Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2

Monocyte/macrophage cell death is an important event during mycobacterial infection. To get insights about the influence of mononuclear phagocyte maturation in this event we compared the response to Mycobacterium tuberculosis (Mtb) infection of fresh isolated monocytes and monocyte-derived macrophages (MDM) from healthy tuberculin positive individuals. Both monocytes and MDM underwent apoptosis, however, there was a higher numbers of apoptotic macrophages with active Caspases 8 and 9. We also compared Mtb-induced cell death in U937 pro-monocytes and PMA-differentiated cells (U937D). In response to Mtb infection, U937D cells underwent apoptosis and promonocytes both apoptosis and necrosis. There were high number of U937D cells producing TNF-α and high number of IL-10⁺ promonocytes. These evidences suggest that U937 could be a valid model to study the mechanisms that rule Mtb-induced cell death. Experiments with the cell line and fresh isolated mononuclear cells with pharmacological inhibitors showed that induction of necrosis involved calcium and cAMP signals resulting in IL-10 production. Necrosis also correlated with Caspase 3, PLA2 activity and bacterial growth. In U937D cells and monocytes from healthy donors there was activation of calcium, TNF-α and Caspase 8 activation and decreased bacterial load. Understanding the mechanisms that control the dichotomy events between apoptosis and necrosis/oncosis associated with cell maturity might open new strategies to better control the course of mycobacterial infections.     

U937 variant cells as a model of apoptosis without cell disintegration

The variant cell line U937_V was originally identified by a higher sensitivity to the cytocidal action of tumor necrosis factor alpha (TNFα) than that of its reference cell line, U937. We noticed that a typical morphological feature of dying U937_V cells was the lack of cellular disintegration, which contrasts to the formation of apoptotic bodies seen with dying U937 cells. We found that both TNFα, which induces the extrinsic apoptotic pathway, and etoposide (VP-16), which induces the intrinsic apoptotic pathway, stimulated U937_V cell death without cell disintegration. In spite of the distinct morphological differences between the U937 and U937_V cells, the basic molecular events of apoptosis, such as internucleosomal DNA degradation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, caspase activation and cytochrome c release, were evident in both cell types when stimulated with both types of apoptosis inducer. In the U937_V cells, we noted an accelerated release of cytochrome c, an accelerated decrease in mitochondrial membrane potential, and a more pronounced generation of reactive oxygen species compared to the reference cells. We propose that the U937 and U937_V cell lines could serve as excellent comparison models for studies on the mechanisms regulating the processes of cellular disintegration during apoptosis, such as blebbing (zeiosis) and apoptotic body formation.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.     

Nanoceria protects from alterations in oxidative metabolism and calcium overloads induced by TNFα and cycloheximide in U937 cells: pharmacological potential of nanoparticles

The present study is aimed to determine the protective effect of a novel nanoparticle with antioxidant properties, nanoceria, on reactive oxygen species (ROS) production, and calcium signaling evoked by the tumor necrosis factor-alpha (TNFα) in combination with cycloheximide (CHX) on apoptosis in the human histiocytic lymphoma cell line U937. Our results show that treatment of U937 cells with 10 ng/mL TNFα in combination with 1 μg/mL CHX led to several Ca²⁺ alterations. These stimulatory effects on calcium signals were followed by intracellular ROS production and mitochondria membrane depolarization, as well as a time-dependent increase in caspase-8 and -9 activities. Our results show that the pretreatment with well known antioxidants such as trolox and N-acetyl cysteine (NAC) partially reduced the apoptotic effects due to the administration of TNFα plus cycloheximide. Furthermore, nanoceria had a stronger protective effect than trolox or NAC. Our findings also suggest that TNFα plus cycloheximide-induced apoptosis is dependent on alterations in cytosolic concentration of calcium [Ca²⁺]_c and ROS generation in human histiocytic U937 cells.     

IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice

Incubation of the human U937 histiocytic lymphoma cell line with granulocyte-macrophage colony stimulating factor (GM-CSF) rendered the cells responsive to induction of TNF by LPS. Treatment with IL-6 reduced TNF production in GM-CSF-primed U937 cells. The inhibitory effect was most pronounced (approximately equal to 80%) when IL-6 was added either along with GM-CSF or within the first 3 h of GM-CSF treatment. Both GM-CSF or IL-6 inhibited [3H]TdR uptake in U937 cells, and simultaneous treatment with GM-CSF and IL-6 resulted in an additive inhibitory effect on cell proliferation. However, the inhibition of TNF production could not be explained by the inhibitory effect of IL-6 on cell growth, nor was it due to a reduction in cell viability. An inhibition of TNF production by IL-6 was also demonstrated in cultured human peripheral blood monocytes. Treatment with IL-6 also resulted in a dose-dependent reduction of the 17-kDa TNF band revealed by SDS-PAGE after labeling monocytes with [35S]cysteine and immunoprecipitation with anti-TNF mAb. In addition, treatment with IL-6 resulted in a reduction of monocyte in vitro cytotoxicity for tumor target cells. Finally, in mice sensitized by the administration of Bacillus Calmette-Guérin, the injection of IL-6 significantly reduced the levels of TNF found in the serum upon challenge with LPS. Inasmuch as TNF is known to be an inducer of IL-6, the inhibitory action of IL-6 on TNF production may represent the negative arm of a regulatory circuit. The inhibitory action of IL-6 on TNF production is consistent with a predominantly antiinflammatory role of IL-6 in the intact organism.     

Epigallocatechin-3-gallate inhibits VCAM-1 expression and apoptosis induction associated with LC3 expressions in TNFα-stimulated human endothelial cells

Tumor necrosis factor alpha (TNF-α) promotes the expression of adhesion molecules and induces endothelial dysfunction, a process that can lead to atherosclerosis. Green tea consumption can inhibit endothelial dysfunction and attenuate the development of arteriosclerosis. The purpose of this study was to examine whether epigallocatechin-3-gallate (EGCG) prevents TNF-α-dependent endothelial dysfunction. Here, we compared the regulatory effects of the green tea components EGCG and l-theanine against TNF-α-induced stimulation of adhesion molecule expression and apoptosis induction, which is associated with autophagy. Monocytic cell adhesion to human endothelial cells was measured using a fluorescently-labeled cell line, U-937. Caspase 3/7 activity was examined with a fluorescent probe and fluorescence microscopy. In addition, we analyzed the expression of several genes by RT-PCR. TNF-α-modulation of LC3 and VCAM1 protein levels were investigated by Western blot (WB). TNF-α induced adhesion of U937 cells to endothelial cells, and gene expression associated with adhesion molecules and apoptosis. On the other hand, EGCG and l-theanine inhibited TNF-α-induced adhesion of U937 cells to endothelial cells and inhibited increases in ICAM1, CCL2 and VCAM1 expression. Furthermore, EGCG and l-theanine inhibited TNF-α-induced apoptosis-related gene expression (e.g., CASP9), and caspase activity while inhibiting TNFα-induced VCAM1, LC3A and LC3B protein expression. Meanwhile, treatment of endothelial cells with autophagy inhibitor 3-methyladenine (3-MA) blocked EGCG-induced expression of CASP9. Together, these results indicate that EGCG can modulate TNF-α-induced monocytic cell adhesion, apoptosis and autophagy. We thus conclude that EGCG might be beneficial for inhibiting TNF-α-mediated human endothelial disorders by affecting LC3 expression-related processes.     

Molecular mechanism of interaction of Mycobacterium tuberculosis with host macrophages under high glucose conditions

Mycobacterium tuberculosis has the potential to escape various cellular defense mechanisms for its survival which include various oxidative stress responses, inhibition of phagosome-lysosomes fusion and alterations in cell death mechanisms of host macrophages that are crucial for its infectivity and dissemination. Diabetic patients are more susceptible to developing tuberculosis because of impairement of innate immunity and prevailing higher glucose levels. Our earlier observations have demonstrated alterations in the protein profile of M. tuberculosis exposed to concurrent high glucose and tuberculosis conditions suggesting a crosstalk between host and pathogen under high glucose conditions. Since high glucose environment plays crucial role in the interaction of mycobacterium with host macrophages which provide a niche for the survival of M. tuberculosis, it is important to understand various interactive mechanisms under such conditions. Initial phagocytosis and containment of M. tuberculosis by macrophages, mode of macrophage cell death, respiratory burst responses, Mycobacterium and lysosomal co-localization were studied in M. tuberculosis H37Rv infected cells in the presence of varied concentrations of glucose in order to mimic diabetes like conditions. It was observed that initial attachment, phagocytosis and later containment were less effective under high glucose conditions in comparison to normal glucose. Mycobacterium infected cells showed more necrosis than apoptosis as cell death mechanism during the course of infection under high glucose concentrations. Co-localization and respiratory burst assay also indicated evasion strategies adopted by M. tuberculosis under such conditions. This study by using THP1 macrophage model of tuberculosis and high glucose conditions showed immune evasion strategies adapted during co-pathogenesis of tuberculosis and diabetes.     

Differential expression of NF-κB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-κB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-κB, pCMV-IκBαM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IκBαM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-α production. Increase in apoptosis of infected THP-1-IκBαM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-κB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-κB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-κB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-κB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.     

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells

Background

The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood.

Methods

In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis.

Results

Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis.

General significance

These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.     

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20295     

Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells.

The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.     

Genetically recessive mutant of human monocytic leukemia U937 resistant to tumor necrosis factor-α-induced apoptosis

Tumor necrosis factor-α (TNF-α) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-α-induced apoptosis, we isolated and characterized a novel TNF-α-resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-α and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-α in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-α-induced apoptosis signaling. In UA cells, TNF-α-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1β converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria. J. Cell. Physiol. 174:179–185, 1998. © 1998 Wiley-Liss, Inc.     

Critical and Independent Role for SOCS3 in Either Myeloid or T Cells in Resistance to Mycobacterium tuberculosis

Suppressor of cytokine signalling 3 (SOCS3) negatively regulates STAT3 activation in response to several cytokines such as those in the gp130-containing IL-6 receptor family. Thus, SOCS3 may play a major role in immune responses to pathogens. In the present study, the role of SOCS3 in M. tuberculosis infection was examined. All Socs3^fl/fl LysM cre, Socs3^fl/fl lck cre (with SOCS3-deficient myeloid and lymphoid cells, respectively) and gp130^F/F mice, with a mutation in gp130 that impedes binding to SOCS3, showed increased susceptibility to infection with M. tuberculosis. SOCS3 binding to gp130 in myeloid cells conveyed resistance to M. tuberculosis infection via the regulation of IL-6/STAT3 signalling. SOCS3 was redundant for mycobacterial control by macrophages in vitro. Instead, SOCS3 expression in infected macrophages and DCs prevented the IL-6-mediated inhibition of TNF and IL-12 secretion and contributed to a timely CD4+ cell-dependent IFN-γ expression in vivo. In T cells, SOCS3 expression was essential for a gp130-independent control of infection with M. tuberculosis, but was neither required for the control of infection with attenuated M. bovis BCG nor for M. tuberculosis in BCG-vaccinated mice. Socs3^fl/fl lck cre mice showed an increased frequency of γδ+ T cells in different organs and an enhanced secretion of IL-17 by γδ+ T cells in response to infection. Socs3^fl/fl lck cre γδ+ T cells impaired the control of infection with M. tuberculosis. Thus, SOCS3 expression in either lymphoid or myeloid cells is essential for resistance against M. tuberculosis via discrete mechanisms.     

Novel piperazine induces apoptosis in U937 cells

The effect of 1,4-bis-(4-(1H-benzo[d]imidazol-2-yl-phenyl)) piperazine (BIPP), a newly synthesized piperazine derivative, on U937 leukemia cell viability was investigated. We show that BIPP induces dose-responsive apoptotic cell death in U937 cells by intrinsic mechanisms of apoptosis. Maximum apoptotic effect of BIPP on U937 cells was observed at 12.8μM. BIPP-induced apoptosis was evident by characteristics such as altered annexin-V binding, caspase activation, loss of mitochondrial membrane potential (MMP) and cytochrome c release. BIPP also differentially activates initiator and effector caspases combined with the loss of MMP strongly suggesting that BIPP causes an intrinsic apoptosis in U937 leukemia cells. Due to our observations that BIPP induces leukemia cell death without significantly affecting normal cells, our data suggests that it may be a potential therapeutic agent for human myeloid leukemia.     

Role of interferon γ and tumour necrosis factor α in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line

Summary In view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocytederived macrophages were activated with interferon (IFN) or tumour necrosis factor (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0±0.7% (mean ± SEM) (conventional [³H]dT uptake assay) and 81.9±5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages.     

Peptides derived from Mycobacterium tuberculosis Rv2301 protein are involved in invasion to human epithelial cells and macrophages

The specific function of putative cut2 protein (or CFP25), encoded by the Rv2301 gene from Mycobacterium tuberculosis H37Rv, has not been identified yet. The aim of this study was to assess some of CFP25 characteristics and its possible biological role in Mycobacterium tuberculosis H37Rv invasion process to target cells. Molecular assays indicated that the gene encoding Rv2301 is present and transcribed in M. tuberculosis complex strains. The presence of Rv2301 protein over the bacilli surface was confirmed by Western blot and immunoelectron microscopy analyses, using goats sera inoculated with synthetic peptides derived from Rv2301 protein. Receptor–ligand binding assays with carcinomic human alveolar basal epithelial cells (A549) and macrophages derived from human histolytic lymphoma monocytes (U937) allowed us to identify five high activity binding peptides (HABPs) in both cell lines, and two additional HABPs only in A549 cells. U937 HABPs binding interactions were characterized by saturation assays, finding dissociation constants (K _d) within the nanomolar range and positive cooperativity (n _H?>?1). Inhibition assays were performed to assess the possible biological role of Rv2301 identified HABPs, finding that some of them were able to inhibit invasion at a 5?μM concentration, compared with the cytochalasin control. On the other hand, HABPs, and especially HABP 36507 located at the N-terminus of the protein, facilitated the internalization of fluorescent latex beads into A549 cells. These findings are of vital importance for the rational selection of Rv2301 HABPs, to be included as components of an antituberculosis vaccine.     

Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.