Ultrastructural changes in the cell envelope of virR-deficient mutant associated with increased vesiculation.

(A) Cryo-electron micrographs of indicated Mtb strains grown in high iron MM. Closed line rectangles were used to calculate grey value profiles of cell envelopes using ImageJ. The dashed line insets within the main micrograph were magnified to show a detailed view of the cell surface. Scale bars are 100 nm in main micrographs and 50 nm in the insets. (B) Density profiles based on grey values of the cross sections marked by solid line rectangles in A. (C) Mean values and standard errors of distances in nm between main cell envelope layers measured in A and C. *P < 0.05 after applying a Tukeýs multiple comparison test. The number of cells analyzed varied from n = 20 (WT), n = 15 (virRmut) and n = 23 (virRmut-C). CM, cytoplasmic membrane; OM, outer membrane; L1, layer 1; L2, layer 2.

Enhanced permeability and vesiculation are linked in the absence of LytR_C solo domain proteins in Mtb.

(A) Uptake of ethidium bromide (EthBr) in the indicated strains, as measured by fluorescence at 590 nm for 65 min. Data are mean and standard deviation of three biological replicates. (B) Sensitivity of the indicated strains to different concentrations of vancomycin as measured by monitoring optical density at 580 nm (OD580) after 7 days. Data are mean and standard deviations of three biological replicated. Errors bars not shown are smaller than symbols. (C) Time course of the incorporation of FDAAs on the indicated strains in 7H9 as measured by fluorescence. Cells were labeled with HADA for 48 h, washed in 7H9 medium and labeled with FDL for 5 h. Ten, cell were fixed and imaged under a fluorescence microscope. Representative images of WT, virRmut and virRmut-Comp strains in 7H9. Data are mean and standard deviations of three biological replicates. *** means P<0.001 and **** means P<0.0001 after applying a One-way ANOVA analysis. The arrow indicates the time at which images were taken. Errors bar not shown are smaller than symbols. (D) immunoblot analysis of cell lysates of two independent conditional mutants (c1 and c2) for virR and cei for the presence of VirR and Cei proteins in cultures incubated with and without anhydrotetracycline (ATc). (E) Nanoparticle tracking analysis (NTA) of EV preparations derived from virR and cei conditional mutants (c2 (virR) and c1 (cei)) obtained from cultures with and without ATc showing number of particles per cm3 determined by Zeta View NTA in three independent EV preparations. Data are presented as mean ± SEM.

Transcriptional profiling of virRmut showcases systemic alterations in cell wall architecture and metabolism.

(A) Experiment design for RNA-seq analyses. RNA was extracted from whole cell extracts from 4 replicate cultures of each strain (WT, virRmut, and virRmut-Comp), and sequenced. (B) PCA plot of the transcriptomics data. The loadings from the two first principal components are plotted. (C) Number of differentially expressed genes between virRmut and either WT (left) or virRmut-Comp (right). (D) Enrichment profiles between sets of genes found up and down-regulated for the contrasts virRmutvs WT and virRmut vs virRmut-Comp. While genes that are differentially expressed in the same direction in both contrasts are mutually enriched, genes whose differential expression goes in different directions in both comparisons are mutually depleted (two-sided Fisher exact test p<0.05 in all four cases). (E) Gene Ontology (GO) enrichments of genes that are 1) simultaneously downregulated in virRmutwith respect to both WT and v virRmut-Comp (left) and 2) simultaneously upregulated (right). GO terms selected for testing included biological process and cell compartment labels containing N > 10 genes, between levels 4-6 of the ontology tree. In this representation, each node in the networks represents a significantly enriched term (terms selected for visualization with FDR=10% and enrichment odds-ratio>2), nodes’ size is proportional to the enrichment significance, and links between terms are added wherever the sets of genes contributing to the enrichments in a connected pair show an intersection larger than 50% of the smallest of the gene sets involved in the pair (see Methods for further details, and supplementary file S2 for an extended list of all the enrichments found).

VirR-dependent genes intersect the regulons of key transcriptional regulators of the responses to stress, dormancy, and cell wall remodeling.

(A) Transcription factors controlling regulons significantly enriched among genes that are 1) simultaneously downregulated in virRmutwith respect to both WT and virRmut -Comp (bottom, 9 TFs) and 2) simultaneously upregulated (top, 1 TF). (B) Differential expression patterns for enriched TFs whose expression is VirR-dependent. (C) Regulatory subnetwork including the genes under transcriptional control of the enriched TFs highlighted in figure 4A that were found to be VirR-dependent. In this visualization, color captures the logFC for the contrast virRmut -WT, and the size of its FDR.

Cell permeability is a major driver of vesicle production in Mtb.

(A) Time course of the uptake of ethidium bromide (EthBr) by Mtb grown in the indicated conditions, as measured by fluorescence at 590=nm for 65 min. Data are mean and standard deviation of three biological replicates. (B) Dot-blot analysis of released EVs in supernatants from Mtb cultures submitted to the indicated conditions. The loading volume was normalized according to CFUs. The graph below represents the quantification of the dotblot using ImageJ. The integrated cell density was normalized to WT values. Data are mean and standard errors from the three biological replicates; **** denotes P<0.0001, *** denotes P=0.0028 after applying a Tuke’s multiple comparison test. (C) Enrichment odds ratios for Fisher exact tests comparing sets of genes up, and down regulated, respectively, in 1) cholesterol-supplemented medium vs. glucose-supplemented medium (Pawełczyk et al., 2021), GEO accession GSE175812); and 2) virRmut vs WT as well as virRmut vs virRmut-Comp. (D) Euler diagram showing the intersection between A) genes upregulated by cholesterol, B) genes down-regulated in virRmut, and C) the DevR regulon. (E) Expression patterns in response to TRZ (1h) and in virRmut for genes involved in metal signaling, metabolism and homeostasis.

VrR regulates the protein content of EVs in Mtb.

(A). Experiment design for proteomics analyses. label-free mass spectrometry data was retrieved for replicates of H37RV (WT), virRmutand virRmut-Comp. For each strain, data corresponding to proteins extracted from either whole-cell extracts (WC) and EVs was produced, totaling 12 samples (2 strains times 2 cell compartments times 3 replicates). (B) PCA plot of the proteomics data. The loadings from the two first principal components, adding up to 50.5% of variance explained, are plotted. (C) Expression patterns of VirR in the proteomics dataset across cell compartments and strains. As expected, while virRmut shows significantly lower levels of VirR protein, the complemented strain does not just restore WT levels of expression, but leads to a strong overexpression of VirR, both in the lysates and the EVs, (D) Bar plots of differentially expressed proteins found between strains in EVs (up: virRmut vs WT; down virRmutvs virRmut-Comp at 5% FDR). (E) Enrichment profiles between sets of proteins found up and down-regulated for the contrasts virRmut vs WT and virRmut vs virRmut-Comp. (F) Gene Ontology enrichments of the downregulated proteins in the VirR mutant strain (left) and the upregulated ones (Right), at the EV location. GO terms selected for testing included biological process and cell compartment labels containing N > 3 genes, between levels 4-6 of the ontology tree. As in figure 3E, terms selected for visualization with FDR=10% and enrichment odds-ratio>2, nodes’ size is proportional to the enrichment significance, and links between terms are added wherever the sets of proteins contributing to the enrichments in a connected pair shows an intersection larger than 50% of the smallest of the gene sets involved in the pair (see Methods for further details, and supplementary file S5 for an extended list of all the enrichments found). (G) Expression patterns of bacterioferritin proteins in the EVs of WT and virRmut. (H) Expression patterns of proteins contributing to enrichments of terms related to mycolic acids biosynthesis. (I) Bottom left: Concentrations of key metabolites in the propionyl-coA detoxification routes. Right: Metabolic pathway of the propionyl-coA detoxification route through the methyl-malonyl route, along with the differential expression statistics between the WT and virRmut strains observed at transcriptomic and proteomic levels for the main enzymes involved. The framed plot represents the quantification of selected metabolites by mass spectrometry.

Lipidomic analysis of cell envelope and EVs from virRmut.

(A) Comparative total lipid analysis of the indicated strains by nano-LC-MS. Lipid species with an abundance higher than 0.5% are shown. Data indicates mean and standard error from three biological replicates. (B) Analysis of polar lipids of the indicated strains by bidimensional (2D)-layer chromatography (TLC) (2D-TLC). Phosphatidyl-myo-inositol dimannosides (Ac1PIM2 and Ac2PIM2, respectively); phosphatidyl-myo-inositol hexamannosides (AcxPIM6); phosphatidylethanolamine (PE); diphosphatidylglycerol (DPG). Phospholipid (P). (C) Analysis of mycolic acid species in the indicated strains by TLC. (D) Analysis of PIDMs and TAGs by TLC. (E) Analysis of apolar lipids of the indicated strains by TLC. Diacylglycerol (DAG); free fatty acid (FF); free mycolic acid (FMA). (F) Analysis of apolar lipids of MEVs isolated from the indicated strains. Triacylglycerols (TAGs); phthiocerol dimycocerosates (PDIMs) (G) Analysis of polar lipids of MEVs isolated from the indicated strains. Phosphatidyl-myo-inositol dimannosides (Ac1PIM2 and Ac2PIM2, respectively); phosphatidyl-myo-inositol hexamannosides (AcxPIM6); diphosphatidylglycerol (DPG).

Chemical and nanometric analysis of isolated cell walls shows higher amounts of PG in the absence of VirR.

(A) Glycosyl composition analysis of isolated cell walls from the indicated strains. Acid hydrolysis prior to GC-MS analysis of isolated cell walls was performed to determine the total amounts of galactose (G), arabinose (A), rhamnose (R), and N-acetylglucosamine (NAGc). The ratio between different sugars is shown to indicate: (i) the relative levels of arabinogalactan (A/G); (ii) the length of the AG polysaccharide chain (G/R); (iii) the amount of PG (NAGc/Rham). Individual measurements are shown from three biological replicates. Data are mean and standard errors. **P < 0.01 after applying a Tukeýs multiple comparison test. (B) Quantification of diaminopimelic acid (DAP) on isolated cell walls from the indicated strains. Data are mean and standard errors from three biological replicates; **** denotes P<0.0001, * denotes P=0.0119 after applying a Tukeýs multiple comparison test. (C) Atomic force microscopy high resolution images taken in liquid from the external surface of purified peptidoglycan from different samples, left to right: WT, virRmut, virRmut-C, the structure of external peptidoglycan layer shows a mesh with pores of different sizes, (D) Graph showing the pore diameter from n = 8 AFM images similar to (C) (each point represent an image) of different samples; black triangles: WT, blue circles: virRmut, red squares: virRmut-C. The pore diameter was calculated from the binary slice from each image containing the greatest number of pores, according to (Pasquina-Lemonche, 2024). (E) The number of pores per surface area from different samples; white triangles: WT, red circles: virRmut, blue squares: virRmut-C, this was calculated from the slice containing the maximum number of pores where the pore size was analyzed, following the method from (Pasquina-Lemonche, 2024). (F) Graph showing the peptidoglycan thickness manually measured using the 1D statistic tools from the open-source program Gwyddion (Nečas and Klapetek, 2012) from AFM images in air containing several fragments of cell wall, each point represents a different peptidoglycan fragment from a different cell. There were three samples analyzed: black WT n=23 peptidoglycan fragments, blue virRmut n=36 and red virRmut-C n=25. Statistics: D) Using a two-tailed t test with Welch’s correction the statistical comparison are: (***) pWT-virRmut = 4.1 10-4 with t = 6.1, df = 7.5, (**) p virRmut -virRmut-C = 0.007 with t = 3.2, df = 13.2, E) Using a two-tailed t test with Welch’s correction the statistical comparison are: (****) pWT-virRmut = 6.6 10-10 with t = 16.3, df = 12.7, (**) p virRmut - virRmut -C = 0.01 with t = 3.0, df = 13.2., F) Using a two-tailed t test with Welch’s correction the statistical comparison are: (****) pWT-virRmut= 2.8 10-18 with t = 17.8, df = 32.3, (****) virRmut - virRmut -C = 3.9 10-17 with t = 16.4, df = 31.8. (G) List of muropeptides identified in the cell walls of Mtb. Features including retention time (RT), observed and expected mass as well as the best match for each peak are shown. Those deacetylated muropeptides are indicated in red. (H) Relative abundance of deacetylated muropeptides in the indicated strains.

VirR interacts with Lcp1.

(A) Blue-native PAGE analysis of recombinant VirR and Lcp1. The molecular mass of markers in kDa are indicated on the left side of the gel. The size of the multimers of VirR and Lcp1 are indicated on the left side of the gel. (B) Upper panel. Representative image of a flotation assay of recombinant VirR alone or in combination with Lcp1 performed on an idioxanol density gradient. The presence of VirR was detected in each fraction by dot-blot using murine polyclonal antibodies raised against VirR. Numbers indicate collected fractions from top (1) to bottom (11). Lower panel. Quantification of the dot-blot by measuring the pixel intensity of the dots using ImageJ. Data are mean and standard error of three independent experiments. (C) Bipartite split-GFP experiment using M. smegmatis D2 expressing plasmids depicted in (Fig S11A), including CtpCNterm, VirR, VirRsol (Δ1-41), Rv3484, Rv3267 (Lcp1), Rv0822, Rv3484LytR_C, Rv3267LytR_C and Rv0822LytR_C. Bacteria were grown in complete 7H9 and fluorescence was recorded by FACS. Pooled data obtained from four to six independent cultures of each strain are shown. Statistical analysis was performed using non-parametric two-sided Mann-Whitney test. ns: not significant. * and ** refer to p-values <0.05 or 0.01, respectively.

Plasmids and primers used in this study.

Gradient mobile phase composition for ion-pairing LC-MS/MS for the quantification of acetyl-CoA, methyl citrate and methylmalonyl-CoA.

Gradient mobile phase composition for ion-pairing LC-MS/MS for the quantification of pyruvate.

Quantifier MRM transitions and collision energies used for acety-CoA, methyl citrate, methylmalonyl-CoA and pyruvate.

Diversity of Mtb LCP proteins.

(A) Domain structure of LCP proteins in Mtb. Proteins are organized according to the presence of (i) both LCP (green) and LytR_C (slight orange) terminal domain (Rv3267, Rv3484 and Rv0822); (ii) the solo LytR_C domain (VirR and Rv2700); and the solo LCP domain (Rv3840). Additional information is provided for Mtb mutants in the indicated genes. (B) Phylogenetic analysis of LCP proteins in Mycobacteria (M. tuberculosis, M. smegmatis, M. marinum and M. leprae). The unrooted tree includes seven separate clusters for proteins with both LCP and LytR_C terminal domains (CpsA1, CpsA2 and CpsA3); with the solo LytR_C terminal domain (Solo_LytRC1 and Solo_LytRC2); with the solo LCP domain; and a separate cluster including the characterized LCP proteins from S. pneumonia and S. aureus. Note the clustering of one of the M. smegmatis LCP proteins with this group. VirR is highlighted in blue.

The enhanced vesiculation phenotype of virRmut occurs in the absence of cell lysis.

(A) Nanoparticle tracking analysis (NTA) of EV preparations derived from WT and the indicated strains showing number of particles per cm3 determined by Zeta View NTA in three independent EV preparations. Data are presented as mean ± SEM. *P ≤ 0.05 (One-way ANOVA with Tukey’s multiple comparisons test). (B) Cell lysis control of virRmut. Shown are immunoblots of cell lysates and culture supernatants (SN) for the indicated strains. The cytoplasmic protein IdeR was detected in cell lysates but not in culture supernatants confirming cell integrity, while the secreted protein Ag85b was readily detected in the culture supernatants. (C) Cell lysis control of Mtb submitted to conditions that alter permeability. Arrows indicate the size of the proteins of interest detected by immunoblot. (D) Alternatively, cell viability was tested by serial dilution and plating onto 7H10 medium. Cultures from B and C were submitted to serial dilutions (10- 1 to 10-5) and plated onto 7H10 after reaching an OD600nm of 0.2.

H2O2 sensitivity assay.

The indicated strains were initially grown on 7H9 medium supplemented with AND to an OD of 0.5. Washed cells were then transferred to the same medium supplemented with and without 13mM H202. After 8 h, cells were diluted down as indicated and spotted onto 7H10 solid medium.

Expression patterns of permeability-dependent genes upregulated in cultures exposed to cholesterol and simultaneously downregulated in virRmut.

Behaviour of virRmutupon conditions that modulate permeability.

(A) Growth curves of virRmut in MM supplemented with either glycerol, cholesterol or glycerol plus TRZ at the indicated concentrations. (B) Dotblot analysis of vesiculation levels of virRmuton the indicated culture media. a, b and c denote biological replicates. Quantification of the dotblot was done by measuring the integrated density of each dot using ImageJ. Data are mean and standard deviations. * p=0.021 after applying a Tuke’s multiple comparison test. (C) Time course of the uptake of ethidium bromide (EthBr) by virRmut grown in the indicated conditions, as measured by fluorescence at 590=nm for 65 min. Data are mean and standard deviation of three biological replicates.

Expression patterns of ribosomal proteins in the EVs from WT, virRmut and virRmut -Comp strains. Proteins shown are downregulated in the vesicles of virRmut with respect to both VirR expressing strains.

Expression patterns in the EVs from WT, virRmut and virRmut -Comp strains of proteins differentially expressed in virRmut that contribute to enrichments of terms related to metabolism of: (A) nucleotides (B) amino-acids (C) oligosaccharides (D) glycogen.

TLC analysis of strains grown in the presence of cholesterol or TRZ.

Analysis of polar lipids, including SL1 and DATs lipids of the indicated strains by TLC. Sulfolipid 1 (SL1); diacylglytrehaloses (DATs); Trehalose dimycolate (TDM).

Additional AFM data.

(A-C) (i) Atomic force microscopy images taken in liquid from a big area showing the sample heterogeneity overview with various peptidoglycan fragments of the following samples: (A) WT, (B) virRmut, (C), virRmut-C. (ii) Atomic force microscopy images zoom in one individual peptidoglycan fragment corresponding to one cell from different samples: (A) WT, (B) virRmut showing the background (Backg.), the external surface (Ext) the internal (Int), (C), virRmut-C, white square showing the size of the zoom images shown in Figure 5 from the external surface of the cells. (D) Curve of grey points showing 255 slices of one AFM image taken using a FIJI macro, in the x axis and the total number of pores in the y axis, the red line is a Gaussian fitted to the curve and the maximum of the Gaussian is marked with a dash red line showing slice 140 in this example corresponding to the slice of the image with the maximum number of pores. (E) Binary slice number 140 from the original AFM image showing the pores in white and the material represented in black. (F) The pores from ‘E’ were analyzed using the FIJI macros where each pore area was measured and plotted in the x axis against the cumulative fraction of total pore area in the y axis as shown in the curve by blue dots. The red dot marks the most likely pore area which corresponds to the half of the cumulative fraction of the total pore area, this area was then used to obtain the pore diameter of the image. The same was repeated with the n = 8 images for each sample to compose the graphs ‘D’ and ‘E’ from Figure 7.

Sensitivity to lysozyme of virRmut.

(A) Analysis of the minimum inhibitory concentration (MIC) of lysozyme of the indicated strains by the resazurin method. (B) Growth of WT and virRmut treated with 0 or 50 µg/ml lysozyme (Lys). Data represents the mean ± SEM of three independent cultures.

Localization of VirR by fluorescence microscopy.

Fluorescence images of a Mtb strain expressing Venus-tagged VirR.

Split-GFP strategy.

(A) Upper panel. Scheme of the plasmid used to test VirR interactions in vivo. Lower panel. Scheme of the potential molecular interaction between VirR and Lcp1 using split-GFP. (B) Gating strategy to analyze split-GFP flow cytometry data. Bacteria were first sorted based on size (FSC) and structure (SSC) to exclude debris (1). Then, aggregates were excluded based on FSC and SSC width (W) and eighth (H) (2,3). GFP signal was then analyzed vs. SSC (4) or mCherry, when relevant (5).