GplR1, an unusual TetR-like transcription factor in Mycobacterium abscessus, controls the production of cell wall glycopeptidolipids, colony morphology, and virulence

Abstract

Mycobacterium abscessus is a major human pathogen, mostly infecting people with pre-existing lung conditions, such as cystic fibrosis. The production of glycopeptidolipids (GPL) is a major determinant of virulence of this bacterium, with clinical isolates that lack GPL generally exhibiting more aggressive clinical behavior. The current paradigm is that GPL production is abolished in vivo via irreversible, spontaneous mutations taking place as part of in-host evolution. Little is known about the mechanisms or extent to which GPL production may be regulated. Here, we describe an unusual TetR-like transcription factor of M. abscessus, mab_1638, that appears to be a strong positive regulator of the entire GPL biosynthesis and export gene cluster through a combination of direct and indirect mechanisms. The inactivation of mab_1638 abolished GPL production, leading to stable rough colony morphology and increased virulence in infection models, characteristics of rough, non-GPL producers. Transcriptome analysis found that the mab_1638 mutant had 118 differentially expressed genes, including the GPL locus and a second, recently described GPL-like locus that produces a related glycosylated lipopeptide called GP8L. Chromatin immunoprecipitation and sequencing revealed a consensus inverted-repeat DNA sequence motif, characteristic of genes regulated by mab_1638. Together, these findings found that mab_1638 encodes a transcription factor required for GPL production and, therefore, has a profound effect on virulence traits. We propose naming this gene GPL regulator 1 (gplR1). This finding raises the important possibility that M. abscessus strains appearing smooth in laboratory growth conditions may nonetheless downregulate GPL-cluster genes in other conditions, including in-patient conditions, and thus acquire the phenotypic characteristics of rough strains.IMPORTANCEMycobacterium abscessus is an important human pathogen, causing disease that is difficult to treat. M. abscessus strains have been observed to have two distinct colony morphologies, smooth and rough, which substantially impact clinical presentation. Rough strains are associated with later-stage, more severe disease and are more virulent in animal models. Smooth morphology is conferred by a molecule called glycopeptidolipid in the outer cell envelope, and rough morphology is known to occur when mutations inactivate genes required for glycopeptidolipid biosynthesis. Little is known about the possibility that glycopeptidolipid production could be regulated. Here, we have identified a transcription factor that is required for glycopeptidolipid biosynthesis, indicating that glycopeptidolipid production is indeed a regulated process and raising the important possibility that strains exhibiting smooth morphology in the lab may downregulate GPL production in the human host, thereby acquiring the virulence properties of rough strains.

Keywords: GPL; Mycobacterium; abscessus; glycopeptidolipid; transcriptional regulation; virulence regulation.

Fig 1

Disruption of mab_1638 causes rough morphology in M. abscessus, due to lack of GPL in the cell envelope. (A) Schematic representation of the transposon insertion location in ATCC19977. (B) PCR confirmation of the insertion in mab_1638. (C) Representative colonies of the indicated strains grown on 7H10 solid media. Images III–V show the mab_1638::tn insertion mutant transformed with the indicated plasmids. In the plasmids harboring mab_1638, the gene was expressed from its native promoter on an integrating plasmid. The plasmid in V expressed mab_1638 with an in-frame deletion of the indicated amino acids. (D) TLC analysis of lipids extracted from the cell walls of the indicated strains. Predicted GPL bands are shown. TDM, trehalose dimycolate.

Fig 2

The mab_1638:tn mutant is hypervirulent in a zebrafish model and behaves similarly to a rough control strain. WT smooth is ATCC19977, and WT rough is ATC19977/CIP104536^T-R. (A) Adult zebrafish were infected by 10⁷ CFU/fish, and survival was followed for 10 days. n = 8 for WT, n = 9 for mab_1638:tn. (B) Bacterial burden was measured on day 14 post-infection by 10⁶ CFU/fish and compared using unpaired t-test. (C) Bacterial burden, as represented by fluorescent pixel count (FPC), was measured on day 3 and day 6 post-infection in embryos infected by ~250 CFU/embryo. (D) The proportion of bacteria found in abscess-like structures (>500 mm², two-dimensional area) was measured on day 6 post-infection in embryos. (C and D) Ordinary one-way ANOVA with Tukey’s multiple comparisons test. (E) Representative embryos from panel D. Red arrows point to clusters of fluorescent bacteria (>500 mm², two-dimensional area), referred to as “abscess-like.”

Fig 3

MAB_1638 negatively impacts expression of GPL biosynthesis and transport genes as well as the GP8L gene cluster. (A) RNAseq revealed genes both up- and downregulated in the mab_1638::tn strain compared to the WT parental strain ATCC19977 (log₂ fold change < −1 or > 1, and adjusted P < 0.05). Genes known or expected to participate in GPL biosynthesis and transport are indicated. (B) Gene set enrichment analysis was used to identify Gene Ontology biological process categories that were disproportionately affected by disruption of mab_1638. “Activated” genes had higher expression in the mab_1638::tn strain, while “Suppressed” genes had lower expression. (C) Diagrams of the GPL biosynthesis and transport gene cluster and a recently described GPL-like gene cluster termed the GP8L cluster, which was also downregulated in the mab_1638::tn strain. Red shadowing indicates downregulated genes. Of the 38 total genes in both clusters, only two genes did not meet the criteria for downregulation and are not shadowed. Genes are not shown to scale. Similarities between genes are indicated by naming and coloration. Bent arrows indicate reported TSSs. (D) Summary of genes predicted to encode transcription factors that were differentially expressed in the mab_1638::tn strain (log₂ fold change < −1 or > 1 and adjusted P < 0.05).

Fig 4

MAB_1638 binding sites are enriched in both up- and downregulated genes and feature a consensus inverted-repeat motif. (A) ChIP-seq was used to identify genomic sites bound by MAB_1638. Genes with annotated TSSs were then stratified based on the presence or absence of MAB_1638 binding sites within 500 nt of their TSSs and how their expression was affected by disruption of mab_1638. Genes that were up- or downregulated in the mab_1638::tn strain were significantly more likely to have MAB_1638 binding sites in their putative regulatory regions than genes that were not differentially expressed (Fisher’s exact test). (B) MEME analysis revealed an inverted-repeat motif present in ~80% of the MAB_1638 binding sites in putative regulatory regions. (C) MAB_1638 binding sites motifs in putative regulatory regions (motif within 500 nt up- or downstream of an annotated TSS) were disproportionately located in the regions ~100 bp upstream of TSSs. (D) Two inverted-repeat MAB_1638 binding site motifs were identified within the GPL biosynthesis locus. Their approximate locations are indicated. (E) Two inverted-repeat MAB_1638 binding site motifs were identified within the GP8L biosynthesis locus. Their approximate locations are indicated. Schematics are not to scale.