The definition of virulence of M. tubercu-losis is not obvious. We believe that viru-lence of M. tuberculosis is related to fac-tors that are crucial for the progression of the disease. Since R. Koch’s publica-tions, it was established that M. tubercu-losis does not secrete toxins. In contrast, M. tuberculosis expresses and secretes several other factors, which are involved in its virulence. The characterization of these factors, generally related to the cell envelope, may be instrumental in the fight against M. tuberculosis. Accord-ingly, these virulence factors are often recognised by the host immune system.
Therefore, their studies may provide op-portunities for a better understanding of the TB pathogenesis. From the immu-nologist’s point of view, secreted viru-lence factors (or those associated with the envelope) are of particular interest, thanks to their ready accessibility to the host immune system. In contrast, the other virulence factors of M. tuberculosis represent less obvious immunological targets. Main virulence factors are sum-marized in the table below (table n° - adapted from reference (44)).
Heparin-binding hemagglutinin (HBHA)
In 1996, Menozzi et al. published the first description of the heparin-bind-ing hemagglutinin (HBHA) (45). This 198 amino-acid residues long protein (8-kDa) mediates the binding of my-cobacteria to epithelial cells, but not to macrophages. HBHA is also able to promote agglutination of rabbit eryth-rocytes, and these properties of agglu-tination and binding to epithelial cells are inhibited by the presence of heparin (sulfated carbohydrates). These later characteristics explain the acronym of HBHA. HBHA is expressed at the surface of M. tuberculosis (figure 9.A.) and M.
bovis BCG where it mediates autologous aggregations, facilitates epithelial colo-nization and finally invasion of alveo-lar epithelial cells by interactions with heparan sulphate proteoglycans (16, 45). HBHA was shown to be involved in extra-pulmonary dissemination. Disrup-tion of the HBHA-encoding gene leads to a significant decrease of the spleen colonization but not of the lung coloni-zation in mice nasally infected with M.
tuberculosis (1). This fact also under-lines the importance of the interaction of M. tuberculosis with non-phagocytic cells in the infectious process, from early seeding to the establishment of latency.
Interactions with sulphated proteogly-cans are not specific for M. tuberculosis.
The use of such ligands to mediate in-vasion of host cells has been demon-strated for several pathogens, including viruses (i.e. human immunodeficiency virus, herpes simplex virus), parasites (i.e. Trypanosoma cruzi) and bacteria (i.e.
Bordetella pertussis, Borrelia burgdorferi and Neisseria gonorrheae) (46).
Table n°2 – Brief overview of main virulence factors Transcriptional regulators
Sigma factors (sigA, sigF, sigE, sigH)
The principal sigma factors is sigA which is necessary for most housekeeping gene transcription. SigF, sigE and sigH are involved in resistance to environmental and oxidative stress.
Response regulators (PhoP, prrA, mprA) PhoP senses Mg++ starvation and could control expression of virulence genes.
Transcriptional regulators (hspR, ideR) hspR is a repressor of key heat-shock genes and ideR is the major regulator of iron uptake and storage genes
Enzymes involved in general cellular metabolism
Amino acid and purine biosynthetic genes (LeuD, TrpD, ProC, PurC)
These enzymes are involved in leucine (LeuD), tryptophan (TrpD), proline (ProC) or purine (PurC) biosynthetic pathways.
Metal uptake
(MgtC, MbtB, nitrate reductase)
MgtC is involved in Mg++ uptake. MbtB encodes for major siderophores and nitrate reductase plays a major role in respiration in the absence of oxygen contributing to the microaerophic capacities of M.
tuberculosis.
Lipid and Fatty acid metabolism (Icl, LipF, phospholipases C)
These enzymes allow M. tuberculosis to exploit resources of fatty acids available in the host environment.
Stress response proteins (KatG, AhpC, SodA, SodC)
KatG and AhpC are catalases that inactivates H2O2 and organic (hydro-) peroxides. KatG is also involved in the activation of the prodrug isoniazid to forme reactive species. Sod molecules are superoxyde dismutases involved in the resistance to respiratory burst produced by phagocytic cells.
Cell envelope function or secretion (CFPs, HspX, ESAT-6, CFP-10, 19 kD-protein, HBHA, Ag85 complex, etc…)
These proteins are exposed to the environment in which M. tuberculosis grows. For the majority, exact functions remain to be determined, other play a role in adhesion (i.e. HBHA) or during latency (i.e. HspX).
Cell surfaces components
(Erp, Mas, FadD26, FadD28, MmpL7, FbpA, MmaA4, PcaA, Lipoarabinomannan)
Some of these components are unique to pathogenic mycobacteria (i.e. Erp, LAM) and therefore constitute excellent targets in the study of M. tuberculosis virulence.
In addition, proteoglycans are ubiqui-tous and expressed by virtually all types of epithelial cells (45, 46).
HBHA is anchored in the outer lipid layer of the mycobacterial surface probably via its hydrophobic N-terminal putative transmembrane domain of 18 amino-acids (47, 48). The external part of HBHA is the C-terminal domain, which is char-acterised by Lys-Pro-Ala-rich repeats and that specifically mediates binding to proteoglycans (figure 9.B.). Between these extremities, there is an α-helical coiled-coil region of 81 AA that medi-ates auto-oligomerization of the HBHA and therefore facilitates mycobacterial aggregation and colonization (48). Ex-pression of surface molecules exposes them to the risk of destruction by pro-teases naturally secreted by the host.
Moreover, in the case of HBHA, its C-ter-minal domain is rich in lysine residues that render it prone to proteolytic deg-radation (49). To avoid destruction of a strategic factor involved in the extrapul-monary dissemination of M. tuberculosis (1), the C-terminal domain of the HBHA is protected by post-translational
meth-ylation of the lysines (50) (figure 9.B.).
Methylation of these lysine residues is a complex event resulting in mono- and di-methylation of some lysines, where-as others remain unmethylated. This process is mediated by one or several methyltransferase(s) produced by the mycobacteria itself. These methyltrans-ferases remain to be identified. In vivo, the methylation appears to be highly dependent on the presence of the trans-membrane domain of HBHA (49, 51).
The methylation of the C-terminal do-main is also important to elicit protec-tive immune responses, as shown both in humans and mice. In humans, only the native, methylated form of HBHA (nHBHA) induces strong interferon-gam-ma (IFN-γ) responses by the peripheral blood mononuclear cells (PBMC) from subjects with latent TB infection (LTBI) (5). The IFN-γ is a Th1 cytokine strongly involved in the protection against infec-tion with M. tuberculosis (5). In contrast, the recombinant form of HBHA (rHBHA) produced by genetically modified E. coli is not methylated because E. coli lacks methyltransferase activity (figure 9.B.)
HBHA KKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVYQK ofM. tuberculosis.This picture represents M. tuberculosis H7Ra after incubation with mAbs that specifically recognize HBHA followed by gold-labelled goat anti-mouse immunoglobulin (x61.00).
Reproduced from Menozzi FD, Rouse JH, Alavi M, Laude-Sharp M, Muller J, Bischoff R, Brennan MJ, Locht C. Identification of a heparin-binding hemagglutinin present in mycobacteria. The Journal of experimental medicine 1996;184:99-1001; Fig 9.B.
Methylation profile of HBHA.Lysines of the C-terminal domain of nHBHA are methylated. In contrast the recombinant HBHA, produced by genetically modified E. coli is not methylated. Adapted from Locht C, Hougardy JM, Place S, Rouanet C, F Mascart. Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis. Tuberculosis 006;86:0-09
(47). The rHBHA is neither able to induce strong IFN-γ secretions by the PBMC from LTBI subjects, nor to afford protec-tion after vaccinaprotec-tion in mice. In con-trast, vaccination with nHBHA confers a level of protection similar to that ob-tained by vaccination with M. bovis BCG (54). These observations support nHBHA as a serious candidate to take in account for future vaccine design against TB.
HBHA-like proteins are also expressed by mycobacteria other than M. tubercu-losis or M. bovis BCG, such as M. avium, M. intracellulare and M. smegmatis. How-ever, these molecules have slightly dif-ferent sequences and display difdif-ferent molecular weights compared to nHBHA expressed by M. tuberculosis (48). For instance, the M. smegmatis HBHA-like protein presents 68 % of of amino acid sequence identity with nHBHA from M.
tuberculosis. Moreover, this protein does not play a role in epithelial adherence, its functions remaining unknown so far (55)
Early-Secreted-Antigenic-Target-6 kDa and Culture-Filtrate-Protein-10 kDa
Culture filtrate proteins (CFP) are found in the culture medium in which M. tu-berculosis has grown. These molecules often induce important immune re-sponses, as observed among M. tubercu-losis-infected subjects. The most studied CFP are the early-secreted-antigenic-target-six kDa (ESAT-6) and the Culture-Filtrate-Protein-ten kDa (CFP-10). ESAT-6 and CFP-10 are encoded by genes that are located in the particular region of difference 1 (RD1) (56, 57) that is absent in all BCG substrains. In contrast, RD1 is expressed by all strains of M. tuberculo-sis and M. bovis. ESAT-6 and CFP-10 are therefore considered as highly specific for M. tuberculosis. As HBHA, both ESAT-6 and CFP-10 induce IFN-γ specific secre-tions and proliferasecre-tions of PBMC from M. tuberculosis-infected subjects (58).
These characteristics are important, as they can be exploited for the develop-ment of diagnostic tools of M. tuberculo-sis infection. In addition, these antigens confer specific protective immunity and may be of interest for new vaccine strat-egies, as reported elsewhere (59-61).
HBHA : key concepts
expressed by the Mycobacterium tuberculosis Complex first described in 1996 by Menozzi F.D. and Locht C.
surface-associated protein of 198 AA (28-kD) methylation of the extracellular domain
mediates autoaggregation and adhesion to epithelial cells induces protection in mice and guinea pigs upon vaccination strong inducer of IFN-γ secretion (human, mouse)
IFN-γ&TB: IFN-γ is a Th1 cytokine. It plays a central role in the acquired resistance to M. tuberculosis. IFN-γ is essentially produced by CD4 Th1, CD8, γδ T cells and NK cells. The main func-tion of the IFN-γ is to activate macro-phages and subsequently, to enhance the killing of intracellular pathogens.
Secretion of IFN-γ alone is necessary but not sufficient for effective control of the infection by M. tuberculosis. In addition, its effect is strongly potenti-ated by TNF-α, another cytokine, and 1,25 hydroxy-vitamin D3. For further details, please refer to the section on
“adaptive immunity” in the “Immunity and Pathogenesis” chapter.
Ag85 complex also stimulates the up-take of mycobacteria by macrophages, by interacting with binding sites on hu-man fibronectin. This results in the en-hancement of complement-mediated phagocytosis by host macrophages.
The Ag85 complex induces protective immunity against TB in rodents, strong T-cell responses (proliferation and IFN-γ production) and humoral responses in PBMC from LTBI subjects (65).
Lipoarabinomanan (LAM), lipomannan (LM) and phosphatidyl-myoinositol mannosides (PIM)
LAM is a complex glycolipid contained in the cell wall of M. tuberculosis that plays important roles in the pathogen-esis of TB. Contrasting with the LAM from fast-growing species (PILAM), LAM expressed by M. tuberculosis is capped by mannosyl residues on its arabinan domain (ManLAM). Man LAM is an im-portant determinant in the modulation of the immune responses, playing a vast array of biological functions (66). For ex-ample, LAM scavenges oxygen radicals, plays a role in M. tuberculosis-induced macrophage apoptosis, causes phago-some maturation arrest and interacts with particular surface receptors, such as DC-SIGN (see paragraph on dendritic cells below). By both inducing the secre-tion of immunomodulatory cytokines (interleukin-10 (IL-10) and Transform-ing-growth-factor-beta (TGF-β)) and inhibiting the release of pro-inflamma-tory cytokine (e.g. secretion of IL-1 and Tumor-necrosis-factor-alpha (TNF-α)), ManLAM also disarms the macrophages.
LM is the biosynthetic precursor of LAM.
PIMs are the anchor motifs of LM and LAM. LM, PILAM and PIMs are
pro-in-Region of difference (RD): Thanks to genomic comparison between myco-bacteria, it was possible to clarify the evolution of the bacteria belonging to the M. tuberculosis Complex. More than 140 genes were identified as main de-terminants for phenotype, host avidity and virulence. Most of these genes are located within chromosomal regions that are referred to as “regions of differ-ence”. Some of these regions were lost during the evolution of several species (virulent or not). However, only RD1 is absent in avirulent strains such as M.
bovis BCG and M. microti. Artificial RD1 expression by M. bovis BCG or M. microti is responsible for a significant increase in virulence. In contrast, expression of 5 other RD by those mycobacteria was not associated with virulence increase. In conclusion, the loss of RD1 expression is one of the major genetic determinants that explains the avirulence of M. bovis BCG and M. microti.
Antigen 85 Complex (62, 63)
The 0/-kDa antigen 85 (Ag85) com-plex is composed of closely related proteins: 85A ( kDa), 85B (0 kDa) and 85C (.5 kDa). These proteins are major secretion products of M. tuberculosis and M. bovis BCG. The proteins of the Ag85 complex represent up to 15 % of the total protein in M. bovis and M. tubercu-losis culture supernatants. Ag85A, B and C are also associated with the mycobac-terial surface and exhibit mycolyltrans-ferase activity. This enzymatic activity is responsible for the covalent attachment of mycolic acids to glycolipids such as α,α’ trehalose dimycolate (TDM or cord factor). Therefore, the Ag85 complex plays a crucial role in the final assembly of the mycobacterial cell wall. Neutrali-zation of the mycolyltransferase activity inhibited cell-wall assembly and susb-sequent cell growth (64). In addition to its role in the cell wall biosynthesis, the
flammatory molecules that are found in variable amounts within the mycobac-terial cell wall. In contrast to ManLAM, LM, PILAM and PIMs are strong inducers of pro-inflammatory cytokines, such as TNF-α, IL- 8 and IL-1, through the Toll-like-receptor (TLR)-MyD88 pathway (see below) (67). LM and PILAM up-regulate the expression of costimula-tory molecules (i.e. CD40 and CD86) and boost the expression of the inducible nitric oxide synthase (67). Therefore, the induction of pro-inflammatory respons-es by PILAM is believed to favour the killing of non-pathogenic mycobacteria for humans, such as M. smegmatis (68). It has therefore been suggested that vari-ation in LAM, LM and PIM proportions could modulate the magnitude of the inflammatory host responses (69).
HSPx or 16-kDa-alpha-crystallin
HspX or α-crystallin is a major antigen of M. tuberculosis that is recognized by the immune system of M. tubercu-losis-infected subjects (70). Hypoxia is thought to be a key signal for inducing HspX expression in vivo, because its ex-pression is greatly induced under anoxic conditions in vitro (71). Therefore, HspX is considered to be a key element in-volved in the promotion of latency of M.
tuberculosis after in vivo infection. (7).
HspX-deficient strains have a reduced ability to grow in macrophages and are therefore, less virulent. Recently, HspX-specific IFN-γ secretions from PBMC of LTBI subjects were shown to be higher than those of TB patients, whereas no difference was observed with ESAT-6 (7). These results offer interesting per-spectives for specific diagnostic assays of LTBI (see below).