in the genome; thus, we analyzed it as a HepR candidate. In vitro enzymatic assay and in vivo gene suppression analysis identified the GGR homolog as HepR and revealed that HepR catalyzes the reduction of ω- and E- prenyl units in Z,E-mixed heptaprenyl diphosphates, and C35 -isoprenoids are mainly biosynthesized using E,E,E-geranylgeranyl diphosphate as a precursor. Thus, it was demonstrated that the Z,E-mixed prenyl reductase family exists in the GGR homologs. To the best of our knowledge, this is the first identification of a new type of Z,E-mixed prenyl reductase with no sequence homology to PR-Dol. The substrate specificity of HepR significantly differed from that of GGR, suggesting that it is a new enzyme. HepR homologs are widely distributed in mycobacterial genomes, and lipid analysis suggests that many strains, including pathogenic species, produce HepR metabolites. The discovery of this new enzyme will promote further research on Z,E-mixed isoprenoids.

Correction: Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0204160.].

Extracellular DNA of slow growers of mycobacteria and its contribution to biofilm formation and drug tolerance.

DNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria's eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as Mycobacterium tuberculosis, M. intracellulare, and M. avium at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in M. tuberculosis and M. intracellulare. In contrast, accumulation of phage DNA in eDNA of M. avium, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria.

Comparative genomic analysis of Mycobacterium intracellulare: implications for clinical taxonomic classification in pulmonary Mycobacterium avium-intracellulare complex disease.

BACKGROUND: Mycobacterium intracellulare is a representative etiological agent of emerging pulmonary M. avium-intracellulare complex disease in the industrialized countries worldwide. The recent genome sequencing of clinical strains isolated from pulmonary M. avium-intracellulare complex disease has provided insight into the genomic characteristics of pathogenic mycobacteria, especially for M. avium; however, the genomic characteristics of M. intracellulare remain to be elucidated. RESULTS: In this study, we performed comparative genomic analysis of 55 M. intracellulare and related strains such as M. paraintracellulare (MP), M. indicus pranii (MIP) and M. yonogonense. Based on the average nucleotide identity, the clinical M. intracellulare strains were phylogenetically grouped in two clusters: (1) the typical M. intracellulare (TMI) group, including ATCC13950 and virulent M.i.27 and M.i.198 that we previously reported, and (2) the MP-MIP group. The alignment of the genomic regions was mostly preserved between groups. Plasmids were identified between groups and subgroups, including a plasmid common among some strains of the M.i.27 subgroup. Several genomic regions including those encoding factors involved in lipid metabolism (e.g., fadE3, fadE33), transporters (e.g., mce3), and type VII secretion system (genes of ESX-2 system) were shown to be hypermutated in the clinical strains. M. intracellulare was shown to be pan-genomic at the species and subspecies levels. The mce genes were specific to particular subspecies, suggesting that these genes may be helpful in discriminating virulence phenotypes between subspecies. CONCLUSIONS: Our data suggest that genomic diversity among M. intracellulare, M. paraintracellulare, M. indicus pranii and M. yonogonense remains at the subspecies or genovar levels and does not reach the species level. Genetic components such as mce genes revealed by the comparative genomic analysis could be the novel focus for further insight into the mechanism of human pathogenesis for M. intracellulare and related strains.

Higher genome mutation rates of Beijing lineage of Mycobacterium tuberculosis during human infection.

Mycobacterium tuberculosis (Mtb) strains of Beijing lineage have caused great concern because of their rapid emergence of drug resistance and worldwide spread. DNA mutation rates that reflect evolutional adaptation to host responses and the appearance of drug resistance have not been elucidated in human-infected Beijing strains. We tracked and obtained an original Mtb isolate of Beijing lineage from the 1999 tuberculosis outbreak in Japan, as well as five other isolates that spread in humans, and two isolates from the patient caused recurrence. Three isolates were from patients who developed TB within one year after infection (rapid-progressor, RP), and the other three isolates were from those who developed TB more than one year after infection (slow-progressor, SP). We sequenced genomes of these isolates and analyzed the propensity and rate of genomic mutations. Generation time versus mutation rate curves were significantly higher for RP. The ratio of oxidative versus non-oxidation damages induced mutations was higher in SP than RP, suggesting that persistent Mtb are exposed to oxidative stress in the latent state. Our data thus demonstrates that higher mutation rates of Mtb Beijing strains during human infection is likely to account for the higher adaptability and an emergence ratio of drug resistance.

Genome-wide identification of essential genes in Mycobacterium intracellulare by transposon sequencing - Implication for metabolic remodeling.

The global incidence of the human nontuberculous mycobacteria (NTM) disease is rapidly increasing. However, knowledge of gene essentiality under optimal growth conditions and conditions relevant to the natural ecology of NTM, such as hypoxia, is lacking. In this study, we utilized transposon sequencing to comprehensively identify genes essential for growth in Mycobacterium intracellulare. Of 5126 genes of M. intracellulare ATCC13950, 506 genes were identified as essential genes, of which 280 and 158 genes were shared with essential genes of M. tuberculosis and M. marinum, respectively. The shared genes included target genes of existing antituberculous drugs including SQ109, which targets the trehalose monomycolate transporter MmpL3. From 175 genes showing decreased fitness as conditionally essential under hypoxia, preferential carbohydrate metabolism including gluconeogenesis, glyoxylate cycle and succinate production was suggested under hypoxia. Virulence-associated genes including proteasome system and mycothiol redox system were also identified as conditionally essential under hypoxia, which was further supported by the higher effective suppression of bacterial growth under hypoxia compared to aerobic conditions in the presence of these inhibitors. This study has comprehensively identified functions essential for growth of M. intracellulare under conditions relevant to the host environment. These findings provide critical functional genomic information for drug discovery.

Adduct Formation of Delamanid with NAD in Mycobacteria.

Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F420)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated Mycobacterium tuberculosis var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene (ndh) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type ndh Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM.

Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis.

Tuberculosis causes the highest mortality among all single infections. Asymptomatic tuberculosis, afflicting one third of the global human population, is the major source as 5-10% of asymptomatic cases develop active tuberculosis during their lifetime. Thus it is one of important issues to develop diagnostic tools for accurately detecting asymptomatic infection. Mycobacterial DNA-binding protein 1 (MDP1) is a major protein in persistent Mycobacterium tuberculosis and has potential for diagnostic use in detecting asymptomatic infection. However, a previous ELISA-based study revealed a specificity problem; IgGs against MDP1 were detected in both M. tuberculosis-infected and uninfected individuals. Although the tertiary structures of an antigen are known to influence antibody recognition, the MDP1 structural details have not yet been investigated. The N-terminal half of MDP1, homologous to bacterial histone-like protein HU, is predicted to be responsible for DNA-binding, while the C-terminal half is assumed as totally intrinsically disordered regions. To clarify the relationship between the MDP1 tertiary structure and IgG recognition, we refined the purification method, which allow us to obtain a recombinant protein with the predicted structure. Furthermore, we showed that an IgG-ELISA using MDP1 purified by our refined method is indeed useful in the detection of asymptomatic tuberculosis.

C-terminal intrinsically disordered region-dependent organization of the mycobacterial genome by a histone-like protein.

The architecture of the genome influences the functions of DNA from bacteria to eukaryotes. Intrinsically disordered regions (IDR) of eukaryotic histones have pivotal roles in various processes of gene expression. IDR is rare in bacteria, but interestingly, mycobacteria produce a unique histone-like protein, MDP1 that contains a long C-terminal IDR. Here we analyzed the role of IDR in MDP1 function. By employing Mycobacterium smegmatis that inducibly expresses MDP1 or its IDR-deficient mutant, we observed that MDP1 induces IDR-dependent DNA compaction. MDP1-IDR is also responsible for the induction of growth arrest and tolerance to isoniazid, a front line tuberculosis drug that kills growing but not growth-retardated mycobacteria. We demonstrated that MDP1-deficiency and conditional knock out of MDP1 cause spreading of the M. smegmatis genome in the stationary phase. This study thus demonstrates for the first time a C-terminal region-dependent organization of the genome architecture by MDP1, implying the significance of IDR in the function of bacterial histone-like protein.

Mycobacterial DNA-binding protein 1 is critical for long term survival of Mycobacterium smegmatis and simultaneously coordinates cellular functions.

Bacteria can proliferate perpetually without ageing, but they also face conditions where they must persist. Mycobacteria can survive for a long period. This state appears during mycobacterial diseases such as tuberculosis and leprosy, which are chronic and develop after long-term persistent infections. However, the fundamental mechanisms of the long-term living of mycobacteria are unknown. Every Mycobacterium species expresses Mycobacterial DNA-binding protein 1 (MDP1), a histone-like nucleoid associated protein. Mycobacterium smegmatis is a saprophytic fast grower and used as a model of mycobacterial persistence, since it shares the characteristics of the long-term survival observed in pathogenic mycobacteria. Here we show that MDP1-deficient M. smegmatis dies more rapidly than the parental strain after entering stationary phase. Proteomic analyses revealed 21 upregulated proteins with more than 3-fold in MDP1-deficient strain, including DnaA, a replication initiator, NDH, a NADH dehydrogenase that catalyzes downhill electron transfer, Fas1, a critical fatty acid synthase, and antioxidants such as AhpC and KatG. Biochemical analyses showed elevated levels of DNA and ATP syntheses, a decreased NADH/NAD+ ratio, and a loss of resistance to oxidative stress in the MDP1-knockout strain. This study suggests the importance of MDP1-dependent simultaneous control of the cellular functions in the long-term survival of mycobacteria.