Virulence, is referred as the ability of a pathogen to cause disease, and for mycobacteria it depends on their ability to reside within host cells and evade the microbicidal mechanisms of macrophages. The outcome of tuberculosis (TB) infection is highly variable and it seems that the closest relationship between the Mycobacterium genre and humans has shaped the mycobacterial genome to encode bacterial factors that reflects a highly evolved and coordinated program of immune evasion strategies that interfere with both innate and adaptive immunity causing disease even in fully immunocompetent host. Although Mycobacterium tuberculosis (MTB) does not have classical virulence factors, it has described many virulence-associated genes and virulence lifestyle genes from Mycobacterium tuberculosis complex (MTBC). In this chapter, we describe the most important gene/molecule involved in the host defense modulation response, also the plethora of strategies to evade immune mechanisms of macrophage. We review the main genes whose inactivation in the mycobacterial genome leads to a measurable loss in virulence in the different validated TB models.
- virulence factor
- cell wall
- immune evasion
Tuberculosis (TB) is mostly caused by
2. Models for measuring MTB virulence
MTB virulence is studied in cell culture and animals models. Thus different parameters of pathogenicity according the TB models are used. A hallmark of MTB pathogenicity is the ability to infect and survive within macrophages; thus, primary macrophages and cell lines are used to analyze the virulence of MTB and mutants at the early stages of infection. Primary macrophages are more representative of the natural
3. Molecules involved in pathogenesis and virulence
3.1. Mycobacterial lipids
Mycobacterial cell wall is rich in lipids and has exceptional physico-chemical properties as a strong impermeability; and even it has peptidoglycan on it, mycobacteria are acid fastness organisms due to the large amount of lipids. In this section, we describe mycobacterial cell wall components and their relation with pathogenicity and virulence, the second part of the chapter is dedicated to study the phenomenon produced by some of the mycobacterial molecules.
3.1.1. Lipoarabinomannan (LAM)
Lipoarabinomannan (LAM) is a glycolipoconjugate composed by an anchor mannosyl phosphate inositol (MPI), a polysaccharide backbone and diverse capping motifs species . Correct translocation of LAM in to the cell wall constitutes an important feature for the mycobacterial stability, the lack of
3.1.2. Lipomannan (LM)
Lipomannan (LM) is a multiglycosylated lipid or polymannosylated Phosphatidylinositol mannoside (PIM). LAM and LM coexist in the mycobacterial cell wall. LM has been considered an innate immunity antigen; tetra-acylated LM activates macrophages using TLR2/TLR4 in a dependent way of MyD88. Di-acylated molecules regulate and inhibit the production of NO secretion and cytokine in macrophages activated by lipopolysaccharide (LPS) . LM purified from
3.1.3. Phosphatidylinositol mannosides (PIMs)
Phosphatidylinositol mannosides (PIMs) constitute a substantial component of the cell envelope, precursor of LAM and LM. PIM has a variable number of mannose units and acylation, virulent species have high order PIM (5 or 6 mannoses) that contribute to the uptake of macrophages by mannose receptor (MR); lower order PIM with few mannoses interact with DC-specific intercellular adhesion molecule-3-grabbing non-integrin DC-SIGN from DC . The acylation state of PIM can induce granuloma formation and cell recruiting in BCG infection; specifically PIM4, PIM6 were used, and the acyl chain was responsible for NKT recruitment . In contrast, glycolipids from MTB as PIM and ManLAM inhibited CD4+ T cell activation by interfering in the phosphorylation and T cell receptor signaling . Host inflammatory response such as TNF, IL-12p40 was inhibited by PIM in murine macrophages through CD14-dependent and CD14-independent mechanisms . PIM induced an increased presence in culture supernatants of alveolar epithelial cells (AEC) of the anti-inflammatory cytokine transforming growth factor beta (TGF-β) and a significant production of reactive oxygen species (ROS) . Diacyl-phosphatidylinositol dimannoside (Ac2PIM2), acylphosphatidylinositol hexamannoside (AcPIM6) and diacylphosphatidylinositol hexamannoside (Ac2PIM6) from virulent MTB stimulate and drive proliferation in bovine PBMC from
3.1.4. Trehalose-6,6´-dimycolate (TDM)
Trehalose-6,6´-dimycolate (TDM) also known as cord factor, is the most abundant and toxic lipid in the mycobacterial cell envelope. TDM is composed by two polar trehalose head group where two mycolic acids (MA) are esterified. MA variations constitute a strong determinant of the inflammatory response of TDM. TDM has biological functions, promoting angiogenesis , inhibits acidification of phagolysosome, prevents Ca2+ dependent phagosome-lysosome fusion and mycobacterial surface lipid removal, which increased trafficking of bacteria to the acidic compartments, causing 99% of killing in macrophages after 3 days of infection . TDM coating beads produce a delayed maturation of phagosomes characterized by a non-acidified and hydrolytically restricted phagosome . Cytokine production in macrophages exposed to TDM has been extensively described; there are a high diversity number of publications about it. The effect of these molecules has been correlated with the innate, early adaptive response (humoral and cellular immunity); to resume: cytokines as IFN-γ, TNF-α, IL-4, IL-6, IL-10; chemokines as MCP1, IL-8, are induced as response to exposition of TDM . Reduction in the expression of MHCII, CD1d, CD80, CD40 and CD96 in the surface of macrophages is induced by the exposure of the cells to TDM . Microspheres coated with TDM showed an increased expression of enzymes and matrix metalloproteinases; molecules associated with tissue remodeling and tissue destruction during caseating granulomas . The inflammatory profile induced by TDM has been related with granuloma development and maintenance, this phenomenon is related with TDM, and is dependent of TNF-α and IL6 expression; also C5a complement factor has been described as part of the granuloma maintenance microenvironment molecule .
3.1.5. Phthiocerol dimycocerosate (PDIM) and phenolic glycolipids (PGL)
Phthiocerol dimycocerosate (PDIM) and phenolic glycolipids (PGL) include a group of related cell wall lipids, non-covalently bounded to the mycobacterial surface. PDIM and PLG are major virulence factors of mycobacteria. PDIM and PGL are molecules required for bacterial duplication during the acute phase . PDIM is involved in mycobacterial resistance to detergents, and also is linked with the permeability and envelope solidity . PDIM is present in
3.2. Secretion systems in mycobacteria
Molecular migration across the mycobacterial cell wall, constitute an important event related with the environment and host cells interaction. Mycobacterial waxy cell envelope controls the molecular movement and the secretion of substances across this structure is dependent of specialized proteins systems, some of these protein structures will be described below.
3.2.1. The twin-arginine transporter (TAT transporter)
The twin-arginine transporter (TAT transporter) is located in the cytoplasmic membrane and transport folded proteins. This system is composed by three membrane proteins named as TATA, TATB and TATC.
3.2.2. The ESX transporter
The ESX transporter has no counterpart in LPS bacteria; it is located in the cytoplasmic membrane and exports and secretes proteins across the mycobacterial cell envelope. ESX genes are encoded at the genome and plasmids  and codify to for ESX type proteins EspA, EspB, EspC, EspG, etc, the secreted proteins ESAT-6 y CFP-10; PE-PPE; and the conserved components EccB, EccC, EccD and MycP . The ESX systems are named as ESX-1 to ESX-5, depending on the variation of the diverse systems and their components.
ESX-1 transporter system is important during mycobacterial infection in MTB and other pathogenic mycobacteria, in BCG the loss of the region of difference 1 (RD1) and the partial loss of the ESX-1 encoding region is related with the attenuation of the strain . ESX-1 allows cytosolic contact and mediates vacuoles rupture ; the protein intervenes in host cell lysis in a contact dependent way, producing gross membrane disruptions . DNA transfer through conjugation is also a function of ESX-1 system ; a phenomenon called “distributive conjugal transfer” that describes a genetic exchange between recipient and donor is dependent of ESX-1 in
ESX-3 is involved in Zn and Fe uptake. ESX-3 proteins: EsxG and EsxH are associated with the (proline-glutamic acid, proline-proline-glutamic acid) PE and PPE secretion . The EsxG and EsxH heterodimer, which harms macrophage phagosome maturation, is secreted by the ESX-3 system  and inhibits the endosomal-sorting complex required for transport (ESCRT) impairing MTB antigen-specific CD4+ activation by macrophages and DC . In
ESX-5 secretion system only present in slow-growing mycobacteria is linked to PPE and PE exportation and pathogenicity. The secretion mechanism of ESX-5 is activated in response to phosphate limitation through phosphate sensing of Pst/SenX3-RegX3 system . In MTB, disruption of ESX-5 showed a strong attenuation, failure in the cell wall integrity and the loss of the secretion of the PPE protein . ESX-5a region, from ESX5 is composed by duplicated genes and had been related with inflammasome activation . Also, mutations in the ESX-5 system components as
3.3. PE proteins: PE-PPE and PE-PGRS
The PE domain permits transportation of proteins, which share the domain. PE-PGRS and PE-PPE interacts with the TLR-2 on DC and macrophages, inducing: cytokine secretion, necrosis and apoptosis and enhance mycobacterial survival . PE-PGRS33 interaction with TLR mediates macrophage entry . PE-PGRS30 mutant showed an attenuated phenotype, specifically inhibits phagosome-lysosome fusion and showed decreased lung colonization and reduced tissue damage . PE-PGRS32 gene is highly conserved in MTB strains, because it has been related with mycobacterial survival in macrophages, persistence and replication . PPE10 has been described as an ESX-5 substrate in pathogenic mycobacteria; mutation of both of them reduced the envelope integrity and mycobacteria hydrophobicity . Co-localization of PE-PGRS33 in the host cell mitochondria induces cell death: necrosis and apoptosis . PE-PGRS47 disruption led to an
MTB genome analysis showed around 90 putative lipoproteins, most of them are part of the mycobacterial cell envelope and the plasma membrane; their function is related with molecular exportation, cell wall homeostasis and nutrient uptake; their presence contribute to host-pathogen interaction.
3.4.1. LpqH (19 kDa protein)
LppX is related with the release of complex lipids to the culture filtrate; LppX structure showed a large cavity that probably binds big motifs as the one present in PDIM. In a mice model, LppX-deficient mutant showed attenuation .
Mpt83 glycosylated lipoprotein related with host cell adhesion, is present in MTB and
LprG also known as P27 lipoprotein, is a ligand of TLR2; inhibits antigen processing in macrophages MHC II . It has a large cavity that binds triacylated agonist of TLR2: LM, LAM and PIM ; and determines LAM envelope localization and control phagosome-lysosome fusion . Expressed in an operon with Rv1410c, binds triacylglyceride (TAG) in the cavity and regulates TAG levels, growth rate and virulence . Involved in cell wall composition, MTB mutant deleted
RpfB multidomain lipoprotein related with resuscitation after mycobacterial dormant state drives in DC Th1-type immunity through interaction with TLR-4 .
LpqS MTB protein conserved in slow-growing pathogenic mycobacteria, is a protein related with survival during latency.
LprN lipoprotein related with cellular entry and survival, is part of the
Lprl lipoprotein present only in bacteria from MTBC, showed upregulation during mycobacterial macrophage infection. Lprl strongly attaches lysozyme, annulling completely their enzymatic activity. Lprl expression in
4. Immune system evasion
Unlike other pathogens, MTB infects and resides within immune cells, this bacterium has the ability to live within the dynamic and heterogeneous environment of macrophage phagosome. Here, the bacilli use a plethora of strategies to evade the microbicidal mechanisms of macrophage, including: phagosome-lysosome fusion, recruitment of hydrolytic lysosomal enzymes, production of reactive oxygen/nitrogen species, antigen presentation and apoptosis. Disruption of those functions in turn disrupts the adaptive immune response. Phagocytosis is an active process that depends on the interaction with various surface receptors expressed on the macrophage such as complement receptor type 3 (CR3), FCγ receptors and lectin receptors and it can be opsonic or non-opsonic. However, non-opsonic phagocytosis of MTB results in higher intracellular survival, although it is difficult to assess if the engagement of specific receptor determines the course of infection . MTB uses PDIM lipids to evade detection by TLRs, thereby preventing mycobacterial delivery into microbicide macrophages expressing iNOS . Moreover, MTB actively blocks the phagosome maturation by their cell wall components or through the secretion of various macromolecules that interferes with this process, which enables bacterial survival in a non-acidified intracellular compartment .
4.1. Phagosome arresting
PtpA and SapM are two phosphatases that contribute with the phagosome arresting. PtpA binds to subunit H of the vacuolar V-ATPase in order to dephosphorylate its substrate, the vacuolar protein sorting 33B (VPS33B) resulting in the exclusion of V-ATPase from mycobacterial phagosome thus inhibiting phagosome acidification . MTB mutant in PtpA was severely attenuated when infecting THP-1 cell line compared with wild type strain, these results show that PtpA is essential for mycobacteria survival within macrophage . SapM is a secretory phosphatase that dephosphorylates phosphatidylinositol 3-phosphate (PI3P) on the phagosome membrane . PI3P is essential for phagosomes to acquire lysosomal constituents; it is involved in the docking of rab effector proteins early endosomal autoantigen 1 (EEA1) and hepatocyte growth factor-regulated tyrosine kinase (HRS) substrate, which are important for phagosome maturation . Disruption of
Ndk is a nucleoside diphosphate kinase with ATP- and GTP-binding activity and it is widely conserved across all the three domains of life. This protein is autophosphorylated and secreted into the culture medium by MTB and possesses GAP activity towards Rho GTPases Rab5 and Rab7, leading to reduced phagolysosome fusion . Besides, Ndk also targets and inactivates the small GTPase Rac1, an essential component of the macrophage NADPH oxidase (NOX2) complex, inactivation of Rac1 was associated with reduced NOX2-mediated production of reactive oxygen species (ROS) and ROS-dependent apoptosis thus contributing significantly to mycobacterial virulence . Another factor crucial for inhibition of phagolysosome fusion is the serine/threonine protein kinase G (PknG). In contrast to other mycobacterial kinases, autophosphorylation on Thr residues at the N terminus of PknG are not involved in the regulation of this kinase; however, it is essential for the capacity of PknG to block lysosomal delivery of mycobacteria and for the bacterial survival in murine BMDM .
The PE_PGRS protein family includes around 60 proteins but the role and function of these proteins remains elusive. Nonetheless, PE-PGRS30 was the first PE-PGRS protein with a certain role in the virulence of MTB. PE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage; and inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the MTB mutant to inhibit phagosome-lysosome fusion .
Several factors have been implicated in phagosome maturation arrest, such as LAM, TDM, LpdC, Zmpq and the Esx-1 secretion system [27, 93, 94, 95]. But more importantly it seems that the requirement for these processes in the different mycobacterial species may not necessarily be identical; for example, BCG is able to arrest phagosome maturation in spite of the absence of RD1 locus, thus phagosome arresting in the case of BCG can be without ESAT-6 and CFP-10 although these proteins are necessary for this arrest in
4.2. Resistance to reactive oxygen and nitrogen species
Upon phagocytosis of mycobacteria, macrophages produce antimicrobial reactive oxygen and nitrogen species (ROS and RNS) via the enzymatic activity of NADPH oxidase (NOX2) and inducible nitric oxide synthase (iNOS), respectively. NOX2 is a multiprotein enzyme complex that assembles and activated in response to phagocytosis. This enzyme complex transfers electrons across the membrane from cytosolic NADPH to molecular oxygen, the reaction produce superoxide anions (O2−) which dismutates into hydrogen peroxide (H2O2) and generates toxic hydroxyl radicals . iNOS is induced upon IFN-γ activation and produces nitrite and nitrate via nitric oxide (NO), this reacts with O2− and forms peroxynitrite (OONO−) . This reactive oxygen and nitrogen intermediates (ROI and RNI) react with a wide range of molecules, such as nucleic acids, proteins, lipids and carbohydrates, thus for intracellular pathogens like MTB survival upon exposure to oxidative stress is critical.
Among the factors that contribute to MTB success as a pathogen are: its ability to survive the redox stress manifested by the host and its capacity synchronize its metabolic pathways and expression of virulence factors. Two component proteins, namely DosS and DosT, are employed by MTB to sense changes in oxygen, nitric oxide and carbon monoxide levels, while WhiB3 and anti-sigma factor RsrA are used to monitor changes in intracellular redox state [98, 99]. Using these and other unidentified redox sensors, mycobacterium orchestrates its metabolic pathways to survive in nutrient deficient, acidic, oxidative, nitrosative and hypoxic environments inside granulomas or infectious lesions . MTB employs versatile machinery of the mycothiol and thioredoxin systems to ensure a reductive intracellular environment for optimal functioning of its proteins even upon exposure to oxidative stress . Mycothiol is a low-molecular-weight thiol and functions like glutathione, the archetypal redox buffer, which is not produced by mycobacteria. Therefore it has antioxidant activity as well as the ability to detoxify a variety of toxic compounds. The thioredoxin (Trx) system is composed of NADPH, thioredoxin reductase (TrxR), and Trx is a small redox protein with two redox-active Cys residues in its active site . Trx is responsible for maintaining a reducing intracellular environment, regenerating the reduced forms of methionine sulfoxide reductase and peroxiredoxins, as well as the redox regulation of enzymes and regulatory proteins by oxidoreduction and the detoxification of ROS . MTB contains three types of Trx, although TrxB is the only one essential to fight against host defenses and for
Catalase peroxidases are enzymes that protect the bacterium from ROS damage and are used to detoxify H2O2, KatG from MTB degrades H2O2 and organic peroxides. Thus the major role of KatG in TB pathogenesis is to catabolize the peroxides generated by the phagocyte NADPH oxidase; although in the absence of this host antimicrobial mechanism, KatG is apparently dispensable . Moreover, KatG also activates the anti-tuberculosis drug isoniazid (INH) converting it to several reactive species that inhibits a mycolic acid biosynthesis . Although isoniazid resistance is multigenic, mutations in
AhpC is a member of the peroxiredoxin family that detoxifies organic peroxides into less reactive alcohol derivatives and confers protection against both oxidative and nitrosative stress. AhpC mutants show an essential role in the resistance to host oxidative agents in the early stages of infection . Besides, phenotypic
Peroxiredoxins (Prx) proteins are multifunctional antioxidant enzymes that reduce and thus detoxify hydroperoxides, organic hydroperoxides and peroxynitrite using electrons from Trx. Five Prx enzymes have been identified in MTB: AhpE, TPx, AhpC, Bcp and BcpB . Tpx is the principal and most effective enzyme involved in the detoxification of H2O2 and peroxynitrite in mycobacteria. In macrophages, the
Deletion of genes that encode methionine sulfoxide reductase (msrA), Mtb proteasome (prcBA), nucleotide excision repair (uvrB) and F-420 biosynthesis (fbiC) are also hyper-susceptible to RNS . Likewise, α-crystalline (HspX), bacterioferritin (bfrB) and the DosR regulon are upregulated by conditions that inhibit aerobic respiration; however, their role in MTB virulence is little understood .
4.3. Inhibition of apoptosis
During MTB infection, several forms of cellular fates have been observed such as necroptosis, apoptosis and autophagy, among those, apoptosis and autophagy have been recognized as innate macrophage defense mechanisms. Apoptosis is highly regulated process where the cytoplasm and other cellular organelles of dying cell are enclosed in membrane bound vesicles called apoptotic bodies. The apoptotic bodies are taken up by macrophages via receptor-mediated phagocytosis in a process defined as efferocytosis without elicit any inflammatory response .
Apoptosis reduces the viability of different mycobacterial species, including MTB; in fact many attenuated strains of mycobacteria induce more apoptosis than their wild type counterparts and exists a reciprocal relationship between virulence and apoptosis . MTB infection mainly results in necrosis, while attenuated mutant strains including BCG and H37Ra primarily induce apoptosis . Most of the factors that have been described as anti-apoptotic molecules play roles in the bacterial redox homeostasis (
The serine/threonine kinase E, pknE contributes to the survival response of MTB by regulating the bacilli machinery to resist apoptosis during nitrate stress. Deletion of
This work was financed by Universidad Técnica de Ambato. Ambato-Ecuador.