Culture Filtrate Protein Research Articles

Two distinct conformational states of Mycobacteriumtuberculosis virulent factor early secreted antigenic target 6kDa are behind the discrepancy around its biological functions.

Early secreted antigenic target 6kDa (ESAT-6) and culture filtrate protein 10kDa (CFP-10) are complex proteins secreted by Mycobacteriumtuberculosis that play a major role in the pathogenesis of tuberculosis. However, studies focusing on the biological functions of ESAT-6 led to discordant results and the role of ESAT-6 remains controversial. In the present study, we aim to address a potential explanation for this discrepancy and to highlight the physiological impact of two conformational states of ESAT-6. Analysis of a recombinant form of ESAT-6 by native gel electrophoresis, size exclusion chromatography and CD spectroscopy revealed that ESAT-6 forms dimers/multimers with higher molecular weight, which disappeared under the action of the detergent amidosulfobetaine-14 (ASB), giving rise to another conformational state of the protein. NMR has further indicated that ASB-treated versus nontreated ESAT-6 adopted distinct structural forms but with no well defined tertiary structure. However, protein-protein docking analysis favored a dimeric state of ESAT-6. Interestingly, the two preparations presented opposing effects on mycobacterial infectivity, as well as macrophage survival, interferon-γ secretion and membrane pore formation. Thereafter, we generated a recombinant form of the physiological heterodimer ESAT-6/CFP-10 that ASB was also able to dissociate and which showed functions similar to those of ESAT-6 dimers/multimers. Our data suggest that, in the absence of CFP-10, the hydrophobic regions of the ESAT-6 can form dimers/multimers, mimicking the ESAT-6/CFP-10 heterodimer, whereas their dissociation generates a protein presenting entirely different activities. Overall, the present study clarifies the intriguing divergences between reports that could be attributed to the ESAT-6 oligomeric state and sheds light on its importance for a better comprehension of the physiopathology of tuberculosis.

Identification of a novel immunodominant antigen Rv2645 from RD13 with potential as a cell-mediated immunity-based TB diagnostic agent

Search for novel specific antigens are urgently needed for the detection of tuberculosis (TB). In this study, we evaluated the diagnostic potential of a novel Mycobacterium tuberculosis (M.tb)-specific candidate antigen (Rv2645) from DNA segment region of differentiation (RD) 13 of M.tb and investigated T-cell recognition during natural infection in humans and experimental mice. Rv2645-specific IFN-γ levels were much higher in the peripheral blood mononuclear cells (PBMCs) of TB patients than that in healthy donors (HDs) (including Bacille Calmette-Guerin (BCG)-vaccinated donors). The enzyme-linked immunospot (ELISPOT) assay with Rv2645 had a high overall agreement (98.0%) with the results from the clinical T-SPOT.TB with 10-kD culture filtrate protein (CFP10) and 6-kD early secreted antigenic target (ESAT6) peptides. The combination of Rv2645 and CFP10-ESAT6 was better than the individual protein, with increased sensitivity and a similar specificity of 96.0% and 98.2%, respectively. Rv2654 also induced M.tb-specific skin-test responses in heat-inactivated M.tb H37Rv immunized mice. Epitope mapping revealed that Rv264530-44 and Rv2645136-143 may be the dominant T-cell and B-cell epitopes, respectively, of Rv2645. This is the first report demonstrating the Rv2654 is a strongly recognized T-cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.

Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis.

A rapid and efficient diagnostic test was developed for the detection of Mycobacterium tuberculosis antigens in serum samples of active tuberculosis (TB) and extrapulmonary TB patients via a liposomal agglutination-based method. A rapid card test has been developed to facilitate the recognition of high-affinity binding rabbit raised purified culture filtrate protein antibodies coupled on the surface of activated liposomal preparation. In the presence of TB antigens, the polyclonal antibodies bound to the liposomal particles demonstrate a visible agglutination reaction. The developed assay was simple, rapid, reliable, sensitive, and specific as a diagnostic test for the detection of antigens in serum samples of clinically confirmed cases of TB within 4-5 minutes' duration. The test was evaluated at different hospitals, medical colleges, and pathology centers, and involved 1483 participants. This investigation was conducted to detect the presence of these antigens during the period of active growth of the microorganism in serum samples for pulmonary TB and processed tissue biopsy for other extrapulmonary TB. Results obtained using this test were compared with acid-fast bacilli smear and culture results. Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application.

Prime-boost vaccination with Bacillus Calmette Guerin and a recombinant adenovirus co-expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis induces robust antigen-specific immune responses in mice.

Tuberculosis (TB) remains to be a prevalent health issue worldwide. At present, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the singular anti-TB vaccine available for the prevention of disease in humans; however, this vaccine only provides limited protection against Mycobacterium tuberculosis (Mtb) infection. Therefore, the development of alternative vaccines and strategies for increasing the efficacy of vaccination against TB are urgently required. The present study aimed to evaluate the ability of a recombinant adenoviral vector (Ad5-CEAB) co-expressing 10-kDa culture filtrate protein, 6-kDa early-secreted antigenic target, antigen 85 (Ag85)A and Ag85B of Mtb to boost immune responses following primary vaccination with BCG in mice. The mice were first subcutaneously primed with BCG and boosted with two doses of Ad5-CEAB via an intranasal route. The immunological effects of Ad5-CEAB boosted mice primed with BCG were then evaluated using a series of immunological indexes. The results demonstrated that the prime-boost strategy induced a potent antigen-specific immune response, which was primarily characterized by an enhanced T cell response and increased production of cytokines, including interferon-γ, tumor necrosis factor-α and interleukin-2, in mice. In addition, this vaccination strategy was demonstrated to have an elevated humoral response with increased concentrations of antigen-specific bronchoalveolar lavage secretory immunoglobulin (Ig)A and serum IgG in mice compared with those primed with BCG alone. These data suggested that the regimen of subcutaneous BCG prime and mucosal Ad5-CEAB boost was a novel strategy for inducing a broad range of antigen-specific immune responses to Mtb antigens in vivo, which may provide a promising strategy for further development of adenoviral-based vaccine against Mtb infection.

Expression of autocrine prolactin and the short isoform of prolactin receptor are associated with inflammatory response and apoptosis in monocytes stimulated with Mycobacterium bovis proteins

Increased levels of prolactin (PRL) have recently been associated with carcinogenesis and the exacerbation of autoimmune diseases, and might be involved in the progression of tuberculosis (TB). To investigate the relationship between PRL and prolactin receptor (PRLr) expression with inflammatory response and apoptosis in monocytes, we used THP-1 cells stimulated with antigens of the Mycobacterium bovis AN5 strain culture filtrate protein (CFP-M. bovis). Western blot (WB), real-time Polymerase chain reaction (PCR), and immunocytochemistry were performed to identify both PRL and PRLr molecules. PRL bioactivity and proinflammatory cytokine detection were assessed. The results showed that PRL and PRLr messenger RNA (mRNA) were synthesized in THP-1 monocytes induced with CFP-M. bovis at peaks of 176- and 404-fold, respectively. PRL forms of 60 and 80kDa and PRLr isoforms of 40, 50, and 65kDa were also identified as time-dependent, while 60-kDa PRL, as well as 40-, and 50-kDa PRLr, were found as soluble forms in culture media and later in the nucleus of THP-1 monocytes. PRL of 60kDa released by monocytes exhibited bioactivity in Nb2 cells, and both synthesized PRL and synthesized PRLr were related with nitrite and proinflammatory cytokine levels proapoptotic activity in CFP-M. bovis-induced monocytes. Our results suggest the overexpression of a full-autocrine loop of PRL and PRLr in monocytes that enhances the inflammatory response and apoptosis after priming with M. bovis antigens.

Combined antigen-specific interferon-γ and interleukin-2 release assay (FluoroSpot) for the diagnosis of Mycobacterium tuberculosis infection.

BackgroundTo evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis.MethodsFollowing ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls.ResultsCorrelation of IFN- γ+ spot-forming cells in EliSpot-IGRA and FluoroSpot were R2 = 0.67 for ESAT-6 and R2 = 0.73 for CFP-10. The number of IL-2- IFN- γ+ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ+ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis.ConclusionsCombined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice.

Filarial infection modulates the immune response to Mycobacterium tuberculosis through expansion of CD4+ IL-4 memory T cells.

Exaggerated CD4(+) T helper 2-specific cytokine producing memory T cell responses developing concomitantly with a T helper 1 response might have a detrimental role in immunity to infection caused by Mycobacterium tuberculosis. To assess the dynamics of Ag-specific memory T cell compartments in the context of filarial infection, we used multiparameter flow cytometry on PBMCs from 25 microfilaremic filarial-infected (Inf) and 14 filarial-uninfected (Uninf) subjects following stimulation with filarial Ag (BmA) or with the M. tuberculosis-specific Ag culture filtrate protein-10 (CFP-10). Our data demonstrated that the Inf group had a marked increase in BmA-specific CD4(+)IL-4(+) cells (median net frequency compared with baseline [Fo] = 0.09% versus 0.01%; p = 0.038) but also to CFP-10 (Fo = 0.16% versus 0.007%; p = 0.04) and staphylococcal enterotoxin B (Fo = 0.49% versus 0.26%; p = 0.04). The Inf subjects showed a BmA-specific expansion of CD4(+)CD45RO(+)IL-4(+) producing central memory (TCM, CD45RO(+)CCR7(+)CD27(+); Fo = 1.1% versus 0.5%; p = 0.04) as well as effector memory (TEM, CD45RO(+)CCR7(-)CD27(-); Fo = 1.5% versus 0.2%; p = 0.03) with a similar but nonsignificant response to CFP-10. In addition, there was expansion of CD4(+)IL-4(+)CD45RA(+)CCR7(+)CD27(+) (naive-like) in Inf individuals compared with Uninf subjects. Among Inf subjects with definitive latent tuberculosis, there were no differences in frequencies of IL-4-producing cells within any of the memory compartments compared with the Uninf group. Our data suggest that filarial infection induces Ag-specific, exaggerated IL-4 responses in distinct T cell memory compartments to M. tuberculosis-specific Ags, which are attenuated in subjects who are able to mount a delayed type hypersensitivity reaction to M. tuberculosis.

Granzyme B-expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques.

The role of neutrophils in tuberculosis (TB), and whether neutrophils express granzyme B (grzB), a pro-apoptotic enzyme associated with cytotoxic T cells, is controversial. We examined neutrophils in peripheral blood (PB) and lung granulomas of Mycobacterium tuberculosis-infected cynomolgus macaques and humans to determine whether mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. We found large numbers of grzB-expressing neutrophils in macaque and human granulomas and these cells contained more grzB+ granules than T cells. Higher neutrophil, but not T cell, grzB expression correlated with increased bacterial load. Although unstimulated PB neutrophils lacked grzB expression, grzB expression increased upon exposure to M.tuberculosis bacilli, M.tuberculosis culture filtrate protein or lipopolysaccharide from Escherichia coli. Perforin is required for granzyme-mediated cytotoxicity by T cells, but was not observed in PB or granuloma neutrophils. Nonetheless, stimulated PB neutrophils secreted grzB as determined by enzyme-linked immunospot assays. Purified grzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration through extracellular matrix and regulating apoptosis or activation in other cell types. These data indicate mycobacterial products and the pro-inflammatory environment of granulomas up-regulates neutrophil grzB expression and suggests a previously unappreciated aspect of neutrophil biology in TB.

Early dynamics of T helper cell cytokines and T regulatory cells in response to treatment of active Mycobacterium tuberculosis infection.

Biomarkers that can identify tuberculosis (TB) disease and serve as markers for efficient therapy are requested. We have studied T cell cytokine production [interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-α] and degranulation (CD107a) as well as subsets of CD4+ T regulatory cells (Tregs) after in-vitro Mycobacterium tuberculosis (Mtb) antigen stimulation [early secretory antigenic target (ESAT)-6, culture filtrate protein (CFP)-10, antigen 85 (Ag85)] in 32 patients with active tuberculosis (TB) disease throughout 24 weeks of effective TB treatment. A significant decline in the fraction of Mtb-specific total IFN-γ and single IFN-γ-producing T cells was already observed after 2 weeks of treatment, whereas the pool of single IL-2+ cells increased over time for both CD4+ and CD8+ T cells. The Treg subsets CD25highCD127low, CD25highCD147++ and CD25highCD127lowCD161+ expanded significantly after Mtb antigen stimulation in vitro at all time-points, whereas the CD25highCD127lowCD39+ Tregs remained unchanged. The fraction of CD25highCD127low Tregs increased after 8 weeks of treatment. Thus, we revealed an opposing shift of Tregs and intracellular cytokine production during treatment. This may indicate that functional signatures of the CD4+ and CD8+ T cells can serve as immunological correlates of early curative host responses. Whether such signatures can be used as biomarkers in monitoring and follow-up of TB treatment needs to be explored further.

ESX/type VII secretion systems of mycobacteria: Insights into evolution, pathogenicity and protection

Pathogenesis of Mycobacterium tuberculosis depends on the secretion of key virulence factors, such as the 6 kDa early secreted antigenic target ESAT-6 (EsxA) and its protein partner, the 10 kDa culture filtrate protein CFP-10 (EsxB), via the ESX-1 secretion system. ESX-1 represents the prototype system of the recently named type VII secretion systems that exist in a range of actinobacteria. The M. tuberculosis genome harbours a total of five gene clusters potentially coding for type VII secretion systems, designated ESX-1 - ESX-5, with ESX-4 being the most ancient system from which other ESX systems seem to have evolved by gene duplication and gene insertion events. The five ESX systems show similarity in gene content and gene order but differ in function. ESX-1 and ESX-5 are both crucial virulence determinants of M. tuberculosis, but with different mechanisms. While ESX-1 is implicated in the lysis of the host cell phagosomes, ESX-5 is involved in secretion of the mycobacteria specific PE and PPE proteins and cell wall stability. Research on type VII secretion systems has thus become a large and competitive research topic that is tightly linked to studies of host-pathogen interaction of pathogenic mycobacteria. Insights into this matter are of relevance for redrawing the patho-evolution of M. tuberculosis, which might help improving current strategies for prevention, diagnostics and therapy of tuberculosis as well as elucidating the virulence mechanisms employed by this important human pathogen.

Immunoreactivity to Culture Filtrate Proteins of Mycobacterium avium Subspecies paratuberculosis in Naturally Infected Goat and Sheep Sera

Mycobacterium avium subspecies paratuberculosis (MAP), the cause of granulomatous chronic enteritis in ruminants ( Johne's disease) is under reported due to difficulties in diagnosing pre-clinical cases. Compromised speci- ficity is a problem due to extensive sharing of antigens /epitopes among MAP and other mycobacterial strains. Culture filtrate (CF) proteins profile of native 'Indian Bison Type' strain of MAP was studied at different harvesting times (2-8 weeks of growth) in Middlebrook 7H9 medium supplemented with ADC, PANTA antibiotics and mycobac- tin J. Analysis of harvested CF proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the greater part of CF proteins had molecular masses (<70 kDa) as 14, 19, 26, 34-41, 52-55, and 70 kDa. Immunoblotting showed reactivity of CF proteins commonly recognised (28, 34-36, 38-42, 45, and 56 kDa) with all four MAP infected goat and sheep sera at 2-8 weeks of growth. Collectively, these immunoreactive MAP CF proteins could be the potential targets for developing diagnostics against Johne's disease with improved sensitivity and high specificity instead of whole cell sonicated crude protoplasmic extracts (PPA).

Evaluation of Recombinant &amp;lt;i&amp;gt;Mycobacterium tuberculosis&amp;lt;/i&amp;gt; Antigens MPT64, CFP10, and ESAT6 for Delayed-Type Hypersensitivity Responses in Guinea Pigs

The tuberculin Purified Protein Derivative (PPD) skin test is widely used; however, the results are often inaccurate. Positive results can be observed in patients with active tuberculosis (TB) as well as in BCG-vaccinated persons and individuals who are infected with mycobacteria but have not developed the disease. MPT64, an antigen secreted from actively growing Mycobacterium tuberculosis and some strains of M. bovis BCG such as BCG Tokyo and BCG Russia, is immunogenic and elicits delayed-type hypersensitivity (DTH) in guinea pigs and humans. This antigen has been used to develop a new skin test for the diagnosis of active TB infection. Two of the antigens encoded by the M. tuberculosis-specific region of difference 1 (RD1, deleted in M. bovis BCG strains), CFP10 (culture filtrate protein 10) and ESAT6 (early secreted antigenic target-6), also induce M. tuberculosis-specific DTH responses. The aim of this study was to investigate the DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo elicited by three purified recombinant proteins (rMPT64, rCFP10 and rESAT6) compared to those elicited by PPD. In this study genes encoding MPT64, CFP10, and ESAT6 were cloned and expressed as recombinant proteins with the addition of a C-terminal His6 tag for ease of purification by Immobilized Metal ion Affinity Chromatography (IMAC). The recombinant proteins (rMPT64, rCFP10, and rESAT6) were purified to homogeneity and were used to elicit DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo. The results showed that rMPT64 elicits a DTH response comparable to that of PPD in M. bovis BCG Tokyo-vaccinated animals. However, M. tuberculosis-infected animals show less reactivity to rMPT64 than they do to PPD. Although single rCFP10 or rESAT6 did not readily elicit a DTH response in M. tuberculosis-infected animals, combining these antigens with rMPT64 led to an increased DTH response, thus enabling the detection of TB infection.

Performance of an interferon-gamma release assay in the diagnosis of tuberculous meningitis in children / Performanţa testului bazat pe eliberarea interferonului gamma în diagnosticul meningitei tuberculoase la copil

AbstractThe new immunodiagnostic tests based on the Mycobacterium tuberculosis specific antigen, early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), showed promising results in the diagnosis of tuberculosis infection. However, there are only few studies in the published literature on performance tests in cerebrospinal fluid. We investigated whether a rapid diagnosis of tuberculous meningitis (TBM) could be established by interferon-γ blood and cerebrospinal fluid (CSF) tests in children.We used the QuantiFERON-TB Gold in Tube test (QFT-IT) on blood and the QuantiFERON-TB Gold test (QFT-G) on the CSF of 63 subjects with TBM (including 25 case of definite TBM and 38 cases of probable TBM) and 62 controls.The CSF analyses indicated possible TBM in 63.4% of cases. The sensitivity of the CSF culture for Mycobacterium tuberculosis was only 39.6%. The sensitivity of the tuberculin skin test (TST) was 49.2% and the specificity was 88.6%. The estimated sensitivities of the QFT-G for the CSF and QFT-IT for the blood in culture confirmed TBM cases (gold standard) were 84% and 80%, respectively. The estimated specificities were 98.2% for the CSF and 87.9% for the blood. This study showed that the sensitivity of QFT for the CSF could be higher than TST and culture and slightly higher in CSF than in blood. The specificity of QFT-G for the CSF was higher those of the TST, but the specificity of QFT-IT is lower.QFT-G of the CSF is a useful diagnostic marker of tuberculosis that may improve the management of TBM, but the test results must be correlated with clinical, radiological and characteristics of CSF. New researches are needed to investigate the performance of QFT-G in the CSF compared with ELISPOT and PCR

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.

Tuberculosis distorts the inhibitory impact of interleukin-10 in HIV infection.

This study aimed to assess how Mycobacterium tuberculosis (MTB) coinfection alters the impact of interleukin-10 in chronic HIV infection. We assessed plasma cytokine levels (interleukin-10, interferon-γ, tumor necrosis factor-α, interleukin-2, interleukin-6 and interleukin-13) in 82 individuals presenting with HIV monoinfection, HIV-LTBI (latent MTB infection) coinfection or HIV-TB (active tuberculosis) coinfection. We also assessed the influence of MTB on the functional impact of interleukin-10 receptor alpha (interleukin-10Rα) blockade on HIV and MTB-specific CD4(+) T cells. Plasma cytokine levels were measured by high sensitivity Luminex. We used an ex-vivo interleukin-10Rα blockade assay to assess if functional enhancement of HIV and MTB-specific CD4(+) T cells was possible following a 48-h stimulation with HIV gag or pooled ESAT-6 (6 kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) peptides. Cell supernatant was collected 48 h after stimulation and the cytokine profile was measured by Luminex. Plasma interleukin-10 levels were elevated in HIV-TB as compared with HIV monoinfection (P < 0.05) and HIV-LTBI (P < 0.05). Plasma interleukin-10 levels correlated to HIV viral load in HIV monoinfection (P = 0.016) and HIV-LTBI (P = 0.042), but not HIV-TB. Ex-vivo blockade of interleukin-10Rα significantly enhanced MTB and HIV-specific CD4(+) T-cell function in HIV-LTBI individuals but not in HIV-TB individuals. Tuberculosis disrupts the correlation between interleukin-10 and markers of HIV disease progression. In addition, HIV-TB is associated with a more inflammatory cytokine milieu compared with HIV monoinfection. Interestingly, interleukin-10Rα blockade can enhance both HIV and MTB-specific T-cell function in HIV-LTBI, but not in HIV-TB coinfection.

Effect of isoniazid on antigen-specific interferon-γ secretion in latent tuberculosis.

Treatment of persons with latent tuberculosis (TB) infection at greatest risk of reactivation is an important component of TB control and elimination strategies. Biomarkers evaluating the effectiveness of treatment of latent TB infection have not yet been identified. This information would enhance control efforts and assist the evaluation of new treatment regimes.We designed a two-group, two-arm, randomised clinical study of tuberculin skin test-positive participants: 26 with documented contact with TB patients and 34 with non-documented contact. Participants in each group were randomly assigned to the immediate- or deferred-isoniazid treatment arms. Assays of in vitro interferon (IFN)-γ secretion in response to recombinant Rv1737 and overlapping synthetic peptide pools from various groups of immunodominant proteins were performed.During isoniazid therapy, a significant increase from baseline in the proportion of IFN-γ responders to the 10-kDa culture filtrate protein, Rv2031, Rv0849, Rv1986, Rv2659c, Rv2693c and the recombinant Rv1737 protein was observed (p⩽0.05). The peptide pool of Rv0849 and Rv1737 recombinant proteins induced the highest percentage of IFN-γ responders after isoniazid therapy.The in vitro IFN-γ responses to these proteins might represent useful markers to evaluate changes associated with treatment of latent TB infection.

CFP-10 from Mycobacterium tuberculosis selectively activates human neutrophils through a pertussis toxin-sensitive chemotactic receptor.

Upon infection with Mycobacterium tuberculosis, neutrophils are massively recruited to the lungs, but the role of these cells in combating the infection is poorly understood. Through a type VII secretion system, M. tuberculosis releases a heterodimeric protein complex, containing a 6-kDa early secreted antigenic target (ESAT-6) and a 10-kDa culture filtrate protein (CFP-10), that is essential for virulence. Whereas the ESAT-6 component possesses multiple virulence-related activities, no direct biological activity of CFP-10 has been shown, and CFP-10 has been described as a chaperone protein for ESAT-6. We here show that the ESAT-6:CFP-10 complex induces a transient release of Ca(2+) from intracellular stores in human neutrophils. Surprisingly, CFP-10 rather than ESAT-6 was responsible for triggering the Ca(2+) response, in a pertussis toxin-sensitive manner, suggesting the involvement of a G-protein-coupled receptor. In line with this, the response was accompanied by neutrophil chemotaxis and activation of the superoxide-producing NADPH-oxidase. Neutrophils were unique among leukocytes in responding to CFP-10, as monocytes and lymphocytes failed to produce a Ca(2+) signal upon stimulation with the M. tuberculosis protein. Hence, CFP-10 may contribute specifically to neutrophil recruitment and activation during M. tuberculosis infection, representing a novel biological role for CFP-10 in the ESAT-6:CFP-10 complex, beyond the previously described chaperone function.

Prolactin modulates cytokine production induced by culture filtrate proteins of M. bovis through different signaling mechanisms in THP1 cells

The immunomodulatory functions of prolactin (PRL) are well recognized. Augmented PRL plasma levels were observed in patients with advanced tuberculosis (TB). Recently, we have reported that LPS and Mycobacterium bovis (M. bovis) induced differential expression of PRL receptor (PRLR) isoforms in THP-1 cells and bovine macrophages, respectively. The aim of this work was to determine whether PRL should be considered as a potential modulator of the signaling pathways and cytokine synthesis, induced by culture filtrate protein (CFP) from M. bovis in THP-1 monocytes. The THP-1 cells were stimulated with PRL (20ng/mL), M. bovis CFP (50μg/mL). PRLR as well as phosphorylated STAT3, STAT5, Akt1/2/3, ERK1/2 and p38 expression were evaluated by Western blot. IL1-β, TNF-α, IL-6, IL-12, IL-8, and IL-10 concentrations were measured by ELISA. Our results demonstrated that the expression pattern of PRLR short isoforms is induced by M. bovis CFP. M bovis CFP induced phosphorylation of Akt2, ERK1/2, p38, STAT3, and STAT5 pathways. In turn, PRL only activated the JAK2/STAT3-5 signaling pathway. However, when combined both stimuli, PRL significantly increased STAT3-5 phosphorylation and downregulated Akt2, ERK1/2, and p38 phosphorylation. As expected, M. bovis CFP induced substantial amounts of IL1-β, IL-6, TNF-α, IL-8, IL-12, and IL-10. However, the PRL costimulation considerably decreased IL1-β, TNF-α, and IL-12 secretion, and increased IL-10 production. This results suggest that up-regulation of IL-10 by PRL might be modulating the pro-inflammatory response against mycobacterial antigens through the MAPK pathway.

Glycosylation and immunoreactivity of the Histoplasma capsulatum Cfp4 yeast-phase exoantigen.

The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome of Histoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells, suggesting its role in pathogenesis. We have generated three monoclonal antibodies as tools for characterization and detection of Cfp4 and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages or during lung infection in a murine model of histoplasmosis. Coinfection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage to Histoplasma yeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well as a strain from Panama. In addition, human immune sera recognize the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.

Interaction of alveolar epithelial cells with CFP21, a mycobacterial cutinase-like enzyme.

Mycobacterium tuberculosis (M. tb), an intracellular pathogen, has the ability to infect alveolar epithelial cells (AEC) also in addition to alveolar macrophages. The virulence of M. tb is attributed to proteins encoded by genomic regions of deletion (RD) and till date 16 such regions (RD1-RD16) have been identified. Culture filtrate protein 21 (CFP21), a RD2 secretory protein, is a cutinase-like enzyme that possesses esterase and lipolytic activity. It is hypothesized that CFP21 could be playing a role in M. tb virulence by disrupting the host cell integrity. In this study, recombinant CFP21 was expressed and purified. The in vitro exposure of type I (WI26) and type II (A549) AEC to CFP21 resulted in a significant decline in their cellular viability by inducing cell apoptosis. However, the cytotoxic effects were more pronounced in WI26 cells than in A549 cells. The analysis of immune responses in CFP21-treated AEC exhibited significant production of reactive oxygen species and anti-inflammatory cytokine TGF-β which indicated oxidative stress-mediated cell death. These results show that CFP21 could play an important role in M. tb pathogenesis by disrupting the host alveolar barrier and thereby facilitating mycobacterial dissemination.