Mycobacterium abscessus Glycopeptidolipid Prevents Respiratory Epithelial TLR2 Signaling as Measured by HβD2 Gene Expression and IL-8 Release

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human β-defensin 2 (HβD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with “masking” of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system

Mycobacterium abscessus (contribution to the study of the cell envelope and the resistance to antibiotic)

PARIS7-Bibliothèque centrale (751132105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Genetic analysis of new 16S rRNA mutations conferring aminoglycoside resistance in Mycobacterium abscessus

OBJECTIVES: We studied the development and fitness cost of 2-deoxystreptamine aminoglycoside resistance of Mycobacterium abscessus. METHODS: Spontaneous 2-deoxystreptamine aminoglycoside-resistant mutants were selected and the frequency of their appearance was determined. The 3′ part of the rrs gene was sequenced to characterize mutations. Additionally, we determined the MICs of aminoglycoside drugs for the different mutants obtained. The dominance/recessivity traits of the different mutations were examined and we explored the potential cost conferred by the mutations selected in vitro on the fitness of these isolates compared with the wild-type strain. RESULTS: The in vitro mutation rate for 2-deoxystreptamine aminoglycoside resistance was ∼10(−7) mutations/cell division. In addition to the known rrs A→G substitution at position 1408 (Escherichia coli numbering), which confers kanamycin resistance (Kan(R)), three new substitutions in rrs were identified in M. abscessus Kan(R) mutants, i.e. T→A at 1406, C→T at 1409 and G→T at 1491. Heterodiploids carrying genomic mutations T→A at 1406 and A→G at 1408 with the wild-type rrs gene carried by the pNBV1 vector showed a resistant phenotype. In contrast, heterodiploids carrying genomic mutations C→T at 1409 and G→T at 1491 with the wild-type rrs gene carried by the pNBV1 vector had a susceptible phenotype. No burden on fitness was observed for the different mutations. CONCLUSION: Mutations in the rrs gene that confer high-level 2-deoxystreptamine aminoglycoside resistance on M. abscessus differ in their dominance/recessivity traits and have no biological cost under our experimental conditions

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

Mycobacterium abscessusis 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 ofM. abscessusgene knockout mutants was not clear. Based on the recent identification of genetic recombineering as the preferred approach, aM. abscessusmutant was generated in which the genemmpL4b, critical to glycopeptidolipid synthesis, was deleted. Compared to the previously well-characterized parental strain 390S, themmpL4Bdeletion 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 ofM. abscessuspathogenesis at the cellular level.</jats:p

TLR2 siRNA treatment decreases TLR2 gene expression in uninfected and <i>M. abscessus</i> -infected A549 cells.

<p>As a first step in assessing the role of TLR2 in respiratory epithelial responses to <i>M. abscessus</i>, the effect of transfection of A549 cells with TLR2 siRNA on TLR2 gene expression was assessed. (A) Uninfected A549 cells were transfected with scrambled RNA or TLR2 siRNA. After 48 h TLR2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative difference in TLR2 gene expression using the A549 monolayers receiving scrambled RNA as the reference, with data presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b>TLR2 transfected cells versus cells receiving scrambled RNA <i>P</i><0.05, <i>t</i>-test. (B) Western blotting of A549 cell extracts from (A) demonstrates decreased TLR2 in cells treated with TLR2 siRNA. (C) A549 cell monolayers were either untreated or transfected with RNA, and either uninfected or challenged with <i>M. abscessus</i> 390SΔ<i>mmpL4b</i>. TLR2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative difference in TLR2 gene expression using uninfected A549 monolayers receiving scrambled RNA as the reference, with data presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> TLR2 siRNA <b>+</b><i>M. abscessus</i> 390SΔ<i>mmpL4b</i> versus scrambled RNA + <i>M. abscessus</i> 390SΔ<i>mmpL4b</i>; <i>P</i><0.05, <i>t</i>-test.</p

A549 cells increase HβD2 gene expression in response to <i>M. abscessus</i> variants lacking GPL, but not the <i>M. abscessus</i> 390S variant expressing GPL.

<p>A549 cell monolayers were uninfected or challenged with <i>M. abscessus</i> variants 390R or 390S. In addition, some uninfected A549 cell monolayers were treated with IL1β or MALP-2 as controls for the ability of A549 cells to upregulate HβD2 gene expression. After 8 hours, HβD2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative fold increase in HBD2 gene expression over that of the untreated group and presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> 390S versus 390R and 390V; <i>P</i><0.05, <i>t</i>-test.</p

Antibody to TLR2 decreases IL-8 release from A549 cells in response to the <i>M. abscessus</i> 390SΔ<i>mmpL4b</i> deletion mutant.

<p>A549 cell monolayers were preincubated with antibody to TLR2 or isotype control antibody and then received no bacteria or were challenged with the <i>M. abscessus</i> 390SΔ<i>mmpL4b</i> deletion mutant. Culture supernates were collected after 8 h and assayed by ELISA for IL-8. Data are means ± SEM of two experiments done in triplicate. * 390SΔ<i>mmpL4b</i> + anti-TLR2 antibody versus 390SΔ<i>mmpL4b</i> alone and 390SΔ<i>mmpL4b</i> + isotype antibody; p<0.01, <i>t</i>-test.</p

A <i>M. abscessus</i> 390SΔ<i>mmpL4b</i> deletion mutant lacking GPL has acquired the ability to stimulate HBD2 gene expression in A549 cells.

<p>(A) A549 cell monolayers were uninfected or challenged with <i>M. abscessus</i> variants 390V, 390S or 390SΔ<i>mmpL4b</i>, a deletion mutant lacking the <i>mmpL4b</i> gene which is a critical component of the GPL biosynthetic pathway. The results of real-time PCR are expressed as the relative fold increase in HβD2 gene expression over that of the untreated group and presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> 390SΔ<i>mmpL4b</i> mutant vs 390S wild type; <i>P</i><0.05, <i>t</i>-test. (B) A549 cell monolayers were uninfected or challenged with <i>M. abscessus</i> 390SΔ<i>mmpL4b</i>, or the complemented 390SΔ<i>mmpL4b</i> mutant. After 8 hours, HβD2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative fold increase in HβD2 gene expression over that of the untreated group and presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> 390SΔ<i>mmpL4b</i> complemented versus 390SΔ<i>mmpL4b</i> mutant; <i>P</i><0.05, <i>t</i>-test.</p

IL-8 release from BEAS 2B cells in response to <i>M. abscessus</i> variants lacking GPL is mediated by TLR2.

<p>(A) BEAS 2B bronchial epithelial cells received no bacteria or were challenged with <i>M. abscessus</i> variants 390R, 390S and 390V. Culture supernates were collected after 8 h and assayed by ELISA for IL-8. Data are means ± SEM of two experiments done in triplicate. * 390S versus 390R and 390V; p<0.01, <i>t</i>-test. (B) BEAS 2B bronchial epithelial cells were preincubated with no antibody, antibody to TLR2 or isotype control antibody. Monolayers then received no bacteria or were challenged with the <i>M. abscessus</i> 390SΔ<i>mmpL4b</i> deletion mutant or the 390SΔ<i>mmpL4b</i> complemented mutant. Culture supernates were collected after 8 h and assayed by ELISA for IL-8. Data are means ± SEM of two experiments done in triplicate. * 390SΔ<i>mmpL4b</i> deletion mutant + anti-TLR2 antibody versus 390SΔ<i>mmpL4b</i> deletion mutant alone and versus 390SΔ<i>mmpL4b</i> deletion mutant + isotype antibody; p<0.01. * 390SΔ<i>mmpL4b</i> complemented mutant versus 390SΔ<i>mmpL4b</i> deletion mutant; p<0.01.</p

TLR2 siRNA treatment decreases HβD2 gene expression in A549 cells challenged with the <i>M. abscessus</i> 390S <i>mmpL4b</i> deletion mutant.

<p>A549 cells were transfected with scrambled RNA or TLR2 siRNA for 48 h with some cell monolayers then challenged with <i>M. abscessus</i> 390SΔ<i>mmpL4b</i>. After 8 h, HβD2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative change in HβD2 gene expression over that of the untreated, uninfected group and presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> TLR2 siRNA <b>+</b><i>M. abscessus</i> 390SΔ<i>mmpL4b</i> versus scrambled RNA + <i>M. abscessus</i> 390SΔ<i>mmpL4b</i>; <i>P</i><0.05, <i>t</i>-test.</p