Cpn60.1 (GroEL1) Contributes to Mycobacterial Crabtree Effect: Implications for Biofilm Formation

Abstract

Biofilm formation is a survival strategy for microorganisms facing a hostile environment. Under biofilm, bacteria are better protected against antibacterial drugs and the immune response, increasing treatment difficulty, as persistent populations recalcitrant to chemotherapy are promoted. Deciphering mechanisms leading to biofilms could, thus, be beneficial to obtain new antibacterial drug candidates. Here, we show that mycobacterial biofilm formation is linked to excess glycerol adaptation and the concomitant establishment of the Crabtree effect. This effect is characterized by respiratory reprogramming, ATP downregulation, and secretion of various metabolites including pyruvate, acetate, succinate, and glutamate. Interestingly, the Crabtree effect was abnormal in a mycobacterial strain deficient for Cpn60.1 (GroEL1). Indeed, this mutant strain had a compromised ability to downregulate ATP and secreted more pyruvate, acetate, succinate, and glutamate in the culture medium. Importantly, the mutant strain had higher intracellular pyruvate and produced more toxic methylglyoxal, suggesting a glycolytic stress leading to growth stasis and consequently biofilm failure. This study demonstrates, for the first time, the link between mycobacterial biofilm formation and the Crabtree effect.

Keywords: Crabtree effect; GroEL1; biofilm; metabolic adaptation; methylglyoxal; mycobacteria.

FIGURE 1

The Δcpn60.1 biofilm defect under standard Sauton’s medium partially resulted from PDIM/PGL alteration. (A) BCG GL2 strains were grown as biofilms in 6% glycerol Sauton’s medium for indicated time points. The ComplΔcpn60.1 is the complemented strain expressing Cpn60.1. (B) Biofilms of BCG Pasteur strains were grown as in (A). PMM50 and PMM137 are PDIM⁻/PGL⁻ and PDIM⁻/PGL⁺, respectively. The experiments were performed at least three times. Representative biofilm pictures are shown.

FIGURE 2

The Δcpn60.1 BCG strain was more susceptible to excess glycerol present in the biofilm medium. (A) Viability of biofilm culture under 6% glycerol Sauton’s medium at days 0 and 25 was determined. ^∗∗p ≤ 0.01 relative to WT at day 25 by unpaired t-test. (B) Washed BCG precultures were subcultured in Sauton’s medium with various glycerol concentrations as indicated for 9 days. Representative pictures are shown. (C) Cells grown in 4, 6, and 8% glycerol Sauton’s medium were disrupted before OD600 determination. ^#p ≤ 0.0001 relative to WT by unpaired t-test. (D) Growth kinetics under 6% glycerol Sauton’s medium measured by OD600 and viability (inset) at 3, 6, and 9 days. ^∗p ≤ 0.05; ^∗∗∗p ≤ 0.001 by unpaired t-test. (E) Representative 25 days biofilms grown in Sauton’s medium with 2 and 4% glycerol as indicated.

FIGURE 3

The Δcpn60.1 strain inhibited WT biofilm and normal growth. (A) Representative 25 days biofilms under 6% glycerol Sauton’s medium are shown. (B) Growth of BCG strains in Sauton’s medium with varying glycerol concentrations as in Figure 2B. The experiments were performed at least three times.

FIGURE 4

The Δcpn60.1’s accumulation of methylglyoxal and rescue of its biofilm by proline-mediated detoxification. (A) MIC of methylglyoxal determined in 7H9 medium with varying amounts of glycerol. (B) Cellular extracts of BCG grown in 6% glycerol Sauton’s medium were determined for methylglyoxal–protein adducts by ELISA. Absorbance at 450 nm was normalized by dividing by protein concentration. ^∗∗p ≤ 0.01 relative to WT by unpaired t-test. (C) WT BCG protein extracts from 6% glycerol Sauton’s medium (±25 mM proline) were quantified for methylglyoxal, followed by data normalization as per protein concentration. ^∗∗p ≤ 0.01 by unpaired t-test. (D) The Δcpn60.1 biofilm was grown in 6% glycerol Sauton’s medium for 25 days with or without 50 mM proline. A control with proline and without glycerol was included.

FIGURE 5

Requirement for Cpn60.1 in the glycerol-triggered ATP downregulation. (A) Mean fold decrease of mycobacterial respiratory proteins NuoA and QcrC relative to 0.2% glycerol Sauton’s medium. ND, no data due to a p> 0.05. (B) BCG strains grown in DTA medium were treated with glycerol for 24 h before ATP measurement. Percentage of ATP decrease was calculated by comparing with 0% glycerol control. ^∗p ≤ 0.05; ^∗∗p ≤ 0.01 and ^∗∗∗p ≤ 0.001 by unpaired t-test. (C) BCG strains were grown in DTA medium ( ± 8% glycerol) to exponential phase before ATP measurement. ^#p ≤ 0.0001 relative to WT (no glycerol) by unpaired t-test.

FIGURE 6

The Δcpn60.1 strain secreted more pyruvate, acetate, and succinate. (A) Proposed pathways generating methylglyoxal, pyruvate, acetate, and succinate. The secretion of pyruvate, acetate, and succinate under excess glycerol is indicated. (B) Pyruvate in the culture filtrates from 6% Sauton’s medium was quantified and normalized by protein concentration. ^∗∗p ≤ 0.01 relative to WT by unpaired t-test. (C) Extracellular acetate was normalized by protein concentration. ^∗p ≤ 0.05 by unpaired t-test. (D) Succinate in the culture filtrates from 6% Sauton’s medium was quantified and normalized by protein concentration. ^∗∗∗p ≤ 0.001 by unpaired t-test. (E) Intracellular pyruvate was measured and normalized by protein concentration. ^∗∗∗p ≤ 0.001 by unpaired t-test.

FIGURE 7

The Δcpn60.1 strain secreted more glutamate than the WT strain. (A) Proposed pathway leading to glutamate/glutamine production and the subsequent secretion of glutamate. (B) M. bovis BCG was grown in 6% glycerol Sauton’s medium for 11 days, and the culture filtrates were determined for glutamate. The experiments were performed three independent times (each in three or four biological replicates). Data from one representative experiment are shown. ^∗∗p < 0.01 relative to WT by unpaired t-test.

FIGURE 8

Metabolic pathways associated with adaptation to excess glycerol and mycobacterial biofilm formation. (A) Slow-growing mycobacteria respond to excess glycerol by reprogramming the respiratory chain, downregulating ATP production, and boosting the glycolysis [see (B) for details]. The pyruvate is proposed to be assimilated into at least four pathways (indicated by box). Proteomics-revealed enzymes participating these pathways were colored (green for upregulation and red for downregulation under excess glycerol). The uptake of glutamine by GlnQ is proposed to occur in the presence of extracellular glutamine. The activation of these pyruvate-assimilating pathways not only prevents the overaccumulation of pyruvate (and thus the toxic methylglyoxal) but also may promote mycobacterial biofilm growth. (B) Both the glycolytic pathway and methylglyoxal detoxification pathway are enhanced in the presence of excess glycerol, leading to enhanced production of the end product pyruvate. Related proteins identified by proteomic analysis were indicated and colored. In particular, PckA, catalyzing predominantly the gluconeogenesis, was downregulated. Mdh, malate dehydrogenase; Fum, fumarate hydratase; Pca, pyruvate carboxylase; Mez, malic enzyme; PDC, pyruvate dehydrogenase complex; CitA, citrate synthase II; Kgd, alpha-ketoglutarate decarboxylase; DlaT, dihydrolipoamide acyltransferase; LpdC, dihydrolipoamide dehydrogenase; GltD, glutamate synthase (small subunit); GlnA1, glutamine synthetase; GlnQ, probable glutamine transporter; Pta, phosphate acetyl-transferase; AckA, acetate kinase; Glpk, glycerol kinase; Pgk, phosphoglycerate kinase; Gpm1, phosphoglycerate mutase 1; PykA, pyruvate kinase; Rv0911, putative glyoxalase; LldD2, lactate dehydrogenase; PckA, phosphoenolpyruvate carboxykinase.