Tuberculosis Culture Filtrate Proteins Research Articles

Predictive value of TNF-\u03b1, IFN-\u03b3, and IL-10 for tuberculosis among recently exposed contacts in the United States and Canada

BackgroundWe examined cytokine immune response profiles among contacts to tuberculosis patients to identify immunologic and epidemiologic correlates of tuberculosis.MethodsWe prospectively enrolled 1272 contacts of culture-confirmed pulmonary tuberculosis patients at 9 United States and Canadian sites. Epidemiologic characteristics were recorded. Blood was collected and stimulated with Mycobacterium tuberculosis culture filtrate protein, and tumor necrosis factor (TNF-α), interferon gamma (IFN-γ), and interleukin 10 (IL-10) concentrations were determined using immunoassays.ResultsOf 1272 contacts, 41 (3.2%) were diagnosed with tuberculosis before or < 30 days after blood collection (co-prevalent tuberculosis) and 19 (1.5%) during subsequent four-year follow-up (incident tuberculosis). Compared with contacts without tuberculosis, those with co-prevalent tuberculosis had higher median baseline TNF-α and IFN-γ concentrations (in pg/mL, TNF-α 129 versus 71, P < .01; IFN-γ 231 versus 27, P < .001), and those who subsequently developed incident tuberculosis had higher median baseline TNF-α concentrations (in pg/mL, 257 vs. 71, P < .05). In multivariate analysis, contact age < 15 years, US/Canadian birth, and IFN or TNF concentrations > the median were associated with co-prevalent tuberculosis (P < .01 for each); female sex (P = .03) and smoking (P < .01) were associated with incident tuberculosis. In algorithms combining young age, positive skin test results, and elevated CFPS TNF-α, IFN-γ, and IL-10 responses, the positive predictive values for co-prevalent and incident tuberculosis were 40 and 25%, respectively.ConclusionsCytokine concentrations and epidemiologic factors at the time of contact investigation may predict co-prevalent and incident tuberculosis.

Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications.

ABSTRACTDirect pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.

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.

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.

Neutrophils in granulomas from Mycobacterium tuberculosis-infected macaques express granzyme B in response to mycobacterial products and pro-inflammatory signals (INC7P.406)

Abstract Inflammation is a critical component of tuberculosis (TB), and may contribute to a poor prognosis. The factors responsible for this inflammation are poorly studied. In addition, the role of neutrophils in TB, and whether neutrophils express granzyme B (grzB), a cytotoxic pro-apoptosis enzyme, is controversial. We examined neutrophils in peripheral blood (PB) and granulomas to determine if mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. PB neutrophils did not express grzB. In contrast, granuloma neutrophils expressed grzB and these cells contained more grzB than T cells. Elevated neutrophil, but not T cell, grzB expression correlated with higher tissue bacterial load. Perforin is required for T cell-mediated cytotoxicity but was not expressed by neutrophils. PB neutrophils upregulated grzB expression when infected with M. tuberculosis or stimulated with M. tuberculosis culture filtrate protein or LPS from E. coli, but not peptide antigens associated with T cell stimulation. Moreover, stimulated PB neutrophils secreted grzB in a perforin-independent process. GrzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration or regulating activation in other cell types. These data indicate M. tuberculosis-specific factors in granulomas upregulate neutrophil grzB expression and suggest a previously unappreciated aspect of neutrophil biology in TB.

Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection

Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen‐specific IFN‐γ and IL‐2‐expressing CD4+ and CD8+ T cells. In exosome‐vaccinated mice, there was a similar TH1 immune response but a more limited TH2 response compared to BCG‐vaccinated mice. Using a low‐dose M. tuberculosis mouse aerosol infection model, exosomes from CFP‐treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell‐free vaccine against an M. tuberculosis infection.

O-linked glycosylation sites profiling in Mycobacterium tuberculosis culture filtrate proteins

Mycobacterium tuberculosis (Mtb) causes tuberculosis, one of the leading causes of fatal infectious diseases worldwide. Cell-cell recognition between the pathogen Mtb and its host is mediated in part by glycosylated proteins. So far, glycoproteins in Mtb are understudied and for only very few glycoproteins glycosylation sites have been described, e.g., alanine and proline rich secreted protein apa, superoxide dismutase SODC, lipoprotein lpqH and MPB83/MPT83. In this study, glycosylated proteins in Mtb culture filtrate were investigated using liquid chromatography-mass spectrometry approaches and bioinformatic analyses. To validate the presence of glycoproteins, several strategies were pursued including collision induced dissociation, high energy collision dissociation and electron transfer dissociation techniques, and bioinformatics analyses involving a neutral loss search for glycosylated moieties. After extensive data curation, we report glycosylation sites for thirteen Mtb glycoproteins using a combination of mass spectrometry techniques on a dataset collected from culture filtrate proteins. This is the first glycoproteomics study identifying glycosylation sites on mycobacterial culture filtrate proteins (CFP) on a global scale. In this study, glycosylation sites in Mtb were characterized by collision-induced dissociation, electron-transfer dissociation and high energy collision dissociation techniques. The identification of glycosylation sites is important for our understanding of the physiology and pathophysiology of Mtb. Glycoproteins are often responsible for protein-protein interactions between host and pathogen and thus represent interesting targets for vaccine development. In addition, our strategy is not limited to Mtb, but could be extended to other organisms. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Syndecan-1 and Heparanase

Syndecan-1 (SDC1) and its endo-beta-D-glucuronidase heparanase (HPA) are implicated in the maintenance of intestinal barrier function, but their detailed functions in Crohn's disease (CD) are not fully investigated. The aim of this study was to determine alteration patterns of SDC1 and HPA and their potential roles in evaluating disease activity and differentiating CD from intestinal tuberculosis (ITB). Tissue and serum specimens were obtained from 89 patients, including 15 patients with functional bowel disorders, 18 active patients with ITB, and 56 patients with CD (remission = 19, active = 37). Basic clinical data were collected and routine blood tests were analyzed. SDC1 and HPA were measured by immunohistochemistry, enzyme-linked immunosorbent assay, reverse transcriptase polymerase chain reaction, and western blot. Colonic epithelial cells were incubated with recombinant HPA, tumor necrosis factor alpha (TNF-α), and mycobacterium tuberculosis culture filtrate protein to detect the alterations of SDC1 and HPA. In the CD group, SDC1 was significantly decreased in mucosa and increased in serum, whereas HPA level in both were elevated. Such alterations were associated with clinicopathological features representing disease activity and injury severity and were not available in functional bowel disorder and ITB groups. Recombinant HPA incubation increased soluble SDC1 in culture supernatants (P = 2 × 10(-4)), and low-dose TNF-α effectively enhanced HPA's activity (P = 3 × 10(-6)). Exogenous TNF-α destroyed cellular SDC1 and raised HPA expressions dose dependently, whereas mycobacterium tuberculosis culture filtrate protein showed no effects. Unique alterations of SDC1 and HPA are shown in both patients with CD and in vitro model. The results indicate SDC1 and HPA are potential markers for CD in evaluating its disease activity and differentiating it from ITB.

Changes in cell-mediated immune response after lung resection surgery for MDR-TB patients

The immune responses of multidrug-resistant tuberculosis (MDR-TB) patients undergoing lung resection surgery were investigated in order to understand the mechanism of strong immune suppression in MDR-TB. We examined changes in cell-mediated immune response (CMI) of a total of sixteen MDR-TB patients, three of them extensively drug-resistant tuberculosis (XDR-TB) patients, after the removal of the heavily diseased lung section. The IFN-γ response to Mycobacterium tuberculosis culture filtrate proteins (Mtb-CFP), one of the most important CMI to defend TB, showed a statistically significant elevation in 2-4 months after operation when compared to the preoperative CMI in patients who were converted into AFB negative and cured in two years' follow-up, suggesting that the recovery of CMI may be one of the key factors in the successful treatment of MDR-TB. Interestingly, IL-10 response to Mtb-CFP was also elevated in 2-4 months after surgery in cured patients although both proliferative response and PBMC composition were not significantly changed. Infection with first- or second-line drugs resistant Mtb reduces the efficiency of chemotherapeutic treatment of MDR-TB to about 50%. Thus, this study suggests that chemotherapeutic treatment of MDR-TB may be more effective when combined with accompanying therapy that increases CMI, includes lung resection surgery.

Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages.

Exosomes are small 30–100 nm membrane vesicles released from hematopoietic and nonhematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present in exosomes inducing these host responses were not defined. This study used LC‐MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis‐infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFPs) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP‐treated J774 cells, the majority of which were also present in exosomes isolated from M. tuberculosis‐infected cells. The exosomes from CFP‐treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.

Immunization of mice with mycobacterial culture filtrate proteins

Culture filtrate proteins were obtained from Mycobacterium tuberculosis cultures after 7 days growth in Proskauer and Beck medium. The protein yield increased substantially to peak about the time the number of viable organisms reached its maximum level (day 8). Examination of the protein concentrate by SDS-PAGE revealed the presence of at least 12 separate protein bands varying from 10 to 90 kD. Mice were injected subcutaneously with 20 micrograms of M. tuberculosis culture filtrate (MTCF) protein suspended in saline or Freund's complete or incomplete adjuvant. The vaccinated mice were subjected to an aerogenic challenge with 10(3) colony-forming unit (CFU) M. tuberculosis Erdman and a significant reduction in the number of viable organisms was observed in the spleens and lungs determined over a 21-day period compared with age-matched normal controls. Mice immunized with the same culture filtrate proteins bound to nitrocellulose particles also showed some resistance to the virulent challenge, suggesting that individual antigens present in the culture filtrate were able to induce a protective T cell-mediated immune response in appropriately immunized mice.

Hypercholesterolemia Impairs Immunity to Tuberculosis

We demonstrate that apolipoprotein E -deficient (ApoE(-/-)) mice are highly susceptible to tuberculosis and that their susceptibility depends on the severity of hypercholesterolemia. Wild-type (WT) mice and ApoE(-/-) mice fed a low-cholesterol (LC) or high-cholesterol (HC) diet were infected with approximately 50 CFU Mycobacterium tuberculosis Erdman by aerosol. ApoE(-/-) LC mice were modestly more susceptible to tuberculosis than WT LC mice. In contrast, ApoE(-/-) HC mice were extremely susceptible, as evidenced by 100% mortality after 4 weeks with tuberculosis. The lung pathology of ApoE(-/-) HC mice was remarkable for giant abscess-like lesions, massive infiltration by granulocytes, elevated inflammatory cytokine production, and a mean bacterial load approximately 2 log units higher than that of WT HC mice. Compared to WT HC mice, the gamma interferon response of splenocytes restimulated ex vivo with M. tuberculosis culture filtrate protein was delayed in ApoE(-/-) HC mice, and they failed to control M. tuberculosis growth in the lung. OT-II cells adoptively transferred into uninfected ApoE(-/-) HC mice had a weak proliferative response to their antigen, indicating impaired priming of the adaptive immune response. Our studies show that ApoE(-/-) deficiency is associated with delayed expression of adaptive immunity to tuberculosis caused by defective priming of the adaptive immune response and that elevated serum cholesterol is responsible for this effect.

Peripheral Blood Gamma Interferon Release Assays Predict Lung Responses andMycobacterium tuberculosisDisease Outcome in Mice

Current diagnostic tests for tuberculosis (TB) are not able to distinguish active disease from latent Mycobacterium tuberculosis infection, nor are they able to quantify the risk of a latently infected person progressing to active TB. There is interest, however, in adapting antigen-specific gamma interferon (IFN-gamma) release assays (IGRAs) to predict disease outcome. In this study, we used the differential susceptibilities of inbred mouse strains to M. tuberculosis infection to evaluate the prognostic capabilities of IGRAs. Using lung and blood cultures, we determined that CBA/J, DBA/2, and C3H/HeJ mice (models of heightened risk of progression to active TB) produced less antigen-specific IFN-gamma in response to M. tuberculosis culture filtrate proteins and early secreted antigenic target-6 than the relatively resistant C57BL/6 mouse strain. Additionally, reduced IFN-gamma secretion in supernatants reflected a reduced frequency of IFN-gamma-responding cells in the lung and blood and not a specific defect in IFN-gamma secretion at the single-cell level. Importantly, detection of antigen-specific IFN-gamma from blood cultures accurately reflected lung responses, indicating that blood can be an appropriate test tissue in humans. Furthermore, reduced antigen-specific IFN-gamma production and low frequencies of IFN-gamma-responding cells from peripheral blood predicted increased risk of TB disease progression across genetically diverse TB disease-susceptible mouse strains, suggesting that similar results may occur in humans. The development of efficacious predictive diagnostic tests for humans would lead to targeted therapy prior to progression to active TB, reducing transmission, incidence, and prevalence rates while maximizing the use of public health resources.

Identification of novel bacterial plasminogen‐binding proteins in the human pathogen Mycobacterium tuberculosis

Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.

A Single Dose of Vitamin D Enhances Immunity to Mycobacteria

Vitamin D was used to treat tuberculosis (TB) in the preantibiotic era. Prospective studies to evaluate the effect of vitamin D supplementation on antimycobacterial immunity have not previously been performed. To determine the effect of vitamin D supplementation on antimycobacterial immunity and vitamin D status. A double-blind randomized controlled trial was conducted in 192 healthy adult TB contacts in London, United Kingdom. Participants were randomized to receive a single oral dose of 2.5 mg vitamin D or placebo and followed up at 6 weeks. The primary outcome measure was assessed with a functional whole blood assay (BCG-lux assay), which measures the ability of whole blood to restrict luminescence, and thus growth, of recombinant reporter mycobacteria in vitro; the readout is expressed as a luminescence ratio (luminescence postinfection/baseline luminescence). IFN-gamma responses to the Mycobacterium tuberculosis antigens early secretory antigenic target-6 and culture filtrate protein 10 were determined with a second whole blood assay. Vitamin D supplementation significantly enhanced the ability of participants' whole blood to restrict BCG-lux luminescence in vitro compared with placebo (mean luminescence ratio at follow-up, 0.57, vs. 0.71, respectively; 95% confidence interval for difference, 0.01-0.25; p=0.03) but did not affect antigen-stimulated IFN-gamma secretion. A single oral dose of 2.5 mg vitamin D significantly enhanced the ability of participants' whole blood to restrict BCG-lux luminescence in vitro without affecting antigen-stimulated IFN-gamma responses. Clinical trials should be performed to determine whether vitamin D supplementation prevents reactivation of latent TB infection. Clinical trial registered with www.clinicaltrials.gov (NCT 00157066).

Mycobacterium tuberculosisESAT-6 antigen immunogenicity in Owl Monkeys

La única vacuna disponible contra la tuberculosis es la cepa Mycobacterium bovis BCG, que ofrece una eficacia protectiva variable (0%-80%), siendo urgente un nuevo agente profiláctico. Se han evaluado diversos candidatos a vacuna contra este patógeno, en los modelos animales de experimentación convencionales (murino, cobayo, conejo), obteniéndose información básica sobre el efecto de la vacuna en la carga bacterial frente a un reto infeccioso, así como también la reducción o prevención de la patología en los pulmones u otros órganos blanco; además de los aspectos relacionados con la respuesta inmune hacia el Mycobacterium tuberculosis. Los primates no humanos tienen ventajas sobre los modelos convencionales en la evaluación de vacunas, de hecho se ha verificado el comportamiento de agentes terapéuticos en humanos después de haber sido medida la capacidad protectiva de éstos en monos con tuberculosis inducida. Los primates más estudiados en la infección por micobacterias son el cynomulgus, y el rhesus, observándose que estos animales mantienen la infección en un estado subclínico, muy similar a la tuberculosis humana donde el 90% de la población infectada mantiene la infección en un estado latente.Dado que el modelo animal debe semejar el comportamiento de las proteínas estudiadas en el ser humano, el mono Aotus puede representar ventajas en investigación de tuberculosis por ser un primate con aproximadamente un 90% de similitud al humano en cuanto a las moléculas del sistema inmune estudiadas hasta hoy. La proteína ESAT-6 de (early secretory antigenic target 6 kD) de Mycobacterium tuberculosis es un componente minoritario del filtrado de cultivo de corto tiempo (CFP), ha sido genética y químicamente caracterizada e induce una potente respuesta inmunogénica del tipo TH1. Este antígeno es secretado durante la fase inicial de crecimiento siendo fuertemente reconocido por animales y humanos infectados por Mycobacterium tuberculosis, este hecho hace que su inclusión en una futura vacuna anti-tuberculosis por subunidades y en pruebas de inmunodiagnóstico.En este trabajo se estudió la respuesta inmune del mono Aotus frente al antígeno micobacteriano ESAT-6 en forma recombinante (rESAT-6). El antígeno fue inmunizado junto con el adyuvante Montanide 720 produciendo una fuerte respuesta humoral y celular, no solo hacia rESAT-6 sino también hacia la proteína nativa presente en el filtrado de medio de cultivo de Mycobacterium tuberculosis. La respuesta celular fue medida por la incorporación de [3H]timidina en ensayos de linfoproliferación. La respuesta humoral se analizó por ensayos de ELISA e inmunoblot, obteniéndose altos títulos de anticuerpos (hasta de 1:12,800) dirigidos rESAT- 6. Se demostró la naturaleza multiepitope de este antígeno ya que en general péptidos que mapean la proteína fueron específicamente reconocidos tanto a nivel celular como humoral. Se observó además variación en la respuesta a la vacunación entre animal y animal, siendo una desventaja para un modelo de experimentación en cuanto a la predicción de la respuesta. Este estudio se constituye en un primer paso de evaluación del mono Aotus como modelo animal en tuberculosis.

A Proteome Reference Map and Proteomic Analysis of Bifidobacterium longum NCC2705

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.

The 19-kDa antigen of Mycobacterium tuberculosis is a major adhesin that binds the mannose receptor of THP-1 monocytic cells and promotes phagocytosis of mycobacteria

Identification of mycobacterial adhesins is needed to understand better the pathogenesis of tuberculosis and to develop new strategies to fight this infection. In this work, THP-1 monocytic cells were incubated with Mycobacterium tuberculosis culture filtrate proteins labelled with biotin and a dominant 19-kDa adhesin was found. This adhesin was characterized as the glycosylated and acylated 19-kDa antigen (Rv 3763). These findings were confirmed in assays with culture filtrate proteins and cell-wall fractions from a recombinant Mycobacterium smegmatis strain that overexpresses the 19-kDa antigen. Further, fluorescent microspheres coated with recombinant culture filtrate proteins adhere to cells in higher numbers than microspheres coated with native M. smegmatis proteins. The binding of the 19-kDa antigen to cells was inhibited with mannose receptor competitor sugars, Ca 2+ chelators and with a monoclonal antibody to the human mannose receptor. Phagocytosis assays showed high-level binding of bacilli to THP-1 cells that was inhibited with α-methyl-mannoside, mannan, EDTA and mAbs to the mannose receptor and to the 19-kDa M. tuberculosis antigen. Immunoprecipitation, cell-surface ELISA and immunostaining confirmed the expression of the mannose receptor by THP-1 cells. In conclusion, here we show that the macrophage mannose receptor, considered a pathogen pattern recognition receptor, may interact with mannose residues of mycobacterial glycoproteins that could promote the phagocytosis of mycobacteria.

Associations between helminth infection and CD4 + T cell count, viral load and cytokine responses in HIV-1-infected Ugandan adults

It has been proposed that helminth infection may exacerbate HIV progression by promoting activation of ‘type 2’ immune responses. To examine this hypothesis, we investigated helminth infection in a cohort of HIV-1-seropositive adults in Entebbe, Uganda, during November 1999 to January 2000. Individuals with helminths were treated. At enrolment, after 5 weeks and after 4 months, CD4 + and CD8 + T cell counts and viral load were measured. Cytokine responses (interferon [IFN]-γ, interleukin [IL]-2, IL-4 and IL-5) to Schistosoma mansoni adult worm antigen (SWA), Mycobacterium tuberculosis culture filtrate proteins (CFPs) and phytohaemagglutinin (PHA) were measured in a whole blood assay. At baseline, CD4 + T cell counts and CD4 +:CD8 + ratios were higher in individuals with helminths than in those without (median CD4 + T cell counts 467/μL and 268/μL, respectively, P = 0.005). Viral load was lower among those with helminths but this was not statistically significant. During follow-up, CD4 + T cell counts and cytokine responses to PHA fell among individuals without helminths. Among those treated for helminths, CD4 + counts remained stable. Viral loads showed a transient increase at 5 weeks, which was more marked among those treated for helminths, but the levels at 4 months were similar to baseline in both groups. Among those with schistosomiasis, IFN-γ and IL-2 responses to CFP, and IL-2 and IL-4 responses to PHA declined but there was a sustained increase in cytokine responses to SWA following treatment. These data do not support the hypothesis that helminth infection exacerbates HIV infection. The possibility that chronic helminth infection may suppress HIV replication and that effects on HIV replication may vary during helminth infection and treatment should be considered.

Cytokine profiles using whole-blood assays can discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population

Whole-blood assays (WB) provide a simple tool for assessing immune cytokine profiles which may be useful laboratory predictors of early disease, aiding the evaluation of new tuberculosis (TB) vaccines and offering insights into disease pathogenesis. Although BCG does not provide protection against pulmonary disease in TB endemic areas, it does modulate immune responses to mycobacterial antigens. It is important, therefore, to evaluate any new tool in an endemic setting in both BCG vaccinees and patients with tuberculosis. We have assessed the optimal conditions in terms of dose and kinetics of those cytokines which are released early (TNF-α, IL6 and TGF-β, IL10) or (interferon [IFN]-γ and IL5) in WB cultures stimulated with mitogens and mycobacterial antigens. Responses were studied in parallel in untreated TB patients and endemic control groups. Optimal responses to LPS (predominantly monocyte-derived) occurred on days 1–2, whereas for PHA (predominantly T-cell-derived), they were on days 3–5. Secreted Mycobacterium tuberculosis culture filtrate proteins (CFP) provided a stronger stimulus for monocyte-derived cytokines compared to PPD, but both antigens were comparable for induction of T-cell cytokines. Using unpaired Student's t-tests, pulmonary tuberculosis patients (P.TB; n=11), in response to CFP, showed higher monocyte-derived IL6 ( p=0.023) and IL10 ( p=0.042) compared to endemic controls (EC; n=13), and significantly suppressed T-cell-derived IFN-γ ( p=0.028) and IL5 ( p=0.012) secretion but increased IL10 ( p=0.047) on day 5, indicating that CFP is a strong stimulus for IL10 secretion in pulmonary TB patients. Extrapulmonary TB patients (E.TB; n=6) showed no elevation of early monocyte-derived cytokines to either PPD or CFP, but showed a marked suppression of the T-cell-derived cytokines IFN-γ (PPD, p=0.015; CFP, p=0.05) and IL5 (PPD, p=0.05; CFP, p=0.015). Cytokine analysis in WB cultures is, therefore, able to discriminate between active tuberculosis infection and nondiseased healthy controls.