The results presented here establish a causal relationship between the fine chemical structure of mycolic acids and innate immune recognition of M. tuberculosis at the earliest period after aerosol infection. Whereas previous studies on the role of pcaA in virulence emphasized the importance of this gene in the persistence of Mtb and the pathology of the later stages of infection, the current study focused on the effects of deletion of pcaA on the earliest events after infection. This has led to the identification of the pcaA-dependent modification of mycolic acids, and in particular of the mycolic acids incorporated into TDM, as a critical proinflammatory lipid modification that regulated host-innate immune recognition during the first week of the murine infection in vivo and the first 24 h of macrophage infection in vitro.
Trehalose dimycolate, also named “cord factor,” has been an intensely studied cell envelope compound of M. tuberculosis
for over 50 yr. TDM was the first virulence determinant proposed for M. tuberculosis
when it was identified in a petroleum ether extract of M. tuberculosis
and found to inhibit the migration of neutrophils (27
). The biologic activity designated cord factor was later identified as TDM and was thought to be responsible both for the cording morphology and mycobacterial virulence. This postulated important role for TDM became less plausible when TDM was isolated from all mycobacteria that produce mycolic acids, most of which are nonpathogenic and do not form serpentine cords (33
). However, interest in TDM has remained intense due to its powerful adjuvant properties, chemical properties when interacting with membranes (23
), and ability to induce granulomatous inflammation in experimental animals that mimics whole Mtb infection (25
By analyzing the activities of purified TDMs in vitro and in vivo, the results from the current study also strongly supported the view that the cyclopropane modification of TDM in the Mtb cell envelope acts directly as an effector of pathogenesis, rather than by inducing indirect effects due to structural modifications of the cell envelope. As such, this study provides proof of principle that the chemical diversity of the Mtb cell envelope has evolved to interact specifically with host cells and not solely as a structural scaffold, as has been noted with other cell envelope mutants with impaired virulence (11
). Cyclopropane modification of membrane lipids has been defined in E. coli
and other bacteria and affects resistance to cold shock and acid (37
). However, the immunomodulatory function for cyclopropane modification of bacterial lipids identified in the current study is a novel function for this chemical entity. M. tuberculosis
expresses a large family of mycolic acid methyl transferases/cyclopropane synthases that modify mycolic acids (39
), two of which are known to be important for pathogenesis (7
). However, the pathogenetic mechanism of cyclopropanation for bacterial virulence or pathogenesis has been unclear. Mycolates are recognized by T cells when presented on CD1, but evidence to date indicates that this recognition is independent of cyclopropane modification (42
). Instead, our findings demonstrated that cyclopropane modification of mycolic acids acted directly to promote the virulent behavior of mycobacteria by modulating innate immune activation of macrophages and potentially other cell types during infection. The particular macrophage receptor molecules responsible for these responses to TDM have not yet been identified, and this important point will require further study.
Our results strongly point to TNF as a key mediator of the effects of the normally cyclopropanated TDM molecules of wild-type Mtb on the host immune response. Thus, the reduction in growth rate of the ΔpcaA
mutant seen in the first week of infection was reversed in TNF-deficient mice, and the difference observed previously in survival of mice infected with wild-type versus ΔpcaA
bacilli was also absent in TNF-deficient mice. Because the ΔpcaA
mutant elicited a markedly reduced TNF response compared with wild-type Mtb, these findings were consistent with the recent proposal that one effect of TNF may actually be to facilitate the growth of the bacilli early in the course of infection (22
). Thus, the reduced stimulation of TNF production by ΔpcaA
TDM leads to less abundance of TNF during initial infection, and reduced bacterial growth in the first week. The critical importance of TNF in antimycobacterial defense is well established in mice (17
) and humans (18
). However, the apparent protective effect of TNF is partially due to the defective immune regulation that results from its absence, leading to massive TH
1 type immune activation, tissue necrosis, and death (19
). A direct role for TNF in antimycobacterial activity of macrophages has been controversial, and recent data suggest that TNF facilitates growth of virulent, but not attenuated strains of Mtb (21
) in cultured human macrophages, suggesting that induction of TNF may be an important virulence strategy of Mtb. Previous studies of Mtb infection in TNF-deficient mice have shown that bacterial burdens are unaffected during the first 2 wk of infection, suggesting either that TNF has no role in early growth of Mtb, or that TNF has equal and opposing effects on bacterial growth during early infection in vivo. In this latter model, loss of a growth promoting effect of TNF in macrophages would be counterbalanced in vivo by loss of a growth restricting effect of TNF produced by other cell types (43
). This model is consistent with the data presented here in which the growth of the pcaA
mutant is restricted in wild-type mice but recovers to wild-type Mtb growth levels in TNF-deficient mice. The data presented here indicate that Mtb has evolved cyclopropane lipid modification to manipulate the host TNF axis. In the case of the ΔpcaA
mutant, defective growth in the first week after aerosol infection and altered innate immune recognition during this period attenuated the later pathology of the infection. As shown in our previous study (8
), this dramatically alters the course of chronic Mtb infection, and thus emphasizes the powerful interrelationship between innate and adaptive immunity in this infection.
Our results expand earlier studies that examined the role of cell envelope lipids in immunopathogenesis of Mtb infection. A clinical strain of Mtb that was hypervirulent for mice induced lower levels of TNF in mouse lung at 28 d (44
) and was hypoinflammatory in cultured macrophages in vitro over the course of a 96-h infection (45
). Recent work indicates that these phenotypes are due to production of phenolic glycolipid by this clinical strain (46
). Thus, the accumulated data indicate the prolonged suppression of host TNF by PGL promotes bacterial virulence, whereas temporally restricted suppression of host TNF during the first weeks of infection through loss of the pcaA
modification of TDM is advantageous to the host. These data are consistent with a model in which structurally distinct lipid components of the cell envelope promote or inhibit host inflammatory responses at distinct time periods during the course of infection for the ultimate purpose of achieving microbial symbiosis.
Our past and present results provide new insight into the relationship between TDM and mycobacterial pathogenesis. Our previous work demonstrated that inactivation of the cyclopropane synthase pcaA
abolished cording and attenuated Mtb in mice (8
). In light of prior work with cord factor these results suggested that the cyclopropane modification of TDM was necessary for the cording morphology and explains the lack of cording of saprophytic mycobacteria that contain TDM because these mycobacteria lack cyclopropane modification of mycolic acids. Our present results indicate that pcaA
modification of TDM with cyclopropyl groups is a proinflammatory modification both in the context of purified glycolipid and whole bacilli. Strikingly, this pathogenetic function of this lipid modification is temporally restricted to early infection. This demonstrates not only that cell envelope glycolipids of Mtb are direct effectors of pathogenesis, but that each cell envelope effector may have distinct functions at restricted time points during infection. Although cyclopropane synthases of Mtb are clearly not essential for in vitro growth and viability, the findings of the current study suggest that pharmacologic inhibition of members of this enzyme family could reverse pathogen-induced immunomodulation, thereby enhancing host immunity and control or eradication of infection.