High-throughput transcriptomics reveals common and strain-specific responses of human macrophages to infection with Mycobacterium abscessus Smooth and Rough variants

Abstract Background During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. Results The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. Conclusions Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.

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Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response

Microbiology ◽

10.1099/mic.0.046557-0 ◽

2011 ◽

Vol 157 (4) ◽

pp. 1187-1195 ◽

Cited By ~ 53

Author(s):

Rachid Nessar ◽

Jean-Marc Reyrat ◽

Lisa B. Davidson ◽

Thomas F. Byrd

Keyword(s):

Pathogenic Bacteria ◽

Biosynthetic Pathway ◽

Gene Knockout ◽

Cellular Level ◽

Mycobacterium Abscessus ◽

Bacterial Gene ◽

Human Macrophages ◽

Knockout Mutants ◽

A Cell ◽

Toll Like Receptor 2

Mycobacterium abscessus is considered to be the most virulent of the rapidly growing mycobacteria. Generation of bacterial gene knockout mutants has been a useful tool for studying factors that contribute to virulence of pathogenic bacteria. Until recently, the optimal genetic approach to generation of M. abscessus gene knockout mutants was not clear. Based on the recent identification of genetic recombineering as the preferred approach, a M. abscessus mutant was generated in which the gene mmpL4b, critical to glycopeptidolipid synthesis, was deleted. Compared to the previously well-characterized parental strain 390S, the mmpL4B deletion mutant had lost sliding motility and the ability to form biofilm, but acquired the ability to replicate in human macrophages and stimulate macrophage Toll-like receptor 2. This study demonstrates that deletion of a gene associated with expression of a cell-wall lipid can result in acquisition of an immunostimulatory, invasive bacterial phenotype and has important implications for the study of M. abscessus pathogenesis at the cellular level.

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High throughput Phenotypic Microarray profiling of Mycobacterium abscessus

Journal of Zankoy Sulaimani - Part A ◽

10.17656/jzs.10720 ◽

2018 ◽

Vol 20 (2) ◽

pp. 9-20

Author(s):

Sirwan Muhsin Muhammed Ameen Ameen ◽

Keyword(s):

High Throughput ◽

Mycobacterium Abscessus ◽

Phenotypic Microarray ◽

Microarray Profiling

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THP-1 and Dictyostelium Infection Models for Screening and Characterization of Anti-Mycobacterium abscessus Hit Compounds

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01601-19 ◽

2019 ◽

Vol 64 (1) ◽

Cited By ~ 2

Author(s):

Adrian Richter ◽

Tirosh Shapira ◽

Yossef Av-Gay

Keyword(s):

Host Cell ◽

Kinase Inhibitors ◽

Screening Method ◽

Inhibitory Effect ◽

Mycobacterium Abscessus ◽

Infection Model ◽

Content Type ◽

Human Macrophages ◽

Human Kinase ◽

Mycobacterial Growth

ABSTRACT !!NCR1!! presents a great challenge to antimycobacterial therapy due to its innate resistance against most antibiotics. M. abscessus is able to grow intracellularly in human macrophages, suggesting that intracellular models can facilitate drug discovery. Thus, we have developed two host cell models: human macrophages for use in a new high-content screening method for M. abscessus growth and a Dictyostelium discoideum infection model with the potential to simplify downstream genetic analysis of host cell factors. A screen of 568 antibiotics for activity against intracellular M. abscessus led to the identification of two hit compounds with distinct growth inhibition. A collection of 317 human kinase inhibitors was analyzed, with the results yielding three compounds with an inhibitory effect on mycobacterial growth, strengthening the notion that host-directed therapy can be applied for M. abscessus.

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Induction of heme oxygenase-1 contributes to survival of Mycobacterium abscessus in human macrophages-like THP-1 cells

Redox Biology ◽

10.1016/j.redox.2015.01.012 ◽

2015 ◽

Vol 4 ◽

pp. 328-339 ◽

Cited By ~ 28

Author(s):

Maher Y. Abdalla ◽

Iman M. Ahmad ◽

Barbara Switzer ◽

Bradley E. Britigan

Keyword(s):

Heme Oxygenase ◽

Mycobacterium Abscessus ◽

Heme Oxygenase 1 ◽

Human Macrophages

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C25-modified rifamycin derivatives with improved activity against Mycobacterium abscessus

10.1101/2021.07.12.452042 ◽

2021 ◽

Author(s):

Laura Paulowski ◽

Katherine S.H. Beckham ◽

Matt D. Johansen ◽

Laura Berneking ◽

Nhi Van ◽

...

Keyword(s):

Antimicrobial Activity ◽

Catalytic Activity ◽

Mycobacterium Abscessus ◽

Synergistic Activity ◽

Intrinsic Resistance ◽

Human Macrophages ◽

Rifamycin Sv ◽

Thermal Profiling ◽

Enzymatic Inactivation ◽

Adp Ribosyltransferase

Infections caused by Mycobacterium abscessus are difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity of M. abscessus to survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin. Unlike rifampicin, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position, is not modified by purified ArrMab. Additionally, we show that the ArrMab D82 residue is essential for catalytic activity. Thermal profiling of ArrMab in the presence of 5j, rifampicin or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin against M. abscessus planktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity against M. abscessus in human macrophages and shows synergistic activity with amikacin and azithromycin.

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Dual β-Lactam Combinations Highly Active against Mycobacterium abscessus Complex In Vitro

mBio ◽

10.1128/mbio.02895-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 17

Author(s):

R. Pandey ◽

L. Chen ◽

C. Manca ◽

S. Jenkins ◽

L. Glaser ◽

...

Keyword(s):

Antibiotic Therapy ◽

Patient Care ◽

Laboratory Finding ◽

Acquired Resistance ◽

Large Family ◽

Mycobacterium Abscessus ◽

Chronic Infections ◽

Content Type ◽

Human Macrophages

ABSTRACT As a consequence of a growing population of immunocompromised individuals, including transplant recipients and cystic fibrosis patients, there has been a dramatic increase in chronic infections caused by Mycobacterium abscessus complex (MABC) strains that are usually recalcitrant to effective antibiotic therapy. The recent rise of macrolide resistance in MABC has further complicated this clinical dilemma, dramatizing the need for novel agents. The repurposing of current antibiotics is one rapid path from discovery to patient care. In this study, we have discovered that dual β-lactams, and specifically the combination of ceftazidime with either ceftaroline or imipenem, are synergistic and have clinically relevant activities, with MIC50s of 0.25 (ceftaroline with 100 µg/ml ceftazidime) and 0.5 µg/ml (imipenem with 100 µg/ml ceftazidime) against clinical MABC isolates. Similar synergy was observed in time-kill studies against the M. abscessus ATCC 19977 strain using clinically achievable concentrations of either imipenem (4 µg/ml) or ceftaroline (2 µg/ml), as the addition of ceftazidime at concentrations of ≥50 µg/ml showed a persistent bactericidal effect over 5 days. Treatment of THP-1 human macrophages infected with three different M. abscessus clinical isolates supported the in vitro findings, as the combination of 100 µg/ml ceftazidime and 0.125 µg/ml ceftaroline or 100 µg/ml ceftazidime and 0.25 µg/ml imipenem dramatically reduced the CFU counts to near baseline levels of infection. This study’s finding that there is synergy between certain β-lactam combinations against M. abscessus infection provides optimism toward identifying an optimum dual β-lactam treatment regimen. IMPORTANCE The emergence of chronic MABC infections among immunocompromised populations and their inherent and acquired resistance to effective antibiotic therapy have created clinical challenges in advancing patients for transplant surgery and treating those with disease. There is an urgent need for new treatment regimens, and the repurposing of existing antibiotics provides a rapid strategy to advance a laboratory finding to patient care. Our recent discoveries that dual β-lactams, specifically the combination of ceftazidime with ceftaroline or ceftazidime with imipenem, have significant in vitro MIC values and kill curve activities and are effective against infected THP-1 human macrophages provide optimism for a dual β-lactam treatment strategy against MABC infections. The unexpected synergistic activities reported in this study create a new path of discovery to repurpose the large family of β-lactam drugs.

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Identification of Proteins Differentially Expressed in Human Monocytes Exposed to Porphyromonas gingivalis and Its Purified Components by High-Throughput Immunoblotting

Infection and Immunity ◽

10.1128/iai.74.2.1204-1214.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 1204-1214 ◽

Cited By ~ 36

Author(s):

Qingde Zhou ◽

Salomon Amar

Keyword(s):

Protein Kinase ◽

Cell Surface ◽

Porphyromonas Gingivalis ◽

High Throughput ◽

Inflammatory Responses ◽

Mitogen Activated Protein Kinase ◽

Antibody Array ◽

Becton Dickinson ◽

Necrosis Factor Alpha ◽

Human Macrophages

ABSTRACT To characterize the roles of Porphyromonas gingivalis and its components in disease processes, we investigated the cytokine profiles induced by live P. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1β [MIP-1β], and MIP-3α) as early as 1 h after exposure to P. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines by P. gingivalis was not triggered simply by bacterial cell surface components, since purified P. gingivalis LPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by live P. gingivalis was significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which live P. gingivalis and its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved in P. gingivalis infection in human macrophages. Exposure of human macrophages to either live P. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially by P. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P. gingivalis infection.

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Impact of relebactam-mediated inhibition of Mycobacterium abscessus BlaMab β-lactamase on the in vitro and intracellular efficacy of imipenem

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz433 ◽

2019 ◽

Author(s):

Eva Le Run ◽

Heiner Atze ◽

Michel Arthur ◽

Jean-Luc Mainardi

Keyword(s):

Second Generation ◽

Antibacterial Activities ◽

Mycobacterium Abscessus ◽

Pulmonary Infections ◽

Human Macrophages ◽

Time Kill ◽

The Impact ◽

Intracellular Proliferation ◽

Lactamase Inhibitor

Abstract Objectives Imipenem is one of the recommended β-lactams for the treatment of Mycobacterium abscessus pulmonary infections in spite of the production of BlaMab β-lactamase. Avibactam, a second-generation β-lactamase inhibitor, was previously shown to inactivate BlaMab, but its partner drug, ceftazidime, is devoid of any antibacterial activity against M. abscessus. Here, we investigate whether relebactam, a novel second-generation inhibitor developed in combination with imipenem, improves the activity of this carbapenem against M. abscessus. Methods The impact of BlaMab inhibition by relebactam was evaluated by determining MICs, time–kill curves and M. abscessus intracellular proliferation in human macrophages. Kinetic parameters for the inhibition of BlaMab by relebactam were determined by spectrophotometry using nitrocefin as the substrate. The data were compared with those obtained with avibactam. Results Combination of relebactam (4 mg/L) with β-lactams led to >128- and 2-fold decreases in the MICs of amoxicillin (from >4096 to 32 mg/L) and imipenem (from 8 to 4 mg/L). In vitro, M. abscessus was not killed by the imipenem/relebactam combination. In contrast, relebactam increased the intracellular activity of imipenem, leading to 88% killing. Relebactam and avibactam similarly potentiated the antibacterial activities of β-lactams although BlaMab was inactivated 150-fold less effectively by relebactam than by avibactam. Conclusions Inhibition of BlaMab by relebactam improves the efficacy of imipenem against M. abscessus in macrophages, indicating that the imipenem/relebactam combination should be clinically considered for the treatment of infections due to M. abscessus.

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