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 Tuke’s 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 both 7H9 and MM as measured by fluorescence. Representative images of both WT and virRmut strains in either 7H9 or MM at 8h after the addition of the FDAA. Data are mean and standard deviations of three biological replicates. 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 and virRmut), and sequenced. Of these 8 samples, 7 passed quality control, (3 WT and 4 virRmut) and were subsequently analyzed. (B) PCA plot of the transcriptomics data. The loadings from the two first principal components are plotted. PC1 (X axis) significantly separates samples across strains (One tailed Mann Whitney test: PC1 in WT vs virRmut: p=2.86 e-02). (C) Number of differentially expressed genes between virRmut and WT. virRmutsignificantly affects expression of a total of 750+913=1663 genes, (Storey-Tibshirani FDR=5%), which represents 41.5% of all genes tested. (D) Gene Ontology (GO) enrichments of downregulated genes in virRmut (left) and upregulated ones (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 table S2 for an extended list of all the enrichments found).

virR regulates the protein content of EVs in Mtb.

(A). Experiment design for proteomics analyses. label-free mass spectrometry data was retrieved for three replicates of H37RV and virRmut. For each strain, in turn, 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 are plotted. PC1 (X axis) significantly separates samples across cell-compartments (One-tailed Mann Withney test: PC1 in EVs vs WC: p= 1.08 e-03), while PC2 separates samples from EVs in virRmut from the rest of samples (one-tailed Mann Withney test: PC2 in virRmut in EV vs all other samples: p=4.55 e-03), indicating a differential peptidic cargo in the vesicles of virRmut. (C) Bar plots of differentially expressed proteins found between strains in in whole cell extracts (left, 19 differentially expressed proteins at 5% FDR), and extracellular vesicles (right, 353 differentially expressed proteins at 5% FDR). (D) 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 3D, 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 table S2 for an extended list of all the enrichments found). (E) Expression patterns of bacterioferritin proteins in the extracellular vesicles of WT and virRmut. (F) Expression patterns of proteins contributing to enrichments of terms related to mycolic acids biosynthesis. (G) 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.

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. (D) Euler diagram showing that 39 out of 48 genes in the DosR regulon are simultaneously repressed in virRmut vs WT, and upregulated in cholesterol vs. glucose enriched medium cultures, suggesting a common activation of the regulon in conditions of low permeability (see also Fig S5). (E) Expression patterns in response to TRZ (1h) and in virRmut for key genes involved in metal signaling, metabolism and homeostasis.

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 Tuke’s 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 Tuke’s 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.

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 S9A), including VirR, VirRsol(Δ1-41), CtpCN, Rv3484, Rv3267 (Lcp1), Rv0822, Rv3484LytR_C, Rv3267LytR_C and Rv0822LytR_C. Bacteria were grown in complete 7H9. Fluorescence was recorded by epifluorescence microscopy. The experiment was performed one time.

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.