Seroreactive Mycobacterial Proteins and Lipid in Cattle with Bovine Tuberculosis

Bovine tuberculosis caused by Mycobacterium bovis is a major economic problem in several countries. Antibody responses are useful indicators of M. bovis infection of cattle. To overcome drawback of serological tests with low sensitivity, identification and characterization of multiple serodiagnostic antigens has been required. In this study, the antigens with strong antibody reactivity were searched using fractionation of M. bovis culture filtrate proteins and probing with sera from M. bovis-infected cattle. Twelve proteins which have not previously been described as serologic targets were identified and six proteins among them were expressed in Escherichia coli. The mycobacterial lipoarabinomannan (LAM) with strong seroreactivity in cattle was identified and purified. IgG and IgA responses against the newly identified proteins, the seroreactive proteins with strong antibody reactivity in human tuberculosis, and LAM were compared in M. bovis-infected and non-infected cattle as well as in field samples. In general, sensitivity of the tested antigens was higher in M. bovis-infected cattle than purified protein derivative (PPD) (+) field samples. Although a diverse reactivity and sensitivity according to the antigens were shown, the diagnostic utility of both IgA and IgG antibody to the antigens was similar in M. bovis-infected cattle but utility of IgG antibody was superior to that of IgA in field samples. The antigen with the highest diagnostic value was LAM in both the groups. Other antigens with considerable diagnostic utility were BCG_3488c, BCG_2330, Antigen 85, HspX, and Rv3593 when considered the sensitivity and area under the receiver characteristic curve (AUC) value. These antigens may be valuable candidates to be included in a cocktail test kit for bovine tuberculosis diagnosis.

Mycobacterial Heparin-binding Hemagglutinin Antigen Activates Inflammatory Responses through PI3-K/Akt, NF-kappaB, and MAPK Pathways

BACKGROUND: Mycobacterium tuberculosis (Mtb) heparin binding hemagglutinin (HBHA) is an Ag known to evoke effective host immune responses during tuberculosis infection. However, the molecular basis of the host immune response to HBHA has not been fully characterized. In this study, we examined the molecular mechanisms by which HBHA can induce the expression of proinflammatory cytokines in macrophages. METHODS: HBHA-induced mRNA and protein levels of proinflammatory cytokines were determined in bone marrow-derived macrophages (BMDMs) using RT-PCR and ELISA analysis. The roles of intracellular signaling pathways for NF-kappaB, PI3-K/Akt, and MAPKs were investigated in macrophage proinflammatory responses after stimulation with HBHA. RESULTS: HBHA robustly activated the expression of mRNA and protein of both TNF-alpha and IL-6, and induced phosphorylation of NF-kappaB, Akt, and MAPKs in BMDMs. Both TNF-alpha and IL-6 production by HBHA was regulated by the NF-kappaB, PI3-K, and MAPK pathways. Furthermore, PI3-K activity was required for the HBHA-induced activation of ERK1/2 and p38 MAPK, but not JNK, pathways. CONCLUSION: These data suggest that mycobacterial HBHA significantly induces proinflammatory responses through crosstalk between the PI3-K and MAPK pathways in macrophages.

Characterization of Immune Responses to Mycobacterium tuberculosis Rv2041c Protein

Tuberculosis, which is caused by Mycobacterium tuberculosis (M. tb), is one of the most important infectious diseases in the world. Although many functional studies have been conducted on M. tb proteins in the post-genomic era, little is known about the function of many proteins expressed specifically during latency. Previously, we reported that Rv2041c from M. tb H37Rv is highly expressed under conditions of low pH and hypoxia, which represent the in vitro mimicry of latent tuberculosis. In the present study, increased expression levels of Rv2041c under hypoxia and low pH in vitro culture was confirmed by RT-PCR. Interestingly, Rv2041c showed significantly increased expression among genes of the same operon and genes belonging to the same functional group. Finally, the immune responses elicited by the recombinant (r) Rv2041c protein were investigated using ex vivo and in vivo models of M. tb infection. A significantly high level of pro-inflammatory cytokines such as TNF-alpha, IL-6, and IL-12p40 was detected in a dose-dependent manner by treatment of murine bone marrow-derived macrophages with rRv2041c protein. In addition, IFN-gamma and TNF-alpha secretion increased after stimulation with purified Rv2041c protein to lymphocytes from latent and active TB mice in a modified Cornell model. In conclusion, our findings suggest that Rv2041c is a new T-cell antigen and could be a potential vaccine candidate against M. tb infection by inducing a strong cellular immune response.

Identification and Diagnostic Utility of Serologic Reactive Antigens from Mycobacterium tuberculosis Sonic Extracts

It is important to identify and to test serologically active antigens, so as to devise a cocktail of the best antigens or peptides. We searched for antigens that have serodiagnostic utility using two-dimensional fractionation of sonic extracts from Mycobacterium tuberculosis and probing with pools of sera from healthy subjects and patients with tuberculosis (TB). Reactive protein spots with patient sera were identified by tandem mass spectrometry. Three proteins, Rv0652, Rv2626c, and Rv3418c, which have not previously been described as serologic targets, were identified. Rv0652 protein among them was expressed in Escherichia coli and serum IgG antibodies against this antigen were measured in 150 patients with pulmonary TB and in 115 healthy subjects. The sensitivity and specificity were 39% and 92%, respectively. These results suggest that a newly identified protein, Rv0652 may be a valuable candidate to be included in a cocktail test kit for TB diagnosis.

Identification of Proteins Induced at Hypoxic and Low pH Conditions in Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis likely reside within a granuloma as a dormant state. An area of necrosis forms at the center of lung granulomas. Within this area, the bacteria are deprived of nutrients and exposed to harsh conditions, including low pH and anoxia. The response of M. tuberculosis to low pH and low oxygen conditions was investigated in both cellular and extracellular proteins by two-dimensional polyacrylamide gel electrophoresis analysis and MALDITOF. Several proteins intensively expressed under low pH and/or hypoxic conditions were found. In the culture filtrate, PhoS1 (Rv0934) and ScoB (Rv2503c) were found in significant amounts under both the low oxygen and acidic stress conditions. These results indeed extend our understanding of acidic response as well as hypoxic in M. tuberculosis and provide an important insight into physiology of the latent bacilli.

The Phospholipase-Protein Kinase C-MEK-ERK Pathway is Essential in Mycobacteria-induced CCL3 and CCL4 Expression in Human Monocytes

BACKGROUND: Little information is available on the identification and characterization of the upstream regulators of the signal transduction cascades for Mycobacterium tuberculosis (M. tbc)-induced ERK 1/2 activation and chemokine expression. We investigated the signaling mechanisms involved in expression of CCL3/MIP-1 and CCL4/MIP-1 in human primary monocytes infected with M. tbc. METHODS: MAP kinase phosphorylation was determined using western blot analysis with specific primary antibodies (ERK 1/2, and phospho-ERK1/2), and the upstream signaling pathways were further investigated using specific inhibitors. RESULTS: An avirulent strain, M. tbc H37Ra, induced greater and more sustained ERK 1/2 phosphorylation, and higher CCL3 and CCL4 production, than did M. tbc H37Rv. Specific inhibitors for mitogen-activated protein kinase (MAPK) kinase (MEK; U0126 and PD98059) significantly inhibited the expression of CCL3 and CCL4 in human monocytes. Mycobacteria-mediated expression of CCL3 and CCL4 was not inhibited by the Ras inhibitor manumycin A or the Raf-1 inhibitor GW 5074. On the other hand, phospholipase C (PLC) inhibitor (U73122) and protein kinase C (PKC)- specific inhibitors (GO6976 and Ro31-8220) significantly reduced M. tbc-induced activation of ERK 1/2 and chemokine synthesis. CONCLUSION: These results are the first to demonstrate that the PLC-PKC-MEK-ERK, not the Ras-Raf-MEK-ERK, pathway is the major signaling pathway inducing M. tbc-mediated CCL3 and CCL4 expression in human primary monocytes.

Mycobacteria-induced Interleukin-12 Expression by Human Monocyte-derived Macrophages Is Negatively Regulated by Phosphatidylinositol 3-kinase and ERK 1/2 Pathways

Both interleukin (IL)-12, an important cytokine skewing the immune response towards a Th1 cytokine profiles, and tumor necrosis factor (TNF)-alpha, are thought to be critical factors in defenses against mycobacteria. In this study, we evaluated the roles of phosphatidylinositol 3-kinase (PI 3-K), and extracellular signal-regulated kinase (ERK) 1/2 pathways in the expression of IL-12 in human monocyte-derived macrophages (MDMs) after stimulation with Mycobacterium tuberculosis H37Rv (M. tbc) or the Triton X-114 solublized proteins (TSP) of M. tbc. Both M. tbc and TSP rapidly phosphorylated ERK 1/2, and Akt in human MDMs. Inhibition of PI 3-K-Akt pathway by specific inhibitors (LY294002 and wortmannin) dramatically increased M. tbc- or TSP-induced IL-12 p40 and p35 mRNA and IL-12 production. In addition, blockade of ERK 1/2 pathway by specific inhibitors (PD98059 and U0126) significantly increased the mRNA levels and cytokine production in M. tbc- or TSP-treated MDMs. On the contrary, M. tbc- or TSP-induced TNF-a production was significantly depressed in human MDMs by pretreatment with inhibitors of PI 3-K or ERK pathways. The M. tbc or TSP stimulation decreased ERK 1/2 phosphorylation by 70% in the presence of wortmannin or LY294002, suggesting that some cross-talk between the PI 3-K-Akt and mitogen-activated protein kinase kinase (MEK)-ERK pathways may be operating in human monocytes during mycobacterial infection. PI 3-K activity is partially required for the M. tbc- or TSP-induced ERK 1/2 phosphorylation. Collectively, these data suggest that the PI 3-K and ERK 1/2 pathways play a central role in the negative regulation of IL-12, but not TNF-a, production by M. tbc.