{"paper_id":"0a978cbc-154d-43c0-b778-4ea2600a8775","body_text":"Proteomic insights into a M. tuberculosis clinical isolate with an increased propensity to form 1 \nviable but non-replicating subpopulations during acid stress 2 \nNastassja L. Kriel1, Julian Coetzee1, Jacoba M. Mouton1, Samantha Sampson1 3 \n 4 \n1DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical 5 \nResearch Council Centre for Tuberculosis Research, Division of Molecular Biology and Human 6 \nGenetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, 7505 Cape 7 \nTown, South Africa. 8 \n 9 \n 10 \n 11 \n 12 \n 13 \n 14 \n 15 \n 16 \n 17 \n 18 \n 19 \n 20 \n 21 \n 22 \n 23 \n*Corresponding author. Mailing address: Division of Molecular Biology and Human Genetics, 24 \nFaculty of Medicine and Health Sciences, Stellenbosch University. P.O. Box 241, Cape Town,8000, 25 \nSouth Africa. Email: nastassja@sun.ac.za 26 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nAbstract 27 \nPhagosome acidification is one of the challenges faced by Mycobacterium tuberculosis  during 28 \ninfection. This intracellular pathogen is known to adapt to its stressful environment through stress 29 \nresponse pathways and by secreting proteins to modify the host immune response for survival and 30 \nproliferation. However, M. tuberculosis also holds the potential to form viable but non-replicating 31 \n(VBNR) and antibiotic tolerant persisters in response to environmental stress, including acid stress. 32 \nIn this study we used a in vitro  acid stress model to stimulate the formation of a VBNR 33 \nsubpopulation in a M. tuberculosis  clinical isolate with an increased propensity to form VBNR 34 \nbacteria. Mass spectrometry-based proteomics was used to characterize the cellular proteome and 35 \nculture filtrate proteome of actively replicating (pH 6,5) and VBNR enriched (pH 4,5) cultures. We 36 \nshow that in response to acid stress, M. tuberculosis S169 increases the expression of known stress 37 \nresponse proteins, including the methyltransferase Rv1405c and the acid stress response two-38 \ncomponent regulatory protein TcrX. Interestingly, we found that the dormancy response regulon 39 \ncomponents were less abundant in acid stressed M. tuberculosis  S169. Our protein aggregation 40 \ncapture culture filtrate proteomic approach revealed that the culture filtrates of low pH stressed M. 41 \ntuberculosis S169 contained less proteins than that of actively replicating cultures. We identified 42 \nseveral proteins previously implicated in M. tuberculosis  persistence, including toxin-antitoxin 43 \nproteins (VapC51 and VapB10), the chorismate mutase (Rv1885c), and several uncharacterized 44 \nproteins. The observed differences identified in the characterisation of this clinical isolate in 45 \ncomparison to published M. tuberculosis H37Rv highlights the need to investigate M. tuberculosis 46 \nclinical isolates for a more representative understanding of the tuberculosis stress response. 47 \nKeywords: Tuberculosis, TB, persister, VBNR, heterogenous, acid stress 48 \n  49 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nAuthor Summary 50 \nTuberculosis is caused by Mycobacterium tuberculosis  and this pathogen can form a subpopulation 51 \nof viable but non-replicating (VBNR) cells that are recalcitrant to antibiotic treatment. These 52 \npersister bacteria increases the risk of treatment failure and tuberculosis recurrence following 53 \ntreatment. Stimulation of a persister population through triggered persister formation can be achieved 54 \nby environmental stress factors such as low pH, nutrient starvation, hypoxia, and antibiotic exposure. 55 \nIn this study we investigate the cellular and culture filtrate proteomes of a high persister forming 56 \nclinical isolate, M. tuberculosis  S169, in response to acid stress. We show that following the 57 \nstimulation of a VBNR subpopulation in response to acid stress, several known acid stress response 58 \nproteins are more abundant in VBNR enriched cultures. Interestingly, we found that stress response 59 \nproteins were less abundant. Using a protein aggregation capture approach we successfully 60 \ncharacterized culture filtrates M. tuberculosis cultures, reducing the bacterial culture amount required 61 \nfor these experiments. Culture filtrates differed between actively replicating and VBNR enriched 62 \ncultures. Several immunogenic proteins were identified in a higher abundance in the culture filtrates 63 \nof VBNR enriched cultures.  64 \n 65 \nIntroduction 66 \nMycobacterium tuberculosis , the pathogen which causes tuberculosis (TB), primarily infects 67 \nmacrophages. During infection, M. tuberculosis is exposed to high levels of reactive oxygen and 68 \nnitrogen intermediates, reduced oxygen and nutrient availability, and  low pH (1). M. tuberculosis 69 \ncan replicate under these conditions or persist in a viable, but non-replicating (VBNR) state (2). 70 \nPersister M. tuberculosis , can coexist with replicating mycobacteria during infection, but these 71 \nbacteria are transiently insensitive to antibiotic treatment due to their non- or slow-replicating nature 72 \n(2,3). These antibiotic recalcitrant bacteria contribute to the length of TB treatment regimens, where 73 \nmultiple antibiotics are required to treat infections for extended periods of time (4,5). Persister 74 \nsubpopulations may resume growth under favourable conditions, increasing the possibility for 75 \nrecurrent disease following treatment (6,7).  76 \nBacterial persisters are thought to arise from spontaneous persistence or triggered persistence (8). 77 \nSpontaneous persistence describes the stochastic formation of persisters at a rate that is constant 78 \nduring growth, accounting for approximately 1% of the bacterial population in stationary phase 79 \n(9,10). Triggered persistence refers to the formation of a persister subpopulation in response to a 80 \nstress signal such as starvation, population density, pH stress, immune factors and drug treatment (8). 81 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nIn other bacterial species, mechanisms of persister formation include metabolic slow down, 82 \nmodulation of nucleoid-associated proteins, expression of drug efflux pumps, activation of toxin-83 \nantitoxin modules, and upregulation of stress response genes (11–15). Similarly, a multiple stress 84 \ndormancy model for M. tuberculosis induced lower energy metabolism, reduced transcription and 85 \ntranslation, and increased expression of stress response genes as mechanisms of dormancy (2). 86 \nSeveral studies have highlighted the upregulation of the dormancy response regulon (DosR) in 87 \nresponse to low oxygen and nitric oxide exposure, further emphasizing the role of stress response 88 \nproteins in bacterial dormancy (16–18). Toxin-antitoxin modules were also more abundant in the 89 \nproteomes of nutrient starved M. tuberculosis, highlighting the cross-species similarities in persister 90 \nlinked pathways (19).  91 \nThe intracellular pathogen M. tuberculosis interacts with the host by presenting various molecules on 92 \nits cell surface, but also through the secretion of molecules (20,21). M. tuberculosis  secretion 93 \nsubstrates play a role in nutrient acquisition, host epigenetic modification, prevention of phagosome 94 \nmaturation, modulation of cytokine response, autophagy, redox regulation, and necrosis and bacterial 95 \ndissemination (20,22). Identification of proteins secreted by M. tuberculosis , especially in response 96 \nto environmental stress, is crucial in understanding how M. tuberculosis subverts killing by host 97 \nmacrophages (23,24). Salmonella persisters have been shown to not only maintain a metabolically 98 \nactive state, but to secrete effector proteins which reprogrammed the host cell polarization form a 99 \npro-inflammatory to anti-inflammatory state (25). Furthermore, advances in immunopeptidomics 100 \nhave highlighted the need to investigate proteins identified in the extracellular region of M. 101 \ntuberculosis by demonstrating that ESX secretion substrates are the prominent source of MHC-I 102 \npresented M. tuberculosis peptides (26). This finding highlights the importance of investigating M. 103 \ntuberculosis secreted proteins as anti-TB vaccine development candidates.  104 \nIn this study we sought to characterize both the cellular proteome and the culture filtrate of a M. 105 \ntuberculosis clinical isolate which has an increased propensity to form VBNR subpopulations (7). 106 \nThis clinical isolate, M. tuberculosis S169, was obtained from an HIV-negative patient who failed 107 \ntreatment following standard 6-month anti-TB treatment (27). M. tuberculosis S169 is susceptible to 108 \nanti-TB treatment and no drug-resistance conferring mutations were identified using whole genome 109 \nsequencing (7). We hypothesize that VBNR M. tuberculosis  may secrete a different subset of 110 \nproteins to that of actively replicating M. tuberculosis . Given the abundance of VBNR M. 111 \ntuberculosis in bacterial culture, we opted to use this clinical isolate with an increased propensity to 112 \nform VBNR M. tuberculosis, to increase the likelihood of identifying VBNR secreted proteins (10). 113 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nWe used a low pH stress model to trigger the formation of a VBNR subpopulation and verified the 114 \nenrichment of a VBNR subpopulation using a dual replication reporter plasmid (28,29). To 115 \ncharacterize the culture filtrates of actively replicating and VBNR enriched cultures, we made use of 116 \na protein aggregation capture approach (30). Our culture filtrate mass spectrometry approach 117 \nrequired smaller amounts of bacterial culture, reducing experimental time, technical variability from 118 \npooling multiple cultures, experimental cost, and in the case of M. tuberculosis, biohazardous risk. 119 \n 120 \nMaterials and Methods 121 \nBacterial culture and acid stress exposure 122 \nM. tuberculosis  clinical isolate S169 obtained from Dr Stephanus Malherbe was collected by the 123 \nCatalysis TB – Biomarker Consortium, Ethical approval granted by Stellenbosch University Human 124 \nResearch Ethics Committee (registration number N10/01/013) (27). Informed consent was obtained 125 \nfrom all study participants (27). All reagents were purchased from Sigma Aldrich unless otherwise 126 \nstated. All experiments were performed in biological triplicate. 127 \nLiquid mycobacterial cultures were grown in Middlebrook 7H9 supplemented with 10% dextrose-128 \ncatalase (DC), 0.2% glucose and 0.05% Tween-80 (7H9-DC). The clinical isolate was transformed 129 \nwith the replication reporter plasmid pTiGc and transformants were cultured in the presence of 25 130 \nµg/ml kanamycin at 37°C. For acid stress, M. tuberculosis S169::pTiGc was sub-cultured in 7H9-DC 131 \npH 6.5 supplemented with 4mM theophylline to an OD 600 of ~1.0 prior to washing with culture 132 \nmedia. Cultures were resuspended in fresh 7H9-DC at pH 6.5 and pH 4.5 and incubated at 37°C for 133 \n48h. Fluorescence dilution was used for the identification of VBNR mycobacteria as previously 134 \ndescribed (28). Aliquots of bacterial culture pre- and post-acid stress were plated to verify bacterial 135 \nviability following acid stress. 136 \nImaging flow cytometry sample preparation, acquisition, and analyses 137 \nCultured M. tuberculosis S169::pTiGc was sonicated for 12 minutes at 36kHz (Zues, Sonicator bath) 138 \nbefore filtering through a 40 µm filter. Cells were fixed with 4% formaldehyde in phosphate buffered 139 \nsaline (PBS) and 0.05% Tween-80 for 30 minutes. Cells were washed and resuspended in PBS prior 140 \nto analysis on the Amnis® ImageStream® X Mark II Imaging Flow Cytometer. A minimum of 20,000 141 \nin-focus events were captured per sample and the data were analysed using IDEAS 6.2 software. 142 \nSignals from bright field, GFP and TurboFP635 were collected from channels 1, 2 and 4, 143 \nrespectively. The spot count feature was used to identify a population of single cells (identified as 1 144 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nobject) after which the GFP best in focus population was selected for viable cells. A histogram of the 145 \nnormalized frequency against the intensity of TurboFP635 was generated for different pH conditions 146 \nand time points and overlapped to demonstrate the formation of a viable, but non-replicating 147 \npopulation.  148 \nVBNR bacteria can be identified by the retention of high TurboFP635. A gate was set on the top 50th 149 \npercentile of the induced TurboFP635 (pH 6.5 0h) culture, termed “high red”. This gate was used to 150 \ndetermine the frequency of “high red” VBNR bacteria in pH 6.5 and pH 4.5 cultures at 48h. The 151 \npercentage of VBNR subpopulation was calculated by the following equation: 152 \n% VBNR subpopulation = % high red pH 4.5 48h - % high red pH 6.5 48h 153 \nProtein extraction 154 \nFollowing acid stress exposure of M. tuberculosis S169::pTiGc, the cell biomass was separated from 155 \nthe culture supernatant by centrifugation at 4000 rpm for 20 minutes.  156 \nCulture supernatants were sterilized by filtering twice using 0.22 µm Steriflip ® filter (Sigma 157 \nAldrich) and kept at under ice-cold conditions. Culture filtrates from pH 4.5 cultures were 158 \nneutralized with sodium hydroxide to a pH of 6.5. All culture filtrates were concentrated using 159 \nAmicon Ultra 3KDa spin columns (Sigma Aldrich) from 25 mL to 500 µL. SDS was added to a final 160 \nconcentration of 10% prior to incubation at 60°C for 1 hour and storage at -20°C.   161 \nCell pellets from 25 mL cultures were stored at -20°C prior to resuspension in 1 mL lysis buffer (20 162 \nmM Tris-HCl, 1% SDS) containing protease inhibitors (Roche c0mpleteTM mini EDTA-free protease 163 \ninhibitor cocktail). Cells were washed once for 2 minutes at 14 000 rpm, 4°C. Cell pellets were 164 \nresuspended in 300 µL lysis buffer containing protease inhibitors and 10 µL DNAseI (RNAse-free) 165 \nprior to cell disruption by mechanical bead-beating (Fast-prep-24 TM, MPbio) using 300 µL acid-166 \nwashed glass beads (425-600 µm, Sigma Aldrich). Bead-beating was performed 8 times for 20 167 \nseconds, at 4 m.s-1 with 1 minute intervals on ice. The cytosolic lysate was cleared by centrifugation 168 \nat 12 000 rpm for 10 minutes at 4°C, the supernatant was recovered then clarified again by 169 \ncentrifugation as before. Clarified supernatants were filtered through 0.22 µm Acrodisks (Sigma 170 \nAldrich) and stored at -20°C. 171 \nMass spectrometry sample preparation 172 \nCulture filtrate and cell lysate proteins were reduced and alkylated prior to capture on MagReSyn ® 173 \nHILIC beads (Resyn Biosciences). Briefly, the concentrated protein sample was reduced with 5 mM 174 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nTCEP at room temperature for 1 hour and subsequently alkylated at 13 mM iodoacetamide for 1 hour 175 \nin the dark. Iodoacetamide was quenched with 11 mM DTT. Cell lysate proteins were digested on 176 \nMagReSyn® HILIC beads as recommended by the manufacturer before storage at -20 °C. 177 \nCulture filtrate proteins were captured onto 150 µL prepared MagReSyn ® HILIC beads, overnight at 178 \n37°C. Beads were washed as per manufacturer recommendations and resuspended in 50 mM TEAB 179 \nwith 1 µg sequence modified trypsin. An on-bead digest was performed at 37°C for 18 hours, 180 \nshaking at 800 rpm. Peptides were recovered and beads were incubated with 100 µL 1% TFA for 30 181 \nseconds at 800 rpm. The 1% TFA solution was recovered and pooled with recovered peptides from 182 \novernight digest. To ensure that bead captured proteins were digested, the protein digest was 183 \nrepeated with 100 µL TEAB and 1 µg sequence modified trypsin as before and recovered peptides 184 \nwere pooled and stored at -20°C. 185 \nRecovered supernatants were dried using a Concentrator plus (Eppendorf) and desalted as previously 186 \ndescribed (31). Peptide concentrations were determined against a 1 mg/mL peptide solution (Pierce) 187 \nusing a spectrophotometer (Jenway 7415). 188 \nLC-MS/MS 189 \nLiquid chromatography was performed on an Ultimate 3000 RSLC equipped with a 20mm × 100 μ m 190 \nC18 trap column (Thermo Scientific) and a CSH 25cm × 75 μ m, 1.7 μ m particle size C18 column 191 \n(Waters). Solvent A (2% acetonitrile:water and 0.1% formic acid) was used to load samples onto the 192 \ntrap column from an autosampler (set to 7°C) at a flow rate of 2µL/min, for 5 minutes before sample 193 \nelution onto the analytical column. A defined flow rate of 300 nL/min with the following gradient 194 \nwith Solvent B (100% acetonitrile, 0.1% formic acid) was used: 5–30% B over 60 min and 30–50% 195 \nB from 60–80 min at 45°C. 196 \nCulture Filtrate samples were analysed in Data Dependent Acquisition (DDA) mode. Cell lysate 197 \nsamples were analysed in Data Independent Acquisition (DIA) mode.  198 \nDDA mass spectrometry analysis was performed using a Orbitrap Fusion TM Tribird TM Mass 199 \nspectrometer (Thermo Scientific) equipped with a Nanospray Flex ionization source on positive 200 \nmode with spray voltage set to 2 kV and ion transfer capillary at 290°C. Spectra were internally 201 \ncalibrated using polysiloxane ions at m/z = 445.12003. For MS1 scans the Orbitrap detector was set 202 \nto a resolution of 60,000 over a scan range of 375–1500 with the AGC target at 4E5, and maximum 203 \ninjection time of 50 ms. Data was acquired in profile more. MS2 acquisitions were performed using 204 \nmonoisotopic precursor selection with ion charges +2 - +7 with an error tolerance of +/-10 ppm. 205 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nPrecursor ions were excluded from repeat fragmentation for 60 s. Precursor ions were selected for 206 \nfragmentation in HCD mode using the quadrupole mass analyser at an HCD energy of 30%. 207 \nFragments ions were detected in the Orbitrap mass analyzer with a resolution of 30,000. The AGC 208 \ntarget was set to 5E4 and a maximum injection time of 60 ms. Data was acquired in centroid mode.  209 \nDIA mass spectrometry analysis was performed on the same instrument as for DDA analysis. The 210 \nresolution of MS1 scans were set to 60,000 over a scan range of 375–1500. The AGC target was set 211 \nto standard and a maximum injection time of 100 ms. Data was acquired in profile more. Precursor 212 \nions were selected for fragmentation in HCD mode using the quadrupole mass analyser at an HCD 213 \nenergy of 30%. Precursor ions were scanned in three windows, 355-555, 555-755, and 755-955 m/z 214 \nand a 10 m/z isolation window with a 1 m/z overlap. Ions were detected in the Orbitrap mass 215 \nanalyzer set to 30,000 resolution and the AGC and maximum injection time set to custom. Data were 216 \nacquired in centroid mode.  217 \nProtein identification 218 \nMaxQuant 2.2.0.0 was used to analyze DDA tandem mass spectrometry data using the M. 219 \ntuberculosis database (UP000001584) downloaded from Uniprot ( https://www.uniprot.org/) in 220 \nFebruary 2023 (32,33). Carbamidomethyl cysteine was set as a fixed modification and oxidated 221 \nmethionine and N-terminal acetylation of proteins were selected as variable modifications. A 222 \nmaximum of 2 missed tryptic cleavages were allowed and proteins were identified with a minimum 223 \nof 1 unique peptide. The protein and peptide false discovery rate (FDR) threshold was less than 0.01. 224 \nRelative quantification was performed for identified protein groups using the MaxQuant LFQ 225 \nalgorithm and the “match between runs” algorithm was used to detect peptides which were not 226 \nselected for MS/MS analysis in other replicate experiments.  227 \nSecreted proteins were identified using LFQ intensity data using Perseus (Figure 3). Potential 228 \ncontaminants, reverse hits, only identified by site potential contaminants, and proteins only identified 229 \nwith one unique peptide were removed. Proteins were considered true identifications if identified in 230 \nat least two of the three biological replicate experiments for a particular condition. Proteins unique to 231 \neither pH 6.5 or pH 4.5, where identifications were only made in one of the two conditions, were 232 \nidentified. For the remaining proteins, the LFQ intensity data was establish if proteins were 233 \nsignificantly differentially abundant in the culture filtrates of pH 4.5 versus pH 6.5 cultures. The data 234 \nwas imputed by replacing missing values from the normal distribution of log2 transformed data for 235 \neach experiment. Statistically significant differentially abundant proteins were identified following a 236 \npaired student’s t-test and a Benjamini-Hochberg FDR correction of 0.05. 237 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nCell lysate DIA data was analysed using FragPipe v22.0 (34–43). The DIA_SpecLib_Quant 238 \nworkflow was selected which built a spectral library using MSFragger-DIA against the M. 239 \ntuberculosis UP000001584 database with added decoy and contaminant proteins using default 240 \nsettings. The library was filtered to 1% FDR at protein and peptide level. The spectral library was 241 \napplied to quantify the DIA data using DIA-NN at 1% FDR. FragPipe-Analyst was used for the 242 \nanalysis and visualization of DIA data using the DIA workflow (44). Variance stabilizing 243 \nnormalization for DIA data was used for normalization and a Perseus-type imputation was selected 244 \nfor differential expression analysis. Differentially expressed genes were identified using a log fold 245 \nchange of 2 and an Benjamini Hochberg corrected p-value of 0.05. Protein groups only identified in 246 \na single biological replicate and contaminant protein groups were excluded from the identified 247 \nprotein list.  248 \nM. tuberculosis gene annotations, protein names and gene ontology information was obtained from 249 \nUniprot ( http://www.uniprot.org/) and KEGG (https://www.genome.jp/kegg/) (33,45). Gene 250 \nontology enrichment analysis was done using the ShinyGO gene-set enrichment tool 251 \n(https://bioinformatics.sdstate.edu/go/) (46). 252 \nResults 253 \nAcid stress promotes the formation of a viable, but non-replicating M. tuberculosis population 254 \nClinical isolate M. tuberculosis  S169 was obtained from a patient who remained culture positive 255 \nfollowing 6 months of TB treatment (27). Whole genome sequencing did not identify any known 256 \nanti-TB treatment resistance conferring mutations and drug susceptibility was confirmed by 257 \nphenotypic testing. Fluorescence dilution demonstrated an increased ability of this isolate to form 258 \nVBNR M. tuberculosis in a macrophage infection model (7,28). We set out to establish if we could 259 \nreplicate the VBNR formation observed for M. tuberculosis S169 in a macrophage infection model 260 \nusing an in vitro  low pH stress model (29). Briefly, M. tuberculosis  S169 transformed with the 261 \nreplication reporter plasmid, pTiGc, was cultured in the presence of theophylline to induce the 262 \nexpression of TurboFP635. Cells were transferred into theophy lline free culture media at either pH 263 \n6.5 or pH 4.5 and incubated for 48h (Figure 1A-B). Imaging flow cytometry demonstrated active 264 \nreplication of M. tuberculosis S169 at pH 6.5 with continued high levels of GFP expression, but 265 \nreduced levels of red fluorescence following the removal of the inducer theophylline (Figure 1C). 266 \nFollowing 48h of acid stress at pH 4.5, M. tuberculosis S169 continued to express high levels of 267 \nGFP, but had reduced red fluorescence intensity, suggesting a decrease in replication (Figure 1C). 268 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\nLow pH stress induced the formation of 17.5% (+/- 4.5) VBNR M. tuberculosis S169, consistent 269 \nwith previous results investigating VBNR subpopulations using a macrophage infection model (7). 270 \nLow pH stress of M. tuberculosis S169 results in down-regulation of DosR 271 \nThe cell biomass recovered from three independent low pH stress experiments was analysed using 272 \nmass spectrometry to investigate the cellular stress response of the high VBNR M. tuberculosis S169 273 \nclinical isolate. We identified 2959 protein groups in the cell lysates of actively replicating and acid 274 \nstressed M. tuberculosis S169 which mapped to 2924 proteins in the KEGG database (Table S1) 275 \nfollowing the removal of contaminant proteins and proteins only identified in a single biological 276 \nreplicate. A comparison of actively replicating and low pH stressed cell lysates revealed that 46 277 \nproteins were only identified in the cell lysates of actively replicating M. tuberculosis S169 (Table 278 \nS2) and an additional 14 proteins were only identified in the cell lysates of VBNR-enriched M. 279 \ntuberculosis S169 (Table S3). Differential analysis of the cell lysate data revealed that 77 proteins 280 \nwere significantly more abundant, and 269 proteins were significantly less abundant (adjusted p-281 \nvalue <0,05, log2 fold change >1) in the cell lysates of low pH stressed M. tuberculosis S169 when 282 \ncompared to that of actively replicating M. tuberculosis S169 (Table S1, Figure 2A).  283 \nGene ontology (GO) enrichment analysis of proteins significantly more abundant in the cell lysates 284 \nof acid stressed cultures did not reveal any significant results. Regardless, the two-component system 285 \nTcrXY component TcrX was significantly more abundant in acid stressed M. tuberculosis  S169 286 \n(Table S1). The TcrXY two component system has previously been shown to be upregulated in 287 \nresponse to acid stress (47). S-adenosyl methionine (SAM)-dependent methyltransferase proteins 288 \nRv1403c and Rv1405c were also significantly more abundant in acid stressed M. tuberculosis S169 289 \n(Table S1). These SAM-dependent methyltransferases have previously been reported to be 290 \nupregulated in response to low pH (48–50).  291 \nA GO enrichment analysis of significantly less abundant proteins in cell lysates of acid stressed 292 \ncultures suggested a down regulation of GO terms associated with universal stress response proteins 293 \n(Figure 2B, Table S4). The dormancy response regulon, under the control of the two-component 294 \nsystem DevR/DevS, has previously been shown to be upregulated in response to acid stress (29,51). 295 \nThe DosR regulon is composed of 47 genes (52). In our study, we identified 38 DosR regulon 296 \nencoded proteins of which 34 were significantly less abundant in the cell lysates of acid stressed M. 297 \ntuberculosis S169 (Table 1).  298 \n 299 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n 300 \n 301 \nTable 1. Abundance of DosR proteins in M. tuberculosis S169 in response to acid stress 302 \nGene Name log2FC adj. pvalue significant Gene Name log2FC adj. pvalue significant \nRv0079 -3.52 0.000511 TRUE fdxA -2.08 0.0308 TRUE \nRv0080 -2.51 0.0029 TRUE Rv2028c -3.43 0.00107 TRUE \nRv0081 -1.12 0.0199 TRUE pfkB -6.13 0.00298 TRUE \nRv0569 -5.16 0.00249 TRUE Rv2030c -4.55 0.00135 TRUE \nnrdZ -2.42 0.0181 TRUE hspX -5.5 0.000573 TRUE \nRv0571c -1.48 0.0378 TRUE acg -4.71 0.00126 TRUE \nRv0572c -1.6 0.0167 TRUE Rv2623 -4.24 0.00342 TRUE \npncB2 -2.18 0.0273 TRUE Rv2624c -2.86 0.00249 TRUE \nRv0574c -1.93 0.0138 TRUE Rv2625c Not detected N/A N/A \nRv1733c Not detected N/A N/A Rv2626c Not detected N/A N/A \nRv1734c Not detected N/A N/A Rv2627c -4.97 0.00257 TRUE \nRv1735c Not detected N/A N/A Rv2628 Not detected N/A N/A \nnarX -2.79 0.00278 TRUE Rv2629 -1.83 0.00497 TRUE \nnarK2 -3.86 0.000717 TRUE Rv2630 -0.0868 0.905 FALSE \nRv1738 -4.67 0.00242 TRUE rtcB Not detected N/A N/A \nRv1812c 0.123 0.148 FALSE Rv3126c Not detected N/A N/A \nRv1813c -2.89 0.00774 TRUE Rv3127 -4.67 0.00316 TRUE \nRv1996 -3.13 0.0104 TRUE Rv3129 Not detected N/A N/A \nctpF -4.78 0.00974 TRUE tgs1 -5.32 0.00156 TRUE \nRv1998c -0.121 0.726 FALSE Rv3131 -4.43 0.00228 TRUE \nRv2003c -1.93 0.012 TRUE devS -1.67 0.0153 TRUE \nRv2004c -3.14 0.00576 TRUE devR -2.11 0.0301 TRUE \nRv2005c -3.32 0.00426 TRUE Rv3134c -5.4 0.00139 TRUE \nRv2006 0.053 0.889 FALSE     \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n12 \n \nCulture filtrates of VBNR enriched cultures showed a higher abundance of lipoproteins 303 \nIn total, we identified 461 protein groups of which 192 protein groups were only identified in the 304 \nculture filtrates of pH 6.5 cultures and 45 protein groups were only identified in the culture filtrates 305 \nof pH 4.5 cultures (Figure 3). Abundance data of protein groups identified in both test conditions 306 \nrevealed that 83 protein groups were differentially abundant between the conditions tested (q-value < 307 \n0.05, FDR 0.05, log2 FC >/<0.05) (Figure 3, Table S5-6). Of the 83 significantly differentially 308 \nabundant proteins, 43 protein groups were less abundant, and 40 proteins were more abundant in the 309 \nculture filtrates of VBNR enriched  M. tuberculosis  S169 (Table S5-6). In total, we identified 275 310 \nprotein groups, which mapped to 274 proteins in the KEGG database, in the culture filtrates of 311 \nactively replicating M. tuberculosis S169 cultures (Table S5). The 128 protein groups identified in 312 \nthe culture filtrates of VBNR-enriched cultures mapped to 128 proteins in the KEGG data base 313 \n(Table S6).  314 \nGO enrichment of proteins identified in the culture filtrates of actively replicating and VBNR 315 \nenriched M. tuberculosis S169 cultures confirmed the enrichment of pathways extracellular region, 316 \nexternal encapsulating structure, and secreted (Figure 4, Table S7-8). Other enriched pathways 317 \nincluded cell wall, cell periphery, plasma membrane, and membrane (Figure 4, Table S7-8). The 318 \nlipoprotein pathway was revealed to be enriched in the culture filtrates of pH 4.5 cultures (Figure 4B, 319 \nTable S8). Several lipoproteins were only identified in VBNR enriched culture filtrates (LpqG, 320 \nDppA, LpqO, FecB2, LppL, LppM, Subl, GlnH, and Rv2585c) or identified with a higher relative 321 \nabundance in pH 4.5 culture filtrates (FecB, LpqB, LprG, and LprA) (Table S6). Zymogen binding, 322 \npreceding the proteolytic cleavage of enzymes to an active state, was also enriched in the culture 323 \nfiltrates of acid-stressed M. tuberculosis  S169 (Figure 4B). Zymogen binding proteins were more 324 \nabundant in the culture filtrates of VBNR enriched cultures, including MetK, LpdC, Mpt64, GroES, 325 \nFbpA and FpbB (Table S6, S8). Proteases were also enriched within the culture filtrates of acid 326 \nstressed M. tuberculosis S169 and included proteases HtrA1, PepA, PepD, Rv3671c, Clp1, Clp2, 327 \nMycP3 and Rv2672 (Table S6, S9).  328 \nDiscussion 329 \nPhagosome acidification is an environmental stress faced by M. tuberculosis during host infection 330 \n(53). In this study, we took advantage of a low pH stress model to trigger the formation of a M. 331 \ntuberculosis VBNR subpopulation (29). To increase the probability of identifying VBNR secreted 332 \nproteins, we made use of a clinical isolate, M. tuberculosis S169, which we previously showed to 333 \nform high proportions of VBNR bacteria (7,27). The fluorescence dilution replication plasmid 334 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n13 \n \nenabled the tracking of bacterial replication in response to low pH stress (Figure 1A) (28,29). 335 \nFollowing acid-stress for 48h at pH 4.5 (Figure 1B), imaging flow cytometry confirmed the 336 \nformation of a large VBNR subpopulation of M. tuberculosis S169 (Figure 1C).  337 \nProteomic characterization of the cell lysate revealed the differential abundance of 346 proteins in 338 \nresponse to acid-stress (Figure 2A, Table S1). Of the 77 proteins significantly more abundant 339 \n(adjusted p-value <0,05, fold change >2) in the cell lysates of acid stressed M. tuberculosis S169, no 340 \nenriched pathways were identified using GO enrichment analysis. However, in agreement with 341 \nprevious reports, the TcrXY two-component system components were more abundant in acid-342 \nstressed M. tuberculosis S169, with the response regulator TcrX significantly more abundant (Table 343 \nS1) (47). TcrX is required for M. tuberculosis  survival during chronic infection (47). Similarly, the 344 \nacid stress-induced methyltransferases Rv1403c and Rv1405c were also significantly more abundant 345 \nin acid stressed M. tuberculosis  S169 (Table S1), as previously reported for M. tuberculosis  346 \n(49,50,54). Interestingly, Rv1405c has also been reported to be upregulated during the enduring 347 \nhypoxic response and nitrosative stress (55,56). Even though Rv1405c is not essential for in vitro 348 \nsurvival, it is required for survival in C57BL/6J mice (57,58). The role of the Rv1405c 349 \nmethyltransferase during infection remains unknown.  350 \nTwo-component systems are required by the bacteria to respond to environmental changes. The PhoP 351 \ncomponent from the PhoPR two-component system is known to positively regulate the aprABC  352 \noperon in response to acidic pH (59–61). In this study, the AprA protein was only identified in acid 353 \nstressed M. tuberculosis S169 (Table S1, S3) and AprB and AprC proteins were not detected (Table 354 \nS1). Interestingly, despite detection of AprA in VBNR enriched cultures, PhoPR components were 355 \nfound to be less abundant in the cell lysates of acid stressed bacteria (Table S1). The two-component 356 \nsystem, KdpD/KdpE, has been suggested to play a role in the evasion of phagocytic killing and 357 \nenabling bacterial persistence (62). In this study, KdpA, KdpB and KdpD were all significantly more 358 \nabundant in the cell lysates of VBNR-enriched cultures (Table S1). KdpE was found to be more 359 \nabundant, but not significantly (Table S1). Interestingly, KdpA has been suggested to be required for 360 \nATP homeostasis and persister formation in Mycobacterium marinum (63).  361 \nIn response to acid stress, 269 cell lysate proteins were significantly less abundant (adjusted p-value 362 \n<0,05, fold change >2) than in the of actively replicating M. tuberculosis S169 (Table S1). Gene 363 \nOntology enrichments revealed a lower abundance of universal stress proteins (Figure 2B, Table S4), 364 \nincluding components from the two-component system DosR, also known as the dormancy response 365 \nregulon. DosR is known to be upregulated in response to hypoxia, starvation and low pH (29,51,64). 366 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n14 \n \nInterestingly, in this study, 34 components of the DosR regulon were significantly less abundant in 367 \nthe acid-stressed M. tuberculosis S169 (Table 1). These results contrast with some previous reports, 368 \nwhich largely show an upregulation of DosR in response to environmental stress. However, a lower 369 \ninduction for DosR in response to low pH has been reported in comparison to hypoxia, starvation 370 \nand stationary phase growth (51). In agreement with our results, DosR components Rv0080, NarX, 371 \nRv2030c, and Rv1813c have previously been shown to be downregulated in response to acid stress 372 \n(51). The DosR regulon is largely down regulated in a M. tuberculosis pellicle biofilm model (65). 373 \nMore recently, another pellicle biofilm study showed the down regulation of DosR genes in five of 374 \nthe six M. tuberculosis lineage 4 clinical isolates studied (66). The clinical isolate investigated in this 375 \nstudy, M. tuberculosis S169, belongs to lineage 4 (7). Interestingly, M. tuberculosis H37Rv, in which 376 \nthe DosR regulon is upregulated in response to low pH, also belongs to lineage 4 (29). These 377 \nfindings highlight the need to investigate the response of M. tuberculosis clinical isolates to 378 \nphysiologically relevant stress conditions for a more comprehensive understanding of the 379 \nmycobacterial stress response.  380 \nRocA, EspA, EspC, Rv2390c, and PE34 were significantly more abundant in the cell lysates of acid 381 \nstressed M. tuberculosis S169, as previously reported (Table S1) (67). ESX-1 is important for M. 382 \ntuberculosis virulence and EspA and EspC are ESX-1 secretion associated proteins (68–71). Several 383 \nother ESX-1 proteins were more abundant in the cell lysates of acid stressed cultures (Table S1) (72–384 \n74). Despite the increased abundance of EspA, EspB, EspD in acid stressed cell lysates, these 385 \nproteins were not detected in the culture filtrates of acid stressed M. tuberculosis S169 (Table S6). 386 \nEspF, EspC, EspH, EspR, and EspK proteins were present in the culture filtrates of actively 387 \nreplicating M. tuberculosis S169 (Table S5). In agreement with previous reports, Rv0516c, LipL, and 388 \nPPE59 were less abundant in response to acid stress (67). Interestingly, PPE22 has not previously 389 \nbeen reported to be upregulated in response to acid stress, however, in this study PPE22 was 390 \nsignificantly more abundant in the cell lysates of acid stressed M. tuberculosis S169 (Table S1) (67). 391 \nMoreso, PPE22 was only identified in the culture filtrates of acid stressed cultures (Table S6). PPE22 392 \nhas been previously been detected in guinea pig lungs at 30 days post infection and more recently 393 \nhas been shown to induce a protective immune response in BALB/c mice, showing promise as a 394 \nvaccine development candidate (75,76). 395 \nSeveral cell division proteins were less abundant in the cell lysates of VBNR-enriched cultures 396 \nincluding WhiB2, MtrA, SepF, FtsZ, FtsK, FtsQ, FtsW, FtsE, CwsA, and CrgA (Table S1). We also 397 \ndetected a lower abundance of DNA replication and repair proteins, including ImuA, RecA, RecR, 398 \nRecN, DnaB, and Rv1277 (Table S1). The downregulation of DNA replication and repair proteins 399 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n15 \n \nand cell division protein aligns with reduced bacterial replication, as observed with the fluorescence 400 \ndilution experiments (Figure 1). Low pH stress induced the formation of a VBNR subpopulation 401 \n(Figure 1C). Interestingly, resuscitation-promoting factors (Rpf) RipA, RipB, and RpfC were 402 \nsignificantly less abundant in the cell lysates of VBNR enriched M. tuberculosis  S169 cultures 403 \n(Table S1). RipB was detected in the culture filtrates of both actively replicating and VBNR enriched 404 \ncultures, but at a significantly lower abundance in VBNR enriched culture filtrates (Table S5-6). 405 \nOther Rpf proteins were identified in the culture filtrates of both test conditions, but with no 406 \nsignificant difference in abundance (Table S9). Rpf muralytic enzymes stimulate growth of dormant 407 \nM. tuberculosis and the loss of Rpfs results in an impaired ability of M. tuberculosis to resuscitate 408 \nfrom a non-culturable state (77).  409 \nCulture supernatants have low protein concentrations, often resulting in the need to pool culture 410 \nsupernatants from multiple cultures to a obtain enough protein. This practice increases the possibility 411 \nof introducing inter-culture variation. To overcome this limitation, we applied a protein aggregation 412 \ncapture approach to study the culture supernatant of a single bacterial culture per replicate 413 \nexperiment. A single culture has the benefit of reducing time, cost, and biohazardous risk in addition 414 \nto limiting technical and biological variation from multiple cultures. Applying this approach, we 415 \nshowed that the culture filtrates of actively replicating M. tuberculosis S169 contained 274 proteins 416 \ncompared to the 128 proteins identified in the culture filtrates of VBNR enriched M. tuberculosis. 417 \nGO pathway enrichment analysis confirmed the enrichment of extracellular region and secreted 418 \npathways (Figure 4). Zymogen binding, lipoprotein, protein folding, and protease pathways were 419 \nenriched from proteins identified in the extracellular fraction of low pH stressed M. tuberculosis 420 \nS169 (Figure 4B). Zymogens are the inactive precursors of enzymes which get converted to active 421 \nforms by proteolysis. Lipoproteins have been implicated in M. tuberculosis virulence and immune 422 \nmodulation (78). Other enriched pathways included the external encapsulating structure, cell 423 \nperiphery, and plasma membrane which may be the result of culturing M. tuberculosis S169 in media 424 \ncontaining the detergent Tween-80 to prevent bacterial clumping. The inclusion of Tween-80 in M. 425 \ntuberculosis culture media has been speculated to result in the solubilization of lipids and the 426 \nshedding of surface adhered molecules (79,80). As indicated by the pathway enrichment analysis, 427 \ncytosolic proteins including RNA polymerase subunits were identified in actively replicating  M. 428 \ntuberculosis S169 culture filtrates (Table S5). Small ribosomal subunits were also identified in the 429 \nculture filtrates of both actively replicating and VBNR enriched M. tuberculosis S169 cultures (Table 430 \nS5 and S6). The identification of these proteins outside the cell may suggest some cell lysis occurred.  431 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n16 \n \nThe culture filtrates of VBNR enriched M. tuberculosis  S169 cultures contained 45 proteins not 432 \nidentified in the extracellular fraction of actively replicating bacteria (Table S6). These proteins and 433 \nthe 83 significantly differentially abundant proteins (40 more abundant and 43 less abundant) 434 \nidentified in the culture filtrates of acid stressed M. tuberculosis S169 suggest that M. tuberculosis 435 \nmay secrete a different subset of proteins in response to low pH stress. The VBNR subpopulation 436 \nonly accounted for 17.5% (+/- 4.5) of the bacterial population investigated at pH 4.5, however, we 437 \nspeculate that VBNR protein secretion contributed to the differences observed in the culture filtrates 438 \nbetween pH 6.5 and pH 4.5 cultures. Although not investigated in this study, differences in the 439 \nproteins found in the extracellular region of M. tuberculosis  S169, may result in a different immune 440 \nresponse during infection. Culture filtrates from VBNR enriched cultures included proteins from 441 \nToxin-antitoxin (TA) systems, VapC51 and VapB10 (Table S6). TA systems have been implicated in 442 \nthe adaptation to environmental stress and bacterial persistence. Type II TA systems are highly 443 \nabundant in M. tuberculosis  genomes (10,81). Interestingly, the chorismate mutase Rv1885c was 444 \nonly identified in the secreted fraction of VBNR enriched cultures, and was recently suggested 445 \ncontribute to Mycobacterium bovis BCG pathogenesis by inhibiting mitochondria-mediated cell 446 \ndeath of macrophages (82). Immunogenic proteins more abundant in the culture filtrates of VBNR 447 \nenriched cultures included FbpA (Mpt44), Mpt53, Mpt64 and Mpt63 (Table S6).   448 \nIn this study we investigated the cellular proteome and the extracellular region of a clinical isolate 449 \nwith an increased propensity to form VBNR bacteria in response to low pH stress (7). We 450 \nacknowledge that our study was limited by only investigating a single clinical isolate, however, this 451 \nisolate was chosen to increase the likelihood of identifying changes in the proteome because of its 452 \nincreased propensity to form VBNR bacteria. We demonstrated that this clinical isolate did form a 453 \nviable but non-replicating population in response to in vitro low pH stress. Cell lysate proteomics 454 \nrevealed increased abundance of known acid stress proteins, however, in contrast to what has been 455 \npublished previously, several proteins of the DosR response regulon were significantly less abundant 456 \nin low pH stressed M. tuberculosis S169. This study highlights the need to investigate the cellular 457 \nresponse of clinical isolates, specifically clinical isolates obtained from individuals with 458 \nunfavourable outcomes, to improve our understanding of factors which may contribute to treatment 459 \nfailure. Using our culture filtrate mass spectrometry approach, we demonstrated that the culture 460 \nfiltrate composition of actively replicating and low pH stressed VBNR enriched cultures had 461 \ndifferent compositions. While we cannot definitively demonstrate secretion of proteins by VBNR 462 \nbacteria, several proteins identified in the culture filtrates of VBNR enriched cultures have 463 \nimplicated roles in bacterial persistence. Importantly, the culture filtrate approached used in this 464 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n17 \n \nstudy has the potential to be used not only to investigate M. tuberculosis  extracellular fractions but 465 \ncan be adapted to study extracellular proteins in other bacteria.  466 \n 467 \nDeclarations 468 \nEthics approval statement 469 \nEthics approval was obtained from the Human Research Ethics Committee (N10/01/013) and the 470 \nBiological and Environmental Safety Committee (BES-2023-13049) at Stellenbosch University.  471 \nConsent for publication 472 \nNot applicable. 473 \nAvailability of data and materials 474 \nImaging flow cytometry data are available from the corresponding author upon request. Mass 475 \nspectrometry proteomics data are available from the ProteomeXchange Consortium via the PRIDE 476 \npartner repository with the identifiers PXD068623 and PXD068720 (83).  477 \nCompeting interests 478 \nAuthors declare that the research reported in this manuscript was completed in the absence of any 479 \ncommercial or financial relationships which could constitute a potential conflict of interest. 480 \nFunding 481 \nThis research was supported by the VALIDATE Network which was funded by Gates Foundation 482 \n(INV-031830) and the South African government through the National Research Foundation of 483 \nSouth Africa (NRF) and the South African Medical Research Council (SAMRC). NK acknowledges 484 \nresearch and salary support from the VALIDATE Network, which was funded by the Gates 485 \nFoundation (INV-031830). SS is funded by the South African Research Chairs Initiative of the 486 \nDepartment of Science and Technology and National Research Foundation (NRF) of South Africa, 487 \naward number UID 86539.  488 \nThe authors are all affiliated with the with the DSI-NRF Centre of Excellence for Biomedical 489 \nTuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; 490 \nDivision of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, 491 \nStellenbosch University, Cape Town. 492 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n18 \n \nAuthors contributions 493 \nNK, JC, JM, and SS assisted with experimental design and conceptualization. NK and JC performed 494 \nthe experimental work and NK analyzed the results. NK drafted the manuscript, tables, and figures. 495 \nAll authors contributed to this manuscript and approved the submitted version. 496 \nAcknowledgements 497 \nWe acknowledge Maré Volk from the Central Analytical Facilities at Stellenbosch University for 498 \ntechnical assistance for mass spectrometry. 499 \nFigure 1A was created in BioRender. Sampson, S. (2025) https://BioRender.com/149jzej. 500 \nFigure 1B was created in BioRender. Sampson, S. (2025) https://BioRender.com/fyvj3w9.  501 \n 502 \nReferences 503 \n1. Gengenbacher M, Kaufmann SHE. 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It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n25 \n \nFigure 1. Acid stress promotes the formation of viable but non-replicating M. tuberculosis. A. 728 \nM. tuberculosis ::pTiGc will constitutively express a GFP (cell viability marker) and express 729 \nTurboFP635 when cultured with theophylline (inducer). Upon removal of theophylline, VBNR or 730 \nslowly replicating M. tuberculosis can be identified through retention of the red fluorescent signal. B. 731 \nM. tuberculosis S169::pTiGc was cultured to an OD 600 ~ 1 prior to transferring into either pH6,5 or 732 \npH4,5 media for 48 hours. Aliquots of cultures were collected before and after stress for imaging 733 \nflow cytometry. Cell pellets were collected for cell lysate proteomics and culture supernatants were 734 \ncollected for culture filtrate proteomics. C. M. tuberculosis  S169::pTiGc was cultured with 735 \ntheophylline and a high intensity of red fluorescence was detected (orange). Following the removal 736 \nof the inducer, actively replicating (pH6,5 cultures) bacteria had a reduction in red fluorescence 737 \n(green), however, pH stressed cultures retained a high red fluorescence intensity (red). Created with 738 \nBioRender.com. 739 \nFigure 2. Proteins identified in the cell lysates of actively replicating and VBNR enriched cell 740 \nlysates. A. Volcano plot representing differential protein abundance for M. tuberculosis S169 cell 741 \nlysate proteins.  Volcano plot generated by FragPipe-Analyst using protein abundance data for 742 \nproteins identified in the cell lysates of actively replicating and acid stressed M. tuberculosis S169. 743 \nFollowing the removal of contaminant proteins and proteins only identified in one biological 744 \nreplicate, 77 proteins were more abundant, and 269 proteins were less abundant in pH 4.5 cell lysates 745 \nwhen compared to pH 6.5 cell lysates (adjusted p-value <0,05, fold change >2). B. Gene ontology 746 \nenrichment of proteins significantly less abundant in the cell lysates of acid stressed M. tuberculosis 747 \nS169. GO enrichment analysis revealed that GO terms associated with universal stress proteins were 748 \nenriched in the 269 proteins found to be significantly less abundant in the cell lysates of acid stressed 749 \nM. tuberculosis S169 when compared to that of actively replicating M. tuberculosis S169. 750 \nFigure 3. Identification of culture filtrate proteins from pH 6.5 and pH 4.5 cultures.  The 751 \ndiagram demonstrates the data analysis of DDA mass spectrometry data for M.  tuberculosis S169 752 \nculture filtrates. Following automated database searching, 461 protein groups were identified in at 753 \nleast two of the three biological replicate experiments in culture filtrate recovered from pH 6.5 and 754 \npH 4.5 cultures. Of these, 192 protein groups were only identified in pH 6.5 cultures filtrates and 45 755 \nproteins were only identified in pH 4.5 culture filtrates. For proteins identified under both test 756 \nconditions, a paired student t-test with a Benjamini-Hochberg correction of 0,05 was used to identify 757 \n83 differentially abundant proteins (log2 FC >/<0,05). In total we identified 275 protein groups in the 758 \nculture filtrates of pH 6.5 culture and 128 protein groups in the culture filtrates of pH 4.5 cultures. 759 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n26 \n \nFigure 4. Enriched pathways of proteins identified in the culture filtrates of actively replicating 760 \nand acid stressed M. tuberculosis  S169. GO enrichment analysis confirmed the enrichment of 761 \nproteins from the extracellular region through enrichment of pathways secreted and extracellular 762 \nregion. A. GO enrichment analysis of proteins from actively replicating M. tuberculosis S169 763 \nrevealed the enrichment of pathways for carbon metabolism, carboxylic acid metabolism, and 764 \noxoacid metabolic process. B. Proteins identified in the extracellular fraction of acid-stressed M. 765 \ntuberculosis S169 revealed the enrichment of pathways for protein folding, lipoprotein, zymogen and 766 \nenzyme binding, and protease. 767 \n  768 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 3, 2025. ; https://doi.org/10.1101/2025.10.03.680234doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}