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 and thus led to stable rough colony morphology, as well as increased virulence in infection models, characteristic of rough, non-GPL-producers. Transcriptome analysis found 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, mab_1638 appears to encode a transcription factor required for production of GPL and therefore having 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.

Figure 1:. Disruption of mab_1638 causes rough morphology in M. abscessus that is 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. A TLC analysis of lipids extracted from the cell walls of the indicated strains. The predicted GPL bands are shown.

Figure 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 by unpaired t test. C: Bacterial burden, as represented by Fluorescent Pixel Count (PFC) 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 was measured on day 6 post infection in embryos. Panels C and D, Ordinary one-way ANOVA with Tukey’s multiple comparisons test. E: Pictures of representative embryos from panel D. the red arrows point to clusters of bacteria (white fluorescence), referred to as “abscess-like”.

Figure 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 ATTC19977 (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 Biology 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 that was also downregulated in the mab_1638::tn strain. Red shadowing indicates downregulated genes. Of the 38 total genes in both clusters, only 2 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.

Figure 4.. MAB_1638 binding sites are enriched in both up- and downregulated genes and feature an inverted-repeat consensus 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.