Activation of ChvG-ChvI regulon by cell wall stress confers resistance to β-lactam antibiotics and initiates surface spreading in Agrobacterium tumefaciens

Abstract

A core component of nearly all bacteria, the cell wall is an ideal target for broad spectrum antibiotics. Many bacteria have evolved strategies to sense and respond to antibiotics targeting cell wall synthesis, especially in the soil where antibiotic-producing bacteria compete with one another. Here we show that cell wall stress caused by both chemical and genetic inhibition of the essential, bifunctional penicillin-binding protein PBP1a prevents microcolony formation and activates the canonical host-invasion two-component system ChvG-ChvI in Agrobacterium tumefaciens. Using RNA-seq, we show that depletion of PBP1a for 6 hours results in a downregulation in transcription of flagellum-dependent motility genes and an upregulation in transcription of type VI secretion and succinoglycan biosynthesis genes, a hallmark of the ChvG-ChvI regulon. Depletion of PBP1a for 16 hours, results in differential expression of many additional genes and may promote a stress response, resembling those of sigma factors in other bacteria. Remarkably, the overproduction of succinoglycan causes cell spreading and deletion of the succinoglycan biosynthesis gene exoA restores microcolony formation. Treatment with cefsulodin phenocopies depletion of PBP1a and we correspondingly find that chvG and chvI mutants are hypersensitive to cefsulodin. This hypersensitivity only occurs in response to treatment with β-lactam antibiotics, suggesting that the ChvG-ChvI pathway may play a key role in resistance to antibiotics targeting cell wall synthesis. Finally, we provide evidence that ChvG-ChvI likely has a conserved role in conferring resistance to cell wall stress within the Alphaproteobacteria that is independent of the ChvG-ChvI repressor ExoR.

Fig 1. The PBP1a depletion fails to form microcolonies independent of flagellar motility.

A. Micrographs of wildtype, PBP1a depletion, and RgsM depletion with or without 1mM IPTG inducer. Each strain was grown to exponential phase, spotted on an ATGN agar pad, allowed to grow for 16 hours, and imaged by DIC microscopy. Scale bar depicts 2μm. The graphic depicts the working model that RgsM _DD-endopeptidase activity is required for incorporation of nascent glycan strands into the preexisting peptidoglycan (PG) macromolecule by PBP1a. RgsM cleaves _DD-crosslinks, PBP1a glycosyltransferase activity incorporates lipid II into the PG glycan strand, PBP1a _DD-transpeptidase activity crosslinks the peptide stem of the nascent PG, fully incorporating it into the macromolecule. EPase, endopeptidase; GTase, glycosyltransferase; TPase, transpeptidase. B. Micrographs of wild type, Δpbp1b1, Δpbp1b2, Δpbp1c, and ΔmtgA. Each strain was grown to exponential phase, spotted on an ATGN agar pad, allowed to grow for 16 hours, and imaged by DIC microscopy. Scale bar depicts 2μm. C. Time-lapse microscopy of the PBP1a depletion grown on an agar pad with or without 1mM IPTG inducer. DIC images were acquired every 10 minutes. Time is shown in hours. For the—PBP1a strain, cells were washed 3X with ATGN media and grown at 28 C with shaking for 4 hours before cells were spotted on an agar pad for imaging.

Fig 2. Analysis of the PBP1a depletion transcriptomes by RNA-seq.

A. Plots comparing Log2Fold Change of the + PBP1a transcriptome to that of the—PBP1a 6-hour transcriptome and to that of the 16-hour depletion. Gray dots represent a single transcript, and the dotted lines represent +/- 2.0 Log2Fold Change threshold. Plots are delimited by chromosome and mega plasmid. B. COG categorical analysis of the 16-hour depletion of PBP1a. Pink, downregulated; Cyan, upregulated.

Fig 3. The response to the depletion of PBP1a mimics transcriptional changes associated with host invasion.

A. Correlation scatterplots depicting relationships between the log2fold-change (L2FC) values in the 6-hour PBP1a depletion and transcriptomic data sets taken under simulated virulence-inducing conditions (AS and AB+AS) and under simulated host-invading conditions (ΔexoR). Each point represents a unique transcript. AS, acetosyrinogone; AB, Agrobacterium minimal media; rho, Spearman correlation coefficient. B. Correlation scatterplots comparing L2FC values of transcripts in the ΔexoR microarray to either the 6-hour or 16-hour PBP1a depletion. Each transcript is colored according to its change in L2FC values from 6 hours of PBP1a depletion to 16 hours of depletion. Gray, no change; Blue, transcript has |L2FC| > 2.0 in the 6-hour but not in the 16-hour depletion; Red, |L2FC| > 2.0 in the 16-hour but not in the 6-hour depletion; Purple, |L2FC| > 2.0 in both the 6-hour and 16-hour depletion.

Fig 4. Succinoglycan overproduction is a conserved response to PBP1a depletion and results in failed microcolony formation.

A. Scatter plots depicting RPKM values of the 6-hour and 16-hour compared to wild type. Each point represents a unique transcript. Points are colored by category. Gold, ChvG-ChvI regulon; Lavender, Motility and Chemotaxis; Green, Type VI Secretion; Blue, Succinoglycan Biosynthesis; Black, mrcA (encoding PBP1a). B. Micrographs of wild type, Δrem, PBP1a replete Δrem, PBP1a depleted Δrem, ΔT6SSpro, PBP1a replete ΔT6SSpro, and PBP1a depleted ΔT6SSpro. Each strain was grown to exponential phase, spotted on a 1% ATGN agar pad containing 1mM IPTG if inducing mrcA, allowed to grow for 16 hours, and imaged by DIC microscopy. The scale bar depicts 2μm. C. Micrographs of wild type, and PBP1a depletion with or without IPTG inducer. Each strain was grown to exponential phase and spotted on a 1% ATGN agar pad containing 25μg/mL calcofluor white and 1mM IPTG if inducing mrcA. Each was allowed to grow for 16 hours and imaged by phase microscopy with and without the DAPI filter for visualizing calcofluor-stained succinoglycan. D. Micrographs of ΔexoA and PBP1a depletion ΔexoA, with or without IPTG inducer. Strains were grown and imaged as described for panel C.

Fig 5. The ChvG-ChvI TCS is conditionally essential under treatment with β-lactam antibiotics.

A. Micrographs of untreated and cefsulodin-treated cells. Wild-type and Δ3pbp cells were grown to exponential phase, spotted on a 1% ATGN agar pad with or without 20 μg/mL cefsulodin and allowed to grow for 16 hours. Each strain was imaged by DIC microscopy. B. Box plots comparing cell length and width between wild-type, PBP1a-depleted, and cefsulodin-treated cells. ns, not significant; ****, p < 0.00005. C. Growth curves of WT, -PBP1a, ΔchvI, and Δ3pbp in the absence (top) and presence of 20 μg/mL cefsulodin (bottom). D. Graph depicting the change in zone of inhibition from wildtype in ΔchvI against ten different antibiotic disks. Error bars represent +/- 1 standard deviation from the mean.

Fig 6. Conservation constraints of ExoR suggest conserved ChvG-ChvI response is independent of ExoR.

Maximum parsimony tree constructed using MUSCLE sequence alignment [67] on the periplasmic regions of ChvG orthologs. In clades that don’t have a ChvG ortholog, the protein with the highest sequence similarity to ChvG was used instead. Conservation of ExoR was calculated using blast max scores from top hits when protein blasting [66] ExoR from Agrobacterium tumefaciens against each species in the tree. Phyre2 [71] predicted structures of periplasmic domains of ChvG orthologs from representatives (bold) in each genus are shown. Conserved structural loops are denoted as L1 and L2.

Fig 7. Activation of ChvG-ChvI can proceed independently of ExoR derepression.

A. Predicted interaction site between ExoR and ChvG through AlphaFold-Multimer structure prediction. Insets show the top-down view of the interaction site with hydrogen bonding and electrostatic surface display. B. Microcolonies of wild type and ΔexoR with and without 20 μg/mL cefsulodin treatment. C. Western blot detection of ExoR proteolysis (ExoR_C20) with anti-FLAG against ExoR-FLAG. D. Working model of activation of ChvG-ChvI in A. tumefaciens. H⁺, free proton representing an acidic environment; β, β-lactam antibiotic.