Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PA, ABL/PI3P, and ABL/PI5P enhance both internalization and intracellular killing of M. abscessus in dTHP-1 cells and monocyte-derived macrophages (MDM) from healthy donors (HD) with pharmacologically inhibited CFTR. (A to C) dTHP-1 cells (A and B) and primary MDM from healthy donors (C), treated or not treated with INH172, were cultured at 5 × 10 5 cells/well in 24-well plates and 2 × 10 5 cells/well in 96-well plates. (A) Cells were stimulated with selected ABL formulations before infection for 30 min and then infected with the M. abscessus reference strain (ATCC 19977). Bacterial uptake was quantified by CFU assay and indicated as phagocytosis index, calculated as the ratio between the CFU obtained immediately after the infection and those from the inoculum. (B and C) Cells, dTHP-1 cells (B) and MDM from healthy donors (C), were exposed or not exposed to INH172, infected with M. Abscessus , and treated for 18 h with the selected ABL formulations. Bacterial growth was assessed by CFU assay. The replication index was calculated as the ratio between the CFU obtained 18 h after infection, in the presence or absence of ABL formulations, and the CFU obtained before the addition of liposomes. The results are shown as the mean ± standard deviation of the values obtained from triplicates of each condition, and panel C is representative of experiments with cells from four different donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Derivative Assay, Cell Culture, Infection, Colony-forming Unit Assay, Liposomes, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P promotes both ROS- and phagolysosome acidification-dependent intracellular M. abscessus killing in CF MDM. (A to C) MDM isolated from CF patients ( n = 17) were plated at the concentration of 1 × 10 6 cells/mL, infected with the M. abscessus reference strain (ATCC 19977), and then stimulated for 18 h with ABL carrying PI5P (A) ( n = 17) in the presence or absence of catalase (PEG-Cat) (B) (representative of n = 4) or concanamycin A (Conc A), a specific inhibitor of vacuolar type H + -ATPase activity (C) (representative of n = 4) at the concentration of 100 U/mL or 1 nM, respectively. Bacterial growth was assessed by CFU assay, and the replication index was calculated as the ratio between the CFU obtained 18 h after infection, in the presence or absence of ABL formulations, and the CFU obtained before the addition of liposomes. (A) Statistical analysis was performed using the two-sided Wilcoxon matched-pair signed rank test ( P = 0.0005). (B and C) The results are shown as the mean ± standard deviation of the values obtained from each condition and are representative of experiments with cells from four different CF patients. *, P < 0.05; **, P < 0.01, ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Isolation, Concentration Assay, Infection, Activity Assay, Colony-forming Unit Assay, Liposomes, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P reduces both bacterial burden and leukocyte recruitment in WT mice infected with M. abscessus . (A) C57BL/6N mice were chronically infected with 10 5 CFU of the M. abscessus reference strain (ATCC 19977) by intratracheal (i.t.) injection and treated after 1 week of infection with ABL loaded with PA, PI3P, or PI5P three times a week for 8, 17, and 29 days. (A and B) After these time points mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). Data from two independent experiments were pooled. The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01).

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Infection, Injection, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P reduces both bacterial burden and leukocyte recruitment in CF mice infected with M. abscessus . (A) WT and CF mice were chronically infected with 10 5 CFU of the M. abscessus reference strain (ATCC 19977) by i.t. injection and treated with ABL/PI5P three times a week for 8 and 29 days. (B and C) Mice were then sacrificed, and BALF and lungs were processed for (B) microbiological analysis and (C) evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment to establish the therapeutic efficacy of liposomes. The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01).

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Infection, Injection, Drug discovery, Liposomes, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P-amikacin combined treatment promotes higher reduction of M. abscessus intracellular growth than single treatments in CF MDM. CF MDM were cultured and infected as described in Materials and Methods. (A and B) Finally, supernatant was collected, cells were lysed, and both were analyzed for extracellular (A) and intracellular (B) bacterial growth. The replication index was calculated as the ratio between the CFU obtained 18 h after infection in the presence or absence of ABL/PI5P and/or amikacin (AMK) and those obtained immediately after infection, before the addition of the stimuli. Results are shown as the mean ± standard deviation of the values obtained from triplicates of each condition and are representative of four CF patients. n.s., not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Cell Culture, Infection, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P-amikacin combined treatment induces both M. abscessus clearance and anti-inflammatory responses in WT mice. (A) WT mice were chronically infected with 10 5 CFU of the M. abscessus reference strain (ATCC 19977) and treated 1 week after infection with AMK and/or ABL/PI5P as described in Materials and Methods. (B and C) After 8 and 29 days of treatment, mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Infection, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

doi: 10.1128/spectrum.02546-21

Figure Lengend Snippet: ABL/PI5P-amikacin combined treatment induces both M. abscessus clearance and anti-inflammatory responses in CF mice. (A) CF mice were chronically infected with 10 5 CFU of the M. abscessus reference strain (ATCC 19977) and treated 1 week after infection with AMK and/or ABL/PI5P as described in Materials and Methods. (B and C) After 29 days of treatment mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Mice were intratracheally infected with M. abscessus (reference strain ATCC 19977) and, starting on day 7 after infection, were inoculated daily, via the i.p. route, with 100 mg/kg body weight of amikacin, whose dose was chosen in preliminary in vivo experiments (Fig. S7) and/or treated 3 times per week for 4 consecutive weeks by intranasal inoculation of 10 5 liposomes.

Techniques: Infection, MANN-WHITNEY

Journal: Journal of Clinical Microbiology

Article Title: Multilocus Sequence Analysis and rpoB Sequencing of Mycobacterium abscessus (Sensu Lato) Strains

doi: 10.1128/jcm.01274-10

Figure Lengend Snippet: FIG. 1. Tree constructed from partial rpoB gene sequences. The tree for all studied strains (n 123) was generated using the neighbor- joining method. Bootstrap support values (%) are indicated for each node. Species assignment of clinical isolates are according to the cri- teria in Ade´kambi et al. (2): f, M. abscessus; ●, M. massiliense; Œ, M. bolletii; numbers in parentheses are the numbers of isolates if there are two or more. Type strains: *, M. abscessus CIP 104536T; **, M. mas- siliense CIP 108297T; ***, M. bolletii CIP 108541T.

Article Snippet: The type strains M. abscessus CIP 104536T (ATCC 19977T), M. massiliense (CIP 108297T), and M. bolletii (CIP 108541T) also were included in the strain collection.

Techniques: Construct, Generated

Journal: Journal of Clinical Microbiology

Article Title: Multilocus Sequence Analysis and rpoB Sequencing of Mycobacterium abscessus (Sensu Lato) Strains

doi: 10.1128/jcm.01274-10

Figure Lengend Snippet: FIG. 2. Trees constructed from the sequences of the eight individual genes included in the final MLSA scheme. The trees for all studied strains (n 123) were generated using the neighbor-joining method. Bootstrap support values (%) at each of the nodes are indicated only for trees showing well-defined clusters. Species assignment of clinical isolates according to the criteria of Ade´kambi et al. (3): f, M. abscessus; ●, M. massiliense; Œ, M. bolletii. Type strains:*, M. abscessus CIP 104536T; **, M. massiliense CIP 108297T; ***, M. bolletii CIP 108541T.

Article Snippet: The type strains M. abscessus CIP 104536T (ATCC 19977T), M. massiliense (CIP 108297T), and M. bolletii (CIP 108541T) also were included in the strain collection.

Techniques: Construct, Generated

Journal: bioRxiv

Article Title: Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

doi: 10.1101/2021.05.10.443476

Figure Lengend Snippet: a ) Volcano plot of differentially expressed genes in M. abscessus ATCC19977 upon exposure to RIF (16μg/mL for 30 mins). Mab_arr and MAB_3189 ( helR ) are the two most highly induced genes and are indicated. b ) Wild type M. abscessus was grown to A 600 of 0.7 and exposed to either 8μg/mL of RIF or 0.5 μg/mL of RBT for 30 mins. The amount of Mab_arr and Mab_helR transcript was determined by qPCR and plotted as fold induction over an unexposed control. Data represents mean ± SD, n=3. SigA was used as an internal normalization control. c ) Average nucleotide identities between eight clinical strains of M. abscessus obtained using FastANI version 1.32. is shown. d ) Expression of Mab_arr and Mab_helR transcript in eight clinical strains of M. abscessus upon exposure to 8μg/mL of RIF determined by qPCR and plotted as fold induction over an unexposed control. Data represents mean ± SD, n=3. SigA was used as an internal normalization control.

Article Snippet: M. abscessus strains (ATCC 19977, clinical strains and ΔMab_helR ) were grown to A 600 of 0.7, exposed to RIF(8μg/ml) or RBT (0.5 μg/ml) for 30mins.

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

doi: 10.1101/2021.05.10.443476

Figure Lengend Snippet: a-b) Growth of ten-fold serial dilutions of M. abscessus ATCC 19977, ΔMab_helR, ΔMab_arr,ΔMab_helR/ ΔMab_arr , and the indicated complementing strains on Middlebrook 7H10 OADC containing indicated concentrations of RIF and RBT. Data is representative of >3 independent experiments. c ) Real time PCR showing the induction over an unexposed control of Mab_arr transcript in wild type M. abscessus and the ΔhelR strains exposed to either 8μg/mL of RIF or 0.5 μg/mL of RBT. Data represents mean ± SD, n=3. SigA was used as an internal normalization control. d ) Growth of ten-fold serial dilutions of wild-type M. tuberculosis mc 2 7000, mc 2 7000 containing a chromosomally integrated copy of helR and mc 2 7000 containing a chromosomally integrated copy of the empty vector (control) on Middlebrook 7H10 OADC/pan 50ng/mL of RIF. Data is representative of 2 independent replicates. e ) Sequence logo of the19bp inverted repeat sequence (RAE) and alignment of upstream sequences of Mab_helR (MAB_3189), Mab_arr, Ms_helR (MSMEG_2174) and Ms_arr . Region of RAE deleted is shaded in grey. f ) Growth of ten-fold serial dilutions of M. abscessus ATCC 19977, ΔMab_helR and complementing strains containing either helR expressed from a native promoter or helR expressed from a promoter lacking one half of RAE on Middlebrook 7H11 OADC containing RIF (16μg/mL).

Article Snippet: M. abscessus strains (ATCC 19977, clinical strains and ΔMab_helR ) were grown to A 600 of 0.7, exposed to RIF(8μg/ml) or RBT (0.5 μg/ml) for 30mins.

Techniques: Real-time Polymerase Chain Reaction, Control, Plasmid Preparation, Sequencing

Journal: bioRxiv

Article Title: Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

doi: 10.1101/2021.05.10.443476

Figure Lengend Snippet: a) Wild type M. abscessus, ΔhelR and complemented strains were grown to A 600 of 0.7 and a 10-fold dilution series was spotted on media lacking antibiotics. No growth defect was observed in the ΔhelR mutant compared to WT bacteria. b) Wild type M. smegmatis, ΔMs_helR and ΔMs_helR complemented with either HelR or Ms_helR were grown to A 600 of 0.7 and a 10-fold dilution series was spotted on media RIF (16μg/mL). Mab_helR can complement the RIF sensitivity of ΔMs_helR .

Article Snippet: M. abscessus strains (ATCC 19977, clinical strains and ΔMab_helR ) were grown to A 600 of 0.7, exposed to RIF(8μg/ml) or RBT (0.5 μg/ml) for 30mins.

Techniques: Mutagenesis, Bacteria

Journal: bioRxiv

Article Title: Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

doi: 10.1101/2021.05.10.443476

Figure Lengend Snippet: a ) Silver stained gel showing RNAP purified from untreated and RIF treated (16μg/mL of RIF for 30 mins) M. abscessus strain in which rpoA was C-terminally tagged with the 10X-his epitope at its native chromosomal location. Purified MabHelR is used as a control. RNAP purified from RIF treated samples was loaded on a preparative SDS PAGE, Coomassie stained and a band corresponding in size to MabHelR was excised and analyzed by LC MS/MS. b ) An M. abscessus strain in which helR was C-terminally tagged with the 3X-FLAG epitope at its native chromosomal location was either untreated or treated with 8-16μg/mL of RIF for 30 mins. Wild-type M. abscessus with an untagged helR gene was used as a control. Samples were normalized by wet cell weight and FLAG tagged proteins were purified using anti-FLAG M2 beads. Samples were analyzed by immunoblotting using anti-FLAG and anti-RpoA antibodies. Purified RNAP was used as a marker. Data is representative of >3 biological replicates. c ) Coomassie stained gel showing elution of proteins using nickel affinity chromatography. Interaction assays were carried out using HelR FLAG and RNAP his followed by binding and elution from a Ni-NTA column. Control samples included either only RNAP his or HelR FLAG. Purified RNAP his or HelR FLAG are included as markers. d ) Multiple-round in vitro transcription assays were performed on the sinP3 promoter using 75nM σ A -RNAP. RIF was added to indicated concentrations for 10 mins at 37°C followed by addition of a 9-or 15-fold molar excess of MabHelR. Transcription was initiated by addition of NTPs and incubated at 37°C for 30 mins. Samples were separated using denaturing PAGE (6% Urea polyacrylamide gel) and the 77nt product was visualized using a Typhoon Imager (GE Healthcare) and quantitated using the Image Quant software. The inhibition of transcription at each RIF concentration is expressed as a ratio of the activity in the absence and presence of RIF.

Article Snippet: M. abscessus strains (ATCC 19977, clinical strains and ΔMab_helR ) were grown to A 600 of 0.7, exposed to RIF(8μg/ml) or RBT (0.5 μg/ml) for 30mins.

Techniques: Staining, Purification, Control, SDS Page, Liquid Chromatography with Mass Spectroscopy, FLAG-tag, Western Blot, Marker, Affinity Chromatography, Binding Assay, In Vitro, Incubation, Software, Inhibition, Concentration Assay, Activity Assay

Journal: bioRxiv

Article Title: Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

doi: 10.1101/2021.05.10.443476

Figure Lengend Snippet: a ) Domain organization of MabHelR showing the location of Walker A and Walker B motifs within domains 1A1 and 1A2. The strictly conserved residues in each motif are underlined and the residues mutated to alanine in the study are in red. b ) ATPase activity of Wt MabHelR is shown in the presence and absence of MabRNAP. MabHelR mutations in either Walker B alone (mWalkerB) or Walker A and B (mWalkerAB) are defective in ATP hydrolysis. Data represents mean ± SD, n=3 c ) Growth of ten-fold serial dilutions of M. abscessus ATCC 19977, ΔMab_helR and ΔMab_helR complemented with either WT helR, helR (mWalkerB) or helR (mWalkerAB) on Middlebrook 7H10-OADC containing RIF (16 μg/mL) or RBT(1.5 μg/mL). Data is representative of at least 3 independent experiments.

Article Snippet: M. abscessus strains (ATCC 19977, clinical strains and ΔMab_helR ) were grown to A 600 of 0.7, exposed to RIF(8μg/ml) or RBT (0.5 μg/ml) for 30mins.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

doi: 10.1101/2021.05.10.443476

Figure Lengend Snippet: b) Immunoblot with anti-FLAG antibodies of M. abscessus ATCC 19977, ΔMab_helR and ΔMab_helR complemented with either WT helR FLAG , helR (ΔPCh1) FLAG , helR (ΔPCh2) FLAG and helR (mPCh) FLAG treated with RIF (16μg/mL) shows that WT and mutant proteins are induced to similar levels in the complementing strains. c) Coomassie stained gel showing elution of proteins using nickel affinity chromatography. Interaction assays were carried out using WT and mutant HelR FLAG with RNAP his as indicated and followed by binding and elution from a Ni-NTA column. Control samples included either only RNAP his or HelR FLAG . Purified RNAP his or HelR FLAG were included as markers.

Article Snippet: M. abscessus strains (ATCC 19977, clinical strains and ΔMab_helR ) were grown to A 600 of 0.7, exposed to RIF(8μg/ml) or RBT (0.5 μg/ml) for 30mins.

Techniques: Western Blot, Mutagenesis, Staining, Affinity Chromatography, Binding Assay, Control, Purification

Journal: bioRxiv

Article Title: Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

doi: 10.1101/2021.05.10.443476

Figure Lengend Snippet: a ) Location of the MabHelR PCh loop (orange) containing helices α16 and α17relative to the active site (AS) residues (gray) in β’ (yellow) and RIF (blue) is shown. Structure of MabHelR was modeled using Phyre and superimposed on the structure of MsHelR bound to RNAP (PDB ID 6YYS) in PyMOL v2.3. 0. The location of RIF was determined from the structure of RIF bound M. smegmatis RNAP (PDB ID 6CCV). Black arrows show the regions of MabHelR PCh loop that are deleted in MabHelRΔPCh1 and magenta arrows show the deleted regions in MabHelR ΔPCh2. Enlarged box below shows the location of the 494-DDED-497 motif at the tip of the PCh loop and the proximity of E496/ D497 to RIF. b ) Growth of ten-fold serial dilutions of M. abscessus ATCC 19977, ΔMab_helR and ΔMab_helR complemented with either WT helR, helR (ΔPCh1) helR (ΔPCh2) and helR (mPCh) on Middlebrook 7H10-OADC containing RIF (16 μg/mL) or RBT(1.5 μg/mL). Data is representative of at least 3 independent experiments. ΔPCh1 corresponds to deletion of residues 467-515, ΔPCh2: deletion of residues 486-505 and mPCh corresponds to point mutations E496A/D496A. c ) Schematic representation of experimental design. RNAP stalling on 5’-bio sinP3 DNA was carried out in the presence of RIF and ATP, the +1 nucleotide. The stalled complexes were tethered on streptavidin magnetic beads. Upon addition of MabHelR, the dissociated RNAP is expected to be present in the eluate and RNAP bound to DNA remains tethered on the beads. d ) Western blot of samples eluted from streptavidin magnetic beads tethered to DNA with RIF trapped RNAP complexes using anti-his antibodies that recognize his-tagged RpoA. Western blots are quantitated using ImageJ. Data represents mean ± SD, n=3.

Article Snippet: M. abscessus strains (ATCC 19977, clinical strains and ΔMab_helR ) were grown to A 600 of 0.7, exposed to RIF(8μg/ml) or RBT (0.5 μg/ml) for 30mins.

Techniques: Magnetic Beads, Western Blot