Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence

Abstract

Genes involved in fatty acid catabolism have undergone extensive duplication in the genus Mycobacterium, which includes the etiologic agents of leprosy and tuberculosis. Here, we show that prokaryotic- and eukaryotic-like isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL) are jointly required for fatty acid catabolism and virulence in Mycobacterium tuberculosis. Although deletion of icl1 or icl2, the genes that encode ICL1 and ICL2, respectively, had little effect on bacterial growth in macrophages and mice, deletion of both genes resulted in complete impairment of intracellular replication and rapid elimination from the lungs. The feasibility of targeting ICL1 and ICL2 for chemical inhibition was shown using a dual-specific ICL inhibitor, which blocked growth of M. tuberculosis on fatty acids and in macrophages. The absence of ICL orthologs in mammals should facilitate the development of glyoxylate cycle inhibitors as new drugs for the treatment of tuberculosis.

Figure 1. Glyoxylate cycle and related metabolic pathways in M. tuberculosis

(a) The beta-oxidation cycle (β-ox) degrades fatty acids to acetyl-CoA (C₂) and propionyl-CoA (C₃). The glyoxylate and methylcitrate cycles are required for anaplerosis and for propionyl-CoA metabolism, respectively, in fatty acid-catabolizing cells. Glyoxylate cycle enzymes are ICL1, ICL2, and MLS; ICL1/ICL2 could also function in the methylcitrate cycle with PRPC and PRPD. Enzymes common to the glyoxylate, methylcitrate, and TCA cycles are ACN, SDH, and FUM. Enzymes common to the glyoxylate and TCA cycles are MDH, MQO, GLTA2, and CITA. Pyruvate is produced from malate by MEZ or from oxaloacetate by sequential action of PCK and PYK; coupled decarboxylation of pyruvate by the pyruvate dehydrogenase complex (PDHC) yields acetyl-CoA. Anaplerosis in carbohydrate-catabolizing cells is by carboxylation of pyruvate or phosphoenolpyruvate (PEP) to oxaloacetate by PCA or PPC, respectively. PPC is absent in M. tuberculosis (dashed line). Gene designations are listed in Supplementary Table 1 online. (b) ICL domain organization. Domains I and III are present in all ICLs. Domain I contains the conserved catalytic motif KKCGH. Domain II is present in fungal and plant ICLs and in mycobacterial ICL2, but absent in mycobacterial ICL1. Domain IV is unique to mycobacterial ICL2. (c) Superimposition of in silico-modeled ICL2 monomer (red) and the X-ray crystal structure of ICL1 monomer (blue). Encircled: ICL catalytic signature motif KKCGH (AA 193-203 in ICL1; AA 213-217 in ICL2). Domain II of ICL2 (AA 269-365) was modeled after the X-ray crystal structure of A. nidulans ICL. Domain IV (not depicted) had no homology to known sequences.

Figure 2. Overlapping roles of ICL1/ICL2 in fatty acid catabolism

(a–f) Growth of bacteria in media containing (a) glycerol, (b) glucose, (c) acetate (C₂), (d) propionate (C₃), (e) butyrate (C₄), or (f) polyoxyethylene sorbitan monolaurate (C₁₂). (g) Growth on [¹⁴C]-palmitate (C₁₆). (h) Growth on DPPC (C₁₆), an abundant phospholipid in the mammalian lung. (a–h) Bacterial strains: wild-type (filled squares), Δicl1 (open circles), Δicl2 (open triangles), Δicl1 Δicl2 (open diamonds), Δicl1 Δicl2 complemented with pICL1 (filled circles) or pICL2 (filled triangles). Results are representative of at least two experiments.

Figure 3. Virulence of ICL-deficient M. tuberculosis in mice

(a–d) Bacterial loads in lungs of C57BL/6 mice. (e) Spleens of C57BL/6 mice 2 wk post-infection. Left to right: splenic bacterial loads (log₁₀ CFU ± SD) were 6.11 ± 0.15 (wild-type), 5.92 ± 0.12 (Δicl1), 6.24 ± 0.08 (Δicl2), 3.58 ± 0.22 (Δicl1 Δicl2). (f) Bacterial loads in lungs of IFN-γ^−/− mice. (g) Lung pathology in C57BL/6 mice 16 wk post-infection. Magnification: 200X. (a–g) Bacterial strains: wild-type (filled squares), Δicl1 (open circles), Δicl2 (open triangles), Δicl1 Δicl2 (open diamonds), Δicl1 Δicl2 transformed with pICL1 (filled circles) or empty vector (cross-marks). Results are representative of two experiments.

Figure 4. Survival of ICL-deficient M. tuberculosis in macrophages

(a–f) Bacterial loads in non-activated (a,c,e) and IFN-γ-activated (b,d,f) mouse bone marrow-derived macrophages. (g,h) Bacterial loads in human blood monocyte-derived macrophages. (a–h) Bacterial strains: wild-type (filled squares), Δicl1 (open circles), Δicl2 (open triangles), Δicl1 Δicl2 (open diamonds), Δicl1 Δicl2 complemented with pICL1 (filled circles) or pICL2 (filled triangles). Results are representative of at least two experiments.

Figure 5. Chemical inhibition of ICL blocks M. tuberculosis growth on fatty acids

(a–d) Growth of wild-type (squares) and Δicl1 Δicl2 (open diamonds) bacteria in media containing (a) glycerol, (b) glucose, (c) propionate (C₃), or (d) polyoxyethylene sorbitan monolaurate (C₁₂), without (filled squares, open diamonds) or with (open squares) addition of 3-NP (0.1 mM). (e,f) Growth of (e) wild-type (open squares) and (f) pICL1-complemented Δicl1 Δicl2 (open circles) bacteria in media containing propionate and 3-NP (0.025, 0.05, 0.1, or 0.2 mM). Results are representative of at least two experiments.

Figure 6. Chemical inhibition of ICL blocks M. tuberculosis growth in macrophages

(a–e) Bacterial loads in M. tuberculosis-infected (a–d) murine bone marrow-derived macrophages and (e) human blood monocyte-derived macrophages without (filled symbols) or with (open symbols) addition of 3-NP (0.2, 1, 5, or 10 mM). Bacterial strains: wild-type (squares), Δicl1 Δicl2 (diamonds), Δicl1 Δicl2 complemented with pICL1 (circles). Results are representative of two experiments.