A hydrolase of trehalose dimycolate induces nutrient influx and stress sensitivity to balance intracellular growth of Mycobacterium tuberculosis

Abstract

Chronic tuberculosis in an immunocompetent host is a consequence of the delicately balanced growth of Mycobacterium tuberculosis (Mtb) in the face of host defense mechanisms. We identify an Mtb enzyme (TdmhMtb) that hydrolyzes the mycobacterial glycolipid trehalose dimycolate and plays a critical role in balancing the intracellular growth of the pathogen. TdmhMtb is induced under nutrient-limiting conditions and remodels the Mtb envelope to increase nutrient influx but concomitantly sensitizes Mtb to stresses encountered in the host. Consistent with this, a ΔtdmhMtb mutant is more resilient to stress and grows to levels higher than those of wild-type in immunocompetent mice. By contrast, mutant growth is retarded in MyD88(-/-) mice, indicating that TdmhMtb provides a growth advantage to intracellular Mtb in an immunocompromised host. Thus, the effects and countereffects of TdmhMtb play an important role in balancing intracellular growth of Mtb in a manner that is directly responsive to host innate immunity.

Figure 1

Rv3451 and Rv3452 are TDM hydrolases in Mtb. (A) Alignment of Rv3451 and Rv3452 protein sequences with Tdmh_Ms, originally marked as Mstdmh (Ojha et al., 2010). The catalytic triad of Ser, Asp and His are indicated. (B) Radio-TLC analysis of lipids in a mixture containing ¹⁴C TDM and lysates from recombinant M. smegmatis, mc²155:Δtdmh_Ms, transformed with either empty plasmid (pJL37), pRv3451 or pRv3452 (also see figure S1). Reactions with purified Tdmh_Ms, and storage buffer were positive and negative controls, respectively. Purified FM was marker. (C) Absence of TDM hydrolyzing activity in the lysates of mc²155:Δtdmh_Ms with five other hydrolases cloned on the same plasmid backbone (pJL37), under constitutive hsp60 promoter, as Rv3451. These plasmids are indicated below each lane. mc²155:Δtdmh_Ms:pRv3451 is positive control. (D) The hydrolase activity of Tdmh_Mtb (Rv3451) in cellular factions of mc²155:Δtdmh_Ms:pRv3451. TL- total lysate; LSP- low speed pellet, obtained from centrifugation of TL at 20,000g for 60 minutes; HSP- high-speed pellet, obtained from centrifugation of low-speed supernatant at 100,000g for 60 minutes; HSS- high-speed supernatant, obtained from the same centrifugation described for HSP. Purified FM was loaded as marker. (Figure 1, related to Figure S1)

Figure 2

Accumulation of TDM in mc²7000:Δtdmh_Mtb. (A) Schematic representation of genomic organization of rv3451 (tdmh_Mtb) and rv3452 (tdmh_Mtb2), and the allelic exchange substrate including upstream (ups) and downstream (ds) regions used for replacement of tdmh_Mtb with hyg^r. (B). Southern blot of BsaAI digested genomic DNA from the parent wild-type (mc²7000) and two hyg^r colonies (Δtdmh_Mtb) probed with ups. (C) A representative radio-TLC of apolar lipids extractable from Mtb strains, mc²7000 and mc²7000:Δtdmh_Mtb at 2, 3, and 4-week stages of biofilms. Purified TDM is the marker. (D) Quantitative analysis of the level of TDM as a percentage of total apolar lipids in mc²7000 (wild-type) and mc²7000:Δtdmh_Mtb (mutant) in 2- and 4- week stages of biofilms (also see figure S2 for complete lipid profile). (Figure 2, related to Figure S2)

Figure 3

Correlation between induced Tdmh_Mtb activity and increased non-selective permeability of mycobacterial envelope under limiting nutrients. (A) Influx of glycerol over two hours in mc²7000 incubated in normal (7H9TyOADC) and nutrient-limiting media (7H9Ty) for indicated time. (B) Regeneration of Mtb(Erd) in 7H9Ty with 0.1% v/v glycerol from a culture maintained in 7H9Ty for indicated time. (C) Accumulation of acetate, phosphorus and glycerol over 30 minutes by mc²155:Δtdmh_Ms transformed with either pJL37, or pRv3451. (D) Accumulation of acetate, phosphorus and glycerol over two hours by cultures of mc²7000, mc²7000:Δtdmh_Mtb, and mc²7000:Δtdmh_Mtb expressing Rv3451 (mc²7000:Δtdmh_Mtb:p51), Rv3452 (mc²7000:Δtdmh_Mtb:p52), or Rv3451-52 (mc²7000:Δtdmh_Mtb:p5152) in nutrient-limiting media (also see Fig. S3). (E) Growth of Mtb(Erd), Δtdmh_Mtb mutant, and the complemented strain (mc²7000:Δtdmh_Mtb:p5152) in 7H9Ty with 0.1% v/v glycerol. (F) Sensitivity of 4-week biofilms of Mtb (Erd), Mtb (Erd):Δtdmh_Mtb, and Mtb (Erd):Δtdmh_Mtb:p5152 to H₂O₂ (40mM) and lysozyme (6μg/mL). (Figure 3, related to Figure S3)

Figure 4

Ectopic induction of TDM hydrolase increases non-selective permeability of Mtb envelope. (A) and (B), Western blot and lipid analysis of acetamide induced expression of tdmh_Ms in mc²7000:pAO10, and the empty plasmid control, mc²7000:pLAM12, in Sauton’s media for time (day) indicated on each lane. Purified TDM and FMs were markers on TLC in panel B. (C) Accumulation of glycerol, phosphorus and acetate over two hours by acetamide induced expression of tdmh_Ms in mc²7000:Δtdmh_Mtb:pAO10, and the empty plasmid control, mc²7000:pLAM12, in Sauton’s media. (D) Sensitivity of the two Mtb strains described in panel E to H₂O₂ (40mM), lysozyme (6μg/mL) and LL37 (20μM). (E) Growth of Mtb (Erd):Δtdmh_Mtb:pAO10 and Mtb(Erd):Δtdmh_Mtb:pLAM12 in media with limiting glycerol (0.1% v/v) and inducer (0.2% v/v acetamide).

Figure 5

The opposing effects of Tdmh_Mtb self-restrict intracellular growth of Mtb in immunocompetent host. (A) Accumulation of radioactivity over a 48-hour period in the intracellular mc²7000, mc²7000: Δtdmh_Mtb, and mc²7000: Δtdmh_Mtb:p5152 when ¹⁴C-acetate was added to the medium of infected RAW264.7 (mean ± SE, n=7). (B) Survival of Mtb (Erd), Mtb (Erd):Δtdmh_Mtb and Mtb (Erd):Δtdmh_Mtb:p5152 in activated BMM from C57BL/6 mice (also see Fig S4C for raw cfu). (C) Sensitivity of intracellular Mtb (Erd), Mtb (Erd):Δtdmh_Mtb and Mtb (Erd):Δtdmh_Mtb:p5152 to 5-day exposure of isoniazid (0.75μg/mL) added to the culture medium of infected primary BMM (C57BL/6) (mean ± SE, n=5). (D) Burden of the three Mtb strains in the lungs of C57BL/6 mice three weeks after aerosolized infection with ~50-100 bacilli/lung. Data represents two independent exposures, each with five animals/per strain (p < 0.0001, Mann-Whitney test). (E) Relative change in intracellular accumulation of radioactivity over a 48-hour period between mc²7000: Δtdmh_Mtb constitutively expressing tdmh_Ms (mc²7000:Δtdmh_Mtb: pYY1) and its corresponding empty vector control, mc²7000: Δtdmh_Mtb:pJL37, when ¹⁴C-acetate was added to the medium of infected RAW264.7 (mean ± SE, n=4). (F) Sensitivity of intracellular Mtb(Erd):Δtdmh_Mtb:pYY1 and Mtb(Erd):Δtdmh_Mtb:pJL37 to 5-day exposure of isoniazid (0.75μg/mL), added to the culture medium of infected primary BMM from C57BL/6 mice (mean ± SE, n=6). (G) Burden of Mtb(Erd):Δtdmh_Mtb:pYY1 and Mtb(Erd):Δtdmh_Mtb:pJL37 in the lung of C57BL/6 mice after three weeks of infection (p < 0.01, Mann-Whitney test). (Figure 5, related to Figure S4)

Figure 6

Tdmh facilitates Mtb growth in MyD88^−/− mice. (A) Growth of Mtb (Erd) or Mtb (Erd):Δtdmh_Mtb and the complemented Δtdmh_Mtb:p5152 strains in activated primary BMMs from MyD88^−/− mice. (B) Accumulation of radioactivity over a 48-hour period in the intracellular mc²7000 and mc²700:Δtdmh_Mtb, when ¹⁴C-acetate was added to the medium of infected primary BMMs from MyD88^−/− mice. (C) & (D) Relative difference between the survival of Mtb (Erd):Δtdmh_Mtb expressing tdmh_Ms (Mtb(Erd): Δtdmh_Mtb pYY1) and its corresponding empty vector control, Mtb (Erd):Δtdmh_Mtb:pJL37, in primary BMMs from C57BL/6 (C), and MyD88^−/− (D) mice. (E) Burden of Mtb(Erd), Mtb(Erd):Δtdmh_Mtb and Mtb(Erd):Δtdmh_Mtb:pYY1 in the lungs of MyD88^−/− mice after 14 days of aerosolized infection (n= 5, p < 0.05, Mann-Whitney test). Mean burden of the three strains after 24 hours of exposure were 93, 89 and 99, respectively. (F) Survival of MyD88^−/− mice infected with each of the three strains described in panel E (n = 8, p < 0.001, Mann-Whitney test). Raw cfu data for panel A, C and D are provided in figures S5A S5B and S5C, respectively. (Figure 6, related to Figure S5)

Figure 7

Schematic representation of Tdmh_Mtb dependent self-balanced growth of intracellular Mtb in immunocompetent host. The status of nutrient availability, stress effect and net growth of Mtb in Mφ at initial (t=0) and later (t>0) phases of infection are represented by green arrow, red arrow and open circle, respectively. Two possible scenarios under which this balance could be perturbed are depicted. Scenario 1 shows the effect of Tdmh_Mtb inactivation on bacterial burden in immunocompetent host. Although the nutrient influx in a Δtdmh_Mtb mutant is likely retarded, this deficiency is outweighed by increased tolerance of the mutant to the host-induced stresses. In scenario 2, reduced intracellular stress in an immunocompromised host can mitigate the effect of Tdmh_Mtb on stress sensitivity, such that Tdmh-dependent nutrient assimilation can accelerate growth of wild-type Mtb in such host.