An unusual genetic switch controls Mycobacterium avium pathogenesis, antibiotic resistance and colony morphology

Abstract

Mycobacterium avium subspecies hominissuis (Mah) is an emerging environmental pathogen highly adapted to a wide range of niches, from treated water systems to mammalian tissues. On solid media, Mah forms two distinct colony morphologies, smooth transparent (SmT) and smooth opaque (SmO). These colony morphologies are representative of broader differential phenotypic states in which SmT cells are virulent and have high resistance to antibiotics while SmO cells are avirulent, antibiotic-sensitive and grow faster than SmT cells in culture. Importantly, Mah interconverts between these two morphotypes but the mechanism of SmT-SmO switching is unknown. Here we show that SmT-SmO switching is governed by a reversible transposition event that regulates expression of a periplasmic lipoprotein, Erp (extracellular repetitive protein). We found that transposition of IS1245, an endogenous insertion sequence, into the erp gene correlated with the SmT-SmO transition, and its precise removal coincided with the switch back to SmT. Genetic analyses showed that erp is required for maintenance of the SmT state and sufficient to drive the switch from SmO to SmT. We also identified a mutation in a periplasmic protease, MarP, that locks Mah in the SmO state and blocks erp-mediated switching to SmT. Our results indicate that Erp and MarP function in a signal transduction pathway that regulates a broad transcriptional response to periplasmic stress. Moreover, identification of components that control Mah colony morphology switching has revealed a potential new strategy for combating the inherent antibiotic resistance of Mycobacterium avium infections.

Figure 1.. Mah clinical strain mc²2500 can adopt transparent the (SmT) and opaque (SmO) morphology.

A. Representative images of SmT and SmO morphology for the Mah strain mc²2500 on plates captured on a scanner with transmitted light, SmT (left) and SmO (right). Single colonies of SmT (i. domed SmT and ii. flat SmT) and SmO were imaged via stereomicroscopy. B. Transmission electron microscopy of individual mc²2500 cells from SmT and SmO colonies. Arrows are used to indicate ILIs. C. Scanning electron microscopy of mc²2500 colony edges of SmT and SmO morphotypes. D. Growth kinetics (OD₆₀₀ over time) of SmT (light blue) and SmO (teal) mc²2500 in liquid media. Each data point is the mean ± standard deviation of three technical replicates. The curve is representative of three biological replicates. E. Dose response curves for SmT and SmO mc²2500 against erythromycin, rifampicin, streptomycin, ethambutol, azithromycin, isoniazid, and chlorine, measured as OD₆₀₀ of the culture grown with the drug at the indicated concentration divided by OD₆₀₀ of the culture grown without the drug. Each data point is the mean ± standard deviation of two technical replicates. The curve is representative of three biological replicates.

Figure 2.. Reversible colony morphology switching depends on the insertion and removal of the transposable element IS1245 from the erp locus.

A. Schematic representation of a switching assay in which a switching lineage is generated by culturing an SmT population and plating to isolate SmO colonies that arise spontaneously. SmO colonies are cultured then treated with erythromycin (ery) before plating to identify rare switching events that revert bacteria to the SmT morphotype. A switching lineage is defined as a full round of SmT to SmO to SmT morphology switching. B. Whole genome sequence alignments of six independent switching lineages of mc²2500. Each genome was aligned to the mc²2500 reference genome. Orange dots above the reference genome denote IS1245 insertions in the reference genome. New insertions of IS1245 (orange boxes), insertions of other mobile genetic elements (blue boxes), large deletions (grey boxes), small insertions or deletions (green boxes), and small nucleotide polymorphisms (purple circles) are marked with their respective genomic position (bp) in each individual genome sequence. Dashed grey box indicates the erp locus in the mc²2500 genome. C. Positions of unique IS1245 insertions in the erp locus derived from independent SmO colonies.

Figure 3.. Erp is a conserved, periplasmic protein that is involved in the maintenance of the SmT morphology.

A. Representative images of erp⁻ and erp⁻ + _perp^WT colony morphology generated by scanning plates with transmitted light. Single colonies of erp⁻ and erp⁻ + _perp^WT were imaged via stereomicroscopy. B. Dose response curves for SmT (light blue), erp⁻ (teal), or erp⁻ + _perp^WT (khaki) against erythromycin and rifampicin measured as OD₆₀₀ of the culture grown with the drug at the indicated concentration divided by OD₆₀₀ of the culture grown without the drug. Each data point is the mean ± standard deviation of two technical replicates. The curve is representative of three biological replicates. C. Volcano plot of differentially expressed genes in erp⁻ + _perp^WT compared to erp⁻. Adjusted p-value ≤ 0.05 and log₂-fold change ≥ 2.0 were considered significant and represented in black and nonsignificant results represented in grey. Select gene categories of interest are highlighted in color and indicated in the legend. D. Volcano plot of differentially expressed genes in erp⁻ + _perp^WT compared to SmT. Adjusted p-value ≤ 0.05 and log₂-fold change ≥ 2.0 were considered significant and represented in black and nonsignificant results represented in grey. E. Schematic representation of conserved protein domains in Erps from different mycobacterial species. The putative lipidation site is denoted with the amino acid. Cladogram (left) indicates the relationship between species based on 16s sequence. F. Stereomicroscopy of single colonies of erp⁻ bacteria complemented with different mycobacterial Erps (species indicated above colony image). G. Schematic representation of Mah Erp mutations and truncations. H. Stereomicroscopy of single colonies of erp⁻ bacteria complemented with Mah Erp mutants. The putative lipidation site is outlined with denoted with the amino acid. I. Western blot of Flag-tagged mutant Erps. GroEL served as a loading control and molecular weight is indicated on the right.

Figure 4.. Mah establishes infection without triggering an overt immune response in a host in contrast to Mtb.

A. Bacterial loads enumerated by colony forming units (CFU) in the spleen, liver, and lung of B6 mice infected with SmT (light blue), erp⁻ (teal), and erp⁻ + _perp^WT (khaki) at indicated time points. Each data point is the mean ± standard deviation of four mice per time and group. Each graph is representative of two independent experiments. B. Bacterial loads enumerated by CFU in murine bone marrow macrophages (BMMs) infected with SmT, erp⁻, and erp⁻ + _perp^WT bacteria at indicated time points. Each data point is the mean ± standard deviation of three technical replicates per time and group. The graph is representative of three independent experiments. C. Venn diagram comparing differentially expressed genes of SmT-, erp⁻-, and Mtb-infected BMMs 24 hours post-infection that are shared between conditions and unique to each condition. Adjusted p-value ≤ 0.05 and log₂-fold change ≥ 1.0 (Mah) and ≥ 2.0 (Mtb) were considered significant. D. Reverse-transcribed quantitative PCR (RT-qPCR) of selected transcripts in SmT Mah, erp⁻ Mah, WT Mtb, and Δ ESX-1 Mtb infected BMMs 24 hours post-infection. mRNA expression levels are normalized to GAPDH and lipopolysaccharide (LPS) stimulation was used as a positive control. Each data point is the mean ± standard deviation of three biological replicates. Transcripts involved in toll-like receptor (TLR) signaling are outlined in red and transcripts involved in the type I interferon (IFN) response are outlined in blue.

Figure 5.. MarP is a periplasmic protease involved in the maintenance of the SmT morphology in addition to erp.

A. Representative plate scans of erp⁻ and SmO (locked) morphology pre- and post-treatment with erythromycin. B. Whole genome sequence alignments of SmT, erp⁻, and SmO (locked). Each genome was aligned to the mc²2500 reference genome. Insertions of IS1245 (orange box) and small nucleotide polymorphisms (SNPs) (purple circle) are marked with their respective genomic location in each individual genome sequence. C. Western blot of MarP in SmT, erp⁻, and marP^G345R. RPOB served as a loading control and molecular weight is indicated on the right. D. Structure alignment of the AlphaFold model of WT Mah MarP (light purple) and Mah MarP^G345R (dark purple) to the crystal structure of Mtb MarP (tan). Insert is an enlargement of the catalytic triad. Catalytic residues are labeled as indicated in the figure legend, and G345R is labeled. E. Representative plate scans of marP^G345R + _pmarP^WT morphology pre- and post-switching (erythromycin treatment). Single colonies of marP^G345R + _pmarP^WT pre-erythromycin treatment and marP^G345R + _pmarP^WT post-antibiotic treatment were imaged via stereomicroscopy. F. Dose response curves for SmT (light blue), erp⁻ (teal), marP^G345R (light purple), and marP^G345R + _pmarP^WT (dark purple) against erythromycin, rifampicin, streptomycin, and isoniazid measured as OD₆₀₀ of the culture grown with the drug at the indicated concentration divided by OD₆₀₀ of the culture grown without the drug. Each data point is the mean ± standard deviation of two technical replicates. The curve is representative of three biological replicates. G. Representative plate scans of erp⁻ + _perp^WT and marP^G345R + _perp^WT morphology.

Figure 6.. Potential mechanisms by which erp, IS1245, and marP mediate the switch between SmT and SmO morphologies.

Top: When both erp and marP are expressed and functional, the SmT morphology is maintained. Within this context, erp may act in a signaling cascade, sensing external changes and inducing large scale transcriptional changes affecting metabolism, membrane transport, and other processes. Additionally, Erp and MarP may interact to promote membrane remodeling both at the transcriptional level and the membrane itself. Altogether, this could contribute to changes at the membrane that result in the SmT colony morphology and enhance Mah’s ability to respond to host immune system or antibiotic challenge. Bottom: When IS1245 inserts itself into the erp locus or when MarP function is disrupted via mutation, the SmO colony morphology is adopted. The absence of Erp or functional MarP and resulting absence of signaling potentially leads to changes in membrane composition and cell wall stress. Cells in this state demonstrate increased growth rate and increased sensitivity to the host immune system and antibiotics. “OM”, outer membrane. “IM”, inner membrane.