Culture Filtrate Protein Research Articles

Mycobacterium avium MAV2054 protein induces macrophage apoptosis by targeting mitochondria and reduces intracellular bacterial growth

Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.

Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv

BackgroundWe have characterized two immunogenic proteins, Rv1197 and Rv1198, of the Esx-5 system of the ESAT-6 family of Mycobacterium tuberculosis H37Rv. MethodsThe complex formation between Rv1197 and Rv1198 was characterized by biophysical techniques. The reactivity of serum from TB patients towards these proteins was characterized by ELISA. Lymphocyte proliferation and cytokine induction were followed in restimulated splenocytes from immunized mice by using MTT assay and CBA flowcytometry, respectively. ResultsRv1197 and Rv1198 strongly interact to form a heterodimeric complex under reducing conditions, which is characterized by a dissociation constant of 97×10−9M and melting temperature, Tm, of 50.5°C. Strong humoral responses to Rv1197, Rv1198, CFP-10 and MoaC1 (Rv3111) antigens were found in Indian patients with active pulmonary tuberculosis (n=44), in comparison to non-infected healthy individuals (n=20). The seroreactivity to Rv1198 was characterized by a sensitivity of 75% and specificity of 90%. In BALB/c mice, immunization with Rv1198-FIA induced a pro-inflammatory response with elevated levels of TNF and IL-6, along with low induction of IFN-γ, IL-2 and IL-10, but no induction of IL-4. ConclusionRv1197 and Rv1198 form a stable complex, which is regulated by the redox state of Rv1198. Rv1198 is immunogenic with highly specific seroreactivity towards TB patients' serum. Rv1198 elicits a pro-inflammatory recall response in immunized mice. General SignificanceThis study characterizes the interaction of Rv1197 and Rv1198, and establishes the immunogenic nature of Rv1198.

Mycobacterium tuberculosis Rv2882c Protein Induces Activation of Macrophages through TLR4 and Exhibits Vaccine Potential.

Macrophages constitute the first line of defense against Mycobacterium tuberculosis and are critical in linking innate and adaptive immunity. Therefore, the identification and characterization of mycobacterial proteins that modulate macrophage function are essential for understanding tuberculosis pathogenesis. In this study, we identified the novel macrophage-activating protein, Rv2882c, from M. tuberculosis culture filtrate proteins. Recombinant Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and express co-stimulatory and major histocompatibility complex molecules via Toll-like receptor 4, myeloid differentiation primary response protein 88, and Toll/IL-1 receptor-domain-containing adaptor inducing IFN-beta. Mitogen-activated protein kinases and NF-κB signaling pathways were involved in Rv2882c-induced macrophage activation. Further, Rv2882c-treated macrophages induced expansion of the effector/memory T cell population and Th1 immune responses. In addition, boosting Bacillus Calmette-Guerin vaccination with Rv2882c improved protective efficacy against M. tuberculosis in our model system. These results suggest that Rv2882c is an antigen that could be used for tuberculosis vaccine development.

Nine things to know about elicitins.

888 I. 888 II. 889 III. 889 IV. 889 V. 891 VI. 891 VII. 891 VIII. 892 IX. 892 X. 893 XI. 893 893 References 893 SUMMARY: Elicitins are structurally conserved extracellular proteins in Phytophthora and Pythium oomycete pathogen species. They were first described in the late 1980s as abundant proteins in Phytophthora culture filtrates that have the capacity to elicit hypersensitive (HR) cell death and disease resistance in tobacco. Later, they became well-established as having features of microbe-associated molecular patterns (MAMPs) and to elicit defences in a variety of plant species. Research on elicitins culminated in the recent cloning of the elicitin response (ELR) cell surface receptor-like protein, from the wild potato Solanum microdontum, which mediates response to a broad range of elicitins. In this review, we provide an overview on elicitins and the plant responses they elicit. We summarize the state of the art by describing what we consider to be the nine most important features of elicitin biology.

Significance of immune response to Mycobacterium tuberculosis culture filtrate protein antigens in cerebrospinal fluid of tuberculous meningitis patients: A search for diagnostic marker

ABSTRACTMycobacterium tuberculosis (H37Ra) culture filtrate proteins (CFP) are explored as a diagnostic marker for tuberculous meningitis (TBM). Cerebrospinal fluid (CSF) samples from patients were categorized as confirmed (n = 47), suspected (n = 20), and non-TBM (n = 25) cases. Immune response by Western blot revealed TBM CSF samples are having heterogeneous response to CFP. CFP ELISA was 92% sensitive and 38.30% specific. ODs of confirmed TBM and non-TBM cases were significantly different (P < 0.0001) and also the suspected TBM and non-TBM cases (P = 0.0001). No significant difference noticed in TBM and suspected TBM (P = 0.90). Thus, CFP can be a better biomarker for the diagnosis of TBM.

Responses to diagnostic tests for bovine tuberculosis in dairy and non-dairy cattle naturally exposed to Mycobacterium bovis in Great Britain

Field surveillance of British cattle using the single intradermal comparative cervical tuberculin (SICCT) test shows a higher incidence rate of bovine tuberculosis (bTB) in dairy compared to beef herds, but a lower probability of post-mortem examination confirmed (PMC) Mycobacterium bovis infection in dairy herds. A cross-sectional study was conducted to compare animal level differences in bTB detection between dairy and non-dairy cattle in Great Britain. During the period from 2002 to 2005, 200 (41% dairy) reactors in the SICCT test (standard interpretation) were randomly selected, and 200 in-contact cattle (43% dairy) were purposively selected from bTB-infected herds. Interferon (IFN)-γ responses in blood to bovine and avian purified protein derivative (PPD), and early secretory antigen target 6 kDa and culture filtrate protein 10 (ESAT-6/CFP10), were measured. The post-mortem examination included gross pathological examination, mycobacterial culture and histopathology. The proportions of cattle positive to ESAT6/CFP10 were 26% (95% confidence interval, CI, 15–39%) in dairy reactors and 62% (95% CI 51–72%) in non-dairy reactors (P < 0.001). PMC risk was 34% (95% CI 24–45%) in dairy reactors and 69% (95% CI 60–78%) in non-dairy reactors (P < 0.001). The odds ratio for PMC risk in dairy reactors compared to non-dairy reactors, after controlling for bTB prevalence, herd size and SICCT test response, was 0.27 (95% CI 0.14–0.53; P < 0.001). In surveillance data, adjusted animal level PMC risks were lower for dairy reactors than for beef reactors aged >2 years (P < 0.001).

Added Value of Long-Term Cytokine Release Assays to Detect Mycobacterium tuberculosis Infection in HIV-Infected Subjects in Uganda.

To investigate whether mycobacterial antigen-induced cytokine secretions are helpful in detecting Mycobacterium tuberculosis (Mtb) infection in a cohort of HIV-infected patients living in a country with a high burden of Mtb and HIV infections, and to determine their predictive value for the development of tuberculosis (TB)-associated immune reconstitution inflammatory syndrome. A total of 352 HIV-infected patients (186 with active TB) were prospectively enrolled when initiating antiretroviral therapy (ART). Sequential blood samples were collected during the first 6 months of ART. Eighty-three HIV-uninfected subjects (39 with active TB) were enrolled as controls. The concentrations of 13 cytokines were measured in supernatants from blood mononuclear cells in vitro stimulated with purified protein derivative (PPD), heparin-binding hemagglutinin (HBHA) or early secreted antigen-6 (ESAT-6) and culture filtrate protein-10 (CFP-10), and results were compared with those of tuberculin skin tests (TST). The best detection of Mtb infection was achieved by ESAT-6/CFP-10-induced interferon-γ concentrations, but results were often negative for patients with CD4 T-cell counts <50 per cubic millimeters. Patients with active TB were identified by high ESAT-6/CFP-10-induced interleukin-6. Conversions of interferon-γ-release assays (IGRA) and TST occurred under ART, and combined TB and antiretroviral treatments of coinfected patients resulted in a decrease of ESAT-6/CFP-10-induced and an increase of HBHA-induced interferon-γ responses. No Mtb antigen-induced cytokines allowed us to predict TB-immune reconstitution inflammatory syndrome or ART-associated TB. In Uganda, ESAT-6/CFP-10-IGRA is better in detecting Mtb infection than TST and, when combined with an HBHA-IGRA, could help to evaluate anti-TB treatment success.

Early Secreted Antigenic Target of 6kDa of Mycobacterium tuberculosis Stimulates Macrophage Chemoattractant Protein-1 Production by Macrophages and Its Regulation by p38 Mitogen-Activated Protein Kinases and Interleukin-4.

Early secreted antigenic target of 6kDa (ESAT-6), the major virulence factor of Mycobacterium tuberculosis, affects host immunity and the formation of granulomas likely through inflammatory cytokines. To understand its role in this regard further, we investigated the effect of ESAT-6 on macrophages by determining the production of macrophage chemoattractant protein (MCP)-1, a major chemokine associated with tuberculosis pathogenesis, by murine bone marrow-derived macrophages (BMDMs) and its regulation by protein kinases and cytokines. The results revealed that ESAT-6, but not Ag85A and culture filtrate protein 10kDa (CFP10), induced MCP-1 production by BMDMs dose and time dependently. Inhibition of p38 but not other mitogen-activated protein kinases (MAPK) and PI3K further enhanced ESAT-6-induced MCP-1 production by BMDMs. Inhibition of p38 MAPK enhanced ESAT-6-induced MCP-1 mRNA accumulation without affecting mRNA stability. ESAT-6 also induced TNF-α from BMDMs and MCP-1 from mouse lung epithelial cells, and these were suppressed by p38 MAPK inhibition, implying cytokine- and cell-specific effect of p38 MAPK inhibition on ESAT-6-induced MCP-1 by macrophages. Pretreatment of BMDMs with IL-4, but not other cytokines (IL-2, IL-10, TNF-α, IFN-γ and IL-1α) further elevated ESAT-6-stimulated MCP-1 production although IL-4 did not induce MCP-1 without ESAT-6. Both p38 MAPK inhibitor and IL-4 did not show additive effect on ESAT-6-induced MCP-1 protein level despite such effect on MCP-1 mRNA level was evident. In conclusion, these results indicate a specific role for both p38 MAPK and IL-4 in ESAT-6-induced MCP-1 production by macrophages and suggest a pathway with significance in tuberculosis pathogenesis.

Effects of Mycobacterium tuberculosis ESAT6-CFP10 Protein on Cell Viability and Production of Nitric Oxide in Alveolar Macrophages

BackgroundMycobacterium tuberculosis is the major pathogen of tuberculosis, which affects approximately one-third of the world’s population. The 6 kDa early secreted antigenic target (ESAT6) and the 10 kDa culture filtrate protein (CFP10), which are secreted by the ESX-1 system in M. tuberculosis, can contribute to mycobacterial virulence.ObjectivesThe aim of this study was to research the effects of M. tuberculosis ESAT6-CFP10 protein on macrophages during a host’s was first and second exposures to M. tuberculosis.Materials and MethodsIn this study, the ESAT6 and CFP10 genes were amplified to create a fusion gene (ESAT6-CFP10) and cloned into the pET-32a(+) and pEGFP-N1 expression vectors, respectively. The recombinant pET-32a(+)-ESAT6-CFP10 plasmid was transformed into the Escherichia coli Origami strain, and the fusion protein was expressed and confirmed by SDS-PAGE and Western blot analysis. The recombinant pEGFP-N1-ESAT6-CFP10 plasmid was transfected into rat alveolar macrophage cells (NR8383). The cell line expressing the ESAT6-CFP10 protein was selected with RT-PCR and designated as NR8383-EC. Finally, the effects of the ESAT6-CFP10 fusion protein on the NR8383 cell line, as well as on the newly constructed NR8383-EC cells, were further assessed.ResultsThe recombinant ESAT6-CFP10 protein was expressed in E. coli and in NR8383 rat alveolar macrophages. This protein affected the proliferation and nitric oxide (NO) generation of the NR8383 and NR8383-EC cells. Although NO generation was inhibited in both cell lines, proliferation was inhibited in NR8383 while it was increased NR8383-EC.ConclusionsThe data indicate that ESAT6-CFP10 could support the survival of M. tuberculosis in the host through altering the host immune response. It also indicates that the host may gain some level of protection from a second exposure to M. tuberculosis, as evidenced by increased proliferation of NR8383-EC.

Cynomolgus macaque neutrophils express IL-4 and IL-10 in response to Mycobacterium tuberculosis bacilli

Abstract Neutrophils are associated with increased disease severity in tuberculosis (TB) but mechanisms underlying their contribution to pathology are poorly understood. In granulomas, neutrophils localize to regions that are adjacent to macrophages, and we hypothesize that neutrophil-expressed cytokines can modify anti-mycobacterial immune responses. To investigate this, we examined pro- and anti-inflammatory cytokine production by neutrophils in Mycobacterium tuberculosis (Mtb) infected macaques, a nonhuman primate that replicates human TB. We found that intact bacteria were potent inducers of anti-inflammatory cytokine expression and viable Mtb elicited IL-10 while dead Mtb induced IL-4 expression. Bacterial stimulation and mycobacterial culture filtrate protein (CFP) induced TNF in peripheral blood neutrophils. Cytokine expression was not induced by stimulation with anti-CD3 antibody, poly I:C, or purified Mtb peptides and was independent of host infection status, suggesting this is an intrinsic property of neutrophils driven by mycobacterial TLR2/4 ligands. There was little polyfunctionality in neutrophils and most cells expressed either IL-4 or IL-10, or these cytokines in combination with TNF. Neutrophils were less efficient than T cells at cytokine production and expressed less cytokine on a per cell basis. Although there are more T cells than neutrophils in granulomas, we found similar frequencies of IL-4 or IL-10 expressing T cells and neutrophils, and identified IL-10+ neutrophils in caseum adjacent to epithelioid macrophages, a region associated with extracellular Mtb. These results suggest neutrophils are underappreciated sources of immunosuppressive cytokines that contribute to poor outcomes in TB.

Using the TBAg/PHA ratio in the T-SPOT(®).TB assay to distinguish TB disease from LTBI in an endemic area.

An important limitation of the T-SPOT(®).TB assay is its inability to distinguish active tuberculosis (TB) from latent tuberculous infection (LTBI). We proposed a new calculation method for the T-SPOT assay and assessed its effect on distinguishing active TB from LTBI. A total of 162 active TB patients and 97 LTBI individuals were diagnosed according to conventional tests and the T-SPOT assay. The results of early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) in T-SPOT cannot be recommended for distinguishing TB from LTBI. The number of phytohaemagglutinin (PHA) spot-forming cells (sfc) in the T-SPOT assay was reduced in active TB patients. The ESAT-6/PHA or CFP-10/PHA ratios in active TB patients were significantly higher than in individuals with LTBI. Using 0.295 as the threshold ratio of Mycobacterium tuberculosis-specific antigen (TBAg) sfc to PHA sfc (TBAg/PHA ratio, the larger of ESAT-6/PHA and CFP-10/PHA), the sensitivity and specificity were 82.1% and 90.7% in distinguishing active TB from LTBI. The TBAg/PHA ratio might also be used to monitor the effect of anti-tuberculosis treatment. Calculating the TBAg/PHA ratio might have the potential to diagnose active TB and distinguish TB disease from LTBI.

Investigation Into the Role of N‐terminal Acetylation of ESAT‐6 in Pathogenesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent for the disease Tuberculosis in humans, is present as a latent infection in approximately one third of the world population. Mtb has become more resilient over the years, as the vaccine, Mycobacterium bovis bacillus Calmette‐Guerin (BCG), has been shown to lose effectiveness 10 years after initial vaccination. Recent research found 6‐kD early secretory antigenic target (ESAT‐6) and 10‐kD culture filtrate protein (CFP‐10) are secreted as a heterodimer by Mtb and are important for virulence. Additionally, ESAT‐6 has been determined to contain membrane lytic activity, while CFP‐10 has been suggested to be a molecular chaperone. Furthermore, membrane lytic ability from ESAT‐6 only occurs after it has dissociated from CFP‐10. Studies suggest ESAT‐6 dissociates from CFP‐10 at low pH to interact with the phagosomal membrane to translocate Mtb into the cytosol of a macrophage, however the mechanism of heterodimer dissociation is unclear. Previous studies have identified and isolated an N‐terminally acetylated ESAT‐6 protein. Binding affinity between CFP‐10 and ESAT‐6 is greatly reduced after acetylation, suggesting that N‐terminal acetylation could be the culprit for the dissociation of the ESAT‐6 and CFP‐10 heterodimer. In this study, we aim to determine whether N‐terminal acetylation of Threonine 2 on ESAT‐6 is required for dissociation of the heterodimer. To test this, three different residues: alanine, glutamine, and arginine, were used to replace Threonine 2. We hypothesized the acetylation‐mimicking residues, alanine and glutamine would contain similar activity to wild‐type ESAT‐6 while acetylation negative control, arginine, would have less activity. Preliminary data, a liposome leakage assay with the dye/quencher pair, 8‐aminonaphthalene‐1,3,6 trisulfonic acid (ANTS) and p‐xylene‐bis‐pyridinium bromide (DPX), revealed these mutations did not affect pore formation of ESAT‐6 alone, but greatly diminished pore formation of the ESAT‐6 complexed with CFP‐10, suggesting alanine and glutamine failed to function as acetylation‐mimicking residues, highlighting the importance of Thr2. Cytotoxicity analysis of post‐macrophage infection using Mycobacterium marinum also revealed decreased activity in Thr2 mutants. Accordingly, if these mutations disrupt dissociation, then decreased translocation to the cytosol would be expected, as such fluorescence electron resonance transfer (FRET) was employed to test this theory. Furthermore, a novel in vitro method to dissociate the heterodimer complex was applied to test whether acetylation is present in wild type and not in proteins containing mutations of Thr2. The resultant proteins were analyzed using narrow range pH gradient two‐dimensional gel electrophoresis (2‐DE). Additionally, 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) reacts with un‐acetylated proteins, and exhibits measurable fluorescence, while N‐terminally acetylated proteins do not, compromising a reliable method for detection of acetylation in these proteins. This study has revealed the physiological importance of N‐alpha‐acetylation of ESAT‐6 in Mtb infection.Support or Funding InformationThis work was supported by the National Institutes of Health Grant 5G12RR008124 from the National Center for Research Resources and Grant 2G12MD007592 from National Institutes on Minority Health Disparities, a component of National Institutes of Health. This Research is also supported by University of Texas at El Paso new faculty start‐up funds.

Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts

Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility to R. solani when expressed in Nicotiana benthamiana. In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806.

Diagnostic performance of interferon-γ release assay for lymph node tuberculosis

The aim of the study was to evaluate the performance of interferon-γ (IFN-γ) release assay (IGRA) (T-SPOT.TB) for patients with suspected lymph node tuberculosis (TB). Of the 405 patients with suspected lymph node TB, enrolled from Beijing Chest Hospital between July 2011 and April 2015, 83 (20.5%) were microbiologically/histopathologically confirmed lymph node TB, and 282 (69.6%) did not have active TB. The remaining 21 inconclusive TB and 19 clinical TB were excluded from the final analysis (9.9%). T-SPOT.TB using peripheral blood mononuclear cells was performed to examine the IFN-γ response to the Mycobacterium tuberculosis–specific antigens early secretory antigenic target 6 and culture filtrate protein 10. The overall sensitivity and specificity for T-SPOT.TB were 90.4% and 70.5%, respectively. Spot-forming cells in the lymph node TB group (184 [48–596/106 peripheral blood mononuclear cells {PBMCs}]) were significantly higher than that in the nonactive TB group (0 [0–41]/106 PBMCs) (P<0.001). These results suggest that the IGRA assay could be a useful aid in the diagnosis of lymph node TB.

Fused Mycobacterium tuberculosis multi-stage immunogens with an Fc-delivery system as a promising approach for the development of a tuberculosis vaccine

Tuberculosis (TB) remains a major health problem worldwide. Currently, the Bacilli Calmette-Guérin (BCG) is the only available licensed TB vaccine, which has low efficacy in protection against adult pulmonary TB. Therefore, the development of a safe and effective vaccine against TB needs global attention. In the present study, a novel multi-stage subunit vaccine candidate from culture filtrate protein-10 (CFP-10) and heat shock protein X (HspX) of Mycobacterium tuberculosis fused to the Fc domain of mouse IgG2a as a selective delivery system for antigen-presenting cells (APCs) was produced and its immunogenicity assessed. The optimized gene constructs were introduced into pPICZαA expression vectors, and the resultant plasmids (pPICZαA-CFP-10:Hspx:Fcγ2a and pPICZαA-CFP-10:Hspx:His) were transferred into Pichia pastoris by electroporation. The identification of both purified recombinant fusion proteins was evaluated by SDS-PAGE and immunoblotting. Then the immunogenicity of the recombinant proteins with and without BCG was evaluated in BALB/c mice by assessing the level of IFN-γ, IL-12, IL-4, IL-17 and TGF-β cytokines. Both multi-stage vaccines (CFP-10:HspX:Fcγ2a and CFP-10:HspX:His) induced Th1-type cellular responses by producing high level of IFN-γ (272pg/mL, p<0.001) and IL-12 (191pg/mL, p<0.001). However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low level of IL-4 (10pg/mL) compared to the CFP-10:HspX:His group. The production of IFN-γ to CFP-10:HspX:Fcγ2a was markedly consistent and showed an increasing trend for IL-12 compared with the BCG or CFP-10:HspX:His primed and boosted groups. Findings revealed that CFP-10:Hspx:Fcγ2a fusion protein can elicit strong Th1 antigen-specific immune responses in favor of protective immunity in mice and could provide new insight for introducing an effective multi-stage subunit vaccine against TB.

Mycobacterium tuberculosis-Specific IL-21+IFN-γ+CD4+ T Cells Are Regulated by IL-12.

In the current study of Mycobacterium tuberculosis (MTB)-specific T and B cells, we found that MTB-specific peptides from early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) induced the expression of IL-21 predominantly in CD4+ T cells. A fraction of IL-21-expressing CD4+ T cells simultaneously expressed Th1 cytokines but did not secrete Th2 or Th17 cytokines, suggesting that MTB-specific IL-21-expressing CD4+ T cells were different from Th1, Th2 and Th17 subpopulations. The majority of MTB-specific IL-21-expressing CD4+ T cells co-expressed IFN-γ and IL-21+IFN-γ+CD4+ T cells exhibited obviously polyfunctionality. In addition, MTB-specific IL-21-expressing CD4+ T cells displayed a CD45RO+CD62LlowCCR7lowCD40LhighICOShigh phenotype. Bcl-6-expression was significantly higher in IL-21-expressing CD4+ T cells than IL-21-CD4+ T cells. Moreover, IL-12 could up-regulate MTB-specific IL-21 expression, especially the frequency of IL-21+IFN-γ+CD4+ T cells. Taken together, our results demonstrated that MTB-specific IL-21+IFN-γ+CD4+ T cells from local sites of tuberculosis (TB) infection could be enhanced by IL-12, which have the features of both Tfh and Th1 cells and may have an important role in local immune responses against TB infection.

Sequence Analysis of the Gene Region Encoding ESAT-6, Ag85B, and Ag85 C Proteins from Clinical Isolates of Mycobacterium tuberculosis

Mycobacterium tuberculosis secreted proteins in culture filtrate and early phase of infection, such as early secretory antigen target 6 (ESAT-6), culture filtrate protein 10 (CFP-10), and antigen 85 complex i.e. Ag85A, Ag85B, and Ag85C which played roles in adherence, invasion, cytolysis, and evading cytosol of macrophage, were virulence factors that determined the immune responses important on pathogenesis of Tuberculosis (TB), including granuloma formation or tissue that determine the degree of disease. The purpose of this research was to analyze the gene region sequence encoding ESAT-6, Ag85B, and Ag85C of Mycobacterium tuberculosis. Mycobacterium tuberculosis strain analyzed was taken from sputum of pulmonary TB patients in East Java, Indonesia. Sequenced DNA analyzed using GENETYX Ver.10. There were no SNPs both inside and outside epitope region of gene encoding ESAT-6, Ag85B, and Ag85C from clinical isolates of Mycobacterium tuberculosis. From this study, it could be concluded that the highly conserved gene region encoding ESAT-6, Ag85B, and Ag85C revealed no sequence polymorphism SNPs in epitope regions among Mycobacterium tuberculosis clinical isolates from sputum specimens of pulmonary TB patients.

Immunogenicity Studies with Microbial Fractions of M. tuberculosis H37Rv Total Culture Filtrate

&lt;p class="1Body"&gt;Current study investigates the whole secretory proteome of &lt;em&gt;Mycobacterium tuberculosis&lt;/em&gt; as culture filtrate fractions to identify immunoprotective protein antigens on the basis of protection studies in animal (mouse and guinea pig) models. Secretory culture filtrate proteins (CFPs) of &lt;em&gt;M. tuberculosis&lt;/em&gt; H&lt;sub&gt;37&lt;/sub&gt;Rv were fractionated into fifteen narrow molecular mass fractions in the order of increasing molecular size (F1-F15) by electroelution. Immunization studies revealed proteins in the molecular weight range of 20-24kDa (F7), 25-30kDa (F8) and 37-42kDa (F11) as key protective fractions against experimental tuberculosis in both the animal (mice and guinea pig) models. Amongst these fractions, F7 imparted even better protection as compared to BCG. Immunological studies with all the fractions demonstrated that although selected three protective fractions were able to induce significant immune responses in both short term culture filtrate (STCF) immunized and Mtb infected animals, there were number of other non-protective fractions also that were inducing higher immune responses either in immunized animals (e.g.F12-F15) or in Mtb challenged animals (e.g.F1-F6). These results demonstrate that only those mycobacterial proteins that are recognized by the host immune system both during immunization and infection can induce significant protection against experimental tuberculosis, however there is no direct correlation between the level of immune responses and degree of protective efficacy.&lt;/p&gt;

Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test.

The tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB), and use of this test has been very valuable in the control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). Recent studies showed that the use of three or four recombinant mycobacterial proteins, including 6-kDa early secretory antigenic target (ESAT6), 10-kDa culture filtrate protein (CFP10), Rv3615c, and Rv3020c, or a peptide cocktail derived from those proteins, in the skin test greatly enhanced test specificity, with minimal loss of test sensitivity. The proteins are present in members of the pathogenic Mycobacterium tuberculosis complex but are absent in or not expressed by the majority of environmental mycobacteria and the BCG vaccine strain. To produce a low-cost skin test reagent, the proteins were displayed at high density on polyester beads through translational fusion to a polyhydroxyalkanoate synthase that mediates the formation of antigen-displaying inclusions in recombinant Escherichia coli. Display of the proteins on the polyester beads greatly increased their immunogenicity, allowing for the use of very low concentrations of proteins (0.1 to 3 μg of mycobacterial protein/inoculum) in the skin test. Polyester beads simultaneously displaying all four proteins were produced in a single fermentation process. The polyester beads displaying three or four mycobacterial proteins were shown to have high sensitivity for detection of M. bovis-infected cattle and induced minimal responses in animals exposed to environmental mycobacteria or vaccinated with BCG.

Effects of crude extracted proteins from supernatant of BCF-P and EGF on acid secretion

In order to compare the in vitro effects of crude extracts of broth culture filtrate protein (BCF-P) from the supernatant of an H. pylori liquid culture and epidermal growth factor (EGF) on acid secretion by isolated rabbit gastric parietal cells. Isolated rabbit gastric parietal cells were incubated with BCF-P or EGF for 1 and 12 h. Morphological changes were assessed by electron microscopy, and 14C-aminopyrine (14C-AP) uptake and H+-K+ATP mRNA expression were measured. At 50 µg/mL, BCF-P stimulated acid secretion by parietal cells (P<0.05) at 1 h but not at 12 h. At 100 µg/mL, BCF-P significantly inhibited histamine-stimulated acid secretion (P<0.05) and decreased H+- K+ATP mRNA expression in parietal cells, but it was reversed after EGF exposure. Crude extracted proteins from BCF-P damaged the inner cell membrane structure of parietal cells, reduced H+-K+ATP gene expression, and inhibited acid secretion, activities that differed markedly from those of EGF.