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Cord factor
Cord factor
Cord factor
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Cord factor
Identifiers
3D model (JSmol)
ChemSpider
  • InChI=1S/C130H250O15/c1-5-9-13-17-21-25-29-33-37-41-43-45-47-49-51-53-57-61-65-71-81-89-97-105-117(131)116(104-96-88-80-74-73-78-86-94-102-114-108-112(114)100-92-84-76-68-63-59-55-40-36-32-28-24-20-16-12-8-4)128(140)142-110-120-122(134)124(136)126(138)130(144-120)145-129-125(137)123(135)121(133)119(143-129)109-141-127(139)115(103-95-87-79-70-64-60-56-52-50-48-46-44-42-38-34-30-26-22-18-14-10-6-2)118(132)106-98-90-82-72-66-69-77-85-93-101-113-107-111(113)99-91-83-75-67-62-58-54-39-35-31-27-23-19-15-11-7-3/h111-126,129-138H,5-110H2,1-4H3
    Key: DJUMKUNMJWRLAX-UHFFFAOYSA-N
  • CCCCCCCCCCCCCCCCCCCCCCCCCC(C(CCCCCCCCCCC1CC1CCCCCCCCCCCCCCCCCC)C(=O)OCC2C(C(C(C(O2)OC3C(C(C(C(O3)COC(=O)C(CCCCCCCCCCCCCCCCCCCCCCCC)C(CCCCCCCCCCCC4CC4CCCCCCCCCCCCCCCCCC)O)O)O)O)O)O)O)O
Properties
C130H250O15
Molar mass 2053.415 g·mol−1
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
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Cording Mycobacterium tuberculosis (H37Rv strain) viewed with fluorescent microscopy

Cord factor, or trehalose dimycolate (TDM), is a glycolipid molecule found in the cell wall of Mycobacterium tuberculosis and similar species. It is the primary lipid found on the exterior of M. tuberculosis cells.[1] Cord factor influences the arrangement of M. tuberculosis cells into long and slender formations, giving its name.[2] Cord factor is virulent towards mammalian cells and critical for survival of M. tuberculosis in hosts, but not outside of hosts.[3][4] Cord factor has been observed to influence immune responses, induce the formation of granulomas, and inhibit tumor growth.[5] The antimycobacterial drug SQ109 is thought to inhibit TDM production levels and in this way disrupts its cell wall assembly.[6]

Structure

[edit]

A cord factor molecule is composed of a sugar molecule, trehalose (a disaccharide), composed of two glucose molecules linked together. Trehalose is esterified to two mycolic acid residues.[7][8] One of the two mycolic acid residues is attached to the sixth carbon of one glucose, while the other mycolic acid residue is attached to the sixth carbon of the other glucose.[7] Therefore, cord factor is also named trehalose-6,6'-dimycolate.[7] The carbon chain of the mycolic acid residues vary in length depending on the species of bacteria it is found in, but the general range is 20 to 80 carbon atoms.[3] Cord factor's amphiphilic nature leads to varying structures when many cord factor molecules are in close proximity.[3] On a hydrophobic surface, they spontaneously form a crystalline monolayer.[9] This crystalline monolayer is extremely durable and firm; it is stronger than any other amphiphile found in biology.[10] This monolayer also forms in oil-water, plastic-water, and air-water surfaces.[1] In an aqueous environment free of hydrophobic surfaces, cord factor forms a micelle.[11] Furthermore, cord factor interlocks with lipoarabinomannan (LAM), which is found on the surface of M. tuberculosis cells as well, to form an asymmetrical bilayer.[1][12] These properties cause bacteria that produce cord factor to grow into long, intertwining filaments, giving them a rope- or cord-like appearance when stained and viewed through a microscope (hence the name).[13]

Evidence of virulence

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Scanning electron micrograph of Mycobacterium tuberculosis

A large quantity of cord factor is found in virulent M. tuberculosis, but not in avirulent M. tuberculosis.[1] Furthermore, M. tuberculosis loses its virulence if its ability to produce cord factor molecules is compromised.[1] Consequently, when all lipids are removed from the exterior of M. tuberculosis cells, the survival of the bacteria is reduced within a host.[14] When cord factor is added back to those cells, M. tuberculosis survives at a rate similar to that of its original state.[14] Cord factor increases the virulence of tuberculosis in mice, but it has minimal effect on other infections.[1]

Biological function

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The function of cord factor is highly dependent on what environment it is located, and therefore its conformation.[15] This is evident as cord factor is harmful when injected with an oil solution, but not when it is with a saline solution, even in very large amounts.[15] Cord factor protects M. tuberculosis from the defenses of the host.[1] Specifically, cord factor on the surface of M. tuberculosis cells prevents fusion between phagosomal vesicles containing the M. tuberculosis cells and the lysosomes that would destroy them.[5][16] The individual components of cord factor, the trehalose sugars and mycolic acid residues, are not able to demonstrate this activity; the cord factor molecules must be fully intact.[5] Esterase activity that targets cord factor results in the lysis of M. tuberculosis cells.[17] However, the M. tuberculosis cells must still be alive to prevent this fusion; heat-killed cells with cord factor are unable to prevent being digested.[16] This suggests an additional molecule from M. tuberculosis is required.[16] Regardless, cord factor's ability to prevent fusion is related to an increased hydration force or through steric hindrance.[5] Cord factor remains on the surface of M. tuberculosis cells until it associates with a lipid droplet, where it forms a monolayer.[15] Then, as cord factor is in a monolayer configuration, it has a different function; it becomes fatal or harmful to the host organism.[18] Macrophages can die when in contact with monolayers of cord factor, but not when cord factor is in other configurations.[1] As the monolayer surface area of cord factor increases, so does its toxicity.[19] The length of the carbon chain on cord factor has also shown to affect toxicity; a longer chain shows higher toxicity.[20] Furthermore, fibrinogen has shown to adsorb to monolayers of cord factor and act as a cofactor for its biological effects.[21]

Cord factor isolated from species of Nocardia has been shown to cause cachexia in mice. Severe muscle wasting occurred within 48 hours of the toxin being administered.[22]

Host responses and cytokines

[edit]

Numerous responses that vary in effect result from cord factor's presence in host cells. After exposure to cord factor for 2 hours, 125 genes in the mouse genome are upregulated.[23] After 24 hours, 503 genes are upregulated, and 162 genes are downregulated.[23] The exact chemical mechanisms by which cord factor acts is not completely known. However, it is likely that the mycolic acids of cord factor must undergo a cyclopropyl modification to lead to a response from the host's immune system for initial infection.[24] Furthermore, the ester linkages in cord factor are important for its toxic effects.[25] There is evidence that cord factor is recognized by the Mincle receptor, which is found on macrophages.[26][27] An activated Mincle receptor leads to a pathway that ultimately results in the production of several cytokines.[28][29] These cytokines can lead to further cytokine production that promote inflammatory responses.[30] Cord factor, through the Mincle receptor, also causes the recruitment of neutrophils, which lead to pro-inflammatory cytokines as well.[31] However, there is also evidence that toll-like receptor 2 (TLR2) in conjunction with the protein MyD-88 is responsible for cytokine production rather than the Mincle receptor.[23]

Cord factor presence increases the production of the cytokines interleukin-12 (IL-12), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor (TNFα), and macrophage inflammatory protein-2 (MIP-2), which are all pro-inflammatory cytokines important for granuloma formation.[16][28][32] IL-12 is particularly important in the defense against M. tuberculosis; without it, M. tuberculosis spreads unhampered.[33][34] IL-12 triggers production of more cytokines through T cells and natural killer (NK) cells, while also leading to mature Th1 cells, and thus leading to immunity.[35] Then, with IL-12 available, Th1 cells and NK cells produce interferon gamma (IFN-γ) molecules and subsequently release them.[36] The IFN-γ molecules in turn activate macrophages.[37]

When macrophages are activated by cord factor, they can arrange into granulomas around M. tuberculosis cells.[15][38] Activated macrophages and neutrophils also cause an increase in vascular endothelial growth factor (VEGF), which is important for angiogenesis, a step in granuloma formation.[39] The granulomas can be formed either with or without T-cells, indicating that they can be foreign-body-type or hypersensitivity-type.[37] This means cord factor can stimulate a response by acting as a foreign molecule or by causing harmful reactions from the immune system if the host is already immunized.[37] Thus, cord factor can act as a nonspecific irritant or a T-cell dependent antigen.[37] Granulomas enclose M. tuberculosis cells to halt the bacteria from spreading, but they also allow the bacteria to remain in the host.[16] From there, the tissue can become damaged and the disease can transmit further with cord factor.[40] Alternatively, the activated macrophages can kill the M. tuberculosis cells through reactive nitrogen intermediates to remove the infection.[41]

Besides inducing granuloma formation, activated macrophages that result from IL-12 and IFN-γ are able to limit tumor growth.[42] Furthermore, cord factor's stimulation of TNF-α production, also known as cachectin, is also able to induce cachexia, or loss of weight, within hosts.[43][44] Cord factor also increases NADase activity in the host, and thus it lowers NAD; enzymes that require NAD decrease in activity accordingly.[3] Cord factor is thus able to obstruct oxidative phosphorylation and the electron transport chain in mitochondrial membranes.[3] In mice, cord factor has shown to cause atrophy in the thymus through apoptosis; similarly in rabbits, atrophy of the thymus and spleen occurred.[45][46] This atrophy occurs in conjunction with granuloma formation, and if granuloma formation is disturbed, so is the progression of atrophy.[46]

Scientific applications and uses

[edit]

Infection by M. tuberculosis remains a serious problem in the world and knowledge of cord factor can be useful in controlling this disease.[24] For example, the glycoprotein known as lactoferrin is able to mitigate cytokine production and granuloma formation brought on by cord factor.[47] However, cord factor can serve as a useful model for all pathogenic glycolipids and therefore it can provide insight for more than just itself as a virulence factor.[11][48] Hydrophobic beads covered with cord factor are an effective tool for such research; they are able to reproduce an organism's response to cord factor from M. tuberculosis cells.[11][48] Cord factor beads are easily created and applied to organisms for study, and then easily recovered.[48]

It is possible to form cord factor liposomes through water emulsion; these liposomes are nontoxic and can be used to maintain a steady supply of activated macrophages.[49] Cord factor under proper control can potentially be useful in fighting cancer because IL-12 and IFN-γ are able to limit the growth of tumors.[50]

See also

[edit]

References

[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Cord factor

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from Grokipedia
Cord factor, also known as 6,6'-dimycolate (TDM), is a constituent of the cell walls of pathogenic mycobacteria, including . It comprises a esterified at the 6 and 6' positions with two long-chain, branched mycolic acids, typically exceeding 70 carbon atoms in length. This structure enables the formation of serpentine, cord-like bacterial aggregates, a hallmark morphological feature first observed in virulent strains and responsible for the compound's name. As a key , cord factor promotes mycobacterial survival within host macrophages by inhibiting phagosome-lysosome fusion and delaying maturation. It elicits formation, the characteristic host response to , through activation of the Syk-Card9 signaling pathway following recognition by the receptor Mincle on innate immune cells. Additionally, cord factor stimulates production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) while contributing to the impermeability of the mycobacterial envelope, which underlies resistance to antibiotics and host defenses. Historically isolated in the 1950s from M. tuberculosis extracts, cord factor has been implicated in various pathological processes, including primary and secondary progression, , and cavitary lesion maintenance that facilitates bacterial transmission. Strains deficient in cord factor, whether through extraction or genetic , exhibit reduced and fail to induce typical granulomatous in animal models. Its dual role as both immunostimulatory and immunosuppressive underscores its importance in the complex interplay between mycobacteria and the host .

Discovery and Nomenclature

Historical Background

The characteristic serpentine cord formation in virulent strains of was first systematically described in 1947 by Gardner Middlebrook and colleagues, who observed these microscopic arrangements of bacterial cells during studies on the morphological differences between virulent and avirulent tubercle bacilli. Through microscopic examination of liquid cultures, they noted that highly virulent strains consistently formed elongated, rope-like cords composed of parallel chains of , while avirulent variants grew in dispersed or clumped patterns without such structures. This led Middlebrook to propose that cord formation was an essential marker of , correlating directly with the pathogen's ability to cause disease in animal models. Building on this, Hubert Bloch isolated a lipid component responsible for inducing cord formation in 1950, extracting it from the cell walls of virulent M. tuberculosis strains using organic solvents. When this lipid extract, termed "cord factor," was added to cultures of non-cord-forming (avirulent) mycobacteria, it prompted the rapid assembly of serpentine cords, confirming its role in mediating this morphological . Bloch's experiments demonstrated that the factor was absent or minimal in avirulent strains, highlighting its specificity to pathogenic mycobacteria and its potential as a determinant. Early investigations into cord factor's pathogenic implications involved injecting the lipid extract into animals, revealing its capacity to elicit granulomatous responses akin to those in infection. In mice and rabbits, subcutaneous or intravenous administration of cord factor produced localized chronic inflammation, , and the development of granuloma-like lesions in the lungs, liver, and other organs, mimicking aspects of mycobacterial disease progression. These findings linked cord factor to the induction of persistent inflammatory foci, underscoring its contribution to the host-pathogen interaction in early pathogenesis. Subsequent chemical analysis in identified cord factor as trehalose dimycolate, a composed of esterified with mycolic acids.

Naming and Synonyms

Cord factor was originally named by Hubert Bloch in 1950 for a lipid fraction extracted from virulent strains of Mycobacterium tuberculosis that promoted the formation of serpentine, cord-like bacterial aggregates observable under microscopy, a morphology linked to enhanced pathogenicity. This designation reflected the distinctive "cording" phenomenon, where bacilli align in parallel chains, distinguishing virulent tubercle bacilli from avirulent variants. In 1956, detailed chemical characterization by Noll, Bloch, Asselineau, and Lederer revealed the structure of this lipid as trehalose 6,6'-dimycolate (TDM), a glycolipid composed of trehalose esterified with two mycolic acid chains, thereby establishing TDM as its primary chemical synonym. This elucidation confirmed the substance's role in mycobacterial cell wall composition and solidified the nomenclature in subsequent biochemical studies. Alternative designations such as trehalose dimycolate or simply dimycolate are commonly employed in scientific literature to emphasize its glycolipid nature, while "mycobacterial cord factor" is used to denote its occurrence in non-tuberculous species, including Mycobacterium leprae and other environmental mycobacteria. These synonyms highlight the compound's broader distribution across the genus Mycobacterium beyond tuberculosis pathogens.

Chemical Structure and Properties

Molecular Composition

Cord factor, chemically known as trehalose 6,6'-dimycolate (TDM), is a glycolipid characterized by a central trehalose moiety—a non-reducing disaccharide composed of two D-glucose units connected via an α,α-1,1-glycosidic bond—esterified at the 6 and 6' hydroxyl positions with two molecules of mycolic acid. This esterification links the hydrophilic trehalose head to the hydrophobic mycolic acid tails, defining its amphipathic nature as a key component of the mycobacterial cell envelope. Mycolic acids, the components of cord factor, are exceptionally long-chain α-branched β-hydroxy fatty acids, typically spanning 70 to 90 carbon atoms in total length, with a shorter α-alkyl branch (C22–C26) and a longer meromycolic chain (C40–C60). These acids feature a conserved β-hydroxy group essential for bond formation and exhibit species-specific variations in the meromycolic chain, including functional groups such as cis- or trans-cyclopropane rings, double bonds in α-mycolates, methoxy groups in methoxy-mycolates, and keto groups in keto-mycolates, which contribute to structural diversity across . For instance, in Mycobacterium tuberculosis, the predominant , α-, methoxy-, and keto-mycolates predominate, influencing the overall composition of cord factor. Due to this heterogeneity in mycolic acid chains and functional groups, the molecular formula of cord factor is variable but can be approximated as C186_{186}H366_{366}O17_{17} for a representative structure, underscoring its classification as a high-molecular-weight with significant content.

Physical and Chemical Characteristics

Cord factor, also known as 6,6'-dimycolate (TDM), exhibits pronounced hydrophobicity primarily due to its long-chain moieties, which render it poorly soluble in but highly soluble in organic solvents such as and mixtures. This amphiphilic character arises from the polar headgroup contrasted against the nonpolar tails, enabling TDM to self-assemble in different environments. In aqueous media, TDM forms micelles with the hydrophilic oriented outward and hydrophobic mycolic acids sequestered internally, while on hydrophobic surfaces like air-water or oil-water interfaces, it organizes into rigid, crystalline that are more stable than those formed by other biological amphiphiles. These formations contribute significantly to the impermeability of the mycobacterial by creating a robust barrier against aqueous penetration. Under physiological conditions, TDM demonstrates high stability, including resistance to , as it requires harsh alkaline conditions to break down into two molecules and one unit. Furthermore, TDM plays a key role in constructing the asymmetrical outer bilayer of mycobacteria, where it intercalates with lipoarabinomannan to form a structured, impermeable layer that enhances overall integrity. Variations in chain lengths and unsaturations further modulate these biophysical properties.

Biosynthesis and Distribution

Biosynthetic Pathway

The biosynthesis of cord factor, also known as 6,6'-dimycolate (TDM), in mycobacteria commences with the production of , a essential for the glycolipid's structure. is primarily synthesized via the OtsA-OtsB pathway, where OtsA (-6-phosphate ) condenses UDP-glucose and glucose-6-phosphate to form trehalose-6-phosphate, which is then dephosphorylated by OtsB (-6-phosphate ). Mycobacteria also utilize the TreY/TreZ pathway, where the enzyme TreY (maltooligosyl ) catalyzes the conversion of the nonreducing terminal α-1,4-linked glucose residues in -like α-glucans into an α-1,1-linked moiety attached to a maltooligosyl chain, and TreZ (maltooligosyl trehalohydrolase) subsequently hydrolyzes the chain to release free . A third pathway involves TreS ( ) converting to . The OtsA-OtsB pathway predominates in mycobacteria, including , while TreY/TreZ is active under conditions where serves as a glucose reservoir, providing the trehalose backbone for TDM assembly. Parallel to trehalose synthesis, mycolic acids—the long-chain fatty acids characteristic of mycobacterial cell walls—are produced through a dedicated polyketide synthase (PKS) machinery. The process involves iterative elongation of meromycolic acid chains by the multifunctional , with activation of the meromycolic acid substrate occurring via FadD32, an acyl-AMP ligase that forms a high-energy acyl-adenylate (acyl-AMP) intermediate to facilitate chain condensation with an α-alkyl branch derived from shorter fatty acids. This activation step is critical, as FadD32 ensures the efficient incorporation of into glycolipids like TDM, and its disruption halts mycolic acid biosynthesis entirely. The assembly of TDM occurs through esterification of two mycolic acid molecules to the 6- and 6'-hydroxyl positions of , catalyzed primarily by the antigen 85 (Ag85) complex of mycolyltransferases (Ag85A, Ag85B, and Ag85C). The reaction proceeds in two stages: first, Ag85 transfers a from an activated donor (such as cell wall-bound mycolates) to the 6-position of , yielding trehalose monomycolate (TMM); second, another is transferred from TMM to the 6'-position of a second TMM molecule, forming the symmetric TDM. The Ag85 enzymes, belonging to the α/β family, utilize a catalytic serine residue to mediate these transacylation reactions in the periplasmic space. Prior to or concurrent with transfer, undergo modifications such as cyclopropanation by enzymes like MmaA4 (cyclopropanase), which introduces trans-cyclopropane rings to fine-tune TDM's immunostimulatory properties; mutants lacking MmaA4 (ΔmmaA4) produce TDM with altered composition, resulting in reduced levels of oxygenated distal modifications and diminished without substantially depleting overall TDM abundance. Genetic of TDM is linked to environmental stresses, particularly in virulent strains like , with adaptations in cell envelope remodeling under hypoxic conditions supporting and cord formation. This ensures adaptive changes in the mycobacterial , with ΔmmaA4 mutants exhibiting perturbed TDM profiles that impair survival in low-oxygen environments.

Occurrence in Mycobacteria

Cord factor, also known as 6,6'-dimycolate (TDM), is a major component of the mycobacterial cell envelope, present in the outer across all species of the genus Mycobacterium. In the pathogenic species , TDM is particularly abundant, representing the most prominent extractable and comprising approximately 2% of the total bacterial dry weight. This is essential for the virulent phenotype in laboratory strains such as H37Rv, where it facilitates the organized aggregation of into serpentine cords visible under , a trait linked to enhanced survival and in host environments. TDM is conserved in non-tuberculous mycobacteria (NTM), including opportunistic pathogens like Mycobacterium avium and , but exhibits species-specific variations in its mycolate moieties—the long-chain fatty acids esterified to the core. These mycolate profiles differ in chain length, branching, and functional groups such as cyclopropanes or hydroxyls, which modulate the glycolipid's ability to promote cord formation and influence overall envelope permeability. For instance, in M. avium, TDM contributes to biofilm-like structures and immune evasion, while in , the more oxygenated mycolates correlate with moderate cording and pulmonary pathogenicity, though less pronounced than in M. tuberculosis. In contrast, avirulent, saprophytic species such as produce TDM at notably lower levels compared to virulent counterparts under similar conditions. The shorter, less complex mycolic acids in M. smegmatis TDM prevent effective intermolecular interactions required for cord assembly, aligning with its non-pathogenic lifestyle and lack of tissue invasion potential. This reduced presence and structural deviation underscore TDM's role in species-specific adaptations within the mycobacterial phylogeny.

Pathogenic Roles

Virulence Mechanisms

Cord factor, also known as trehalose dimycolate (TDM), plays a critical role in the intracellular survival of Mycobacterium tuberculosis by interfering with phagosome maturation in host macrophages. Specifically, TDM inhibits the fusion of phagosomes containing the bacteria with lysosomes, preventing acidification and degradation of the pathogen within the phagolysosome. This mechanism allows M. tuberculosis to persist intracellularly, evading lysosomal killing and promoting bacterial replication inside the host cell. Studies have demonstrated that TDM disrupts normal trafficking events, including interactions with SNARE proteins, which are essential for phagosome-lysosome fusion. In animal models of , TDM contributes significantly to bacterial and survival. Mutants deficient in TDM production or modification exhibit markedly reduced pathogenicity. For instance, the M. tuberculosis ΔmmaA4 strain, which lacks proper oxygenation of mycolic acids incorporated into TDM, shows attenuated growth in lungs. This attenuation leads to prolonged host survival, with infected mice living up to 450 days versus 225 days for wild-type infections, highlighting TDM's essential role in establishing persistent infection. TDM also modulates host cell death pathways to favor bacterial persistence. By upregulating anti-apoptotic proteins such as in macrophages, TDM inhibits pro-inflammatory , which would otherwise promote and immune activation. This shift allows infected cells to survive longer, reducing immune clearance and enabling M. tuberculosis to replicate without triggering robust adaptive responses. Such modulation supports the pathogen's strategy to maintain a non-inflammatory environment conducive to chronic infection.

Cord Formation and Toxicity

Cord factor, or trehalose 6,6'-dimycolate (TDM), induces the characteristic serpentine cord structures in virulent mycobacteria through hydrophobic interactions between the long-chain moieties esterified to the core. These interactions dominate the molecular surface, with mycolic acids comprising approximately 70% of the exposed area, promoting linear aggregation of bacterial cells into stable, rope-like formations that enhance persistence in host environments. This cording is prominently observed in virulent strains such as H37Rv under nutrient-rich conditions, such as saline media, where TDM facilitates cell alignment and biofilm-like organization. In contrast, avirulent or saprophytic strains exhibit reduced cording due to alterations in TDM composition, underscoring its role as a determinant. In its configuration, cord factor exerts potent on host macrophages by disrupting mitochondrial function, leading to impaired electron transport and subsequent . This toxicity integrates TDM into cellular membranes, inducing swelling and loss of mitochondrial integrity, which compromises energy production and triggers necrotic pathways. The presentation is critical, as it exposes the hydrophobic mycolic tails to interact directly with host lipid bilayers, amplifying damage compared to micellar forms that are relatively inert. The dependency on mycolic acid chain length further modulates both cord stability and toxicity, with longer chains (>C60) in virulent mycobacteria enhancing these effects relative to shorter variants in saprophytes. Virulent strains feature mycolic acids of 60–90 carbons, often oxygenated, which promote tighter hydrophobic packing in TDM, stabilizing cord architectures and increasing membrane-disruptive potential against macrophages. Saprophytic species, with chains typically limited to 60–62 carbons, produce less stable cords and exhibit diminished toxicity, highlighting how chain elongation correlates with enhanced pathogenicity.

Host Immune Interactions

Recognition by Host Receptors

Cord factor, also known as trehalose-6,6'-dimycolate (TDM), is primarily recognized by the host immune system through the C-type lectin receptor Mincle (encoded by CLEC4E), a pattern recognition receptor expressed on myeloid cells such as macrophages and dendritic cells. Mincle directly binds the trehalose-mycolate motif of TDM with high affinity, enabling specific detection of this mycobacterial glycolipid on the bacterial cell surface. This interaction is calcium-dependent and involves the carbohydrate recognition domain of Mincle, where the trehalose head group coordinates with a conserved Ca2+ ion via an EPN motif, while the mycolate lipid tails engage hydrophobic pockets in the receptor. Upon binding TDM, Mincle associates with the γ-chain (FcRγ) to recruit and activate the spleen (SYK), initiating downstream signaling through the CARD9 adaptor protein and leading to and NFAT activation. This pathway is enhanced by cooperative interactions with other host receptors, including (TLR2), which senses mycobacterial lipoproteins and amplifies Mincle-mediated responses in macrophages, and DC-SIGN (CD209), which can facilitate TDM presentation on dendritic cells to promote SYK-dependent signaling. Such synergies ensure robust innate immune activation without relying solely on Mincle, particularly in diverse cellular contexts. The molecular details of Mincle-TDM recognition have been elucidated by crystal structures of Mincle ectodomains complexed with TDM analogs or related ligands, reported in 2013 studies, which highlight key residues like Arg183 for lipid anchoring and explain the receptor's specificity for trehalose-based glycolipids. Evolutionarily, Mincle is conserved across mammals, but human polymorphisms in CLEC4E, such as single nucleotide variants rs10841845 and rs10841847, modulate receptor function and are associated with altered susceptibility to tuberculosis, with certain alleles conferring protection against pulmonary infection in population studies. These genetic variations underscore Mincle's role in host-pathogen adaptation. This initial recognition by Mincle primes pathways that culminate in cytokine release, contributing to antimycobacterial defenses.

Cytokine Responses and Pathological Effects

Cord factor, or trehalose 6,6'-dimycolate (TDM), triggers rapid upregulation of pro-inflammatory in host cells following exposure. In murine macrophages, TNF-α expression increases approximately 3-fold within 2 hours of TDM stimulation and remains elevated at 24 hours, contributing to the initiation of inflammatory cascades. Similarly, IL-6 and IL-12 production is induced in macrophages and dendritic cells, peaking within the first 24 hours and promoting the recruitment of immune cells essential for formation. These drive the organization of structures in the lungs, mimicking early pathological responses in . In mouse models, TDM exposure leads to distinct pathological effects, including mediated by TNF-α, characterized by significant body and systemic wasting. occurs through of thymocytes, reducing size and impairing T-cell development. Neutrophilic is prominent in the lungs, with neutrophils enhancing formation and exacerbating tissue damage via Mincle-dependent signaling. Genome-wide analysis reveals 125 genes upregulated more than 1.5-fold (P < 0.05) at 2 hours post-exposure, expanding to 503 genes by 24 hours, reflecting a broadening inflammatory . TDM contributes to chronic inflammation in by sustaining cytokine-driven responses that perpetuate granulomatous . When injected intraperitoneally and intravenously in mice, purified TDM elicits lung inflammation, vascular occlusion, hemorrhage, and granuloma-like structures that closely resemble those in active , including hypercoagulopathy and long-term tissue remodeling. This model demonstrates TDM's potency, as low doses (10 μg) suffice to induce sustained leukocyte infiltration and elevated levels of IL-10, IL-12p40, and , underscoring its role in mimicking full outcomes.

Biomedical Applications

Research Tools

Cord factor, also known as trehalose 6,6'-dimycolate (TDM), serves as a key experimental reagent in laboratory research on mycobacterial interactions with host cells, particularly through its incorporation into model systems that replicate aspects of the bacterial . Researchers coat hydrophobic latex or polystyrene beads with purified TDM extracted from mycobacteria such as or M. tuberculosis to simulate the glycolipid-rich outer envelope, enabling controlled studies of , uptake, and dynamics in macrophages. For instance, TDM-coated beads delay phagosome maturation by retaining early endosomal markers like the while slowing acquisition of lysosomal markers such as LAMP1, mirroring the intracellular survival strategies of live mycobacteria. These bead models have been instrumental in dissecting receptor-mediated , revealing that TDM promotes Fcγ receptor engagement and Mincle-dependent uptake without altering surface marker expression on macrophages like MHCII or CD80. TDM can also be formulated into liposomes, forming stable vesicles that incorporate the alongside other mycobacterial lipids or phospholipids to more closely model the fluid mosaic structure of the mycomembrane. These TDM liposomes facilitate and uptake assays by providing a three-dimensional, curved surface that promotes interactions with host receptors, allowing quantitative assessment of internalization rates in cell lines such as RAW264.7 macrophages. Unlike rigid beads, liposomal models better capture the dynamic fluidity of mycobacterial envelopes, aiding investigations into how TDM influences membrane rigidity and endocytic trafficking during host-pathogen encounters. Synthetic analogs of TDM, featuring precisely defined mycolate chain lengths (e.g., C22-C26 saturated or branched variants), have been developed to probe ligand-receptor interactions in binding assays, offering greater control over structural variables than natural extracts. These analogs, such as dibehenate (TDB) or acylated esters with uniform acyl chains, bind avidly to the receptor Mincle on macrophages, with dissociation constants in the micromolar range, as determined by and reporter cell assays. Such tools have elucidated the minimal structural requirements for Mincle activation, showing that shorter mycolate chains reduce binding affinity while maintaining presentation. In applications, TDM-coated surfaces or clickable photocrosslinking probes enable the isolation and identification of host interacting proteins, with established protocols dating back to the early 2000s for bead-based pull-downs and evolving into advanced chemical by the 2010s. For example, TDM immobilized on beads or magnetic surfaces captures Mincle and associated adaptor proteins like FcRγ from lysates, followed by to map interaction networks; more recently, photoactivatable TDM mimics have identified SNARE proteins (e.g., VAMP3, syntaxin-6) as direct binders that regulate fusion. These methods, often using , have revealed over 800 differentially regulated host proteins in TDM-exposed cells, prioritizing those involved in endosomal trafficking and . TDM tools also briefly reference granuloma-like structures , linking protein isolations to downstream pathological effects.

Therapeutic and Diagnostic Uses

Cord factor, also known as trehalose 6,6'-dimycolate (TDM), has shown promise as an adjuvant in formulations due to its ability to enhance Th1 immune responses. In (TB) , synthetic analogues like trehalose 6,6'-dibehenate (TDB) incorporated into cationic liposomes (e.g., CAF01) stimulate robust Th1 and Th17 T cell responses when combined with subunit antigens such as H1, providing protection against challenge comparable to BCG in mouse models. This adjuvanticity is mediated through Mincle receptor activation and FcRγ–Syk–Card9 signaling, promoting antigen-specific IFN-γ production and lung-resident memory T cells. In BCG-based formulations, TDB boosts Th1/Th17 responses, improving overall against TB. For cancer applications, components of TDM induce Th1-biased responses with IL-12 and IFN-γ production in tumor vaccination models, suppressing tumor growth and enhancing cytotoxic T lymphocyte activity in preventive and therapeutic settings using ovalbumin antigens in mice. Clinical evaluation of TDM analogues in adjuvants like CAF01 has advanced to phase I trials for TB subunit post-2015, demonstrating strong Th1 responses with minimal adverse effects in humans. As a diagnostic marker, TDM and its mycolic acid constituents can be detected in using liquid chromatography- (LC-MS), offering a sensitive approach to identify M. tuberculosis . This method achieves 94% sensitivity and 93% specificity for s in adult TB samples, enabling distinction of virulent strains based on profiles such as alpha-, methoxy-, and keto- variants that differ across mycobacterial . Untargeted further reveals TDM-related biomarkers in clinical samples, supporting rapid TB diagnosis even in low-burden pediatric cases where is limited. Compared to acid-fast (AFB) , which has only 23% positivity in children and moderate sensitivity in adults, -based TDM detection provides superior accuracy without relying on bacterial morphology or viable counts. Therapeutic targeting of TDM focuses on disrupting its interactions to mitigate in TB. hybrids like isoniazid-nicotinic acid derivatives (e.g., INH-D2) inhibit TDM-induced by blocking Syk/PI3K pathways, reducing TNF-α, IL-6, infiltration, and hypoxia in models, thereby alleviating pulmonary damage. Prenylated from , such as sophoraflavanone G, act as Mincle-Syk-Erk inhibitors to suppress TDM-stimulated and production (e.g., TNF-α, IL-6, ), decreasing and recruitment and nearly eliminating at 200 mg/kg doses in mice. These approaches exploit TDM's role in excessive while preserving anti-mycobacterial immunity. For anti-cancer uses, TDM's induction of IL-12 and IFN-γ in tumors supports immune activation, as seen in models where adjuvants limit tumor progression through enhanced Th1 responses.

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