All the strains representing four sublineages of lineage 2 of M. tuberculosis
with the Beijing spoligotype were capable of growing and causing lung pathology in guinea pigs exposed to low-dose aerosol infection. The ability of mycobacteria to grow in the lungs over time is the most conventional measure of strain virulence. While differences between the four sublineages were not overt, members of the RD207 sublineage, consisting of strains more likely to cause secondary clinical cases, caused more-severe pathology in these animals than members of the RD142 or RD181 sublineage, with the fourth sublineage, RD150, showing an intermediate pattern. All strains tested were capable of inducing TH1 immunity. In comparison with the RD207 sublineage, the RD142 strains showing the highest IFN-γ responses were associated with mild inflammation and reduced regulatory Foxp3+
expression. This finding suggests that the RD142 strains are more immunogenic, and it is consistent with the ability of the guinea pigs to control and contain these particular strains more quickly. In addition, all strains induced some degree of regulatory host molecules (Foxp3, TGF-β, and IL-17), which seems to be a general property of the Beijing strains analyzed in animal studies to date (32
). Members of the RD207 sublineage gave the highest signals.
Animals infected with the RD207 sublineage showed both a significant drop in activated CD4+ CD45hi T cells in the lungs as the infections progressed and a concomitant large rise in markers associated with regulatory T cell influx into the lungs. Together, these data indicate that different sublineages of lineage 2 do not behave in a comparable manner in the animal model but instead have some observable differences in their degree of immunopathology and capacity to generate protective and/or regulatory immunity. Hence, this supports the hypothesis, albeit cautiously made, that the RD207 sublineage of lineage 2 may be associated with distinct clinical and pathological properties and that these properties may influence the transmission capacity of isolates within patient communities.
To explore the possible mechanisms for differences observed across sublineages, we analyzed the whole-genome sequences of the strains included in this study. Each of the sublineages had specific mutations that, based on their SIFT values, suggested an impact on gene function. The strains from sublineage RD207 had a mutation in Rv0989c (grcC2
), a diphosphate synthase required for cell wall biosynthesis (27
). These strains also had a mutation in the gene Rv2959c, encoding a methyltransferase involved in the biosynthesis of phenolglycolipid, which is considered a virulence factor (39
). It is tantalizing to speculate that a mutation in this gene may render the bacteria more virulent, as observed in the epidemiologic analysis (more secondary cases) and in the animal model (more necrosis and inflammation).
The strains from sublineage RD150 had mutations that may have an impact on the function of four interesting genes. Rv0577, a gene restricted to members of the M. tuberculosis
), has been used for diagnostic purposes (45
). The protein encoded by Rv0577 may regulate innate and adaptive immunity by interacting with Toll-like receptor 2 (8
). Rv1009 (rpfB
) is one of the most immunogenic resuscitation-promoting factors (40
), and deletion of this gene has been associated with delayed reactivation from chronic tuberculosis in mouse (50
). Rv1638 (uvrA
) is part of the nucleotide excision repair system which counteracts the deleterious effects of DNA lesions (41
) and is essential for Mycobacterium smegmatis
to survive under conditions of hypoxia and low carbon source (13
). Rv2416c (eis
) encodes a secretory protein that enhances intracellular survival of M. tuberculosis
in monocytes and contributes to its pathogenicity (53
). A study demonstrated that Eis impaired the host defense against tuberculosis by disturbing the cross regulation of T cells, producing an imbalance between TH1 and TH2 responses, which could be a factor in the pathogenesis of tuberculosis (22
The strains from sublineage RD142 have mutations in three interesting genes for which other polymorphisms have been described. Rv0989c (grcC2
) had a different mutation in RD142 strains than in the RD207 strains discussed previously. Rv1811 (mgtC
) encodes a virulence factor required to survive in macrophages and under conditions with low Mg2+
). Different mutations in this gene have been found in strains from the Euro-American lineage (spoligotype Haarlem) (2
). Rv1317c (alkA
) is part of the AdaA-AlkA adaptive response in M. tuberculosis
, and multiple mutations have been found in strains from lineage 4 (Euro-American), lineage 2 (the same mutation has been found previously in the W-Beijing 210 strain, which belongs to the RD142 sublineage), and in Mycobacterium bovis
). It has been suggested that a defective adaptive response by these genes will confer a selective advantage to M. tuberculosis
The strains from RD181 are a paraphyletic group, defined as a group of organisms which includes the most recent common ancestor of all of its members but not all of the descendants of that most recent common ancestor. In this particular case, RD181 strains share an ancestor, but the group also includes RD150 and RD142 strains. This implies that SNPs shared by all RD181 strains are also present in RD150 and RD142 strains and that there were not common nsSNPs exclusive for all RD181 strains.
Although some of these mutations may explain the pathological and immunological differences observed, there are a multitude of factors that can influence the transmission and pathogenic capabilities of a given isolate, such as host and environmental factors (5
), HIV coinfection (42
), and the concentration of organisms in environmental air (17
). Until recently, the only bacterial factor considered was the presence of drug resistance; some studies have suggested that M. tuberculosis
resistant to isoniazid is less transmissible (52
) and less pathogenic than fully susceptible organisms (7
), although an earlier study in our laboratory (33
) investigating the virulence of multidrug-resistant isolates did not show much evidence of loss of virulence of these strains. More recent studies suggest that different groups of isolates of M. tuberculosis
may contribute to different clinical outcomes (14
). As noted recently (52
), the application of new molecular typing techniques has increased both our knowledge of bacterial factors and the identification of separate lineages of isolates. It is overly optimistic to expect that the myriad of factors can be modeled in animals such as the guinea pig used here, but such models can provide clues. Like humans, the guinea pig undergoes a process of granulomatous inflammation and necrosis when infected with M. tuberculosis
, and the differing degrees to which this occurs may be an indicator of the virulence of the infecting isolate (36
). Moreover, by applying new flow cytometric techniques (30
) and RT-PCR methods, one can detect differences in the expression of protective immunity (RD142 strains clearly generated the strongest response, suggesting that they are of increased immunogenicity), as well as the induction of signals consistent with the generation of regulatory T cells (which we found here to be highest in animals infected with the RD207 strains).
Most studies to date of the pathogenicity of strains with the guinea pig model have tended to focus on the Beijing strains, and far less is known about other lineages or families of strains. There is a growing concern that the newly emerging isolates of M. tuberculosis
in general are more pathogenic, and this may have a serious impact on vaccine effectiveness. Not only is there a suspicion that BCG may actually have selected for the more virulent strains (1
), but recent data (32
) show that BCG is only transiently protective against Beijing strains and cannot overcome the induction of regulatory T cells. Since some new-generation vaccines are also based on BCG (4
), this raises the real possibility that such vaccines will not work (32
). While as yet unproven, the induction of regulatory T cells by these pathogenic strains, coupled with dampening or loss of protective immunity but continuance of TH17 responses (as seen here), may drive the degree of severity of lung pathology, which in turn will enable bacilli to escape the lungs and then potentially be transmitted.
One of the primary public health strategies to control tuberculosis is the evaluation of persons in close contact with an infectious tuberculosis patient (contact investigation) to identify secondary cases of active tuberculosis and latent tuberculosis infection. The bacterial factors governing transmissibility and pathogenicity of M. tuberculosis
are poorly understood. Therefore, additional clinical and animal studies such as ours may serve to identify factors (like the features of the exposure or the immune status of the exposed person) that suggest a situation in which there is a greater risk of developing active tuberculosis. In these cases, the evaluation of contacts should be undertaken with great urgency. Also, we have discovered mutations likely to be functional in genes that are currently being used for diagnostic purposes (Rv0577 and Rv1009) (10
) or as candidates for subunit vaccines (Rv1009). These polymorphisms may limit their efficacy as diagnostic or vaccine targets.
In conclusion, the current molecular method-based sublineage classification appears to be associated biologically with clinical and pathological consequences, and differences between sublineages, particularly in the context of loss of protective immunity and increased lung damage, may favor or influence the capacity of these isolates to be transmitted within communities.