Gallein and isoniazid act synergistically to attenuate Mycobacterium tuberculosis growth in human macrophages

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug resistance. Increased intracellular polyphosphate (polyP) in Mtb enhances resistance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb's ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB.


Introduction
Tuberculosis (TB) remains a significant global public health challenge, and in 2022, the causative bacterium Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths worldwide [1].During infection, Mtb encounters a variety of stressors originating from the host, and in response, employs adaptive physiological mechanisms to endure these stresses, promoting both resistance to antibiotics and the development of drug resistance [2][3][4][5][6].
These complexities not only demand prolonged treatment regimens but also contribute to the emergence of drug-resistant Mtb strains [1].Notably, resistance to the primary antibiotic, isoniazid (INH), is a prevalent form of monoresistance in Mtb, which is associated with treatment failures and the emergence of multidrug-resistant TB [1].Mtb's resistance to the majority of antibiotics is attributed to the thickening of the cell envelope [7,8], the activation of enzymes that modify antibiotics or their targets, and the action of efflux pumps [9,10].These mechanisms collectively reduce the efficacy of antibiotics.Polyphosphate (polyP) is a chain of phosphate residues and is present in all kingdoms of life [11].PolyP metabolism has been linked to the virulence of pathogens such as Mtb, Burkholderia mallei, Pseudomonas aeruginosa, Salmonella enterica, and Shigella flexneri [12][13][14][15].A highly conserved bacterial enzyme, polyphosphate kinase (PPK), synthesizes polyP from ATP, while polyP levels are regulated by the action of exopolyphosphatase (PPX), an enzyme that removes terminal phosphate residues from a polyP chain [16].The Mtb genome encodes two PPKs, PPK1 (Rv2984) and PPK2 (Rv3232c), as well as two PPXs, PPX1 (Rv0496) and PPX2 (Rv1026) [17].Pathogenic bacteria lacking PPK or having reduced PPK levels exhibit defects in stress response, quorum sensing, growth, survival, and virulence [13,[18][19][20][21][22][23][24].For instance, intracellular polyP is necessary for the survival of Mtb in host cells [14,22,25,26], and deletion of PPK1 in M. smegmatis attenuates the survival of ingested M. smegmatis in human macrophages [27].
Conversely, increased intracellular polyP in Mtb causes increased resistance to antibiotics [14,26,[28][29][30].In addition to intracellular polyP, bacteria also have extracellular polyP.The pathogenic bacterium Neisseria gonorrhoeae has polyP in its capsule, and the polyP potentiates resistance to antimicrobials [23,24].We observed that both Mtb and M. smegmatis accumulate extracellular polyP [27].Treatment of Mtb-infected macrophages with a polyP-degrading recombinant exopolyphosphatase (ScPPX) reduced the Mtb burden in macrophages, suggesting that both intracellular and extracellular polyP potentiate the survival of Mtb in host cells [27].
Given the absence of PPK enzymes in humans [31], bacterial PPKs could serve as potential targets for antituberculosis therapeutics.The small molecule gallein [32] is a broad-spectrum PPK inhibitor [33,34], and in this report, we find that gallein strongly potentiates the ability of INH to inhibit Mtb growth alone and in human macrophages.

Results
Gallein enhances the INH-mediated inhibition of Mtb growth PPK1 and PPK2, enzymes which synthesize polyP, are both necessary for Mtb viability [21,35].Ellagic acid derivatives from a medicinal plant inhibit PPK1 in Pseudomonas aeruginosa [36].Gallein, a small molecule with similarity to ellagic acid, was identified as a potent inhibitor of both PPK1 and PPK2 in P. aeruginosa [33].Gallein also inhibits Gβγ subunit signaling in mammalian cells [37,38].To determine if gallein affects Mtb, cells were cultured in the presence of different concentrations of gallein.Gallein at 0.005 µM, 0.05 µM, or 0.5 µM did not significantly affect Mtb growth, as assessed by an increase in OD 600 values (Figures S1 A-C), while 5 µM gallein slightly slowed growth, and 50 µM gallein inhibited growth by approximately 80% (Figures 1 A and B).In the absence of gallein, 1 µg/ml INH caused a partial but not complete reduction in Mtb growth (Figure S1 D), and this effect was potentiated by 5 and 50 µM gallein (Figures 1 A and B).The addition of 0.005 µM, 0.05 µM, or 0.5 µM gallein did not significantly enhance the effect of 1 µg/ml INH (Figures S1 A-C).suggest that INH increases total, cell surface, and extracellular polyP, and that gallein blocks this effect except for total polyP at day 5.

Gallein prevents INH-induced thickening of the Mtb cell envelope
When exposed to INH, Mtb undergoes cell envelope thickening and reduces cell envelope permeability as an adaptive response to withstand INH [28].To investigate whether gallein can reverse the effects of INH on cell envelope morphology, Mtb was treated with 1 µg/ml INH and/or 5 µM gallein for 14 days, and the Mtb cell wall was visualized using transmission electron microscopy (TEM).As previously observed [28], INH increased Mtb cell envelope thickness (Figures 3 A and B).Gallein alone did not significantly affect cell envelope thickness.For cells exposed to both INH and gallein, the average cell envelope thickness was comparable to control cells, but there were two distinct populations of Mtb cells (Figures 3 A and B).For 43.0 ± 2.6% (mean ± SEM, n=3) of the Mtb in the presence of INH and gallein, there was a detectable cell envelope, while the remaining Mtb had no detectable cell envelope (Figures 3 A and B).These results suggest that in the absence of INH, gallein does not inhibit growth by affecting cell envelope thickness, and that the combination of INH and gallein causes the formation of two populations of cells, one with a detectable cell envelope (which for many of these cells is thicker than that of control cells), and one with a compromised cell envelope.

Gallein and INH work synergistically to downregulate key metabolic pathways
To evaluate the impact of gallein on Mtb metabolism, Mtb were exposed to 1 µg/ml INH and/or 5 µM gallein for 24 hours, then incubated with a viability dye for 12 hours, and the resulting fluorescence signal was measured.The conversion of the non-fluorescent dye resazurin to the fluorescent resorufin product serves as a measure of metabolism [43,44], and is also used to assess the susceptibility of Mtb to antimicrobial compounds [45].In comparison to the control, Mtb exposed to INH showed increased metabolic activity (Figure S4A).Gallein decreased metabolic activity in the presence or absence of INH (Figure S4A).S4B).This observation suggests that the metabolic changes induced by gallein and INH were more substantial than those caused by gallein or INH alone (Figure S4B).A heatmap also indicated that although INH alone and gallein alone have some effect on metabolites, the combination of gallein and INH has a more profound effect (Figure 4A).As previously observed using a variety of INH concentrations [47], 1 µg/ ml INH significantly reduced the levels of nicotinamide adenine dinucleotide (NAD + ) (Figure 4B and Table 1).Other workers also found that 6.4 µg/ml INH alters levels of many metabolites [46].We observed that gallein significantly reduced the levels of deoxythymidine diphosphate (dTDP) and biotin (Figure 4C and Table 1).The combination of gallein and INH significantly reduced the levels of 37 metabolites (Figure 4D and Table 1) including NAD + and dTDP but not biotin (Figure 4E).These pathways are detailed in

Discussion
Bacteria resist antibiotics by activating drug efflux pumps and/or enzymes which modify the antibiotic or its target [7].Cellular metabolic rearrangements can also cause antibiotic resistance [46,[48][49][50][51][52].In this report, we show that the the effects of PPK deletion [33].Our findings suggest that gallein exhibits a greater effectiveness on Mtb compared to P. aeruginosa.Mtb can differentiate into heterogeneous populations, such as non-replicating persisters and growing bacteria with the capacity to become persisters [55].Mtb treated with INH causes a rapid killing of cells followed by a reduction in the killing rate as the number of persister cells increases [56,57].In Clostridiodes difficile, a gram-positive bacteria, some cells in a population stochastically become antibiotic tolerant persisters [58].C. difficile forms metabolically dormant spores that resist antibiotic pressure and persist in the host [59].Two distinct morphotypes of C.
difficile spores exist, one with thick outer surface layer and the other with thin outer surface layer

Cell culture
Human peripheral blood was collected from healthy volunteers who gave written consent, and with specific approval from the Texas A&M University human subjects institutional review board.Peripheral blood mononuclear cells (PBMCs) were purified as previously described [68]. The

Bacterial survival assay
To determine the effect of INH and/or Gallein on the survival of Mtb in human macrophages, human macrophages (from blood monocytes cultured with GM-CSF for 6 days were mixed with Mtb following [27], in the absence or in the presence of 1 µg/ml INH and/or 5 µM Gallein.At day 7, after isolating monocytes from donor blood, after removing loosely adhered cells as described above, 200 µL RBCSGLP (RBCSG containing 50 μg/mL leucine and 50 μg/mL pantothenate) were added to macrophages in each well in 96-well, tissue-culture-treated, polystyrene plates (# 353072, Corning) and incubated for 30 minutes at 37 °C.Meanwhile, 1 mL of Mtb from a log phase culture was washed twice with RBCSGLP without GM-CSF by centrifugation at 12,000 x g for 2 minutes in a microcentrifuge tube, resuspended in 1 mL of RBCSGLP, and the OD 600 of 100 µl of the culture in a well in a 96-well plates (# 353072 Corning) was measured as above.200 µl of RBCSGLP was used as a blank.The bacteria were diluted to an OD 600 of 0.5 (~10 7 Mtb/ mL) in RBCSGLP.Mtb (~1 µl) was added to macrophages in each well such that there were ~5 bacteria per macrophage, considering ~20% of the blood monocytes converted to macrophages in the presence of GM-CSF [75].The bacteria-macrophage co-culture plate was centrifuged at 500 x g for 3 minutes with a Multifuge X1R Refrigerated Centrifuge (Thermo Scientific, Waltham, MA) to synchronize phagocytosis of the bacteria, and incubated for 2 hours at 37 °C.The supernatant medium was removed by gentle pipetting and was discarded.
200 µL of PBS warmed to 37 °C was added to the co-culture in each well, cells were gently washed to remove un-ingested extracellular bacteria, the PBS was removed, and 200 µL of RBCSGLP with GMCSF in the absence or in the presence of 1 µg/ml INH and/or 5 µM gallein was added to the cells.After 2 hours, cells were washed twice with PBS as above.200 µL of RBCSGLP with GMCSF in the absence or in the presence of 1 µg/ml INH and/or 5 µM gallein was then added to the cells.After 4 and/or 48 hours of infection, macrophages were washed as above with PBS, the PBS was removed, and cells were lysed using 100 µL 0.1% Triton X-100 (Alfa Aesar, Ward Hill, MA) in PBS for 5 minutes at room temperature by gentle pipetting, and 10 µl and 100 µL of the lysates were plated onto agar plates (as described above for Mtb culture).The Mtb containing agar plates were incubated for 3 to 4 weeks or until the Mtb colonies appeared.Bacterial colonies obtained from plating 10 µl and 100 µl lysates were manually counted, the number of viable ingested bacterial colonies per 10 µl and 100 µl lysates was calculated, and the number of viable ingested bacteria colony forming units (cfu) per ml of lysate was then calculated, which corresponds to the number of viable ingested bacteria in ~2 x 10 5 macrophages.To calculate the percent of control, cfu/ml of the control was considered 100%.

PolyP assays, RNA extraction, and quantitative reverse transcription PCR
Log phase Mtb cultures were prepared similarly to the growth assays in 6-well, tissue culture-treated plates (# 353046, Corning), with the exception that 5 µM gallein and/or 1 µg/ml INH were used.Plates were incubated in a container humidified with wet paper towels at 37 °C for 24 hours.After incubation, 100 µl of cells were transferred to a 96-well, black/clear, tissueculture-treated, glass-bottom plate (# 353219, Corning) for imaging as described below.The remaining cells were transferred to a 15-ml conical tube, harvested by centrifugation at 4000 x g for 10 minutes, and 4.5 ml of the supernatant was transferred to a new 15-ml conical tube for extracellular polyP measurement.Cell pellets were further processed for RNA extraction, following the procedure outlined below.PolyP levels in the supernatant were assessed by adding 25 µg/ml of DAPI (Biolegend) (from a stock of 2 mg/ml) and measuring fluorescence at 415 nm excitation and 550 nm emission, as previously described [76].PolyP standards (Sodium Polyphosphates, Glassy, Spectrum, New Brunswick, NJ) at concentrations of 0, 0.5, 1, 10, 100, 200, and 500 µg/ml were prepared in 7H9S.The polyP content was normalized to the total protein content, which was determined from the cell lysates as described below.
For RNA extraction, cell pellets obtained from the treatments described above were resuspended in 250 μL of GITC lysis buffer (containing 4 M guanidine isothiocyanate and 50 mM Tris-HCl at pH 7).The cells were lysed by incubating at 95 °C for 10 minutes.Ten microliters of the lysates were used to determine the total protein content using a Bradford assay.The remaining lysates were used to extract RNA using an RNA extraction kit (Zymo Research, Irvine, CA).
Complementary DNA (cDNA) was synthesized from 2 µg of RNA using the Maxima H Minus First Strand cDNA Synthesis kit (Thermo Scientific).Quantitative PCR was performed using SYBR GreenER™ qPCR SuperMix Universal reagent (Thermo Scientific), following the manufacturer's instructions using a QuantStudio (TM) 6 Flex thermal cycler (Thermo Scientific).
The levels of Mtb's ppk1 and ppk2 mRNAs were determined using the gene-specific primers listed in Table 3.

Fluorescence microscopy
To determine the localization of polyP in Mtb, 100 µl of Mtb cells from the growth assay on Day 21 or the growth assay on Day 1, 5, or 14 were transferred to a 96-well, black/clear, tissueculture-treated, glass-bottom plate (# 353219, Corning) or cells smears were prepared on Superfrost micro glass slides (Cat#48311-703, VWR) as described previously [77].The cells were fixed with 4% (wt/vol) paraformaldehyde (Cat#19210, Electron Microscopy Sciences, Hatfield, PA) in PBS for 10 minutes.After fixation, the cells were washed two times with 300 µl of PBS, blocked with 1 mg/ml BSA (Thermo Scientific) in PBS, and then stained with 10 µg/ml of GFP-PPX (provided generously by Dr. Ursula Jacob from the University of Michigan) in PBS/0.1% Tween 20 (PBST; Fisher Scientific) [42].Following staining, the Mtb cells were washed three times with PBST, and 200 µl of PBS was added to the well, or coverslips were mounted on slides with smears with Vectashield hard set mounting medium (Cat# H-1800, Vector Labs, Burlingame, CA) and left to dry overnight in darkness.Images of Mtb were captured using a 100× oilimmersion objective on a Nikon Eclipse Ti2 (Nikon, Kyoto, Japan), and image deconvolution was performed using the Richardson-Lucy algorithm [78] in NIS-Elements AR software (Nikon).The integrated fluorescence density was measured in randomly selected individual cells manually using the freehand selection feature in Fiji (ImageJ) [79].

Transmission electron microscopy
Mtb cells treated with 5 µM gallein and/or 1 µg/ml INH for 14 days were prepared as described for the growth assays above.A volume of 100 µl of cells in 7H9S was fixed by adding an equal volume of 2× fixative, which contained 84 mM NaH 2 PO 4 , 68 mM NaOH, 4% paraformaldehyde (Cat#19210, Electron Microscopy Sciences), and 1% glutaraldehyde (Cat#0875, VWR).The samples were gently rocked for 1 hour and then stored at 4 ˚C.Sample preparation for TEM imaging was performed by the Texas A&M University Microscopy and Imaging Center Core Facility's staff (RRID: SCR_022128).Briefly, on the following day, the fixed samples were collected by centrifugation for 5 minutes at 14,000 × g.Subsequently, they were postfixed and stained for 2 hours with 1% osmium tetroxide in 0.05 M HEPES at pH 7.4.
The samples were then collected by centrifugation and washed with water five times, and dehydrated with acetone according to the following protocol: 15 minutes in 30%, 50%, 70%, and 90% acetone each, followed by three changes of 100% acetone, each lasting 30 minutes.During the final wash step, a minimal amount of acetone was retained, just enough to cover the pellets, to prevent rehydration of the samples.Subsequently, the samples were infiltrated with modified minutes at 200 W (vacuum at 20 inches Hg).This was followed by four cycles of 100% resin for 5 minutes each at 200 W (vacuum at 20 inches Hg).The resin was then removed, and the sample fragments were transferred to BEEM conical-tip capsules that were prefilled with a small amount of fresh resin.More resin was added to fill the capsules, and they were left to stand upright for 30 minutes to ensure that the samples sank to the bottom.The samples were polymerized at 65°C for 48 hours in an oven and then left at room temperature (RT) for an additional 24 hours before sectioning.Sections of 70 to 80 nm thickness were obtained using a Leica UC/FC7 ultramicrotome (Leica Microsystems), deposited onto 300-mesh copper grids, and stained with uranyl acetate-lead citrate.Grids were imaged using a JEOL 1200 EX TEM operating at 100 kV.Cell wall thickness was measured using ImageJ.

Metabolic activity assay
Cell proliferation and viability in Mtb can be assessed by incubating the cells with resazurin reagent for 6-12 hours, and monitoring the color change from blue to purple [80].Metabolically active cells transform the non-fluorescent blue dye (resazurin) into a fluorescent pink product (resorufin), while inactive cells rapidly lose their metabolic capacity and, consequently, do not generate a fluorescent signal [43].Mtb from the log phase culture were prepared as previously described for the proliferation assay, with 100 µl of cells being prepared per well in 96-well, tissue culture-treated plate (# 353072, Corning).Mtb was treated with 5 µM gallein and/or 1 µg/ml INH, and the plate containing the Mtb was incubated at 37 °C for 24 hours.Cells were then incubated with prewarmed Deep Blue Cell Viability resazurin dye (Biolegend) to a final concentration of 10% in each well for 12 hours [80].The fluorescence signal was then measured using a microplate reader following the manufacturer's protocol.

Metabolomics
Mtb cells from log-phase cultures were prepared as described for the growth assays, with the exception that 10 ml of culture was prepared.The Mtb cells were treated with 5 µM gallein and/or 1 µg/ml INH.After 24 hours, 10 ml of the culture was collected by centrifugation at 4000 x g for 10 minutes at 4 °C.The cells were then washed twice with 10 ml of chilled (0-4°C) phosphate-buffered saline (PBS) to prevent metabolite contamination from the culture media.The cells were resuspended in 10 ml of PBS [81], and 100 µl of this suspension was transferred to a 96-well plate to measure the OD 600 .The remaining cell suspension was centrifuged again at 4000 x g for 10 minutes at 4 °C to collect the cell pellets for metabolite extraction.After the final wash, excess PBS was carefully removed from all the samples.The day before the assay, 10 ml of extraction solvent (acetonitrile (BDH83639.400,VWR): methanol (BDH20864.400,VWR):water classes of metabolites in the samples using an untargeted metabolomics approach at the UTSW metabolomics core facility (https://www.utsouthwestern.edu/research/corefacilities/metabolomics/).Metabolites detected in at least three biological replicates were considered for further analysis.The peak area of metabolites was normalized to the total protein content determined from Mtb lysates, and pathway enrichment analysis was conducted using the online analytical tool Metaboanalyst (www.metaboanalyst.ca)and the BioCyc Database, employing the Mtb H37Rv reference genome [83].

Statistical analysis
Statistical analyses were performed using Prism 10 (GraphPad Software, Boston, MA) or Microsoft Excel.P < 0.05 was considered significant.ppk2 cDNA were quantified by quantitative PCR using gene-specific primers (Supplemental Table 1).The cDNA level from untreated Mtb (Control) was set to 1.All values are mean ± SEM of three independent experiments.Table S1: Oligonucleotides for quantitative real-time polymerase chain reaction (qPCR).
Infection of host cells with Mtb leads to a resistance of the Mtb to INH[4,40,41], and increased intracellular polyP in Mtb causes increased resistance to antibiotics[14,26,[28][29][30].To determine if exposure of Mtb to INH potentiates accumulation of polyP(28,29), Mtb cells were cultured in the presence of INH.After exposure to INH for 21 days, the Mtb cells were fixed without permeabilizing the cells and then stained with the GFP-tagged polyP binding domain of Escherichia coli PPX (GFP-PPX)[42].For unknown reasons, the cells showed a wide range of staining intensities (Figures2 A and B).INH concentrations from 0.1 to 100 µg/ml increased the average amount of cell-surface polyP (Figures2 A and B).A 1 day exposure of Mtb to 1 µg/ml INH also increased cell-surface and total cellular polyP (Figures2 C -E).We previously observed that Mtb cells accumulate extracellular polyP[27], and at 1 day INH also increased the accumulation of extracellular polyP (Figure2 F).At 5 and 14 days, 1 µg/ml INH significantly increased cell-surface, total cellular, and extracellular polyP (Figures2 G -L).At 14 days, Mtb controls had, using an arbitrary cutoff, 23 % of cells with low levels of cell surface polyP, cells treated with gallein in the presence or absence of INH had 43% of cells with low levels of cell surface polyP, but none of the cells treated with INH had low levels of cell surface polyP (FigureS2A).Together, these findings suggest that INH induces the accumulation of cellular, extracellular, and cell surface polyP.Gallein prevents the INH-induced accumulation of extracellular and cell surface polyPOne possible mechanism for the effect of gallein on Mtb growth is that gallein, by inhibiting PPKs, decreases polyP levels.At 1 day, in the absence of INH, gallein did not decrease cell surface polyP, but did block the INH-induced increase in cell surface polyP, bringing the cell surface polyP levels to levels comparable to control cells (Figure 2 D).Gallein decreased cellular polyP in both the absence and presence of INH (Figure 2 E).Gallein also decreased extracellular polyP in both the absence and presence of INH (Figure 2 F).INH and gallein did not significantly affect levels of Mtb ppk1 and ppk2 mRNAs (Figure S3), suggesting that the effects on polyP levels are not mediated by changes in the levels of these mRNAs.At 5 days, gallein significantly decreased cell surface polyP in both the absence or in the presence of INH (Figure 2 G).At 5 days, gallein decreased cellular polyP in the absence of INH (Figure 2 H).Gallein also decreased extracellular polyP in both the absence and presence of INH (Figure 2 I).In the presence of INH, gallein decreased extracellular polyP to levels comparable to control cells (Figure 2 I).At 14 days, gallein significantly decreased cell surface polyP in both the absence or in the presence of INH (Figure 2 J).At 14 days, gallein did not significantly decrease cellular polyP in the absence of INH (Figure 2 K), but significantly decreased cellular polyP in the presence of INH (Figure 2 K).Gallein decreased extracellular polyP in both the absence and presence of INH (Figure 2 L).At 14 days, in the absence or presence of INH, gallein decreased extracellular polyP to an undetectable level (Figure 2 L).At 14 days, in the presence of the combination of INH and gallein, cellular debris was visible, indicating cell death (Figure S2 B).For unknown reasons, in control cells, cellular and extracellular polyP levels tended to decrease with the age of the cultures.Together, these results Guanosine GO:0042453 -deoxyguanosine metabolic process guanine and guanosine salvage II 2-deoxy-D-ribose 5phosphate GO:0019692 -deoxyribose phosphate metabolic process 2'-deoxy-α-D-ribose 1-phosphate degradation UDP-α-D-glucose GO:0006011 -UDP-glucose metabolic process trehalose biosynthesis I glycerol 2-phosphate GO:0006072 -glycerol-3-phosphate metabolic process cytidine-5'-diphosphate-glycerol biosynthesis NAD+ GO:0019674 -NAD metabolic process NAD Metabolism dCDP GO:0046087 -cytidine metabolic process CDP--NADP metabolic process NAD(P)/NADPH interconversion 5-oxo-L-proline 5-oxo-L-proline metabolism 5-oxo-L-proline metabolism N-acetyl-L-glutamine N-acetyl-L-glutamine N-acetyl-L-glutamine dAMP GO:0046033 -AMP metabolic process purine deoxyribonucleosides salvage Methylmalonate semialdehyde 2-methyl-3-oxopropanoate methylmalonate semialdehyde an N-acyl-L-aspartate N-acyl-L-aspartate an N-acyl-L-aspartate INH + Gallein NADP+ GO:0006739 -NADP metabolic process NAD phosphorylation and dephosphorylation Adenosine 3',5'bisphosphate GO:0046031 -ADP metabolic process mycobacterial sulfolipid biosynthesis 2'-deoxycytidine GO:0047844 -deoxycytidine deaminase activity superpathway of pyrimidine deoxyribonucleosides degradation Trehalose GO:0005991 -trehalose metabolic process Trehalose Biosynthesis L-proline GO:0006560 -proline metabolic process L-proline Degradation D-ribose 5-phosphate GO:0043456 -regulation of pentosephosphate shunt pentose phosphate pathway (non-oxidative branch) I UMP GO:0046049 -UMP metabolic process UMP Biosynthesis Pyridoxine GO:0008614 -pyridoxine metabolic process vitamin B6 degradation I Metabolites were assigned Gene Ontology (GO) terms and classified into metabolic pathways.Table 2. Characterization of metabolites significantly altered in Mtb treated with the combination of INH and gallein compared to control.5-oxo-L-proline D-ribose 5-phosphate Fermentation 2-deoxy-D-ribose 5acid and Lipid Degradation 217 Metabolites were grouped based on their involvement in metabolic pathways.
antibiotic INH causes Mtb to increase the accumulation of cell surface, cellular, and extracellular polyP.Gallein, a bacterial PPK 1 and 2 inhibitor, inhibits Mtb cell surface, cellular, and extracellular polyP accumulation in both the presence and absence of INH, and in the presence of INH inhibits Mtb cell envelope formation in some but not all Mtb cells.Both INH and gallein have modest effects on metabolite levels, but the combination of INH and gallein strongly reduces levels of metabolites in several metabolic pathways.Possibly as a consequence of the effects of gallein on polyP levels, cell envelope formation, and metabolites, gallein inhibits Mtb growth in both in vitro culture and in human macrophages, and strongly potentiates INH inhibition of Mtb growth in in vitro culture and in human macrophages (Figure 5).We observed a significant increase in cellular, extracellular, and cell surface polyP in response to INH.Compared to log-phase cells, stationary phase Mtb have more polyP and are more resistant to killing by INH [27, 53], and capsular polyP accumulation protects N. gonorrhoeae from antibiotics [23, 24].Combining these observations, one possibility is that Mtb increase polyP in response to INH as a protective measure.The antibiotic rifampicin causes Mtb to thicken its capsular outer layer and increase the net negative charge of the cell surface to reduce rifampicin permeability [54].It is possible that the INH-induced increase in the accumulation of the highly negatively charged polyP on the cell surface might similarly increase the net negative charge of the cell surface, reducing permeability to INH.At 5 µM, gallein inhibits extracellular polyP levels in Mtb and inhibits INH-induced increases in cell surface and extracellular polyP.Gallein treatment of P. aeruginosa required concentrations exceeding 25 µM to reduce intracellular polyP accumulation, and 100 µM to mimic

[ 60 ,
61].Multi-drug resistant Mtb have thicker cell envelopes[62].INH caused many, but not all, Mtb cells to have thickened cell envelopes.In response to INH and gallein, Mtb formed two distinct populations of cells: one with cell envelope thicknesses comparable to INH-treated cells, and the other with no discernible cell envelope.One possibility is that under combined INH and gallein pressure, either stochastic differentiation or asymmetric cell division gives rise to ~43% of cells with an intact cell envelope that have the potential to become slow growing persisters, and ~ 57% of cells with compromised cell envelope that eventually die, and this might be the reason we observed no net increase in Mtb cell growth in the presence of 50 µM gallein and INH.For control Mtb cells, there was a large variation in cell surface polyP levels, and INH increased both the average cell surface polyP levels and cell envelope thickness.At day 14, although there was a clear bimodal distribution of cell envelope thicknesses in the presence of INH and gallein, there was no observable bimodal distribution of cell-surface polyP levels.This suggests that there is not a strict correlation between cell-surface polyP level and cell envelope thickness, and that the effect of combined INH and gallein on cell wall thicknesses may be due to effects on additional pathways in addition to cell surface polyP.Mtb uses the glucose disaccharide trehalose as a core component of cell surface glycolipids important for virulence, and during the transition into a non-replicating persister phase, it metabolizes trehalose to generate ATP[63][64][65].In Mtb exposed to the combination of INH and gallein, the significant reduction in levels of trehalose and components necessary for trehalose biosynthesis, such as UDP-α D-glucose[66] suggests that the combination of INH and gallein compromises trehalose-dependent cell-surface glycolipid, and thus cell envelope, formation as we observed under TEM, and by decreasing trehalose levels, reduces this source of energy to prevent persister formation.Gallein significantly reduced the levels of biotin.Mtb relies on biotin synthesis for its survival during infection, and the disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest[67].This suggests that gallein may inhibit Mtb growth by promoting cell death rather than merely inhibiting growth.The combination of gallein and INH resulted in decreased levels of metabolites associated with several key metabolic pathways.It is plausible that the synergistic action of INH and gallein on Mtb growth is not solely due to the reduced integrity of the cell envelope, but also to the disruption of other metabolic pathways.In conclusion, the combination of INH and gallein affects several aspects of Mtb physiology, and gallein thus potentiates INH antibiotic effects on Mtb.Because they are not present in mammalian cells [31], PPKs are attractive target for suppressing Mtb growth and elimination.

( 40 :
40:20)) was prepared and stored at -70°C.On the day of the experiment, heavy amino acid standards (Metabolomics amino acid mix standard, Cat# MSK-A2-1.2,Cambridge Isotope Laboratories, Tewksbury, MA) were added as a spike to the extraction solvent to 5 µM final concentration.To halt bacterial metabolism, the Mtb pellets were immediately suspended in 200 µl of spiked extraction solvent that had been pre-cooled on dry ice[46].Mtb disruption was achieved using a Mini-beadbeater-16 (BioSpec Products, Bartlesville, OK).The Mtb samples (200 µl) were placed in 2 ml Polypropylene Microvials (Cat#10832, BioSpec Products) containing approximately 70 µl of 0.1 mm Zirconia/Silica beads (Cat# 11079101z, BioSpec Products).Beadbeating was performed in three cycles of 1 minute at 3,450 oscillations/min, with 2 minutes of cooling on ice between cycles[82].Subsequently, the microvials containing the disrupted cells and zirconium beads were incubated at -20°C for 20 minutes.They were then clarified by centrifugation at 8,000 x g for 15 minutes at 4 degrees Celsius.The resulting supernatant was filtered through a 0.22 µm filter (Cat# UFC30GV25, Merck Millipore, Cork, IRL) into 0.2 ml glass Stepvial inserts (Cat# 200 238, ThermoScientific).These step vials were placed inside 0.25 ml polypropylene vials with polypropylene caps with PTFE/silicone septa (Cat# 200 410, ThermoScientific).The pellets containing beads were resuspended in 100 µl of Radioimmunoprecipitation assay buffer (RIPA) (Cat#89900, Thermo Scientific) containing 1X protease and phosphatase inhibitor cocktail (Cat#1861281, Thermo Scientific), incubated on ice for 15 minutes, clarified by centrifugation at 10,000 x g for 5 minutes at 4 o C, and 25 µl of the supernatant was used to determine the protein amount following the manufacturer's instructions using a Pierce BCA Protein Assay Kit (Cat#23225, Thermo Scientific).A vial containing a quality control mixture pool of 12.5 µl from each sample (150 µl total) was prepared along with the other samples.These vials were sealed with Parafilm and stored at -80 °C before the untargeted metabolomics analysis[81].Samples were analyzed with a Shimadzu high-performance liquid chromatography (HPLC) (Nexera X2 LC-30AD, Kyoto, Japan) coupled to a Sciex TripleTOF 6600 high-resolution mass spectrometer (HRMS) for the separation and detection of various

Figure 1 :
Figure 1: Gallein potentiates the ability of INH to inhibit Mtb growth both in in vitro culture and

Figure 5 :
Figure 5: INH and gallein synergistically alter cellular homeostasis.A) Mtb under no antibiotic

Figure S4 :
Figure S4: Gallein inhibits Mtb metabolic activity.(A) Mtb, treated without or with 1 µg/ml INH Mtb for 2 hours, and the non-ingested Mtb were removed.The macrophages were then incubated with gallein and/or INH, and at 4 and 48 hours after adding Mtb the macrophages were lysed with a detergent (0.1% Triton X-100) that does not kill Mtb, and the bacteria were plated and colonies were counted.At 48 but not 4 hours, INH decreased the viability of ingested Mtb (Figures1 C and D).At both times, gallein decreased ingested Mtb viability compared to control, and in the presence of INH significantly decreased the viability of ingested Mtb, with no detected surviving bacteria at 48 hours (Figures1 C and D).Together, these results [39]etermine if the reduced growth with INH and/or 5 and 50 µM gallein causes a permanent effect on Mtb, the day 14 cultures were washed and resuspended in medium without INH or gallein and the growth was monitored (FigureS1 E).Surprisingly, exposure to INH caused Mtb cells to grow faster and exposure to gallein caused a slight reduction in growth.Together, these data indicate that a 14 day exposure of Mtb to 1 µg/ml INH and 5 or 50 µM gallein is bacteriostatic but not bactericidal.In patients with tuberculosis, Mtb bacteria can be ingested by macrophages, and are able to survive inside the macrophages[39].To determine if gallein affects the ability of Mtb to survive in macrophages, human macrophages, derived from circulating monocytes from healthy donors, were incubated with suggest that for both free Mtb and Mtb in macrophages, gallein inhibits growth and enhances INH's ability to inhibit growth.

Table 2
and include nucleoside and nucleotide biosynthesis and degradation, carrier, cofactor, and vitamin biosynthesis, amino acid biosynthesis and degradation, carbohydrate biosynthesis and degradation, aminoacyl tRNA charging, cell structure biosynthesis, metabolic regulator biosynthesis, fermentation, and fatty acid and lipid degradation pathways.Gallein, INH, or the combination of gallein and INH, did not significantly upregulate any detectable metabolite.These data indicate that gallein and INH work synergistically to impact the majority of key metabolic pathways, contributing to the inhibition of Mtb growth.211

Table 1 . Metabolites significantly altered in Mtb treated with INH and/or gallein, as 212 compared to the control.
All cultures were incubated at 37 °C in a humidified incubator.These supplemented cultures are hereafter referred to as 7H9S or 7H10S.Mtb culture plate was prepared as described method.However, in this case, each well of a type 353046, 6 well, tissue culture-treated plate (Corning) contained a final OD 600 of 0.01 in 5 ml.Mtb was incubated with gallein at concentrations of 0.005, 0.05, 0.5, 5, or 50 µM and/or 1 µg/ml INH.A [73,74]ne (Lonza)), and where indicated containing 25 ng/mL human granulocyte-macrophage colony-stimulating factor (GM-CSF) ((# 572903, Biolegend, San Diego, CA) at 37 °C in a humidified chamber with 5% CO 2 in 96-well, tissue-culture-treated, polystyrene plates (type 353072, Corning) with 2 x 10 5 cells in 200 µL in each well.At day 7, loosely adhered cells were removed by gentle pipetting and removing the medium, and fresh RBCSG containing GM-CSF (as described above) was added to the cells to a final volume of 200 µL per well, and Mtb survival assays were performed as described below.The attenuated (mc-ΔleuDΔpanCD) Biosafety Level-2 strain of Mtb, which is a derivative of the H37Rv strain[69], was a gift from Dr. Jim Sacchettini, Texas A&M University, CollegeStation, TX and is referred to as Mtb in this paper.This strain was cultured according to the (Beantown Chemical, Hudson, NH) either in a rotator (10 rpm) or on plates with 7H10 agar (BD) and the above additives.obtainedfromlog-phaseliquidculture, as described above, were washed twice with 10 ml of 7H9S by centrifugation at 4000 x g for 10 minutes.The optical density at 600 nm (OD 600 ) was measured in a well of a 96-well, tissue-culture-treated, polystyrene plate (type 353072, Corning) at 600 nM with a Synergy Mx monochromator microplate reader (BioTek, Winooski, VT).The Mtb were then resuspended in an appropriate volume of 7H9S to achieve a final OD 600 of 1.Ten microliters of Mtb with an OD 600 of 1 were added to each well to reach a final OD 600 of 0.01 in 1 ml.The plates were subsequently incubated in a container with humidity provided by wet paper towels at 37 °C in a humidified incubator.On the 21st day, the Mtb culture was gently resuspended, and 100 µl of the cells were transferred to a 96-well, tissue culture-treated plate (# 353072, Corning).The OD 600 was measured using a microplate reader.Given that we observed complete inhibition of Mtb growth with 100 µg/ml INH in our experimental setup, we opted to utilize 1 µg/ml INH for all our assays.This choice aligns with previous reports from other research groups, which had documented the resistance of Mtb to concentrations of INH greater than 1 µg/ml[73,74].To investigate the impact of gallein (3',4',5',6'-Tetrahydroxyspiro[isobenzofuran-1(3H),9'-(9H)xanthen]-3-one) (Cat#3090, Tocris, Minneapolis, MN) and/or INH on Mtb growth, a To determine the viability of Mtb cells pre-exposed to INH and/or gallein for 14 days, 200 µl of cells from above experiments were washed twice with 1 ml of 7H9S, resuspended in 200 µl of 7H9S without INH and gallein 96-well plates ((# 353072, Corning), and the OD 600 of the cells was measured daily for 14 days, and the Mtb growth curves were generated as a percentage of the OD 600 on day 0.