Proton Motive Force-Disrupting Antimycobacterial Guanylurea Polymer.

Mycobacterial infectious diseases, including tuberculosis (TB), severely threaten global public health. Nonreplicating Mycobacterium tuberculosis (Mtb) is extremely difficult to eradicate using current TB drugs that primarily act on replicating cells. Novel TB drugs acting on unconventional targets are needed to combat TB efficiently. Although membrane-disrupting antimicrobial peptides and their synthetic mimics exhibit the potential to kill persisters, the lack of microbe selectivity, especially toward mycobacteria, has been a concern. Here, we report that the recently developed poly(guanylurea)-piperazine (PGU-P) shows fast and selective mycobactericidal effects. Using a nonpathogenic model organism, Mycobacterium smegmatis (Msm), we have found that the mycobactericidal effects of PGU-P are correlated to the disruption of the mycobacterial membrane potential and bioenergetics. Accordingly, PGU-P also potentiates bedaquiline, an oxidative phosphorylation-targeting TB drug disturbing mycobacterial bioenergetics. Importantly, PGU-P also exhibits a promising activity against pathogenic Mtb with a minimum inhibitory concentration of 37 µg/mL. Our results support that PGU-P is a novel class of antimycobacterial biomaterial, and the unique structural feature can contribute to developing novel antimycobacterial drugs.

Crystal structure of a hemerythrin-like protein from Mycobacterium kansasii and homology model of the orthologous Rv2633c protein of M. tuberculosis.

Pathogenic and opportunistic mycobacteria have a distinct class of non-heme di-iron hemerythrin-like proteins (HLPs). The first to be isolated was the Rv2633c protein, which plays a role in infection by Mycobacterium tuberculosis (Mtb), but could not be crystallized. This work presents the first crystal structure of an ortholog of Rv2633c, the mycobacterial HLP from Mycobacterium kansasii (Mka). This structure differs from those of hemerythrins and other known HLPs. It consists of five α-helices, whereas all other HLP domains have four. In contrast with other HLPs, the HLP domain is not fused to an additional protein domain. The residues ligating and surrounding the di-iron site are also unique among HLPs. Notably, a tyrosine occupies the position normally held by one of the histidine ligands in hemerythrin. This structure was used to construct a homology model of Rv2633c. The structure of five α-helices is conserved and the di-iron site ligands are identical in Rv2633c. Two residues near the ends of helices in the Mka HLP structure are replaced with prolines in the Rv2633c model. This may account for structural perturbations that decrease the solubility of Rv2633c relative to Mka HLP. Clusters of residues that differ in charge or polarity between Rv2633c and Mka HLP that point outward from the helical core could reflect a specificity for potential differential interactions with other protein partners in vivo, which are related to function. The Mka HLP exhibited weaker catalase activity than Rv2633c. Evidence was obtained for the interaction of Mka HLP irons with nitric oxide.

The Rv2633c protein of Mycobacterium tuberculosis is a non-heme di-iron catalase with a possible role in defenses against oxidative stress.

The Rv2633c gene in Mycobacterium tuberculosis is rapidly up-regulated after macrophage infection, suggesting that Rv2633c is involved in M. tuberculosis pathogenesis. However, the activity and role of the Rv2633c protein in host colonization is unknown. Here, we analyzed the Rv2633c protein sequence, which revealed the presence of an HHE cation-binding domain common in hemerythrin-like proteins. Phylogenetic analysis indicated that Rv2633c is a member of a distinct subset of hemerythrin-like proteins exclusive to mycobacteria. The Rv2633c sequence was significantly similar to protein sequences from other pathogenic strains within that subset, suggesting that these proteins are involved in mycobacteria virulence. We expressed and purified the Rv2633c protein in Escherichia coli and found that it contains two iron atoms, but does not behave like a hemerythrin. It migrated as a dimeric protein during size-exclusion chromatography. It was not possible to reduce the protein or observe any evidence for its interaction with O2 However, Rv2633c did exhibit catalase activity with a kcat of 1475 s-1 and Km of 10.1 ± 1.7 mm Cyanide and azide inhibited the catalase activity with Ki values of 3.8 µm and 37.7 µm, respectively. Rv2633c's activity was consistent with a role in defenses against oxidative stress generated during host immune responses after M. tuberculosis infection of macrophages. We note that Rv2633c is the first example of a non-heme di-iron catalase, and conclude that it is a member of a subset of hemerythrin-like proteins exclusive to mycobacteria, with likely roles in protection against host defenses.

Correction: Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006389.].

Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.

Signals modulating the production of Mycobacterium tuberculosis (Mtb) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of Mtb virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed Mtb in vitro, intraphagosomal and infected host expression analyses. Our Mtb expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found in vivo to establish Mtb infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that Mtb infection inhibited the macrophage G1/S transition, and polyketides under WhiB3 control arrested the macrophages in the G0-G1 phase. Notably, infection with the Mtb whiB3 mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by Mtb WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type Mtb. Thus, our findings demonstrate that Mtb WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby Mtb modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle.

Species Typing of Nontuberculous Mycobacteria by Use of Deoxyribozyme Sensors.

BACKGROUND: Nontuberculous mycobacteria (NTM) species are a rising threat, especially to patients living with pulmonary comorbidities. Current point-of-care diagnostics fail to adequately identify and differentiate NTM species from Mycobacterium tuberculosis (Mtb). Definitive culture- and molecular-based testing can take weeks to months and requires sending samples out to specialized diagnostic laboratories. METHODS: In this proof-of-concept study, we developed an assay based on PCR amplification of 16S ribosomal RNA (rRNA) rrs genes by using universal mycobacterial primers and interrogation of the amplified fragments with a panel of binary deoxyribozyme (BiDz) sensors to enable species-level identification of NTM (BiDz-NTMST). Each BiDz sensor consists of 2 subunits of an RNA-cleaving deoxyribozyme, which form an active deoxyribozyme catalytic core only in the presence of the complimentary target sequence. The target-activated BiDz catalyzes cleavage of a reporter substrate, thus triggering either fluorescent or colorimetric (visually observed) signal depending on the substrate used. The panel included BiDz sensors for differentiation of 6 clinically relevant NTM species (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium kansasii, and Mycobacterium gordonae) and Mtb. RESULTS: Using the fluorescent BiDz-NTMST assay, we successfully identified the species of 38 clinical isolates. In addition, a subset of strains was tested with visual BiDz sensors, providing proof-of-concept for species typing of NTM by the naked eye. CONCLUSIONS: The BiDz-NTMST assay is a novel platform for rapid identification of NTM species. This method is highly specific and significantly faster than current tools and is easily adaptable for onsite diagnostic laboratories in hospitals or clinical laboratories.

Plakinamine P, A Steroidal Alkaloid with Bactericidal Activity against Mycobacterium tuberculosis.

Tuberculosis is the leading cause of death due to infectious disease worldwide. There is an urgent need for more effective compounds against this pathogen to control the disease. Investigation of the anti-mycobacterial activity of a deep-water sponge of the genus Plakina revealed the presence of a new steroidal alkaloid of the plakinamine class, which we have given the common name plakinamine P. Its structure is most similar to plakinamine L, which also has an acyclic side chain. Careful dissection of the nuclear magnetic resonance data, collected in multiple solvents, suggests that the dimethyl amino group at the 3 position is in an equatorial rather than axial position unlike previously reported plakinamines. Plakinamine P was bactericidal against M. tuberculosis, and exhibited moderate activity against other mycobacterial pathogens, such as M. abscessus and M. avium. Furthermore, it had low toxicity against J774 macrophages, yielding a selectivity index (SI, or IC50/MIC) of 8.4. In conclusion, this work provides a promising scaffold to the tuberculosis drug discovery pipeline. Future work to determine the molecular target of this compound may reveal a pathway essential for M. tuberculosis survival during infection.

Evidence for Inhibition of Topoisomerase 1A by Gold(III) Macrocycles and Chelates Targeting Mycobacterium tuberculosis and Mycobacterium abscessus.

Mycobacterium tuberculosis and the fast-growing species Mycobacterium abscessus are two important human pathogens causing persistent pulmonary infections that are difficult to cure and require long treatment times. The emergence of drug-resistant M. tuberculosis strains and the high level of intrinsic resistance of M. abscessus call for novel drug scaffolds that effectively target both pathogens. In this study, we evaluated the activity of bis(pyrrolide-imine) gold(III) macrocycles and chelates, originally designed as DNA intercalators capable of targeting human topoisomerase types I and II (Topo1 and Topo2), against M. abscessus and M. tuberculosis We identified a total of 5 noncytotoxic compounds active against both mycobacterial pathogens under replicating in vitro conditions. We chose one of these hits, compound 14, for detailed analysis due to its potent bactericidal mode of inhibition and scalable synthesis. The clinical relevance of this compound was demonstrated by its ability to inhibit a panel of diverse M. tuberculosis and M. abscessus clinical isolates. Prompted by previous data suggesting that compound 14 may target topoisomerase/gyrase enzymes, we demonstrated that it lacked cross-resistance with fluoroquinolones, which target the M. tuberculosis gyrase. In vitro enzyme assays confirmed the potent activity of compound 14 against bacterial topoisomerase 1A (Topo1) enzymes but not gyrase. Novel scaffolds like compound 14 with potent, selective bactericidal activity against M. tuberculosis and M. abscessus that act on validated but underexploited targets like Topo1 represent a promising starting point for the development of novel therapeutics for infections by pathogenic mycobacteria.

Exploitation of Mangrove Endophytic Fungi for Infectious Disease Drug Discovery.

There is an acute need for new and effective agents to treat infectious diseases. We conducted a screening program to assess the potential of mangrove-derived endophytic fungi as a source of new antibiotics. Fungi cultured in the presence and absence of small molecule epigenetic modulators were screened against Mycobacterium tuberculosis and the ESKAPE panel of bacterial pathogens, as well as two eukaryotic infective agents, Leishmania donovani and Naegleria fowleri. By comparison of bioactivity data among treatments and targets, trends became evident, such as the result that more than 60% of active extracts were revealed to be selective to a single target. Validating the technique of using small molecules to dysregulate secondary metabolite production pathways, nearly half (44%) of those fungi producing active extracts only did so following histone deacetylase inhibitory (HDACi) or DNA methyltransferase inhibitory (DNMTi) treatment.

Selective Killing of Dormant Mycobacterium tuberculosis by Marine Natural Products.

The dormant phenotype acquired by Mycobacterium tuberculosis during infection poses a major challenge in disease treatment, since these bacilli show tolerance to front-line drugs. Therefore, it is imperative to find novel compounds that effectively kill dormant bacteria. By screening 4,400 marine natural product samples against dual-fluorescent M. tuberculosis under both replicating and nonreplicating conditions, we have identified compounds that are selectively active against dormant M. tuberculosis This validates our strategy of screening all compounds in both assays as opposed to using the dormancy model as a secondary screen. Bioassay-guided deconvolution enabled the identification of unique pharmacophores active in each screening model. To confirm the activity of samples against dormant M. tuberculosis, we used a luciferase reporter assay and enumerated CFU. The structures of five purified active compounds were defined by nuclear magnetic resonance (NMR) and mass spectrometry. We identified two lipid compounds with potent activity toward dormant and actively growing M. tuberculosis strains. One of these was commercially obtained and showed similar activity against M. tuberculosis in both screening models. Furthermore, puupehenone-like molecules were purified with potent and selective activity against dormant M. tuberculosis In conclusion, we have identified and characterized antimycobacterial compounds from marine organisms with novel activity profiles which appear to target M. tuberculosis pathways that are conditionally essential for dormancy survival.

Dragmacidin G, a Bioactive Bis-Indole Alkaloid from a Deep-Water Sponge of the Genus Spongosorites.

A deep-water sponge of the genus Spongosorites has yielded a bis-indole alkaloid which we have named dragmacidin G. Dragmacidin G was first reported by us in the patent literature and has recently been reported by Hitora et al. from a sponge of the genus Lipastrotheya. Dragmacidin G is the first in this series of compounds to have a pyrazine ring linking the two indole rings. It also has a rare N-(2-mercaptoethyl)-guanidine side chain. Dragmacidin G shows a broad spectrum of biological activity including inhibition of methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis, Plasmodium falciparum, and a panel of pancreatic cancer cell lines.

Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents.

Bacterial topoisomerase functions are required for regulation of DNA supercoiling and overcoming the DNA topological barriers that are encountered during many vital cellular processes. DNA gyrase and topoisomerase IV of the type IIA bacterial topoisomerase family are important clinical targets for antibacterial therapy. Topoisomerase I, belonging to the type IA topoisomerase family, has recently been validated as a potential antitubercular target. The topoisomerase I activity has been shown to be essential for bacterial viability and infection in a murine model of tuberculosis. Mixture-based combinatorial libraries were screened in this study to identify novel bacterial topoisomerase I inhibitors. Using positional-scanning deconvolution, selective small-molecule inhibitors of bacterial topoisomerase I were identified starting from a polyamine scaffold. Antibacterial assays demonstrated that four of these small-molecule inhibitors of bacterial topoisomerase I are bactericidal against Mycobacterium smegmatis and Mycobacterium tuberculosis The MICs for growth inhibition of M. smegmatis increased with overexpression of recombinant M. tuberculosis topoisomerase I, consistent with inhibition of intracellular topoisomerase I activity being involved in the antimycobacterial mode of action.

DNA Antenna Tile-Associated Deoxyribozyme Sensor with Improved Sensitivity.

Some natural enzymes increase the rate of diffusion-limited reactions by facilitating substrate flow to their active sites. Inspired by this natural phenomenon, we developed a strategy for efficient substrate delivery to a deoxyribozyme (DZ) catalytic sensor. This resulted in a three- to fourfold increase in sensitivity and up to a ninefold improvement in the detection limit. The reported strategy can be used to enhance catalytic efficiency of diffusion-limited enzymes and to improve sensitivity of enzyme-based biosensors.

Synthesis and antitubercular activity of 1,2,4-trisubstitued piperazines.

Parallel solid phase synthesis offers a unique opportunity for the synthesis and screening of large numbers of compounds and significantly enhances the prospect of finding new leads. We report the synthesis and antitubercular activity of chiral 1,2,4-trisubstituted piperazines derived from resin bound acylated dipeptides against Mycobacterium tuberculosis strain H37Rv.

Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection.

Intracellular pathogens such as Mycobacterium tuberculosis have evolved strategies for coping with the pressures encountered inside host cells. The ability to coordinate global gene expression in response to environmental and internal cues is one key to their success. Prolonged survival and replication within macrophages, a key virulence trait of M. tuberculosis, requires dynamic adaptation to diverse and changing conditions within its phagosomal niche. However, the physiological adaptations during the different phases of this infection process remain poorly understood. To address this knowledge gap, we have developed a multi-tiered approach to define the temporal patterns of gene expression in M. tuberculosis in a macrophage infection model that extends from infection, through intracellular adaptation, to the establishment of a productive infection. Using a clock plasmid to measure intracellular replication and death rates over a 14-day infection and electron microscopy to define bacterial integrity, we observed an initial period of rapid replication coupled with a high death rate. This was followed by period of slowed growth and enhanced intracellular survival, leading finally to an extended period of net growth. The transcriptional profiles of M. tuberculosis reflect these physiological transitions as the bacterium adapts to conditions within its host cell. Finally, analysis with a Transcriptional Regulatory Network model revealed linked genetic networks whereby M. tuberculosis coordinates global gene expression during intracellular survival. The integration of molecular and cellular biology together with transcriptional profiling and systems analysis offers unique insights into the host-driven responses of intracellular pathogens such as M. tuberculosis.

Orphan response regulator NnaR is critical for nitrate and nitrite assimilation in Mycobacterium abscessus.

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab's ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR's contribution to pathogenesis.

Detection of Mtb and NTM: preclinical validation of a new asymmetric PCR-binary deoxyribozyme sensor assay.

Tuberculosis (TB) and infectious diseases caused by non-tuberculous mycobacteria (NTM) are global concerns. The development of a rapid and accurate diagnostic method, capable of detecting and identifying different mycobacteria species, is crucial. We propose a molecular approach, the BiDz-TB/NTM, based on the use of binary deoxyribozyme (BiDz) sensors for the detection of Mycobacterium tuberculosis (Mtb) and NTM of clinical interest. A panel of DNA samples was used to evaluate Mtb-BiDz, Mycobacterium abscessus/Mycobacterium chelonae-BiDz, Mycobacterium avium-BiDz, Mycobacterium intracellulare/Mycobacterium chimaera-BiDz, and Mycobacterium kansasii-BiDz sensors in terms of specificity, sensitivity, accuracy, and limit of detection. The BiDz sensors were designed to hybridize specifically with the genetic signatures of the target species. To obtain the BiDz sensor targets, amplification of a fragment containing the hypervariable region 2 of the 16S rRNA was performed, under asymmetric PCR conditions using the reverse primer designed based on linear-after-the-exponential principles. The BiDz-TB/NTM was able to correctly identify 99.6% of the samples, with 100% sensitivity and 0.99 accuracy. The individual values of specificity, sensitivity, and accuracy, obtained for each BiDz sensor, satisfied the recommendations for new diagnostic methods, with sensitivity of 100%, specificity and accuracy ranging from 98% to 100% and from 0.98 to 1.0, respectively. The limit of detection of BiDz sensors ranged from 12 genome copies (Mtb-BiDz) to 2,110 genome copies (Mkan-BiDz). The BiDz-TB/NTM platform would be able to generate results rapidly, allowing the implementation of the appropriate therapeutic regimen and, consequently, the reduction of morbidity and mortality of patients.IMPORTANCEThis article describes the development and evaluation of a new molecular platform for accurate, sensitive, and specific detection and identification of Mycobacterium tuberculosis and other mycobacteria of clinical importance. Based on BiDz sensor technology, this assay prototype is amenable to implementation at the point of care. Our data demonstrate the feasibility of combining the species specificity of BiDz sensors with the sensitivity afforded by asymmetric PCR amplification of target sequences. Preclinical validation of this assay on a large panel of clinical samples supports the further development of this diagnostic tool for the molecular detection of pathogenic mycobacteria.

Mycobacterial trehalose dimycolate reprograms macrophage global gene expression and activates matrix metalloproteinases.

Trehalose 6,6'-dimycolate (TDM) is a cell wall glycolipid and an important virulence factor of mycobacteria. In order to study the role of TDM in the innate immune response to Mycobacterium tuberculosis, microarray analysis was used to examine gene regulation in murine bone marrow-derived macrophages in response to 90-µm-diameter polystyrene microspheres coated with TDM. A large number of genes, particularly those involved in the immune response and macrophage function, were up- or downregulated in response to these TDM-coated beads compared to control beads. Genes involved in the immune response were specifically upregulated in a myeloid differentiation primary response gene 88 (MyD88)-dependent manner. The complexity of the transcriptional response also increased greatly between 2 and 24 h. Matrix metalloproteinases (MMPs) were significantly upregulated at both time points, and this was confirmed by quantitative real-time reverse transcription-PCR (RT-PCR). Using an in vivo Matrigel granuloma model, the presence and activity of MMP-9 were examined by immunohistochemistry and in situ zymography (ISZ), respectively. We found that TDM-coated beads induced MMP-9 expression and activity in Matrigel granulomas. Macrophages were primarily responsible for MMP-9 expression, as granulomas from neutrophil-depleted mice showed staining patterns similar to that for wild-type mice. The relevance of these observations to human disease is supported by the similar induction of MMP-9 in human caseous tuberculosis (TB) granulomas. Given that MMPs likely play an important role in both the construction and breakdown of tuberculous granulomas, our results suggest that TDM may drive MMP expression during TB pathogenesis.

Deoxyribozyme cascade for visual detection of bacterial RNA.

In the blink of the eye: a cascade of two deoxyribozymes was designed for rapid visual detection of bacterial 16S rRNA. The detection limit is 12.5 ng by the naked eye, with the ability to differentiate between closely related pathogenic and nonpathogenic species.

Implementation of a mycobacterial CRISPRi platform in Mycobacterium abscessus and demonstration of the essentiality of ftsZMab.

Mycobacterium abscessus (Mab) is a highly drug-resistant non-tuberculous mycobacterial species that causes debilitating TB-like pulmonary infections. The lack of genetic tools has hampered characterization of its extensive repertoire of virulence factors, antimicrobial resistance mechanisms, and drug targets. In this study, we evaluated the performance of a mycobacterial single plasmid CRISPRi-dCas9 system optimized for M. tuberculosis and M. smegmatis for inducible gene silencing in Mab. The efficacy of CRISPRi-mediated repression of two antibiotic resistance genes (blaMab, whiB7Mab) and two putative essential genes (ftsZMab,topAMab) was determined by measuring mRNA transcript levels and phenotypic outcomes. While our results support the utility of this mycobacterial CRISPRi dCas9Sth1 single-plasmid platform for inducible silencing of specific target genes in Mab, they also highlighted several caveats and nuances that may warrant species-specific optimization for Mab. We observed overall lower levels of gene repression in Mab including variable silencing of different target genes despite use of PAMs of similar predicted strength. In addition, leaky gene repression in the absence of inducer was noted for some genes but not others. Nonetheless, using CRISPRi we demonstrated the silencing of multiple target genes and validated ftsZMab as an essential gene and promising drug target for the first time.