Accelerated immunopathological response of mice infected with Mycobacterium tuberculosis disrupted in the mce1 operon negative transcriptional regulator

Mycobacterium tuberculosis causes a variety of clinical outcomes determined by host as well as bacterial factors. M. tuberculosis disrupted in the mce1 operon causes increased mortality in immunocompetent mice. This operon is negatively regulated by mce1R (Rv0165c). We studied the role of mce1R in infection outcome in mice. At 5 x 10(4) tail vein infectious dose, the median survival time (MST) of mice infected with the mce1R mutant M. tuberculosis H37Rv was 293 days, while mice infected with the wild-type H37Rv survived more than 350 days (P < 0.0001). At a higher dose (5 x 10(6)), the MST of mutant-infected mice was 32 days, compared with 127 days for wild type-infected mice (P < 0.0001). With either tail vein or aerosol infection, mutant-infected mice developed larger granulomatous lesions in their lungs than mice infected with the wild type. Mutant-infected mice were unable to control the bacterial burden in the first 4 weeks of infection, but even after achieving control later, these mice succumbed to granulomatous pneumonia. These observations suggest that the early deregulated expression of the mce1 operon products determines later granulomatous tissue response. mce1 operon may homeostatically regulate the cell wall architecture in vivo that elicits a steady-state granuloma tissue response permitting M. tuberculosis to establish a long-term infection.     

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen’s persistence.     

CCR5-deficient mice control Mycobacterium tuberculosis infection despite increased pulmonary lymphocytic infiltration

The control of Mycobacterium tuberculosis infection is dependent on the development of an adaptive immune response, which is mediated by granulomas. The granuloma is a dynamic structure that forms in the lung and consists primarily of macrophages and lymphocytes. For this structure to be effective in containment of the bacillus, it must develop in an organized and timely manner. The formation of the granuloma is dependent on recruitment of activated cells through adhesion molecules and chemokines. M. tuberculosis infection causes an increase in the expression of beta-chemokines CCL3, CCL4, and CCL5, and their receptor CCR5, in the lungs. In this study, we demonstrate that CCR5-transgenic knockout mice were capable of recruiting immune cells to the lung to form granulomas. CCR5(-/-) mice successfully induced a Th1 response and controlled infection. Unexpectedly, M. tuberculosis infection in these mice resulted in greater numbers of lymphocytes migrating to the lung and higher levels of many inflammatory cytokines, compared with wild-type mice, without apparent long-term detrimental effects. In the absence of CCR5, there were more dendritic cells in the lung-draining lymph nodes and more primed T lymphocytes in these mice. Bacterial numbers in the lymph nodes were also higher in CCR5(-/-) mice. Therefore, CCR5 may play a role in the migration of dendritic cells to and from the lymph nodes during M. tuberculosis infection.     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

(1)H NMR-based metabolomic profiling in mice infected with Mycobacterium tuberculosis

Tuberculosis (TB) is one of three major infectious diseases, and the control of TB is becoming more difficult because of the emergence of multidrug-resistant and extensively drug-resistant strains. In this study, we explored the (1)H NMR-based metabolomics of TB using an aerobic TB infection model. Global profiling was applied to characterize the responses of C57Bl/6 mice to an aerobic infection with virulent Mycobacterium tuberculosis (MTB). The metabolic changes in organs (i.e., the lung, the target organ of TB, and the spleen and liver, remote systemic organs) and in serum from control and MTB-infected rats were investigated to clarify the host-pathogen interactions in MTB-infected host systems. Principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots showed distinct separation between control and MTB-infected rats for all tissue and serum samples. Several tissue and serum metabolites were changed in MTB-infected rats, as compared to control rats. The precursors of membrane phospholipids, phosphocholine, and phosphoethanolamine, as well as glycolysis, amino acid metabolism, nucleotide metabolism, and the antioxidative stress response were altered based on the presence of MTB infection. This study suggests that NMR-based global metabolite profiling of organ tissues and serum could provide insight into the metabolic changes in host infected aerobically with virulent Mycobacterium tuberculosis.     

Salmonellosis in mice infected with Mycobacterium tuberculosis BCG. I. Role of endotoxin in infection

In agreement with previous observations, mice dying of salmonellosis were found to have a relatively constant number of Salmonella cells in their carcasses (ca. 5 x 10(9)). This number was not the result of bacterial overgrowth in moribund animals and therefore appears to be related to lethality. Similar numbers of salmonellae were recovered from the carcasses of infected mice which had previously been rendered hyperreactive to endotoxin either by infection with M. tuberculosis BCG or by adrenalectomy. In BCG mice, desensitization to endotoxin did not occur during the infection and, therefore, at death these mice contained a number of bacteria which would be equivalent to 1,000 ld(50) of endotoxin. Although the number of bacteria recovered from normal mice is roughly equivalent to a lethal quantity of endotoxin, this is obviously not the case in hyperreactive mice. Therefore, the relationship between lethality and 5 x 10(9) salmonellae cannot be explained by their endotoxin content. Nevertheless, when hyperreactive BCG mice are challenged parenterally with Salmonella, the endotoxin content of the inoculum may markedly influence the course of the infection.     

Molecular and functional analysis of the mce4 operon in Mycobacterium smegmatis

Mycobacterium smegmatis contains 6 homologous mce (mammalian cell entry) operons which have been proposed to encode ABC‐like import systems. The mce operons encode up to 10 different proteins of unknown function that are not present in conventional ABC transporters. We have analysed the consequences of individually deleting each of the genes of the mce4 operon of M. smegmatis, which mediates the transport of cholesterol. None of the mce4 mutants were able to grow in cholesterol suggesting that all these genes are required for its uptake and that none of them can be replaced by the homologous genes of the other mce operons. This result suggests that different mce operons do not provide redundant capabilities and that M. smegmatis, in contrast with Mycobacterium tuberculosis, is not able to use alternative systems to import cholesterol in the analysed culture conditions. Either deletion of the entire mce4 operon or single point mutations that eliminate the transport function cause a phenotype similar to the one observed in a mutant lacking all 6 mce operons suggesting a pleiotropic role for this system.     

Enhanced macrophage activity in granulomatous lesions of immune mice challenged with Mycobacterium tuberculosis

In this study, we evaluated the cellular influx and cytokine environment in the lungs of mice made immune by prior vaccination with Mycobacterium bovis bacillus Calmette-Guérin compared with control mice after infection with Mycobacterium tuberculosis to characterize composition of protective lesions in the lungs. Immune mice controlled the growth of the M. tuberculosis challenge more efficiently than control mice. In immune animals, granulomatous lesions were smaller and had a more lymphocytic core, less foamy cells, less parenchymal inflammation, and slower progression of lung pathology than in lungs of control mice. During the chronic stage of the infection, the bacterial load in the lungs of immune mice remained at a level 10 times lower than control mice, and this was associated with reduced numbers of CD4P(+P) and CD8P(+P) T cells, and the lower expression of protective (IL-12, IFN-gamma), inflammatory (TNF-alpha), immunoregulatory (GM-CSF), and immunosuppressive (IL-10) cytokines. The immune mice had higher numbers of CD11b- CD11c(high) DEC-205(low) alveolar macrophages, but lower numbers of CD11b+ CD11c(high) DEC-205(high) dendritic cells, with the latter expressing significantly lower levels of the antiapoptotic marker TNFR-associated factor-1. Moreover, during the early stage of chronic infection, lung dendritic cells from immune mice expressed higher levels of MHC class II and CD40 molecules than similar cells from control mice. These results indicate that while a chronic disease state is the eventual outcome in both control and immune mice infected with M. tuberculosis by aerosol exposure, immune mice develop a protective granulomatous lesion by increasing macrophage numbers and reduced expression of protective and inflammatory cytokines.     

Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice

Endotoxin from Gram-negative bacteria bound to CD14 signals through Toll-like receptor (TLR) 4, while components of Gram-positive bacteria, fungi, and Mycobacterium tuberculosis (M.tb.) preferentially use TLR2 signaling. We asked whether TLR4 plays any role in host resistance to M.tb. infection in vivo. Therefore, we infected the TLR4 mutant C3H/HeJ mice and their controls, C3H/HeN mice, with M.tb. by aerosol. TLR4 mutant mice had a reduced capacity to eliminate mycobacteria from the lungs, spread the infection to spleen and liver, with 10-100 times higher CFU organ levels than the wild-type mice and succumbed within 5-7 mo, whereas most of the wild-type mice controlled infection and survived the duration of the experiment. The lungs of TLR4 mutant mice showed chronic pneumonia with increased neutrophil infiltration, reduced macrophages recruitment, and abundant acid-fast bacilli. Furthermore, the pulmonary expression of TNF-alpha, IL-12p40, and monocyte chemoattractant protein 1 was significantly lower in C3H/HeJ mice when compared with the wild-type controls. C3H/HeJ-derived macrophages infected in vitro with M.tb. produced lower levels of TNF-alpha. Finally, the purified mycobacterial glycolipid, phosphatidylinositol mannosides, induced signaling in both a TLR2- and TLR4-dependent manner, thus suggesting that recognition of phosphatidylinositol mannosides in vivo may influence the development of protective immunity. In summary, macrophage recruitment and the proinflammatory response to M.tb. are impaired in TLR4 mutant mice, resulting in chronic infection with impaired elimination of mycobacteria. Therefore, TLR4 signaling is required to mount a protective response during chronic M.tb. infection.     

Mycobacterium tuberculosis infection in complement receptor 3-deficient mice

Complement receptor type 3 (CR3) present on macrophages is used by Mycobacterium tuberculosis as one of its major phagocytic receptors. In this study, we examined the in vivo significance of CR3-mediated phagocytosis on the pathogenesis of disease caused by M. tuberculosis. The outcome of tuberculous infection in mice deficient in the CD11b subunit of CR3 (CR3-/-) on a mixed 129SV and C57BL background and control wild-type counterparts was comparable with respect to survival, bacterial burden, granulomatous lesion development, and cytokine expression in the spleen and lungs. M. tuberculosis infection was also examined in CR3-/- mice on C57BL/6 and BALB/c backgrounds and was found to be similar. In conclusion, our results suggest that in the absence of CR3, M. tuberculosis is able to gain entry into host cells via alternative phagocytic receptors and establish infection. The data also indicate that absence of CR3 does not alter disease course in either the relatively resistant C57BL/6 or the relatively susceptible BALB/c strains of mice.     

IRF1 as a potential biomarker in Mycobacterium tuberculosis infection

Pulmonary tuberculosis (PTB) is a major global public health problem. The purpose of this study was to find biomarkers that can be used to diagnose tuberculosis. We used four NCBI GEO data sets to conduct analysis. Among the four data sets, GSE139825 is lung tissue microarray, and GSE83456 , GSE19491 and GSE50834 are blood microarray. The differential genes of GSE139825 and GSE83456 were 68 and 226, and intersection genes were 11. Gene ontology (GO) analyses of 11 intersection genes revealed that the changes were mostly enriched in regulation of leucocyte cell-cell adhesion and regulation of T-cell activation. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs revealed that the host response in TB strongly involves cytokine-cytokine receptor interactions and folate biosynthesis. In order to further narrow the range of biomarkers, we used protein-protein interaction to establish a hub gene network of two data sets and a network of 11 candidate genes. Eventually, IRF1 was selected as a biomarker. As validation, IRF1 levels were shown to be up-regulated in patients with TB relative to healthy controls in data sets GSE19491 and GSE50834 . Additionally, IRF1 levels were measured in the new patient samples using ELISA. IRF1 was seen to be significantly up-regulated in patients with TB compared with healthy controls with an AUC of 0.801. These results collectively indicate that IRF1 could serve as a new biomarker for the diagnosis of pulmonary tuberculosis.     

Enhanced effect of BCG vaccine against pulmonary Mycobacterium tuberculosis infection in mice with lung Th17 response to mycobacterial heparin‐binding hemagglutinin adhesin antigen

Although the BCG vaccine can prevent tuberculosis (TB) in infants, its ability to prevent adult pulmonary TB is reportedly limited. Therefore, development of a novel effective vaccine against pulmonary TB has become an international research priority. We have previously reported that intranasal vaccination of mice with a mycobacterial heparin‐binding hemagglutinin adhesin (HBHA) plus mucosal adjuvant cholera toxin (CT) enhances production of IFN‐γ and anti‐HBHA antibody and suppresses extrapulmonary bacterial dissemination after intranasal infection with BCG. In the present study, the effects of intranasal HBHA + CT vaccine on murine pulmonary Mycobacterium tuberculosis (Mtb) infection were examined. Intranasal HBHA + CT vaccination alone failed to reduce the bacterial burden in the infected lung. However, a combination vaccine consisting of s.c. BCG priming and an intranasal HBHA + CT booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14 compared with BCG vaccine alone. Further, it was found that intranasal HBHA + CT vaccine enhanced not only IFN‐γ but also IL‐17A production by HBHA‐specific T cells in the lung after pulmonary Mtb infection. Therefore, this combination vaccine may be a good candidate for a new vaccine strategy against pulmonary TB.     

Aggravated infection in mice co-administered with Mycobacterium tuberculosis and the 27-kDa lipoprotein

We have previously reported that mice immunized with the mycobacterial 27-kDa lipoprotein were more susceptible to Mycobacterium tuberculosis (Mtb) challenge. We also showed that 27-kDa lipoprotein abrogated the protection afforded by the BCG vaccine when administrated together, suggesting that the 27-kDa lipoprotein may modulate the course of experimental mycobacterial infection. In this study, we address the role of the 27-kDa lipoprotein in modulating the immune response to mycobacteria. Our results show that co-administration of BALB/c mice with Mtb and the 27-kDa lipoprotein (Mtb+27kDa), but not its non-acylated form, increases the susceptibility of mice to Mtb infection. Significantly lower DTH reaction and splenocyte proliferation to PPD stimulation were also observed in Mtb+27kDa-infected mice compared to Mtb-infected mice. Furthermore, during infection, splenocytes and purified T cells lost their ability to proliferate in response to concanavalin A stimulation more rapidly in the Mtb+27kDa-infected mice, which was accompanied by high IFN-gamma and NO production, but low TNF-alpha secretion levels. Addition of L-NMMA, anti-IFN-gamma and anti-TNF-alpha antibodies restored in vitro proliferative responses of T cells from Mtb+27kDa-infected mice. Short-term L-NMMA treatment of Mtb+27kDa-infected mice prevented the 27-kDa-mediated immunosuppression and increase in susceptibility to Mtb. Altogether, these data suggest that the 27-kDa lipoprotein plays a role in Mtb infection by inducing increased suppression of the immune response due to elevated NO production.     

ST2 deficient mice display a normal host defense against pulmonary infection with Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths world-wide every year. Successful host defense mainly depends on a strong Th type 1 response. We investigated the role of T1/ST2 (recently identified as the receptor for IL-33), a typical Th2 marker in the assumption that a shift towards a beneficial Th1 response would occur in the absence of ST2. For this, ST2 KO and WT mice were intranasally infected with a virulent strain of M. tuberculosis (150 CFU). In line with our hypothesis, ST2 KO animals displayed increased numbers of lymphocytes infiltrating the lung after 2 weeks of infection, increased IFNγ production by splenocytes in ST2 KO mice early in infection and enhanced lung IFNγ levels at the chronic phase of the disease. However, we did not detect any differences between ST2 KO and WT mice in mycobacterial loads in lungs or liver after M. tuberculosis infection. The pulmonary inflammatory response, as measured by relative lung weights, cytokine and chemokine levels as well as histopathological analysis, was similar in ST2 KO and WT mice. These data suggest that apart from inducing a modest shift towards the Th1 response, the role of ST2 during murine M. tuberculosis infection is limited.     

IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung

IL-23 is required for the IL-17 response to infection with Mycobacterium tuberculosis, but is not required for the early control of bacterial growth. However, mice deficient for the p19 component of IL-23 (Il23a(-/-)) exhibit increased bacterial growth late in infection that is temporally associated with smaller B cell follicles in the lungs. Cxcl13 is required for B cell follicle formation and immunity during tuberculosis. The absence of IL-23 results in decreased expression of Cxcl13 within M. tuberculosis-induced lymphocyte follicles in the lungs, and this deficiency was associated with increased cuffing of T cells around the vessels in the lungs of these mice. Il23a(-/-) mice also poorly expressed IL-17A and IL-22 mRNA. These cytokines were able to induce Cxcl13 in mouse primary lung fibroblasts, suggesting that these cytokines are likely involved in B cell follicle formation. Indeed, IL-17RA-deficient mice generated smaller B cell follicles early in the response, whereas IL-22-deficient mice had smaller B cell follicles at an intermediate time postinfection; however, only Il23a(-/-) mice had a sustained deficiency in B cell follicle formation and reduced immunity. We propose that in the absence of IL-23, expression of long-term immunity to tuberculosis is compromised due to reduced expression of Cxcl13 in B cell follicles and reduced ability of T cells to migrate from the vessels and into the lesion. Further, although IL-17 and IL-22 can both contribute to Cxcl13 production and B cell follicle formation, it is IL-23 that is critical in this regard.