The scenario of chemical reactions prompted by the infection by Mycobacterium tuberculosis is huge. The infection generates a localized inflammatory response, with the recruitment of neutrophils, monocytes, and T-lymphocytes. Consequences of this immune reaction can be the eradication or containment of the infection, but these events can be deleterious to the host inasmuch as lung tissue can be destroyed. Indeed, a hallmark of tuberculosis (TB) is the formation of lung cavities, which increase disease development and transmission, as they are sites of high mycobacterial burden. Pulmonary cavitation is associated with antibiotic failure and the emergence of antibiotic resistance. For cavities to form, M. tuberculosis induces the overexpression of host proteases, like matrix metalloproteinases and cathepsin, which are secreted from monocyte-derived cells, neutrophils, and stromal cells. These proteases destroy the lung parenchyma, in particular the collagen constituent of the extracellular matrix (ECM). Namely, in an attempt to destroy infected cells, the immune reactions prompted by mycobacterial infections induce the destruction of vital regions of the lung, in a process that can become fatal. Here, we review structure and function of the main molecular actors of ECM degradation due to M. tuberculosis infection and the proposed mechanisms of tissue destruction, mainly attacking fibrillar collagen. Importantly, enzymes responsible for collagen destruction are emerging as key targets for adjunctive therapies to limit immunopathology in TB.

Introduction

Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis, is a global health threat, as it infects about one-third of the human population with ∼1.7 million people worldwide dying annually [1]. Currently available drugs are only partially effective, mainly due to the countless ability of M. tuberculosis to develop resistance [2]. Only ∼10% of infected people develop TB, because in most healthy individuals, the immune defense system is sufficient to prevent disease development [3].

The capacity of M. tuberculosis to cause disease is strongly connected with its ability to escape immune defense mechanisms [3]. Infection is initiated upon inhalation of droplets containing a few bacteria, which are internalized through phagocytosis by the alveolar macrophages, to form the phagosome. Then, activated alveolar macrophages transfer phagocytosed M. tuberculosis to the destructive environment of lysosomes [4]. However, M. tuberculosis has a great ability to survive in harsh conditions and has developed many tools to skip the lysosome. For instance, once inside phagosomes, M. tuberculosis secretes the phosphatase SapM and the serine/threonine kinase PknG to prevent phagosome–lysosome fusion [5]. In addition, M. tuberculosis is also able to replicate in host macrophages and to disseminate from this site of primary infection to potentially any organ, through its adhesin HBHA [610]. Even more, it can persist in the host for decades in a state of low metabolism, denoted as dormancy, and possibly reactivate through a mechanism mediated by resuscitation promoting factors [1125] and Ser/Thr kinases [2629].

M. tuberculosis infection of macrophages drives a localized inflammatory response with recruitment of neutrophils, and T-lymphocytes, organizing themselves into the structure of a granuloma (Figure 1) [30]. After the activation of T-lymphocytes, different types of cytokines are produced, mainly including interleukin 2 and interferon gamma. The pathophysiological hallmark of TB is caseous necrosis, with lesions appearing like cheesy material. Caseous necrosis is connected to another feature of pathogenesis, named as lung cavitation, which allows M. tuberculosis to leak into bronchial cavities and facilitate expectoration of bacilli. Indeed, disease development and transmission is strongly increased by the formation of lung cavities, as they are the site of very high mycobacterial burden [31]. However, despite the importance of cavitation in the development of the disease as well as in the generation of antimicrobial resistance, dissection of the precise sequence of events leading to cavitation is hitherto unknown.

Mycobacterium tuberculosis causes granuloma formation and matrix destruction.

Figure 1.
Mycobacterium tuberculosis causes granuloma formation and matrix destruction.

MMP activity results in matrix degradation with the release of matrix degradation products and erosion of the granuloma leading to dissemination of the mycobacteria.

Figure 1.
Mycobacterium tuberculosis causes granuloma formation and matrix destruction.

MMP activity results in matrix degradation with the release of matrix degradation products and erosion of the granuloma leading to dissemination of the mycobacteria.

Connective tissue fibers, especially collagen, maintain the normal arrangement of lung structure. Consistently, a typical lung cavity generated by M. tuberculosis consists of an external zone of collagen and internal part of softening caseum. An initial explanation of cavitation was liquefaction, whereby the region of caseous necrosis liquefies and develops into an environment with a high oxygen content, favoring the intense multiplication of tubercle bacilli [32] (Figure 1). However, the lipid contents of the internal caseum do not have enzymatic activity and are unable to break down the triple helical structure of collagen, which provides the tensile strength of the lung [32]. Therefore, cavitation must involve extracellular matrix (ECM) breakdown.

To accomplish this selfish plan of cavity formation, M. tuberculosis induces the overexpression of host proteases, like matrix metalloproteinases (MMPs) [33] and cathepsin [31]. Proteases, in particular MMPs, secreted from monocyte-derived cells, neutrophils, and stromal cells, are involved in both cell recruitment and tissue damage (Figure 1). This article reviews the complex scenario of molecular actors involved in ECM destruction, with a particular focus collagen structure and degradation and the critical role of enzymes responsible for collagen destruction, as they are emerging as key targets for adjunctive therapies to limit immunopathology in TB.

Molecular actors in TB cavitation

The ECM and the collagen fiber

Given the central role of ECM degradation of the alveolar wall for TB cavitation, it is important to describe its molecular features. ECM is a highly dynamic environment, as it constantly undergoes remodeling and degradation during vital physiological processes such as angiogenesis, wound healing, and development. The importance of the ECM is witnessed by the wide range of syndromes arising from genetic abnormalities in ECM proteins [34]. ECM is mainly composed of water, proteins, and polysaccharides, with a heterogeneous composition and topology, depending on the tissue. Specifically, it contains two main classes of macromolecules: proteoglycans and fibrous proteins (Figure 2) [34,35]. The main fibrous ECM proteins are collagens, elastins, fibronectins, and laminins, forming unique fibrous network providing stability and structural support to surrounding cells [36]. Of these proteins, collagen is the most abundant within the interstitial ECM and constitutes up to 30% of the total protein mass of a multicellular animal, as it has a multiplicity of roles, in providing tensile strength, regulating cell adhesion, supporting chemotaxis and migration, and directing tissue development [37]. There are at least 16 types of collagen, but 80–90% of the collagen in the body consists of types I, II, and III (Table 1), which are essential structural components of all connective tissues such as the cartilage, bone, skin, tendons, and ligaments [38,39].

The extra cellular matrix (ECM) is a complex and dynamic environment.

Figure 2.
The extra cellular matrix (ECM) is a complex and dynamic environment.

A simplified scheme showing main components of ECM.

Figure 2.
The extra cellular matrix (ECM) is a complex and dynamic environment.

A simplified scheme showing main components of ECM.

Table 1
Major collagen molecules
Type Molecule composition Structural features Representative tissues 
Fibrillar collagens 
 I [α1(I)]2[α2(I)] 300-nm-long fibrils Skin, tendon, bone, ligaments, dentin, interstitial tissues 
 II [α1(II)]3 300-nm-long fibrils Cartilage, vitreous humor 
 III [α1(III)]3 300-nm-long fibrils; often with type I Skin, muscle, blood vessels 
 V [α1(V)]3 390-nm-long fibrils with globular N-terminal domain; often with type I Similar to type I; also cell cultures, fetal tissues 
Fibril-associated collagens 
 VI [α1(VI)][α2(VI)] Lateral association with type I; periodic globular domains Most interstitial tissues 
 IX [α1(IX)][α2(IX)][α3(IX)] Lateral association with type II; N-terminal globular domain; bound glycosaminoglycan Cartilage, vitreous humor 
Sheet-forming collagens 
 IV [α1(IV)]2[α2(IV)] Two-dimensional network All basal laminaes 
Type Molecule composition Structural features Representative tissues 
Fibrillar collagens 
 I [α1(I)]2[α2(I)] 300-nm-long fibrils Skin, tendon, bone, ligaments, dentin, interstitial tissues 
 II [α1(II)]3 300-nm-long fibrils Cartilage, vitreous humor 
 III [α1(III)]3 300-nm-long fibrils; often with type I Skin, muscle, blood vessels 
 V [α1(V)]3 390-nm-long fibrils with globular N-terminal domain; often with type I Similar to type I; also cell cultures, fetal tissues 
Fibril-associated collagens 
 VI [α1(VI)][α2(VI)] Lateral association with type I; periodic globular domains Most interstitial tissues 
 IX [α1(IX)][α2(IX)][α3(IX)] Lateral association with type II; N-terminal globular domain; bound glycosaminoglycan Cartilage, vitreous humor 
Sheet-forming collagens 
 IV [α1(IV)]2[α2(IV)] Two-dimensional network All basal laminaes 

Collagen types I, II, III, and V form fibers, whereas type IV collagen forms a two-dimensional reticulum. In fibrous collagens, collagen molecules are hierarchically organized. Indeed, they form side-by-side interactions to pack together and form long thin fibrils of similar structure that are then assembled together to form larger collagen fibers (Figure 3). Collagen fibrils exhibit an axial stagger of ∼234 amino acid residues (∼67 nm), whereas the diameter of fibrils varies depending on the tissue, even for a specific type of collagen [40]. The precise properties of the different fibrous collagens are due to the different ability of the rod-like triple helices to form side-by-side interactions generating the fibrils.

The hierarchical structure of type I collagen fiber.

Figure 3.
The hierarchical structure of type I collagen fiber.

Collagen triple helices form side-by-side interactions to generate long thin fibrils, that are then assembled to form larger collagen fibers.

Figure 3.
The hierarchical structure of type I collagen fiber.

Collagen triple helices form side-by-side interactions to generate long thin fibrils, that are then assembled to form larger collagen fibers.

Molecular features of collagen

Type I collagen is the most abundant collagen of the human body. It is a heterotrimeric molecule composed of two α1 and one α2 left-handed polyproline II-like chains that are assembled into a right-handed supercoiled triple helix [4154]. Each chain contains precisely 1050 amino acids forming a characteristic right-handed triple helix. All collagens were shown to contain three-strands and form a similar triple helix; unique properties of each collagen type are due to segments that interrupt the triple helix, forming other three-dimensional structures [39].

Type I collagen is synthesized as procollagen, containing both amino-terminal and carboxyl-terminal pro-peptide sequences (Figure 4). Post-translational modifications of the procollagen polypeptide include hydroxylation, glycosylation, and disulfide-bond formation between the C-terminal pro-peptides of three procollagens. This latter modification aligns the chains in register and initiates the formation of the triple helix, in a zipper-like process toward the N-terminus. Once the triple helix is formed, pro-peptide sequences are proteolytically cleaved by proteases which recognize specific sequences in N- and C-terminal regions of collagen, denoted as telopeptides (Figure 4). These telopeptides, spanning from 9 to 25 residues, contain the unusual amino acid hydroxylysine and play a crucial role in directing fibrillogenesis by forming covalent cross-links between hydroxylysine residues of adjacent collagen molecules [55]. Indeed, it has been demonstrated that isolated N-terminal telopeptides accelerate the nucleation step in fibrillogenesis of intact or modified collagen lacking telopeptides [55,56].

Molecular features of procollagen.

Figure 4.
Molecular features of procollagen.

A drawing of the entire structure of premature type I collagen including the N- and C-terminal pro-peptides, the telopeptide regions, and the triple helical region.

Figure 4.
Molecular features of procollagen.

A drawing of the entire structure of premature type I collagen including the N- and C-terminal pro-peptides, the telopeptide regions, and the triple helical region.

The collagen triple helix structure at an atomic level

Collagen-like molecules have been found in lower eukaryotic, prokaryotic, and viral genomes [57,58]. The complexity of collagen molecule and its fibrous nature prevent detailed investigations on the full-length protein [43,59]. To overcome this limitation, many diversified strategies have been adopted [39,51,60,61]. Most of the available structural information on the triple helix has been essentially achieved through the characterization of peptide models in triple helix conformation [39,44,51,6267]. These studies showed that the three parallel polypeptide strands of each collagen molecule adopt a left-handed polyproline II-type (PPII) helical conformation and coil about each other with a one-residue stagger to form a right-handed triple helix (Figure 5A). The tight packing of PPII helices within the triple helix mandates that every third residue is Gly, resulting in a repeating X-Y-Gly sequence, where X and Y can be any amino acid. The Gly residue in the collagen repeating triples is also required for the formation of hydrogen bonds linking the backbone NH of glycine with a carbonyl group (C=O) in an adjacent polypeptide (Figure 5B). Although all types of amino acids may be located at positions X and Y of the triplets, they are frequently occupied by imino acids, (2S)-proline (Pro, 28%), and (2S,4R)-4-hydroxyproline (Hyp, 38%), respectively. The high prevalence of imino acids in collagen triplets, with Pro-Hyp-Gly as the most common (10.5%), is explained by the strong contribution that these residues give to the triple helix stability. The role of imino acids in triple helix stabilization has been the subject of intense research activities that has been reviewed and discussed in several literature reports [39,46,49,51,52,62,63,66,6873]. High-resolution studies have also shown (i) that the positions X and Y of the triple helix are not conformationally equivalent and (ii) triple helix geometrical parameters may locally change to generate helices with variable symmetries ranging from 75 or to 107 [39,44,51,54,62,63,67,7477,78].

Structure of the collagen triple helix.

Figure 5.
Structure of the collagen triple helix.

(A) Cartoon representation of the structure of the triple helical collagen-like peptide (Pro-Pro-Gly)10. (B) Stick representation showing the typical intermolecular hydrogen bonding interactions between the nitrogen of the Gly residue and the carbonyl group of the residue located in the X position of an adjacent chain. Further contribution to the triple helix stabilization is occasionally provided by intermolecular interactions established by specifics side chains [39,44,51,62,63,7981].

Figure 5.
Structure of the collagen triple helix.

(A) Cartoon representation of the structure of the triple helical collagen-like peptide (Pro-Pro-Gly)10. (B) Stick representation showing the typical intermolecular hydrogen bonding interactions between the nitrogen of the Gly residue and the carbonyl group of the residue located in the X position of an adjacent chain. Further contribution to the triple helix stabilization is occasionally provided by intermolecular interactions established by specifics side chains [39,44,51,62,63,7981].

Further insights from crystallographic structures of the collagen triple helix

The vital importance of collagen as a scaffold demands a pool of essential characteristics, including thermal stability, mechanical strength, and the ability to engage in specific interactions with other biomolecules. Importantly, such properties are crucial for the structural integrity of the collagen triple helix, as triple helix defects are associated with severe pathogeneses like osteogenesis imperfecta (OI) [82]. The most common mutations causing structural collagen defects are single-nucleotide variants, substituting the glycine within the Gly-X-Y repeat with a bulkier or charged residue. The determination of single crystal X-ray structures of collagen-like models have been a real breakthrough, since they also evidenced the structural effects of disease causative mutations, like the Gly-to-Ala mutation found in OI (Figure 6). Indeed, the X-ray structure of a peptide hosting this mutation showed a local untwisting of the triple helix occurring upon Gly mutation to alanine, where the characteristic hydrogen bonds of collagen triple helices are mediated by interstitial water molecules [45,49,5054,66,67,77,8390,91,92]. Usually, other layers of water molecules are bound to those of the first shell to generate complex networks that are able to connect different triple helices in the crystalline state. This high hydration has been identified as a reason for the stabilization of the triple helix induced by the presence of Hyp in the Y position. However, the initially proposed model based on an essential role of water molecules in stabilization [88,92] has progressively declined, as hypotheses relying on stereoelectronic effect [66,93] and/or intrinsic imino acid propensities have been proposed [52,53]. Studies carried out by using host–guest model polypeptides have led to the definition of a reliable scale of amino acid/imino acid propensities for collagen triple helix [39,80,94,95]. Furthermore, extensive analyses carried out on host–guest peptides containing proline derivatives have clearly demonstrated that the frequency and the location of specific diastereoisomer within the collagen sequence is related to the role played by these imino acids in triple helix stabilization [60].

Collagen triple helix and collagen receptors.

Figure 6.
Collagen triple helix and collagen receptors.

Four representative triple helix crystal structures, showing (A) the effects of Gly-Ala mutation, responsible for OI, on the collagen triple helix structure (PDB code: 1cgd), (B) a complex of the triple helix with a bacterial adhesin, CNA (PDB code: 2f6a), (C) with human Discoidin Domain Receptor 2 (PDB code: 2wuh) and (D) with Integrin Alpha2Beta1 (PDB code: 1dzi).

Figure 6.
Collagen triple helix and collagen receptors.

Four representative triple helix crystal structures, showing (A) the effects of Gly-Ala mutation, responsible for OI, on the collagen triple helix structure (PDB code: 1cgd), (B) a complex of the triple helix with a bacterial adhesin, CNA (PDB code: 2f6a), (C) with human Discoidin Domain Receptor 2 (PDB code: 2wuh) and (D) with Integrin Alpha2Beta1 (PDB code: 1dzi).

There are up to date ∼70 structures of collagen triple helices in the Protein Databank (PDB), including triple helices with different sequences and many structures of complexes between the triple helix peptides and their biological cognates. Indeed, the collagen triple helix is the target of several human receptors binding the ECM. Crystal structures have been determined for complexes of the triple helix with integrin [96,97], DDR2 discoidin domain [98], the Von Willebrand factor A3 domain [99], complement C1s, MASP-1 CUB2 domain (PDB code: 3pob) [100], matrix metalloproteinase MMP-1 and MMP-12 [33], and the chaperone Hsp47/SERPINH1 [101] (Figure 6). Playing such a central role, the collagen triple helix is also exploited by pathogens as a target in the host–pathogen interaction involving extracellular pathogens, such as enterococci, staphylococci, and streptococci [96,102106]. Adherence is mediated by protein adhesins of the MSCRAMM (microbial surface components recognizing adhesive matrix molecules) family. These adhesive proteins from Gram-positive bacteria do not share sequence homology with the collagen-binding I domains of integrins and do not require metal ions for collagen binding. Consistently, these proteins employ a collagen-binding mechanism that is drastically different from that of the collagen-binding integrins, since they do not recognize specific collagen sequences, but target the triple helix structure.

Interestingly, collagen-like triple helices have been identified also in prokaryotic genomes [107117], although prokaryotic collagens do not contain hydroxyproline, as bacteria lack the prolyl-hydroxylase enzyme necessary for post-translational modification of Pro to Hyp. Best-characterized prokaryotic collagens are the two collagen-like proteins, Scl1 and Scl2, which have been demonstrated to be simultaneously expressed on the cell surface of Streptococcus pyogenes and to be able to promote bacterial adhesion to the host [107,108,118120].

Structure and role of collagen proteases in collagen degradation governing TB cavitation

Molecular features of the MMPs

Collagen triple helix is extremely stable to degradation by most proteases. Among the few enzymes that are able to degrade the triple helix are MMPs, which are also known as matrixins. MMPs are a family of zinc-dependent proteases that can collectively degrade, in addition to collagen, all components of the ECM. Excessive MMP activity is implicated in diverse pulmonary pathologies characterized by ECM destruction. MMP-1, MMP-13, and MMP-14 are known to be up-regulated in rabbit cavitary tissue [121]. MMP-12 is highly expressed and up-regulated in cavity tissue, although the significance of this finding is unclear as MMP-12 is not induced in human TB [122]. However, despite the potentially key role of MMPs in lung matrix destruction in human TB, mechanisms resulting in tissue damage have not been defined.

Human MMP proteases to date are classified based on their preferred substrate and cellular localization (Table 2). Among MMPs, MMP-1, MMP-8, and MMP-13 are the classical collagenases, able to cleave interstitial fibrillar collagen. Apart from these enzymes, MMP-2 (gelatinase A) and MMP-14 are also able to initiate the breakdown of collagen fibrils. These collagenases cleave the triple helical collagen approximately three-quarters away from the N-terminus, resulting in three-quarters and one quarter length fragments that are unstable at body temperature and undergo denaturation, rendering them susceptible to other nonspecific tissue proteinases.

Table 2
Features of main human MMPs and available structural information in the PDB
Name Location Substrates/PFAM architecture PDB codes 
MMP1 Secreted Col I, II, III, VII, VIII, X, gelatin 1CGE 
MMP2 Secreted Gelatin, Col I, II, III, IV, Vii, X 1CK7 
MMP3 Secreted Col II, IV, IX, X, XI, gelatin IB3D 
MMP7 Secreted Fibronectin, laminin, Col IV, gelatin IMMP 
MMP8 Secreted Col I, II, III, VII, VIII, X, aggrecan, gelatin 2OY4 
MMP9 Secreted Gelatin, Col IV, V 5UE3 
MMP10 Secreted Col IV, laminin, fibronectin, elastin 1Q3A 
MMP11 Secreted Col IV, fibronectin, laminin, aggrecan NA 
MMP12 Secreted Elastin, fibronectin, Col IV 1JIZ 
MMP13 Secreted Col I, II, III, IV, IX, X, XIV, gelatin 4FU4 
MMP14 Membrane Gelatin, fibronectin, laminin 1BQQ, 3C7X 
MMP15 Membrane Gelatin, fibronectin, laminin NA 
MMP16 Membrane Gelatin, fibronectin, laminin 1RM8 
Name Location Substrates/PFAM architecture PDB codes 
MMP1 Secreted Col I, II, III, VII, VIII, X, gelatin 1CGE 
MMP2 Secreted Gelatin, Col I, II, III, IV, Vii, X 1CK7 
MMP3 Secreted Col II, IV, IX, X, XI, gelatin IB3D 
MMP7 Secreted Fibronectin, laminin, Col IV, gelatin IMMP 
MMP8 Secreted Col I, II, III, VII, VIII, X, aggrecan, gelatin 2OY4 
MMP9 Secreted Gelatin, Col IV, V 5UE3 
MMP10 Secreted Col IV, laminin, fibronectin, elastin 1Q3A 
MMP11 Secreted Col IV, fibronectin, laminin, aggrecan NA 
MMP12 Secreted Elastin, fibronectin, Col IV 1JIZ 
MMP13 Secreted Col I, II, III, IV, IX, X, XIV, gelatin 4FU4 
MMP14 Membrane Gelatin, fibronectin, laminin 1BQQ, 3C7X 
MMP15 Membrane Gelatin, fibronectin, laminin NA 
MMP16 Membrane Gelatin, fibronectin, laminin 1RM8 

Under normal physiological conditions, MMP activity is tightly regulated at various stages: during transcription, proteolytic processing of their inactive pro forms, zymogens, as well as by inhibition of enzyme activity by endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs). MMPs share a common domain architecture, including a pro-peptide, a catalytic domain, and the haemopexin-like C-terminal domain, which is linked to the catalytic domain by a flexible hinge region (Figure 7).

A collection of human MMP proteases.

Figure 7.
A collection of human MMP proteases.

Domain organization of main MMPs according to the PFAM database.

Figure 7.
A collection of human MMP proteases.

Domain organization of main MMPs according to the PFAM database.

They are all synthesized as inactive zymogens, as the pro-peptide must be removed to activate the enzyme. The pro-peptide contains a conserved cysteine residue that interacts with the zinc ion in the active site and prevents binding and cleavage of the substrate. X-ray crystallographic structures of several MMP catalytic domains have been determined so far (Table 2).

The structure of the catalytic domain is conserved among known MMPs. This metalloproteinase domain is ∼160 amino acid residues in length with the catalytic zinc ion residing in the C-terminal segment of this domain. The active site is a large groove, which hosts three histidine residues (found in the conserved sequence HExxHxxGxxH) that bind a catalytically important Zn2+ ion (Figure 8). The catalytic domain is connected to the C-terminal domain by a flexible hinge or linker region of up to 75 amino acids long (Figure 8). The proteolytic activity of the enzyme resides in the catalytic domain, but it requires the C-terminal domain, to cleave the three chains of the triple helical collagen. Indeed, although the catalytic domain can also cleave many noncollagenous proteins, its activity on native triple helical collagen is negligible in the absence of the C-terminal domain. This domain, structurally similar to the serum protein hemopexin, forms a β-propeller structure with four blades linked by disulfide bonds between blade I and blade IV (Figure 8). Interestingly, crystal structures of all catalytic domains so far determined show a peculiar feature: The catalytic cleft is too narrow to accommodate a triple helical collagen molecule. Therefore, MMP-1 must unwind triple helical collagen locally before peptide bond hydrolysis.

Molecular structure of MMP1.

Figure 8.
Molecular structure of MMP1.

(A) Ribbon representation of active MMP1 (PDB code: 1cge) including the catalytic (orange) and the HPX (purple) domains. (B) A detail of the catalytic site showing complexation of a zinc ion by the three catalytic histidines.

Figure 8.
Molecular structure of MMP1.

(A) Ribbon representation of active MMP1 (PDB code: 1cge) including the catalytic (orange) and the HPX (purple) domains. (B) A detail of the catalytic site showing complexation of a zinc ion by the three catalytic histidines.

Mechanism of collagen triple helix engagement and distortion by MMPs

How collagenolytic MMPs might unwind and digest the triple helix continues to be an intriguing open question, albeit hypotheses of mechanical manipulations that could separate the chains twisted together have been proposed. The crystal structure of human MMP-1 in complex with a collagen triple helical peptide shows extensive interaction of all three collagen chains with both the catalytic and the C-terminal HPX domain. This feature explains the strict requirement of the HPX domain for efficient collagenolysis [33]. In the crystal structure, the collagen peptide is an uninterrupted triple helix ∼115 Å in length (Figure 9) bent by ∼10° near the Cat–HPX junction, whereas the MMP-1 structure adopts a more closed conformation compared with free MMP1. This feature, together with the interdomain flexibility of MMP-1 demonstrated by NMR studies [123], suggested that by clamping both the catalytic site cleft and the HPX domains, MMP-1 bends and unfolds the collagen triple helix [33]. Interestingly, MMP-1, even inactivated, is capable of presenting a collagen triple helix sufficiently unwound to another MMP or non-collagenolytic protease for successful digestion.

Molecular interaction of the collagen triple helix with MMP1 domains.

Figure 9.
Molecular interaction of the collagen triple helix with MMP1 domains.

(Top) Ribbon representation of the structure of the complex between MMP1 and the collagen triple helix peptide (PDB code: 2mqs). Arrows highlight the positions of the catalytic site and of the scissible bond interacting with the HPX domain, located ∼25 Å from one another. Zinc ions are drawn in blue. (Bottom) A possible mechanism of triple helix hydrolysis by MMPs, upon distortion of the triple helix at the scissible bond due to the interactions with the HPX domain and sandwiching of MMP-1 to bring the catalytic site (blue) on top of the scissible bond (arrow).

Figure 9.
Molecular interaction of the collagen triple helix with MMP1 domains.

(Top) Ribbon representation of the structure of the complex between MMP1 and the collagen triple helix peptide (PDB code: 2mqs). Arrows highlight the positions of the catalytic site and of the scissible bond interacting with the HPX domain, located ∼25 Å from one another. Zinc ions are drawn in blue. (Bottom) A possible mechanism of triple helix hydrolysis by MMPs, upon distortion of the triple helix at the scissible bond due to the interactions with the HPX domain and sandwiching of MMP-1 to bring the catalytic site (blue) on top of the scissible bond (arrow).

The next question is, however, how does the catalytic site reach the scissible bond of the collagen triple helix, which is located close to the HPX domain and ∼25 Å from MMP-1 catalytic site? The side-by-side organization of MMP-1 catalytic and HPX domains in the crystal structure is in apparent contrast with a sandwich organization of catalytic and HPX domain on the triple helix (Figure 9) [124]. However, paramagnetic NMR studies have suggested the possibility that the catalytic domain folds over the scissile bond region bound near the HPX to sandwich the triple helix between these domains (Figure 9) [124]. Consistently, the long and flexible linker between the catalytic and Hpx domains (Figure 9) suggests that they are potentially free enough to reorient between the side-by-side orientations observed for MMP-1 and a sandwich arrangement. Interestingly, the HPX domain is absent in MMP-7, MMP-23, and MMP-26 (Figure 7), this indicating that the mechanism of collagen triple helix recruitment prior to hydrolysis is not shared by all MMPs.

Cathepsin K in cavitary collagenolysis

Apart from MMPs, other type I collagenases were found to be strongly associated with TB pathology. Using a rabbit model, most abundantly expressed proteins in cavity tissues were identified as (i) cathepsin K (CTSK), which can cleave both the alpha-1 and alpha-2 subunits of type I collagen; (ii) mast cell chymase-1 (CMA1), which cleaves procollagen during the initiation of collagen fibril synthesis, and (iii) MMP-1, MMP-13, and MMP-14. These findings suggested a previously unidentified role for non-MMP collagenolysis in active TB, implicating CTSK [31]. Notably, the levels of expression of CTSK were found to be higher than those of MMP-1, with elevated levels not only on the lesions but also in the plasma in active pulmonary TB [31]. These findings suggest that collagen degradation mediated by CTSK plays an important role in cavity formation, and confirm that collagen destruction is the key mechanism underlying cavity formation [31].

Human CTSK is a unique papain-like cysteine protease, as it is the sole to be able to cleave the collagen triple helix at multiple sites [125]. Indeed, it is the major collagen enzyme responsible for osteoclast-mediated collagen breakdown in bones and a key pharmaceutical target for the treatment of osteoporosis. Indeed, excessive CTSK activity in humans is associated with osteoporosis, arthritis, and bone cancers [126]. On the other hand, CTSK deficiency leads to a skeletal dysplasia characterized by dwarfism and osteosclerosis, denominated pycnodysostosis [127,128]. In this disease, osteoclasts and fibroblasts have been shown to accumulate undigested collagen fibrils in endosomal and lysosomal compartments [127].

CTSK is synthesized as an inactive proenzyme containing a 15-amino acid signal sequence, a 99-amino acid pro-peptide, and the catalytic domain (Figure 10A). The pro-peptide contains a conserved N-glycosylation site, which may target the inactive proenzyme to lysosomes via the mannose 6-phosphate receptor pathway. The catalytic domain is structurally formed by two domains, a left domain (L) and a right domain (R), linked by an interdomain two-strand beta sheet. This central domain creates a V-shaped cleft containing the catalytic residues (Cys25 and His159) (Figure 10B). These structural features are widely conserved among all papain-like cysteine proteases [129] and do not provide mechanistic insights into the collagenolytic activity of CTSK. In addition, the crystal structure of CTSK has evidenced that, similar to MMPs, the catalytic cleft of the enzyme, ∼5 Å wide, is not compatible with the collagenolytic activity of the enzyme, because of the larger dimension of the triple helical collagen molecules (∼15-Å wide). However, different from MMPs, CTSK lacks any protein domains implicated in collagen unwinding or specific binding. This feature has stimulated several studies to understand the mechanism of collagen fiber degradation by CTSK [130].

Structural organization of the CTSK protease.

Figure 10.
Structural organization of the CTSK protease.

(A) Schematic representations CTSK domains, as defined by the PFAM database. (B) Cartoon representation of the crystal structure of CTSK (PDB code: 3C9E). The catalytic Cys25 is highlighted by an arrow.

Figure 10.
Structural organization of the CTSK protease.

(A) Schematic representations CTSK domains, as defined by the PFAM database. (B) Cartoon representation of the crystal structure of CTSK (PDB code: 3C9E). The catalytic Cys25 is highlighted by an arrow.

Mechanism of collagen triple helix engagement and distortion by cathepsin K

An important information for the currently believed mechanism of triple helix engagement and distortion by CTSK is that the catalytic activity of CTSK on fiber collagen requires the presence of glycosaminoglycans (GAGs) with which it forms high molecular mass complexes [131,132]. Without GAGs, CTSK only works as a gelatinase and telopeptide cleaving activity. Also, a key achievement has been the crystal structure of a complex between an inactive mutant of CTSK and chondroitin 4-sulfate (C4-S), showing the binding of multiple CTSK molecules to a single GAG chain, denoted by the authors as ‘beads-on-a-strand’ [133]. This observation has suggested that the collagenase activity of CTSK requires more copies of the enzyme [133]. Combining X-ray crystallography, molecular modeling, mutagenesis, and electron microscopy, a mechanism of collagen fiber degradation by CTSK was identified [130]. Specifically, CTSK was crystallized in the presence of either C4-S or dermatan sulfate, the most common GAGs associated with collagen fibers in bone. These studies evidenced two important GAG-binding regions, rich in positively charged residues, formed by residues that are unique to CTSK among cysteine cathepsins (K119, K122, R123, R127, and K40, K41, R108, R111, K214). Importantly, the interactions of the GAGs with CTSK in these structures revealed a dimeric organization of the enzyme held together by protein–protein as well as protein–GAG contacts. Docking of CTSK dimer–GAG complex interactions with the collagen suggested that specific interactions with protein regions and with the GAG molecule play a role in collagen unwinding. Finally, scanning electron microscopy demonstrated that CTSK molecules localized at the gap regions of fiber collagen. This was confirmed by Edman degradation, showing that CTSK initial cleavage sites are located at the N-terminal region of tropocollagen molecules [130]. Based on these results, the current hypothesis for collagen fiber hydrolysis by CTSK involves the recruitment of a GAG molecule by a collagen triple helix, followed by the binding to a CTSK dimer (Figure 11). The collagen-binding event to CTSK dimers distorts the triple helices at the N-terminal side of tropocollagen molecules, thus allowing for proteolytic action by CTSK [130].

Mechanism of triple helix degradation by CTSK.

Figure 11.
Mechanism of triple helix degradation by CTSK.

(Left) A triple helix binds a GAG molecule. This complex binds two CTSK molecules through positively charged patches, the GAG-binding regions. This binding distorts the triple helix and makes it prone to be hydrolyzed by CTSK.

Figure 11.
Mechanism of triple helix degradation by CTSK.

(Left) A triple helix binds a GAG molecule. This complex binds two CTSK molecules through positively charged patches, the GAG-binding regions. This binding distorts the triple helix and makes it prone to be hydrolyzed by CTSK.

Enzymes responsible for collagen destruction as promising targets for adjunctive TB therapies

The development of new or re-purposed drugs for TB treatment is needed to accomplish the Sustainable Development Goals, which aims to reduce 90% of TB incidence rate by 2030 [1]. Enzymes responsible for collagen destruction are emerging as key targets for adjunctive therapies to limit immunopathology in TB. Inhibition of MMPs using the broad-spectrum MMP inhibitor Marimastat was shown to reduce both granuloma formation and bacterial load in the Mtb-infected tissue model [134]. In addition, both Marimastat and other MMP inhibitors specific for MMP9 or MMP2/MMP9 are able to increase the killing activity of the frontline anti-TB drugs isoniazid (INH) and rifampicin (RIF) [135]. These studies also showed that treatment with MMP inhibitors impacts granuloma morphology and stabilizes the blood vessels that irrigate the infection site. Consistently, the combination of either INH or RIF with MMP inhibitors increases drug delivery and/or retention in the lung, resulting in increased drug efficacy. Altogether, these studies underline the importance of exploiting strategies to improve the efficacy of existing drugs by the addition of collagenase inhibitors to current multi-drug regimens. Further studies aimed at improving efficacy and specificity of collagenase inhibitors would greatly contribute to the development of efficient TB regimens.

Conclusion perspective

In recent years, extensive studies were performed, aimed at the understanding of biochemical steps involved in the process of collagen degradation responsible for lung cavity formation due to M. tuberculosis infection. To this aim, important contributions have come from the structural determination of all players involved in this process.

The collagen triple helix has been the object of decades of studies, as its structure could not be derived by fiber diffraction studies. The impact of structural studies of collagen models has been impressive. The crystal structure determination of several models has been obtained, which have unambiguously shown structural features of the triple helix and its triple helical symmetry. Also, studies of host guest peptides have allowed the identification of determinants of triple helix stability, an important characteristic of collagen molecules, which confer tensile strength. Consistently, distortions of the triple helix structure due to single-point mutations have been associated to important diseases, like osteogenesis imperfecta. In M. tuberculosis, the process of collagen triple helix degradation to form cavities is still highly debated. After tons of theories, it appears clear that this process is due to MMPs and the cathepsin CTSK, which are secreted from monocyte-derived cells, neutrophils, and stromal cells. The current knowledge of mechanisms of collagen unwinding and degradation by these proteases, described here, have a great potential for the setup of novel therapies. Indeed, all recent studies have demonstrated that the elucidation of chemical processes involved in collagen degradation represents a key strategic point to develop protease inhibitors for adjunctive therapies to limit immunopathology in TB. Strategies targeting patients with cavitary TB have the potential to improve cure rates and reduce disease transmission.

Abbreviations

     
  • C4-S

    chondroitin 4-sulfate

  •  
  • CTSK

    cathepsin K

  •  
  • ECM

    extracellular matrix

  •  
  • GAGs

    glycosaminoglycans

  •  
  • INH

    isoniazid

  •  
  • MMPs

    matrix metalloproteinases

  •  
  • OI

    osteogenesis imperfecta

  •  
  • PDB

    Protein Databank

  •  
  • PPII

    polyproline II-type

  •  
  • RIF

    rifampicin

  •  
  • TB

    tuberculosis

Acknowledgments

All the authors acknowledge the MRC project MR/R000255/1 and FEP Regione Campania 2007–2013 Misura 3.5 and the COST action CA16231, ENOVA - European Network of Vaccine Adjuvants.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors equally contributed to this work.