Disruption of nontuberculous mycobacteria biofilms induces a highly vulnerable to antibiotic killing phenotype.

Objectives: Structural or mucus hypersecretory pulmonary diseases such as cystic fibrosis (CF), wherein viscous mucus accumulates and clearance functions are impaired, predispose people to lung infection by inhaled bacteria that form biofilm aggregates. Nontuberculous mycobacteria (NTM), primarily Mycobacterium abscessus and Mycobacterium avium, are the growing cause of these lung infections and are extremely challenging to treat due to antibiotic recalcitrance. Better therapeutic approaches are urgently needed. We developed a humanized monoclonal antibody (HuTipMab) directed against a biofilm structural linchpin, the bacterial DNABII proteins, that rapidly disrupts biofilms and generates highly vulnerable newly released bacteria (NRel). Methods: HuTipMab's ability to recognize HupB, NTM's DNABII homologue was determined by ELISA. Relative ability of HuTipMab to disrupt biofilms formed by lab-passaged and clinical isolates of NTM was assessed by CLSM. Relative sensitivity of NTM NRel to antibiotic killing compared to when grown planktonically was evaluated by plate count. Results: HuTipMab recognized HupB and significantly disrupted NTM biofilms in a time- and dose-dependent manner. Importantly, NTM NRel of lab-passaged and clinical isolates were now highly sensitive to killing by amikacin and azithromycin. Conclusions: If successful, this combinatorial treatment strategy would empower existing antibiotics to more effectively kill NTM newly released from a biofilm by HuTipMab and thereby both improve clinical outcomes and perhaps decrease length of antibiotic treatment for people that are NTM culture-positive.

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.

Development of a new cellular immunological detection method for tuberculosis based on HupB protein induced IL-6 release test.

Objective: As a virulence factor, HupB plays important roles in the survival of MTB after infection and modulates the host immune response. In the current study, we aim to explore a new cellular immunological detection method for tuberculosis infection detection based on HupB protein. Methods: HupB was used to stimulate PBMCs extracted from pulmonary tuberculosis (PTB) patients, and secreted cytokines was examined. Then, we constructed a single center and a multi-center clinical trials to collect PBMCs from PTB patients, nPTB patients, or healthy volunteers to verify our findings. Results: Cytokine's screening illustrated that IL-6 was the only cytokine released after HupB stimulation. Single-center and multi-center clinical trials showed that HupB stimulation significantly increased the level of IL-6 in the supernatant of PBMCs from PTB patients. Then we compared the specificity and sensitivity of HupB induced IL-6 release assay with ESAT-6 and CFP10 induced interferon γ release assay (IGRA), and found in smear positive PTB patients, the specificity and sensitivity of HupB induced IL-6 release assay was better than IGRA, and in smear negative PTB patients, the sensitivity was better. Combination of both assays provided an improved specificity and sensitivity for tuberculosis diagnosis. Conclusion: This study explored an immunological detection method for tuberculosis infection cells based on HupB protein-induced IL-6 release test, which can be used to enhance the diagnosis diagnostic accuracy of TB.

Mycobacterial IHF is a highly dynamic nucleoid-associated protein that assists HupB in organizing chromatin.

Nucleoid-associated proteins (NAPs) crucially contribute to organizing bacterial chromatin and regulating gene expression. Among the most highly expressed NAPs are the HU and integration host factor (IHF) proteins, whose functional homologues, HupB and mycobacterial integration host factor (mIHF), are found in mycobacteria. Despite their importance for the pathogenicity and/or survival of tubercle bacilli, the role of these proteins in mycobacterial chromosome organization remains unknown. Here, we used various approaches, including super-resolution microscopy, to perform a comprehensive analysis of the roles of HupB and mIHF in chromosome organization. We report that HupB is a structural agent that maintains chromosome integrity on a local scale, and that the lack of this protein alters chromosome morphology. In contrast, mIHF is a highly dynamic protein that binds DNA only transiently, exhibits susceptibility to the chromosomal DNA topology changes and whose depletion leads to the growth arrest of tubercle bacilli. Additionally, we have shown that depletion of Mycobacterium smegmatis integration host factor (msIHF) leads to chromosome shrinkage and replication inhibition.

HupB, a nucleoid-associated protein, is critical for survival of Mycobacterium tuberculosis under host-mediated stresses and for enhanced tolerance to key first-line antibiotics.

To survive and establish its niche, Mycobacterium tuberculosis (Mtb) engages in a steady battle against an array of host defenses and a barrage of antibiotics. Here, we demonstrate that Mtb employs HupB, a nucleoid-associated protein (NAP) as its key player to simultaneously battle and survive in these two stress-inducing fronts. Typically, NAPs are key to bacterial survival under a wide array of environmental or host-mediated stresses. Here, we report that for Mtb to survive under different macrophage-induced assaults including acidic pH, nutrient depletion, oxidative and nitrosative stresses, HupB presence is critical. As expected, the hupB knockout mutant is highly sensitive to these host-mediated stresses. Furthermore, Mtb aptly modulates HupB protein levels to overcome these stresses. We also report that HupB aids Mtb to gain tolerance to high levels of rifampicin (RIF) and isoniazid (INH) exposure. Loss of hupB makes Mtb highly susceptible to even short exposures to reduced amounts of RIF and INH. Overexpressing hupB in Mtb or complementing hupB in the hupB knockout mutant triggers enhanced survival of Mtb under these stresses. We also find that upon loss of hupB, Mtb significantly enhances the permeability of its cell wall by modulating the levels of several surface lipids including phthiocerol dimycocerosates (PDIMs), thus possibly influencing overall susceptibility to host-mediated stresses. Loss of hupB also downregulates efflux pump expression possibly influencing increased susceptibility to INH and RIF. Finally, we find that therapeutic targeting of HupB with SD1, a known small molecule inhibitor, significantly enhances Mtb susceptibility to INH and THP-1 macrophages and significantly reduces MIC to INH. Thus, our data strongly indicate that HupB is a highly promising therapeutic target especially for potential combinatorial shortened therapy with reduced INH and RIF doses.

Approaches for targeting the mycobactin biosynthesis pathway for novel anti-tubercular drug discovery: where we stand.

INTRODUCTION: Several decades of antitubercular drug discovery efforts have focused on novel antitubercular chemotherapies. However, recent efforts have greatly shifted toward countering extremely/multi/total drug-resistant species. Targeting the conditionally essential elements inside Mycobacterium is a relatively new approach against tuberculosis and has received lackluster attention. The siderophore, Mycobactin, is a conditionally essential molecule expressed by mycobacteria in iron-stress conditions. It helps capture the micronutrient iron, essential for the smooth functioning of cellular processes. AREAS COVERED: The authors discuss opportunities to target the conditionally essential pathways to help develop newer drugs and prolong the shelf life of existing therapeutics, emphasizing the bottlenecks in fast-tracking antitubercular drug discovery. EXPERT OPINION: While the lack of iron supply can cripple bacterial growth and multiplication, excess iron can cause oxidative overload. Constant up-regulation can strain the bacterial synthetic machinery, further slowing its growth. Mycobactin synthesis is tightly controlled by a genetically conserved mega enzyme family via up-regulation (HupB) or down-regulation (IdeR) based on iron availability in its microenvironment. Furthermore, the recycling of siderophores by the MmpL-MmpS4/5 orchestra provides endogenous drug targets to beat the bugs with iron-toxicity contrivance. These processes can be exploited as chinks in the armor of Mycobacterium and be used for new drug development.

Th2 immune response by the iron-regulated protein HupB of Mycobacterium tuberculosis.

BACKGROUND: HupB is an iron-regulated protein essential for the growth of Mycobacterium tuberculosis inside macrophages. To investigate if HupB induced a dominant Th2 type immune response, we studied the effect of rHupB on PBMCs from TB patients and by infecting mouse macrophages with wild type and hupB KO mutants. METHODS: PBMCs from pulmonary TB (n = 60), extra pulmonary TB (n = 23) and healthy controls (n = 30) were stimulated with purified HupB and the cytokines secreted were assayed. The sera were screened for anti-HupB antibodies by ELISA. Mouse macrophages cell line (RAW 264.7) was infected with wild type, hupB KO and hupB-complemented strains of M. tuberculosis grown in high and low iron medium and the expression of cytokines was assayed by qRT-PCR. RESULTS: Murine macrophages infected with the hupB KO strain produced low levels of the pro-inflammatory cytokines IFN-γ, TNF-α, IL-1, and IL-18 and high levels of IL-10. HupB induced IL-6 and IL-10 production in PBMCs of TB patients and down-regulated IFN-γ and TNF-α production. The influence of HupB was remarkable in the EPTB group. CONCLUSION: HupB shifted the immune response to the Th2 type. Low IFN-γ and elevated IL-10 in EPTB patients is noteworthy.

Iron uptake and transport by the carboxymycobactin-mycobactin siderophore machinery of Mycobacterium tuberculosis is dependent on the iron-regulated protein HupB.

Iron-starved Mycobacterium tuberculosis utilises the carboxymycobactin-mycobactin siderophore machinery to acquire iron. These two siderophores have high affinity for ferric iron and can withdraw the metal ion from insoluble iron hydroxides and iron-binding proteins. We first reported HupB, a multi-functional mycobacterial protein to be associated with iron acquisition in M. tuberculosis. This 28 kDa cell wall protein, up regulated upon iron limitation functions as a transcriptional activator of mycobactin biosynthesis and is essential for the pathogen to survive inside macrophages. The focus of this study is to understand the role of HupB in iron uptake and transport by the carboxmycobactin-mycobactin siderophore machinery in M. tuberculosis. Experimental approaches included radiolabelled iron uptake studies by viable organisms and protein-ligand binding studies using the purified HupB and the two siderophores. Uptake of 55Fe-carboxymycobactin by wild type M. tuberculosis (WT M.tb.H37Rv) and not by the hupB KO mutant (M.tb.ΔhupB) showed that HupB is necessary for the uptake of ferri-carboxymycobactin. Additionally, the radiolabel recovery was high in HupB-incorporated liposomes upon addition of the labelled siderophore. Bioinformatic and experimental studies using spectrofluorimetry, CD analysis and surface plasmon resonance not only confirmed the binding of HupB with ferri-carboxymycobactin and ferri-mycobactin but also with free iron. In conclusion, HupB is established as a ferri- carboxymycobactin receptor and by virtue of its property to bind ferric iron, functions as a transporter of the ferric iron from the extracellular siderophore to mycobactin within the cell envelope.

Addressing the Possibility of a Histone-Like Code in Bacteria.

Acetylation was initially discovered as a post-translational modification (PTM) on the unstructured, highly basic N-terminal tails of eukaryotic histones in the 1960s. Histone acetylation constitutes part of the "histone code", which regulates chromosome compaction and various DNA processes such as gene expression, recombination, and DNA replication. In bacteria, nucleoid-associated proteins (NAPs) are responsible these functions in that they organize and compact the chromosome and regulate some DNA processes. The highly conserved DNABII family of proteins are considered functional homologues of eukaryotic histones despite having no sequence or structural conservation. Within the past decade, a growing interest in Nε-lysine acetylation led to the discovery that hundreds of bacterial proteins are acetylated with diverse cellular functions, in direct contrast to the original thought that this was a rare phenomenon. Similarly, other previously undiscovered bacterial PTMs, like serine, threonine, and tyrosine phosphorylation, have also been characterized. In this review, the various PTMs that were discovered among DNABII family proteins, specifically histone-like protein (HU) orthologues, from large-scale proteomic studies are discussed. The functional significance of these modifications and the enzymes involved are also addressed. The discovery of novel PTMs on these proteins begs this question: is there a histone-like code in bacteria?

G-Quadruplex-Forming DNA Aptamers Inhibit the DNA-Binding Function of HupB and Mycobacterium tuberculosis Entry into Host Cells.

The entry and survival of Mycobacterium tuberculosis (Mtb) within host cells is orchestrated partly by an essential histone-like protein HupB (Rv2986c). Despite being an essential drug target, the lack of structural information has impeded the development of inhibitors targeting the indispensable and multifunctional C-terminal domain (CTD) of HupB. To bypass the requirement for structural information in the classical drug discovery route, we generated a panel of DNA aptamers against HupB protein through systemic evolution of ligands by exponential (SELEX) enrichment. Two G-quadruplex-forming high-affinity aptamers (HupB-4T and HupB-13T) were identified, each of which bound two distinct sites on full-length HupB, with an estimated KD of ∼1.72 µM and ∼0.17 µM, respectively, for the high-affinity sites. While HupB-4T robustly inhibited DNA-binding activity of HupB in vitro, both the aptamers recognized surface-located HupB and significantly blocked Mtb entry into THP-1 monocytic cells (p < 0.0001). In summary, DNA aptamers generated in this study block DNA-binding activity of HupB, inhibit virulent Mtb infection in host cells, and demonstrate aptamers to be inhibitors of HupB functions. This study also illustrates the utility of SELEX in developing inhibitors against essential targets for whom structural information is not available.

Comprehensive relative quantitative metabolomics analysis of lycopodium alkaloids in different tissues of Huperzia serrata.

Qian ceng Ta, the whole plant of Huperzia serrata, is an important landscape and medicinal herbs and contains abundant bioactive lycopodium alkaloids. Although the structures of more than 100 lycopodium alkaloids in Huperzia serrata have been isolated and identified, the content and distribution of these alkaloids in different tissues are still unclear. In current study, an ultra-performance liquid chromatography-mass spectrometry based comprehensive metabolomics strategy was developed, including the extraction, separation, identification, and statistical analysis. The results showed that different types lycopodium alkaloids could be separated at different time-windows, which was helpful for further metabolite identification. Peak4388 and peak3954 were metabolite biomarkers for the different tissues according to the principle component analysis and partial least squares-discriminant analysis model. A computational tool based in-house database was also built up and used for putative identification. Of the 2354 true peaks after four-step filtration, 118 peaks were putatively identified as lycopodium alkaloids by using in-house database, and four of which was identified by authentic standards. Alternatively, another computational software was used to predict the fragmentation pattern, to dereplicate the structure of identified peaks, and identified the peak3585 to N-methylhuperzine A. The integration of both computational tools could be used for more metabolites identification.

The Origin of Chromosomal Replication Is Asymmetrically Positioned on the Mycobacterial Nucleoid, and the Timing of Its Firing Depends on HupB.

The bacterial chromosome undergoes dynamic changes in response to ongoing cellular processes and adaptation to environmental conditions. Among the many proteins involved in maintaining this dynamism, the most abundant is the nucleoid-associated protein (NAP) HU. In mycobacteria, the HU homolog, HupB, possesses an additional C-terminal domain that resembles that of eukaryotic histones H1/H5. Recently, we demonstrated that the highly abundant HupB protein occupies the entirety of the Mycobacterium smegmatis chromosome and that the HupB-binding sites exhibit a bias from the origin (oriC) to the terminus (ter). In this study, we used HupB fused with enhanced green fluorescent protein (EGFP) to perform the first analysis of chromosome dynamics and to track the oriC and replication machinery directly on the chromosome during the mycobacterial cell cycle. We show that the chromosome is located in an off-center position that reflects the unequal division and growth of mycobacterial cells. Moreover, unlike the situation in E. coli, the sister oriC regions of M. smegmatis move asymmetrically along the mycobacterial nucleoid. Interestingly, in this slow-growing organism, the initiation of the next round of replication precedes the physical separation of sister chromosomes. Finally, we show that HupB is involved in the precise timing of replication initiation.IMPORTANCE Although our view of mycobacterial nucleoid organization has evolved considerably over time, we still know little about the dynamics of the mycobacterial nucleoid during the cell cycle. HupB is a highly abundant mycobacterial nucleoid-associated protein (NAP) with an indispensable histone-like tail. It was previously suggested as a potential target for antibiotic therapy against tuberculosis. Here, we fused HupB with enhanced green fluorescent protein (EGFP) to study the dynamics of the mycobacterial chromosome in real time and to monitor the replication process directly on the chromosome. Our results reveal that, unlike the situation in Escherichia coli, the nucleoid of an apically growing mycobacterium is positioned asymmetrically within the cell throughout the cell cycle. We show that HupB is involved in controlling the timing of replication initiation. Since tuberculosis remains a serious health problem, studies concerning mycobacterial cell biology are of great importance.

Histone methyltransferase SUV39H1 participates in host defense by methylating mycobacterial histone-like protein HupB.

Host cell defense against an invading pathogen depends upon various multifactorial mechanisms, several of which remain undiscovered. Here, we report a novel defense mechanism against mycobacterial infection that utilizes the histone methyltransferase, SUV39H1. Normally, a part of the host chromatin, SUV39H1, was also found to be associated with the mycobacterial bacilli during infection. Its binding to bacilli was accompanied by trimethylation of the mycobacterial histone-like protein, HupB, which in turn reduced the cell adhesion capability of the bacilli. Importantly, SUV39H1-mediated methylation of HupB reduced the mycobacterial survival inside the host cell. This was also true in mice infection experiments. In addition, the ability of mycobacteria to form biofilms, a survival strategy of the bacteria dependent upon cell-cell adhesion, was dramatically reduced in the presence of SUV39H1. Thus, this novel defense mechanism against mycobacteria represents a surrogate function of the epigenetic modulator, SUV39H1, and operates by interfering with their cell-cell adhesion ability.

HupB Is a Bacterial Nucleoid-Associated Protein with an Indispensable Eukaryotic-Like Tail.

In bacteria, chromosomal DNA must be efficiently compacted to fit inside the small cell compartment while remaining available for the proteins involved in replication, segregation, and transcription. Among the nucleoid-associated proteins (NAPs) responsible for maintaining this highly organized and yet dynamic chromosome structure, the HU protein is one of the most conserved and highly abundant. HupB, a homologue of HU, was recently identified in mycobacteria. This intriguing mycobacterial NAP is composed of two domains: an N-terminal domain that resembles bacterial HU, and a long and distinctive C-terminal domain that contains several PAKK/KAAK motifs, which are characteristic of the H1/H5 family of eukaryotic histones. In this study, we analyzed the in vivo binding of HupB on the chromosome scale. By using PALM (photoactivated localization microscopy) and ChIP-Seq (chromatin immunoprecipitation followed by deep sequencing), we observed that the C-terminal domain is indispensable for the association of HupB with the nucleoid. Strikingly, the in vivo binding of HupB displayed a bias from the origin (oriC) to the terminus (ter) of the mycobacterial chromosome (numbers of binding sites decreased toward ter). We hypothesized that this binding mode reflects a role for HupB in organizing newly replicated oriC regions. Thus, HupB may be involved in coordinating replication with chromosome segregation.IMPORTANCE We currently know little about the organization of the mycobacterial chromosome and its dynamics during the cell cycle. Among the mycobacterial nucleoid-associated proteins (NAPs) responsible for chromosome organization and dynamics, HupB is one of the most intriguing. It contains a long and distinctive C-terminal domain that harbors several PAKK/KAAK motifs, which are characteristic of the eukaryotic histone H1/H5 proteins. The HupB protein is also known to be crucial for the survival of tubercle bacilli during infection. Here, we provide in vivo experimental evidence showing that the C-terminal domain of HupB is crucial for its DNA binding. Our results suggest that HupB may be involved in organizing newly replicated regions and could help coordinate chromosome replication with segregation. Given that tuberculosis (TB) remains a serious worldwide health problem (10.4 million new TB cases were diagnosed in 2015, according to WHO) and new multidrug-resistant Mycobacterium tuberculosis strains are continually emerging, further studies of the biological function of HupB are needed to determine if this protein could be a prospect for novel antimicrobial drug development.

Molecular detection of Isoniazid, Rifampin and Ethambutol resistance to M. tuberculosis and M. bovis in multidrug resistant tuberculosis (MDR-TB) patients in Pakistan.

The various aspects of MDR-TB, type of pathogen, different drug sensitive methods and mutation (s) in specific genes were determined. The histone-like protein (hupB) gene of M. tuberculosis was targeted by using primer sets: N & S and M & S (produced 645 bp & 318 bp fragment respectively). The most significant risk factors were the poverty and male gender of ages 11-25 years. All samples were detected as M. tuberculosis. By Drug Proportion method, the highest percentage (37%) was found resistant to only Rifampin. By MGIT method, the highest percentage (82.2%) was found resistant with the triple combination (Rifampin-RIF + Isoniazid-INH + Ethambutol-EMB) of the drugs. The highest mutations (76.92%) were found in gene rpoB (codon 531) in MDR TB patients. By, MAS-PCR, the highest percentage (34%) were found resistant to combination (INH + RIF) of the drugs. Minimum samples were resistant to RIF and RIF + INH drugs by MGIT, while proportionate results were observed from MAS-PCR and DP. Moreover, by MAS-PCR mutation in gene embB (306) caused EMB resistance (51.64%). We found that M. tuberculosis was the main cause of MDR-TB. Our findings may further be used for an early diagnosis of multi-drug resistant tuberculosis.

Functional insights from a comparative study on the dynamics of Antigen85 proteins and MPT51 from Mycobacterium tuberculosis.

Antigen85 (Ag85) proteins of Mycobacterium tuberculosis are mycolyl transferases that aid in cell wall biosynthesis. MPT51 (Ag85D) is closely related to Ag85 proteins. We have performed a comparative molecular dynamics (MD) simulation study of Ag85 proteins (Ag85A, Ag85B, and Ag85C) and MPT51. We observe that helix α5, ß7-α9 loop, and N-terminal region of helix α9 of Ag85 proteins are mobile, suggestive of lid like movement over the active site. Further, in Ag85B, we observe the proposed scooting mode of the hydrophobic gating residue Phe232. Our simulations also show a similar scooting mode for Phe232 of Ag85A and Trp158 of Ag85C. We also found aromatic residue clusters at the ends of the hydrophobic channel of Ag85 proteins, which may have functional significance. Although MPT51 lacks the tunnel, it has the aromatic clusters. The aromatic cluster region has the ability to bind trehalose. From an immunoinformatics study, a promiscuous linear epitope was identified in MPT51 which could be useful in subunit vaccine studies. Recent studies have shown that a mycobacterial protein HupB, interacts with Ag85 proteins and has a regulatory role in cell wall biogenesis, with implications in growth rate and latency. We performed molecular docking studies of HupB protein with Ag85 proteins and predicted potential sites of interaction in Ag85 proteins. The insights gained through the current study can potentially pave way for newer therapeutic interventions. Graphical Abstract Dynamics of antigen85 proteins and MPT51 from Mycobacterium tuberculosis.