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[Preprint]. 2024 Sep 13:rs.3.rs-4999164.
doi: 10.21203/rs.3.rs-4999164/v1.

Trehalose catalytic shift is an intrinsic factor in Mycobacterium tuberculosis that enhances phenotypic heterogeneity and multidrug resistance

Hyungjin Eoh  1 , Jae Jin Lee  1 , Daniel Swanson  2 , Sun-Kyung Lee  3 , Stephanie Dihardjo  1 , Gi Yong Lee  1 , Gelle Sree  1 , Emily Maskill  2 , Zachary Taylor  4 , Michael Van Nieuwenhze  5 , Abhyudai Singh  6 , Jong-Seok Lee  7 , Seok-Yong Eum  3 , Sang-Nae Cho  8 , Benjamin Swarts  2
Affiliations

Trehalose catalytic shift is an intrinsic factor in Mycobacterium tuberculosis that enhances phenotypic heterogeneity and multidrug resistance

Hyungjin Eoh et al. Res Sq. .

Abstract

Drug-resistance (DR) in many bacterial pathogens often arises from the repetitive formation of drug-tolerant bacilli, known as persisters. However, it is unclear whether Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), undergoes a similar phenotypic transition. Recent metabolomics studies have identified that a change in trehalose metabolism is necessary for Mtb to develop persisters and plays a crucial role in metabolic networks of DR-TB strains. The present study used Mtb mutants lacking the trehalose catalytic shift and showed that the mutants exhibited a significantly lower frequency of the emergence of DR mutants compared to wildtype, due to reduced persister formation. The trehalose catalytic shift enables Mtb persisters to survive under bactericidal antibiotics by increasing metabolic heterogeneity and drug tolerance, ultimately leading to development of DR. Intriguingly, rifampicin (RIF)-resistant bacilli exhibit cross-resistance to a second antibiotic, due to a high trehalose catalytic shift activity. This phenomenon explains how the development of multidrug resistance (MDR) is facilitated by the acquisition of RIF resistance. In this context, the heightened risk of MDR-TB in the lineage 4 HN878 W-Beijing strain can be attributed to its greater trehalose catalytic shift. Genetic and pharmacological inactivation of the trehalose catalytic shift significantly reduced persister formation, subsequently decreasing the incidence of MDR-TB in HN878 W-Beijing strain. Collectively, the trehalose catalytic shift serves as an intrinsic factor of Mtb responsible for persister formation, cross-resistance to multiple antibiotics, and the emergence of MDR-TB. This study aids in the discovery of new TB therapeutics by targeting the trehalose catalytic shift of Mtb.

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Figures

Figure 1
Figure 1. Metabolic networks of DS-TB and DR-TB clinical isolates altered by trehalose supplementation.
(A) 2D (left panel) and 3D (right panel) principal component analysis (PCA) of the metabolome profiles of DS- (red), RSR- (green), MDR- (blue), XDR- (magenta), and TDR-TB (light blue) clinical isolates cultured in m7H9 containing 20 mM trehalose. (B) Targeted metabolomics analysis focusing on intermediates in trehalose metabolism, glycolysis, and the pentose phosphate pathway. PEP, phosphoenolpyruvate. Data points represent the average of 15 biological replicates ± s.e.m. **, P < 0.05; ns, not significant by Student’s t-test. (C) qRT-PCR analysis of treSin TB clinical isolates. **P < 0.01, as determined by Student’s t-test.
Figure 2
Figure 2. Trehalose catalytic shift as an adaptive strategy for the emergence of drug-resistant mycobacterial mutants.
(A) The rates at which the indicated mycobacterial strains acquired resistance to RIF (left panel) or INH (right panel) per generation were measured using the classical fluctuation assay. All values represent the average of 25 biological replicates ± s.e.m. *, P < 0.01 as determined by Student’s t test. (B) Colony formation in a spot assay. Ten colonies from RIF-resistant bacilli obtained from (A, left panel) were spotted on m7H10 containing either no RIF or 25 μg/mL RIF. WT, naïve drug-sensitive M. smegmatis bacilli. (C) Coculture of wildtype M. smegmatis expressing green fluorescence protein (GFP) and ItreSSM expressing red fluorescence protein (RFP) was subjected to intermittent exposure to RIF over a total of 5 cycles, referred to as G0-G5 cultures. The relative enrichment of wildtype and ItreSSM in G0 to G5 cultures was calculated via flow cytometry and is represented as a percentage. Gray bar represents ItreSSM; black bar represents wildtype.
Figure 3
Figure 3. Biochemical and metabolic properties of RIF-resistant M. smegmatis (FluxRIF) bacilli.
(A) Targeted metabolomics profiles. The analysis focused on intermediates in trehalose metabolism, glycolysis, and the pentose phosphate pathway in naïve M. smegmatis bacilli and ten selected FluxRIF bacilli. (B) Fold change of treS mRNA expression of naïve M. smegmatis and FluxRIF bacilli under RIF treatment conditions relative to untreated conditions. (C) Membrane potential (ΔΨm) of naïve M. smegmatis and FluxRIF bacilli was monitored after treatment with 30 μg/mL RIF by flow cytometry. The percentage of bacilli exhibiting high and low ΔΨm of each strain was calculated relative to the untreated condition. (D) Intrabacterial ATP concentration in FluxRIF bacilli was measured, and its abundance relative to naïve bacilli is represented as a fold change. (E) Relative RIF permeability and EtBr permeability kinetics of naïve M. smegmatis or FluxRIF bacilli were assessed. All values represent the average of biological replicates ± s.e.m. *, P < 0.05; **, P<0.01; ****, P<0.001; ns, not significant by Student’s t-test.
Figure 4
Figure 4. Enhanced metabolic heterogeneity of mycobacterial cells due to trehalose catalytic shift activity.
RMR-tre fluorescence dye labeling pattern are shown for (A) wildtype M. smegmatis, (B) ItreSSM, and (C) pTreSSM (M. smegmatis overexpressing TreS) following treatment with a sublethal dose of RIF. Bacilli located in the R1 area are defined as the RMR-trehigh population. Additionally, the RMR-tre fluorescence dye labeling patterns for (D) a selected FluxRIF bacillus and (E) its CRISPRi treS knockdown strain after ATc treatment was depicted.
Figure 5
Figure 5. Mathematical modeling of the role of trehalose catalytic shift in the emergence of drug tolerance and drug resistance in Mtb.
(A) The schematic diagram illustrates the phenotypic transitioning model, where drug-sensitive mycobacterial bacilli (gray) evolve into drug-resistant bacilli (black) via repetitively forming drug-tolerant bacilli (red). In this model, mycobacterial bacilli can reversibly switch between drug-sensitive and drug-tolerant states. Once a bacillus becomes drug-tolerant, it remains in that state for multiple generations before reverting to a drug-sensitive state. Following prolonged antibiotic exposure, each drug-tolerant bacillus irreversibly achieves permanent drug resistance. To assess the impact of the trehalose-catalytic shift on the frequency of each step in phenotypic transitioning, wildtype M. smegmatis and ItreSSM were utilized in a fluctuation assay as depicted in Fig. S6A. (B) The rates forming drug-resistant mutants in wildtype and ItreSSM against RIF were calculated using the classical fluctuation assay and the Lea-Coulson method (m/Nt where m is the number of resistant colonies and Nt is the total input).
Figure 6
Figure 6. The role of trehalose catalytic shift in the emergence of antibiotic cross-resistance.
(A) IC50 values for INH (left panel) and BDQ (right panel) were assessed in naïve M. smegmatis, FluxRIF, and ItreSFlux bacilli, both with and without ATc treatment. (B) A spot assay was performed on m7H10 containing 10X MIC INH or BDQ, using naïve M. smegmatis, FluxRIF, and ItreSFlux bacilli. (C) IC50 values for BDQ were determined for naïve M. smegmatis and two selected FluxINH bacilli. (D) A spot assay on m7H10 containing 10X MIC of BDQ (left panel) was conducted with naïve M. smegmatis and FluxINH bacilli. The right panel displayed the average diameters and the standard deviation of colonies formed on m7H10 containing 10X MIC BDQ.
Figure 7
Figure 7. Characterization of HN878’s trehalose catalytic shift activity.
(A) The expression of treS mRNA in HN878 and lineage 4 strains (e.g., H37Rv, Erdman, and CDC1551) was measured before and after treatment with RIF. The closed black circles represent the changes in mRNA levels following RIF treatment, with fold changes depicted relative to untreated counterparts. (B) Growth kinetics of HN878 and lineage 4 strains on m7H10 containing trehalose as the sole carbon source were shown. The impact of ValA on the growth of HN878 is also illustrated. All values represent the average of triplicates ± s.e.m. **, P<0.01 by Student’s t-test. (C) RIF treatment induced changes in the levels of trehalose, glucose 6P, and pentose 5P in HN878 and lineage 4 strains, relative to the no-treatment condition. (D) The effects of ValA (left panel) or CRISPRi-mediated treS inactivation (right panel) on the rates at which HN878 and lineage 4 clinical strains acquired RIF resistance per generation were measured using the classical fluctuation assay. (E) The impact of ValA on IC50 values of RIF against the indicated Mtb clinical strains is shown: HN878 (~62 ng/mL), ERD (~31 ng/mL), CDC1551 (~22 ng/mL) and HN878 treated with ValA (~22 ng/mL). All values are the average of biological triplicates ± s.e.m. *, P<0.05, **, P<0.01; ***, P<0.001; ns, not significant (by Student’s t-test).
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