scholarly journals Treatment of Virulent Mycobacterium tuberculosis and HIV Coinfected Macrophages with Gallium Nanoparticles Inhibits Pathogen Growth and Modulates Macrophage Cytokine Production

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Seoung-ryoung Choi ◽  
Bradley E. Britigan ◽  
Prabagaran Narayanasamy
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Growth Inhibition ◽  
Cytokine Production ◽  
Virulent Strain ◽  
Enzymatic Reactions ◽  
Macrophage Infection ◽  
Content Type ◽  
Cytokine Analysis ◽  
Promising Therapeutic Approach ◽  
Gallium Nanoparticles

ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a global threat. The course of TB is negatively impacted by coexistent infection with human immunodeficiency virus type 1 (HIV). Macrophage infection with these pathogens modulates their production of pro- and anti-inflammatory cytokines, which could play a crucial role in pathogenesis. Despite the important role of macrophages in containing infection by a variety of microbes, both HIV and M. tuberculosis infect and replicate within these cells during the course of HIV-M. tuberculosis coinfection. Both M. tuberculosis and HIV require iron for growth and replication. We have previously shown that gallium encapsulated in nanoparticles, which interferes with cellular iron acquisition and utilization, inhibited the growth of HIV and an attenuated strain of M. tuberculosis within human monocyte-derived macrophages (MDMs) in vitro. Whether this was true for a fully virulent strain of M. tuberculosis and whether gallium treatment modulates cytokine production by HIV- and/or M. tuberculosis-infected macrophages have not been previously addressed. Therefore, coinfection of MDMs with HIV and a virulent M. tuberculosis strain (H37Rv) was studied in the presence of different gallium nanoparticles (GaNP). All GaNP were readily internalized by the MDMs, which provided sustained drug (gallium) release for 15 days. This led to significant growth inhibition of both HIV and M. tuberculosis within MDMs for up to 15 days after loading of the cells with all GaNP tested in our study. Cytokine analysis showed that HIV-M. tuberculosis coinfected macrophages secreted large amounts of interleukin 6 (IL-6) and IL-8 and smaller amounts of IL-1β, IL-4, and tumor necrosis factor alpha (TNF-α) cytokines. However, all GaNP were able to regulate the release of cytokines significantly. GaNP interrupts iron-mediated enzymatic reactions, leading to growth inhibition of HIV-M. tuberculosis coinfection in macrophages, and also modulates release of cytokines that may contribute to HIV-TB pathogenesis. IMPORTANCE GaNP interrupts iron-mediated enzymatic reactions, leading to growth inhibition of virulent HIV-M. tuberculosis coinfection in macrophages, and also modulates release of cytokines that may contribute to HIV-TB pathogenesis. Macrophage-targeting GaNP are a promising therapeutic approach to provide sustained antimicrobial activity against HIV-M. tuberculosis coinfection.

scholarly journals Transcriptional Profiling of Mycobacterium tuberculosis Exposed toIn VitroLysosomal Stress

2016 ◽  
Vol 84 (9) ◽  
pp. 2505-2523 ◽  
Author(s):  
Wenwei Lin ◽  
Paola Florez de Sessions ◽  
Garrett Hor Keong Teoh ◽  
Ahmad Naim Nazri Mohamed ◽  
Yuan O. Zhu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Metabolic Reprogramming ◽  
Human Macrophage ◽  
Host Immune System ◽  
Activated Macrophages ◽  
Intrinsic Resistance ◽  
Macrophage Infection ◽  
Content Type

Increasing experimental evidence supports the idea thatMycobacterium tuberculosishas evolved strategies to survive within lysosomes of activated macrophages. To further our knowledge ofM. tuberculosisresponse to the hostile lysosomal environment, we profiled the global transcriptional activity ofM. tuberculosiswhen exposed to the lysosomal soluble fraction (SF) prepared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation conditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory conditions that led to the absence of apparent mycobacterial replication,M. tuberculosisexpressed a unique transcriptome with modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy response, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific responses, we identified theglgE-mediated 1,4 α-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin systems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A meta-analysis including previously reported transcriptomic studies in macrophage infection andin vitrostress models was conducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding geneRv1258cwas selected for validation. AnM. tuberculosis ΔRv1258cmutant was constructed and displayed increased susceptibility to killing by lysosomal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macrophage cell line THP-1.

scholarly journals The Effect of Hyperglycaemia on In Vitro Cytokine Production and Macrophage Infection with Mycobacterium tuberculosis

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0117941 ◽  
Author(s):  
Ekta Lachmandas ◽  
Frank Vrieling ◽  
Louis G. Wilson ◽  
Simone A. Joosten ◽  
Mihai G. Netea ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cytokine Production ◽  
Macrophage Infection

scholarly journals Biphasic Dynamics of Macrophage Immunometabolism during Mycobacterium tuberculosis Infection

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Lanbo Shi ◽  
Qingkui Jiang ◽  
Yuri Bushkin ◽  
Selvakumar Subbian ◽  
Sanjay Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune Cells ◽  
Aerobic Glycolysis ◽  
Defense Responses ◽  
Bioactive Lipids ◽  
Macrophage Infection ◽  
Mycobacterium Tuberculosis Infection ◽  
Metabolic Switch ◽  
Content Type ◽  
M1 Polarization

ABSTRACT Macrophages are the primary targets of Mycobacterium tuberculosis infection; the early events of macrophage interaction with M. tuberculosis define subsequent progression and outcome of infection. M. tuberculosis can alter the innate immunity of macrophages, resulting in suboptimal Th1 immunity, which contributes to the survival, persistence, and eventual dissemination of the pathogen. Recent advances in immunometabolism illuminate the intimate link between the metabolic states of immune cells and their specific functions. In this review, we describe the little-studied biphasic metabolic dynamics of the macrophage response during progression of infection by M. tuberculosis and discuss their relevance to macrophage immunity and M. tuberculosis pathogenicity. The early phase of macrophage infection, which is marked by M1 polarization, is accompanied by a metabolic switch from mitochondrial oxidative phosphorylation to hypoxia-inducible factor 1 alpha (HIF-1α)-mediated aerobic glycolysis (also known as the Warburg effect in cancer cells), as well as by an upregulation of pathways involving oxidative and antioxidative defense responses, arginine metabolism, and synthesis of bioactive lipids. These early metabolic changes are followed by a late adaptation/resolution phase in which macrophages transition from glycolysis to mitochondrial oxidative metabolism, with a consequent dampening of macrophage proinflammatory and antimicrobial responses. Importantly, the identification of upregulated metabolic pathways and/or metabolic regulatory mechanisms with immunomodulatory functions during M1 polarization has revealed novel mechanisms of M. tuberculosis pathogenicity. These advances can lead to the development of novel host-directed therapies to facilitate bacterial clearance in tuberculosis by targeting the metabolic state of immune cells.

scholarly journals The Polyphosphate Kinase Gene ppk2 Is Required for Mycobacterium tuberculosis Inorganic Polyphosphate Regulation and Virulence

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Yu-Min Chuang ◽  
Deborah A. Belchis ◽  
Petros C. Karakousis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Interleukin 2 ◽  
Fold Increase ◽  
Polyphosphate Kinase ◽  
Macrophage Infection ◽  
Inorganic Polyphosphate ◽  
Kinase Gene ◽  
Content Type ◽  
Cytokines And Chemokines ◽  
P Content

ABSTRACT The Mycobacterium tuberculosis gene Rv3232c/MT3329 (ppk2) encodes a class II polyphosphate kinase, which hydrolyzes inorganic polyphosphate (poly P) to synthesize GTP. We assessed the role of ppk2 in M. tuberculosis poly P regulation, antibiotic tolerance, and virulence. A ppk2-deficient mutant (ppk2::Tn) and its isogenic wild-type (WT) and complemented (Comp) strains were studied. For each strain, the intrabacillary poly P content, MIC of isoniazid, and growth kinetics during infection of J774 macrophages were determined. Multiplex immunobead assays were used to evaluate cytokines elaborated during macrophage infection. The requirement of ppk2 for M. tuberculosis virulence was assessed in the murine model. The ppk2::Tn mutant was found to have significantly increased poly P content and a 4-fold increase in the MIC of isoniazid relative to the WT and Comp strains. The ppk2::Tn mutant showed reduced survival at day 7 in activated and naive J774 macrophages relative to the WT. Naive ppk2::Tn mutant-infected macrophages showed increased expression of interleukin 2 (IL-2), IL-9, IL-10, IL-12p70, and gamma interferon (IFN-γ) relative to WT-infected macrophages. The ppk2::Tn mutant exhibited significantly lower lung CFU during acute murine infection compared to the control groups. ppk2 is required for control of intrabacillary poly P levels and optimal M. tuberculosis growth and survival in macrophages and mouse lungs. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a highly successful human pathogen because it has developed mechanisms to multiply and survive in the lungs by circumventing the immune system. Identification of virulence factors responsible for M. tuberculosis growth and persistence in host tissues may assist in the development of novel strategies to treat TB. In this study, we found that the mycobacterial enzyme polyphosphate kinase 2 (PPK2) is required for controlling intracellular levels of important regulatory molecules and for maintaining susceptibility to the first-line anti-TB drug isoniazid. In addition, PPK2 was found to be required for M. tuberculosis growth in the lungs of mice, at least in part by suppressing the expression of certain key cytokines and chemokines by inactivated lung macrophages.

scholarly journals A New ESX-1 Substrate inMycobacterium marinumThat Is Required for Hemolysis but Not Host Cell Lysis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Rachel E. Bosserman ◽  
Kathleen R. Nicholson ◽  
Matthew M. Champion ◽  
Patricia A. Champion
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Response ◽  
Secretion System ◽  
Mycobacterial Species ◽  
Macrophage Infection ◽  
Content Type ◽  
Family Protein ◽  
Species Specific ◽  
System 1 ◽  
Insight Into

ABSTRACTThe ESX-1 (ESAT-6 system 1) secretion system plays a conserved role in the virulence of diverse mycobacterial pathogens, including the human pathogenMycobacterium tuberculosisandM. marinum, an environmental mycobacterial species. The ESX-1 system promotes the secretion of protein virulence factors to the extracytoplasmic environment. The secretion of these proteins triggers the host response by lysing the phagosome during macrophage infection. Using proteomic analyses of theM. marinumsecretome in the presence and absence of a functional ESX-1 system, we and others have hypothesized that MMAR_2894, a PE family protein, is a potential ESX-1 substrate inM. marinum. We used genetic and quantitative proteomic approaches to determine if MMAR_2894 is secreted by the ESX-1 system, and we defined the requirement ofMMAR_2894for ESX-1-mediated secretion and virulence. We show that MMAR_2894 is secreted by the ESX-1 system inM. marinumand is itself required for the optimal secretion of the known ESX-1 substrates inM. marinum. Moreover, we found that MMAR_2894 was differentially required for hemolysis and cytolysis of macrophages, two lytic activities ascribed to theM. marinumESX-1 system.IMPORTANCEBothMycobacterium tuberculosis, the cause of human tuberculosis (TB), andMycobacterium marinum, a pathogen of ectotherms, use the ESX-1 secretion system to cause disease. There are many established similarities between the ESX-1 systems inM. tuberculosisand inM. marinum. Yet the two bacteria infect different hosts, hinting at species-specific functions of the ESX-1 system. Our findings demonstrate that MMAR_2894 is a PE protein secreted by the ESX-1 system ofM. marinum. We show that MMAR_2894 is required for the optimal secretion of mycobacterial proteins required for disease. Because theMMAR_2894gene is not conserved inM. tuberculosis, our findings demonstrate that MMAR_2894 may contribute to a species-specific function of the ESX-1 system inM. marinum, providing new insight into how theM. marinumandM. tuberculosissystems differ.

scholarly journals Integration of Metabolomics and Transcriptomics Reveals a Complex Diet of Mycobacterium tuberculosis during Early Macrophage Infection

mSystems ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Michael Zimmermann ◽  
Maria Kogadeeva ◽  
Martin Gengenbacher ◽  
Gayle McEwen ◽  
Hans-Joachim Mollenkopf ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Flux Balance Analysis ◽  
Intracellular Pathogens ◽  
Macrophage Infection ◽  
Content Type ◽  
Flux Balance ◽  
Genome Wide ◽  
A Genome ◽  
Host Pathogen ◽  
Balance Analysis

ABSTRACT The nutrients consumed by intracellular pathogens are mostly unknown. This is mainly due to the challenge of disentangling host and pathogen metabolism sharing the majority of metabolic pathways and hence metabolites. Here, we investigated the metabolic changes of Mycobacterium tuberculosis, the causative agent of tuberculosis, and its human host cell during early infection. To this aim, we combined gene expression data of both organisms and metabolite changes during the course of infection through integration into a genome-wide metabolic network. This led to the identification of infection-specific metabolic alterations, which we further exploited to model host-pathogen interactions quantitatively by flux balance analysis. These in silico data suggested that tubercle bacilli consume up to 33 different nutrients during early macrophage infection, which the bacteria utilize to generate energy and biomass to establish intracellular growth. Such multisubstrate fueling strategy renders the pathogen’s metabolism robust toward perturbations, such as innate immune responses or antibiotic treatments. Nutrient acquisition from the host environment is crucial for the survival of intracellular pathogens, but conceptual and technical challenges limit our knowledge of pathogen diets. To overcome some of these technical roadblocks, we exploited an experimentally accessible model for early infection of human macrophages by Mycobacterium tuberculosis, the etiological agent of tuberculosis, to study host-pathogen interactions with a multi-omics approach. We collected metabolomics and complete transcriptome RNA sequencing (dual RNA-seq) data of the infected macrophages, integrated them in a genome-wide reaction pair network, and identified metabolic subnetworks in host cells and M. tuberculosis that are modularly regulated during infection. Up- and downregulation of these metabolic subnetworks suggested that the pathogen utilizes a wide range of host-derived compounds, concomitant with the measured metabolic and transcriptional changes in both bacteria and host. To quantify metabolic interactions between the host and intracellular pathogen, we used a combined genome-scale model of macrophage and M. tuberculosis metabolism constrained by the dual RNA-seq data. Metabolic flux balance analysis predicted coutilization of a total of 33 different carbon sources and enabled us to distinguish between the pathogen’s substrates directly used as biomass precursors and the ones further metabolized to gain energy or to synthesize building blocks. This multiple-substrate fueling confers high robustness to interventions with the pathogen’s metabolism. The presented approach combining multi-omics data as a starting point to simulate system-wide host-pathogen metabolic interactions is a useful tool to better understand the intracellular lifestyle of pathogens and their metabolic robustness and resistance to metabolic interventions. IMPORTANCE The nutrients consumed by intracellular pathogens are mostly unknown. This is mainly due to the challenge of disentangling host and pathogen metabolism sharing the majority of metabolic pathways and hence metabolites. Here, we investigated the metabolic changes of Mycobacterium tuberculosis, the causative agent of tuberculosis, and its human host cell during early infection. To this aim, we combined gene expression data of both organisms and metabolite changes during the course of infection through integration into a genome-wide metabolic network. This led to the identification of infection-specific metabolic alterations, which we further exploited to model host-pathogen interactions quantitatively by flux balance analysis. These in silico data suggested that tubercle bacilli consume up to 33 different nutrients during early macrophage infection, which the bacteria utilize to generate energy and biomass to establish intracellular growth. Such multisubstrate fueling strategy renders the pathogen’s metabolism robust toward perturbations, such as innate immune responses or antibiotic treatments.

scholarly journals Subcellular Partitioning and Intramacrophage Selectivity of Antimicrobial Compounds against Mycobacterium tuberculosis

2017 ◽  
Vol 61 (3) ◽  
Author(s):  
Michael D. Schump ◽  
Douglas M. Fox ◽  
Carolyn R. Bertozzi ◽  
Lee W. Riley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Performance ◽  
Axenic Culture ◽  
Bacterial Pathogen ◽  
Drug Distribution ◽  
Broth Culture ◽  
Infection Model ◽  
Moderate Activity ◽  
Macrophage Infection ◽  
Content Type

ABSTRACT The efficacy of antimicrobial drugs against Mycobacterium tuberculosis, an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. However, inside infected macrophages, bacteria encounter an environment which differs substantially from broth culture and are subject to important host-dependent pharmacokinetic phenomena which modulate drug activity. Here, we describe how pH-dependent partitioning drives asymmetric antimicrobial drug distribution in M. tuberculosis-infected macrophages. Specifically, weak bases with moderate activity against M. tuberculosis (fluoxetine, sertraline, and dibucaine) were shown to accumulate intracellularly due to differential permeability and relative abundance of their ionized and nonionized forms. Nonprotonatable analogs of the test compounds did not show this effect. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. We further demonstrate that the efficacy of a clinically used compound, clofazimine, is augmented by pH-based partitioning in a macrophage infection model. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis.

scholarly journals Inhibition of CorA-Dependent Magnesium Homeostasis Is Cidal in Mycobacterium tuberculosis

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Yumi Park ◽  
Yong-Mo Ahn ◽  
Surendranadha Jonnala ◽  
Sangmi Oh ◽  
Julia M. Fisher ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Discovery ◽  
Macrophage Infection ◽  
Content Type ◽  
Magnesium Homeostasis ◽  
Direct Binding ◽  
Pore Opening

ABSTRACT Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.

scholarly journals Persistent Infection by a Mycobacterium tuberculosis Strain That Was Theorized To Have Advantageous Properties, as It Was Responsible for a Massive Outbreak

2015 ◽  
Vol 53 (11) ◽  
pp. 3423-3429 ◽  
Author(s):  
Laura Pérez-Lago ◽  
Yurena Navarro ◽  
Pedro Montilla ◽  
Iñaki Comas ◽  
Marta Herranz ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Virulent Strain ◽  
General Assumption ◽  
Host Population ◽  
Resistance Mutations ◽  
Single Nucleotide ◽  
Content Type ◽  
Treatment Interruptions ◽  
Highly Virulent

The strains involved in tuberculosis outbreaks are considered highly virulent and transmissible. We analyzed the case of a patient in Madrid, Spain, who was persistently infected over an 8-year period by the same BeijingMycobacterium tuberculosisstrain. The strain was responsible for a severe outbreak on Gran Canaria Island. The case provides us with a unique opportunity to challenge our assumptions aboutM. tuberculosisBeijing strains. No clinical/radiological findings consistent with a virulent strain were documented, and thein vitrogrowth rate of the strain in macrophages was only moderate. No secondary cases stemming from this prolonged active case were detected in the host population. The strain did not acquire resistance mutations, despite constant treatment interruptions, and it remained extremely stable, as demonstrated by the lack of single-nucleotide-polymorphism (SNP)-based differences between the sequential isolates. Our data suggest that the general assumption aboutM. tuberculosisBeijing strains having advantageous properties (in terms of virulence, transmissibility, and the tendency to acquire mutations and resistance) is not always accurate.

scholarly journals Mycobacterium tuberculosis Toxin CpnT Is an ESX-5 Substrate and Requires Three Type VII Secretion Systems for Intracellular Secretion

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
B. Izquierdo Lafuente ◽  
R. Ummels ◽  
C. Kuijl ◽  
W. Bitter ◽  
A. Speer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cells ◽  
Secretion Systems ◽  
Macrophage Infection ◽  
Content Type ◽  
Secretion Pathway ◽  
Terminal Domain ◽  
Type Vii Secretion ◽  
Type Vii Secretion System ◽  
Axenic Conditions

ABSTRACT CpnT, a NAD+ glycohydrolase, is the only known toxin that is secreted by Mycobacterium tuberculosis. CpnT is composed of two domains; the C-terminal domain is the toxin, whereas the N-terminal domain is required for secretion. CpnT shows characteristics of type VII secretion (T7S) substrates, including a predicted helix-turn-helix domain followed by a secretion motif (YxxxE). Disruption of this motif indeed abolished CpnT secretion. By analyzing different mutants, we established that CpnT is specifically secreted by the ESX-5 system in Mycobacterium marinum under axenic conditions and during macrophage infection. Surprisingly, intracellular secretion of CpnT was also dependent on both ESX-1 and ESX-4. These secretion defects could be partially rescued by coinfection with wild-type bacteria, indicating that secreted effectors are involved in this process. In summary, our data reveal that three different type VII secretion systems have to be functional in order to observe intracellular secretion of the toxin CpnT. IMPORTANCE For decades, it was believed that the intracellular pathogen M. tuberculosis does not possess toxins. Only fairly recently it was discovered that CpnT is a potent secreted toxin of M. tuberculosis, causing necrotic cell death in host cells. However, until now the secretion pathway remained unknown. In our study, we were able to identify CpnT as a substrate of the mycobacterial type VII secretion system. Pathogenic mycobacteria have up to five different type VII secretion systems, called ESX-1 to ESX-5, which play distinct roles for the pathogen during growth or infection. We were able to elucidate that CpnT is exclusively secreted by the ESX-5 system in bacterial culture. However, to our surprise we discovered that, during infection studies, CpnT secretion relies on intact ESX-1, ESX-4, and ESX-5 systems. We elucidate for the first time the intertwined interplay of three different and independent secretion systems to secrete one substrate during infection.

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