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Selection and identification of specific glycoproteins and glycan biomarkers of macrophages involved in Mycobacterium tuberculosis infection
Abstract
Macrophages are the primary host target cells of Mycobacterium tuberculosis (M.tb). However, little is known about the changes of membrane glycopatterns of macrophages in response to M. tb infection. Using lectin microarrays we compared the differential expression of glycopatterns of macrophages upon stimulation with the heat-inactivated virulent M.tb H37Rv or attenuate M.tb H37Ra. We found that widespread alteration of macrophage membrane glycopatterns were induced by the heat-inactivated virulent M. tb H37Rv, as shown by the significantly changed binding abilities of 11 lectins (sugar binding proteins) among 40 lectins tested. The binding ability of the lectin ABA to macrophages showed the greatest increase after virulent M. tb H37Rv treatment, which suggests that the expression of N-acetyl-d-lactosamine (ABA binding ligand Galβ1-3GalNAc, O-link glycan) is mainly increased on macrophages during virulent M.tb infection. Addition of ABA blocked the attachment/engulfment of M. tb H37Rv, but not H37Ra, to macrophages. Further, increased glycosylated CD44, one of ABA-binding glycoproteins on macrophages, was identified by pull-down assays with ABA-agarose, followed by mass spectrometry and western blotting. ABA directly binds with Galβ1-3GalNAc-glycosylated CD44 on macrophage, and inhibits M. tb mannose-capped lipoarabinomannan (ManLAM) binding to glycosylated CD44. Moreover, ABA increases IL-6, but reduces IL-10 production of ManLAM-treated macrophages and inhibits M. tb H37Rv-induced necrosis in macrophages. Our study will help to reveal the mechanism of pathogenicity and virulence of M. tb from a new perspective and provide a potential new diagnostic and therapeutic strategy for tuberculosis based on glycopatterns, ABA and its ligand Galβ1-3GalNAc-glycosylated CD44 target molecule on macrophage.
... BMDMs were prepared as we previously described (Tang et al., 2017). Briefly, bone marrow cells isolated from the femurs and tibias of BALB/c mice were treated with ACK Lysis Buffer (Beyotime, Shanghai, China) and cultured in DMEM (Gibco, UK) supplemented with 10% FBS (Gibco), 1% Penicillin-Streptomycin Solution, and 50 ng/ml macrophage colony stimulating factor (M-CSF, Peprotech) for 6 days to induce differentiation into BMDM (the adherent cells). ...
... Intracellular IFN-γ expression of CD3 + CD4 + T cells was determined by staining with APC-anti-CD3, FITC-anti-CD4, PE-anti-IFN-γ (Biolegend, CA, USA). All procedures were performed as we previously described (Tang et al., 2017;Yuan et al., 2019) and analyzed on BD Accuri C6 Flow cytometer (BD Biosciences). ...
... As the central defensive mediators of the immune response, macrophages can be transformed into a growth-permissive state in response to different stimuli of M.tb, like Mannose-capped lipoarabinomannan (ManLAM) and phenolic glycolipid (PGL) (Cambier et al., 2014(Cambier et al., , 2017. Previously, we have confirmed that ManLAM promotes apoptosis, M2 polarization and inhibits antigen-presenting activity of macrophages (Sun et al., 2016;Pan et al., 2017;Tang et al., 2017). In line with these work, DNA aptamer targeting to ManLAM has a strong potential for use as an immune enhancer of BCG in mice and monkeys models, and clinical findings also revealed the potential of ManLAM for TB diagnosis (Sun et al., 2016;Tang et al., 2016). ...
As the first line defensive mediators against Mycobacterium tuberculosis (M.tb) infection, macrophages can be modulated by M.tb to influence innate and adaptive immunity. Recently, we have identified several potential immunodominant T-cell antigens from the region of deletion (RD) of M.tb H37Rv, including Rv1768 from RD14. In this study, we further determined that Rv1768 was highly conserved among virulent M.tb strains and mainly distributed as a secreted protein. Exposure to recombinant purified Rv1768 (rRv1768) induced apoptosis of bone marrow derived macrophages (BMDMs) but showed no dose-dependent manner. Regarding macrophage activation, significant higher levels of iNOS and pro-inflammatory cytokines (like IL-6 and TNF-α) were detected in rRv1768-challenged BMDMs, whereas arginase 1 (Arg1) expression was markedly decreased. Meanwhile, MHC-II expression and antigen presentation activity of BMDMs were also enhanced by rRv1768 stimulation, leading to significantly increased IFN-γ expression of CD4⁺ T cells isolated from H37Rv-infected mice. It is worthy to note that Rv1768-induced IFN-γ production of peripheral blood mononuclear cells (PBMCs) and Rv1768-specific immunoglobulins was specifically observed in H37Rv-infected mice, but not BCG-infected or normal mice. Analysis of clinical blood samples further revealed that Rv1768 had a higher sensitivity and specificity (91.38 and 96.83%) for tuberculosis diagnosis than the results obtained from clinical CFP10 and ESAT6 peptides (CE)-based enzyme-linked immunospot (ELISPOT) assay. The area under ROC curve of Rv1768 was 0.9618 (95% CI: 0.919–1.000) when cutoff value set as 7 spots. In addition, Rv1768-specific IgG and IgM also exhibited moderate diagnostic performance for tuberculosis compared with CE specific antibodies. Our data suggest that Rv1768 is an antigen that strongly activates macrophages and has potential to serve as a novel ELISPOT-based TB diagnostic agent.
... Its precursors, including LM, phosphatidyl-myoinositol mannosides (PIMs), have been shown to have potential regulatory effects in vitro experiments on immune cells (Dao et al. 2004;Gilleron et al. 2003). Mannose-capped lipoarabinomannan (ManLAM) is a major cell wall lipoglycan and an important immunomodulatory component of mycobacteria (Kang et al. 2005;Sun et al. 2016;Tang et al. 2017;Gringhuis et al. 2009;Yonekawa et al. 2014;Yuan et al. 2019;Torrelles and Schlesinger 2010) (Table 11.3). Bacterial ManLAM can also be secreted and recognized by macrophages and DCs via pattern recognition receptors, including MR, Toll-like receptor 2 (TLR2), DC-SIGN, CD1d, sphingosine-1phosphate receptor 1 (S1P1), Dectin-2, and CD44, and triggers several cell signaling pathways (Sun et al. 2016;Yonekawa et al. 2014;Pan et al. 2014;Osanya et al. 2011;Zajonc et al. 2006). ...
... In addition, using lectin microarrays it was shown that infection of virulent M. tb H37Rv, but not attenuated M. tb H37Ra, induces increased Galβ1-3GalNAc-glycosylated CD44 on macrophage (Tang et al. 2017). Addition of ABA lectin blocked the attachment/engulfment of M. tb H37Rv to macrophage. ...
Glycosylation plays an important role in infectious diseases. Many important interactions between pathogens and hosts involve their carbohydrate structures (glycans). Glycan interactions can mediate adhesion, recognition, invasion, and immune evasion of pathogens. To date, changes in many protein N/O-linked glycosylation have been identified as biomarkers for the development of infectious diseases and cancers. In this review, we will discuss the principal findings and the roles of glycosylation of both pathogens and host cells in the context of human important infectious diseases. Understanding the role and mechanism of glycan-lectin interaction between pathogens and hosts may create a new paradigm for discovering novel glycan-based therapies that can lead to eradication or functional cure of pathogens infection.
... Immunoglobin G (IgG), a highly abundant glycoprotein in serum, is known to mediate a variety of immune responses. IgG protein glycosylation has been implicated to be associated with TB pathogenesis and inflammation (Pilkington et al. 1995;Shinzaki et al. 2012;Lu et al. 2016;Hare et al. 2017;Tang et al. 2017;Pagan et al. 2018), and there has been an increasing interest in the analysis of the N-glycan profile of human IgG in healthy and TB disease states. However, few systematical studies have been reported for the potential application of serum IgG agalactosylation for TB auxiliary diagnosis in larger samples of TB patients. ...
... Human M. tb H37Rv, H37Ra and nonvirulent M. bovis Bacille Calmette-Guérin (BCG) strains were maintained on 7H9 middle brook liquid medium and harvested while in the log phase of growth (Tang et al. 2017). Female C57BL/6 mouse (8 weeks old) were provided by the Animal Laboratory Center of Wuhan University. ...
Tuberculosis (TB) is the leading infectious cause of mortality worldwide, especially in developing countries. However, effective means for TB diagnosis, especially for bacillus negative (Bn) TB laboratory diagnosis are urgently needed. In the present study, serum IgG from each tuberculosis patients and healthy controls was purified using affinity chromatography. The samples were then analyzed using mass spectrometry (MS) and ultra performance liquid chromatography (UPLC) methods. We quantitatively assessed the changes of serum IgG galactosylation in 567 human serum samples including 377 pulmonary TB patients and 190 healthy donors (HDs). We found significantly more agalactosylated (G0) vs monogalactosylated (G1) and digalactosylated (G2) N-glycans of IgG in TB patients, including smear negative TB patients, than in HDs. The detection rate of TB diagnostic performance by MS for IgG-Gal-Ratio G0/(G1 + G2 × 2) is 90.48% for bacillus positive (Bp) and 73.16% for Bn TB patients. Further, combination of MS method with other routine laboratory TB diagnostic methods significantly increased the detection rate to 91.01%~98.39%. Similar results were observed in Mycobacterium tuberculosis (M.tb) infection mouse models. The decrease in galactosylation of IgG in TB patients were also qualitatively confirmed using specific lectin blot assay. Using above techniques we can discriminate the content of IgG G0 with terminal N-acetylglucosamine and IgG-Gal-Ratio G0/(G1 + G2 × 2) between TB patients and HDs. Our data suggest that quantitative analysis of serum-based IgG-Gal-Ratio G0/(G1 + G2 × 2) could be used for TB auxiliary diagnosis with high effectiveness and feasibility, and its combination with other routine laboratory TB diagnostic methods could remarkably improve the detection rate.
... The MPI anchors of ManLAM are recognized by CD1 (including CD1b and CD1d), Toll like receptor 2 (TLR2), dendritic cell immunoactivating receptor (DCAR), mannose-binding protein (MBP), and lactosylceramide enriched lipid rafts in the plasma membrane ( Figure 1) [10][11][12][13][14][15][16]. Other receptors and molecules that bind to ManLAM include pulmonary surfactant protein A (PS-A), Dectin 2 and CD44 [17][18][19][20][21], however, the ManLAM binding domains that interact with these molecules remain unknown. ...
... Early studies observed increased tumour necrosis factor (TNF) production by both human and murine macrophages following ManLAM stimulation [32], while later studies demonstrated that ManLAM inhibits various interferon γ (IFN-γ)-mediated microbicidal and tumoricidal activities, and triggers macrophages to produce anti-inflammatory cytokine IL-10 [19,26,33,34]. Previously, we reported that ManLAM recognized by CD44 causes the upregulation of IL-1β, IL-12 and iNOS (inducible nitric oxide synthase) expression in macrophages, indicating that ManLAM-CD44 signalling enhances M1 macrophage polarization [19,20]. However, the binding of ManLAM to MR might lead to IL-10 production and impair M1 polarization [19]. ...
Mannose-capped lipoarabinomannan (ManLAM) is a high molecular mass amphipathic lipoglycan identified in pathogenic Mycobacterium tuberculosis (M. tb) and M. bovis Bacillus Calmette-Guérin (BCG). ManLAM, serves as both an immunogen and a modulator of the host immune system, and its critical role in mycobacterial survival during infection has been well-characterized. ManLAM can be recognized by various types of receptors on both innate and adaptive immune cells, including macrophages, dendritic cells (DCs), neutrophils, natural killer T (NKT) cells, T cells and B cells. MamLAM has been shown to affect phagocytosis, cytokine production, antigen presentation, T cell activation and polarization, as well as antibody production. Exploring the mechanisms underlying the roles of ManLAM during mycobacterial infection will aid in improving tuberculosis (TB) prevention, diagnosis and treatment interventions. In this review, we highlight the interaction between ManLAM and receptors, intracellular signalling pathways triggered by ManLAM and its roles in both innate and adaptive immune responses.
... M. tb H37Rv (strain American Type Culture Collection (ATCC) 93 009) and Mycobacterium bovis BCG (ATCC strain 35 734) were purchased from the Beijing Biological Product Institute (Beijing, China) (Sun et al. 2016;Tang et al. 2017). The mycobacterial strain was grown in Middlebrook 7H9 broth (BD Biosciences, NJ, USA) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, BD Biosciences, NJ, USA) and 0.05% Tween-80. ...
... C57BL/6 mice were purchased from Wuhan Centers for Disease Prevention and Control. To harvest murine resident peritoneal macrophages, thioglycolate-elicited macrophages were prepared by injecting mice with 3.5 mL of 3% sterile thioglycolate media (BD Biosciences, NJ, USA) (Tang et al. 2017). After 5 days, peritoneal cells were harvested by lavage and cultured overnight. ...
Macrophages are the primary host target cells of Mycobacterium tuberculosis (M. tb). As a subunit of immunoregulatory cytokines IL-27 and IL-35, Epstein–Barr virus-induced gene 3 (EBI3) has typically been explored as the secreted form and assessed in terms of its effects triggered by extracellular EBI3. However, little is known about intracellular EBI3 function. In the current study, we report that EBI3 production by macrophages is elevated in TB patients. We further demonstrate that increased EBI3 accumulates in virulent M. tb-treated murine macrophages. Eukaryotic translation elongation factor 1-alpha 1 (eEF1A1) binds to intracellular EBI3 to reduce Lys48 (K48)-linked ubiquitination of EBI3, leading to EBI3 accumulation. Moreover, the intracellular EBI3 inhibits caspase-3-mediated apoptosis in M. tb-treated macrophages. Herein, we propose a novel mechanism for accumulating intracellular EBI3 and its regulation of macrophage apoptosis in response to virulent M. tb.
... Recent studies have demonstrated that glycans or glycoconjugates might serve as biomarkers in diagnosis of multiple diseases, especially in infectious diseases. Tang et al. [25] found that N-acetyl-d-lactosamine was mainly increased on macrophages during virulent Mycobacterium tuberculosis infection and could serve as a potential novel diagnostic and therapeutic biomarker. Moreover, Wang et al. [26] demonstrated that O-glycosylation of CtxB-BCAL2737a could be used to detect anti-O-glycan antibodies in human serum samples from patients with Burkholderia-associated infections. ...
Post-neurosurgical bacterial meningitis (PNBM) is one of the severe complications in patients receiving neurosurgical procedures. Recent studies have found microbe-related glycans play important roles in adhesion, invasion, and toxicity toward innate immunological reactions. In this study, we aimed to investigate the glycomic profile and its potential diagnostic efficacy in post-neurosurgical bacterial meningitis (PNBM) patients with hemorrhagic stroke. A total of 136 cerebrospinal fluid (CSF) samples were recruited and divided into a PNBM group and a non-PNBM group based on the clinical diagnostic criteria. A lectin biochip-based method was established for the detection of glycans in CSF. The clinicopathological data and biochemical parameters in CSF from all patients were analyzed. Two models for multivariate analysis investigating glycan changes in the CSF were conducted, aiming at determining the specific expression and diagnostic efficacy of lectin-probing glycans (LPGs) for PNBM. In univariate analysis, we found that 8 out of 11 LPGs were significantly correlated with PNBM. Model 1 multivariate analysis revealed that PNA (p = 0.034), Jacalin (p = 0.034) and LTL (p = 0.001) were differentially expressed in the CSF of PNBM patients compared with those of non-PNBM patients. Model 2 multivariate analysis further disclosed that LTL (p = 0.021) and CSF glucose (p < 0.001) had independent diagnostic efficacies in PNBM, with areas under the curve (AUC) of 0.703 and 0.922, respectively. In summary, this study provided a new insight into the subject of CSF glycomics concerning bacterial infection in patients with hemorrhagic stroke.
... Researchers should also seek to create bispecific engineered lectins (i.e. similar to bispecific antibodies) that are capable of binding two distinct targets (e.g. one that binds glycans on target cells and another that binds glycans on phagocytes for opsonization (Tang et al. 2017;Park et al. 2021)) in order to boost the killing mechanism of lectins. Scientific research on therapeutic approaches targeting malignancies that integrates both cancer glycomics and cancer genomics has become imperative in fighting humans' most dreaded diseases. ...
Unlike cytotoxic chemotherapy, cancer immunotherapy offers targeted therapies that exploit the effector mechanisms of the immune system to combat cancer. However, most therapeutic strategies have so far focused predominantly on the orchestration of the adaptive immune responses to anti-cancer immunotherapies. Unfortunately, the emergence of resistance and associated severe toxicities rendered this modality of treatment imperfect. Because of their complex nature and the late ability to selectively separate distinct innate immune responses, the enormous potential of innate immunity as an immunotherapy was largely neglected. Recently, the growing demand to find alternatives to adaptive immunity-based immunotherapy concurred with growing appreciation of the innate immune effectors contributions to anti-tumor immunity. In particular, the innate immunity anti-infective responses overlap with those that target cancer indicating that these responses can readily be manipulated to design new therapeutic approaches. The paradigm of lectin pathway in recognition of distinct ‘non-self’ (antigenic) glycans on the surface of pathogenic microbes in concert with cancer’s indigenous aberrant (antigenic) glycans render lectin pathway a canonical component of innate immune system that can be extrapolated to cancer immunotherapy. By virtue of recent advances in lectin engineering, the encouraging results of using engineered lectins as anti-viral agents can be replicated in cancer immunotherapy.
... A lectin microarray was produced using 95 lectins (Raybiotech Lectin Array 95 kit, Cat. No. GA-Lectin-95, RayBiotech, Inc.) with different binding preferences covering N-and O-linked glycans and analyzed as previously described (33,34). Whole-cell lysates labeled with fluorescent dye Cy3 (GE Healthcare, UK) were applied to the lectin microarrays, which were then scanned with a Genepix 4000B confocal scanner (Axon Instruments, CA, USA). ...
Alterations in glycosylation regulate fundamental molecular and cellular processes of cancer, serving as important biomarkers and therapeutic targets. However, the potential association and regulatory mechanisms of E6 oncoprotein on glycosylation of cervical cancer cells are still unclear. Here, we evaluated the glycomic changes via using Lectin microarray and determined the corresponding enzymes associated with endogenous high-risk HPV16 E6 expression in cervical cancer cells. α-2,6 sialic acids and the corresponding glycosyltransferase ST6GAL1 were significantly increased in E6 stable-expressing HPV⁻ cervical cancer C33A cells. Clinical validation further showed that the expression of ST6GAL1 was significantly increased in patients infected with high-risk HPV subtypes and showed a positive association with E6 in cervical scraping samples. Interfering ST6GAL1 expression markedly blocked the oncogenic effects of E6 on colony formulation, proliferation, and metastasis. Importantly, ST6GAL1 overexpression enhanced tumorigenic activities of both E6-positive and E6-negative cells. Mechanistical investigations revealed that E6 depended on activating YAP1 to stimulate ST6GAL1 expression, as verteporfin (inhibitor of YAP1) significantly suppressed the E6-induced ST6GAL1 upregulation. E6/ST6GAL1 triggered the activation of downstream cGMP/PKG signaling pathway and ODQ (inhibitor of GMP production) simultaneously suppressed the oncogenic activities of both E6 and ST6GAL1 in cervical cancer cells. Taken together, these findings indicate that ST6GAL1 is an important mediator for oncogenic E6 protein to activate the downstream cGMP/PKG signaling pathway, which represents a novel molecular mechanism and potential therapeutic targets for cervical cancer.
... Mannose, one of the natural ligands for the MR, is a monosaccharide, which is expressed in the envelope or cytoplasm of macrophages (23,24), and plays a key role in human metabolism, especially in the glycosylation of proteins (25). Mannose is required for N-glycosylation and glycophospholipid anchoring synthesis. ...
Background:
Mannose receptor (MR) is an immune adhesion molecule and is mainly expressed in macrophages and nonmature dendritic cells. The ligand mannose, one of the natural ligands of MR, is a monosaccharide, which is localized in the envelope or cytoplasm of macrophages. The aim of this study was to investigate expression of MR and its ligand mannose in tumor tissues of primary advanced gastric cancer and to evaluate the predictive and prognostic value of the positive cells in gastric cancer patients.
Methods:
Histochemical staining for Narcissus pseudonarcissus lectin (NPL) and immunohistochemical envision two-step assay for MR were used to detect expression of NPL and MR in primary advanced gastric adenocarcinoma tissues. Adjacent non-cancerous gastric tissues of the patients were used as controls. Relationship of NPL and MR expression in the tumor tissues with clinicopathological features and survival time of the gastric cancer patients were analyzed.
Results:
Numbers of NPL+ and MR+ macrophages in stromal tissues of gastric cancer were significantly higher than those in the adjacent non-cancerous gastric tissues (P=0.006; P<0.001). NPL expression in the primary tumor tissues was significantly more dominant than that in the adjacent non-cancerous gastric tissues (P=0.003). Expression of both the molecules in macrophages in tumor tissues was negatively correlated (r=-0.363, P=0.009). TNM stage of the patients was closely correlated to number of MR+ macrophages and NPL expression in the stromal tissues of gastric cancer (P=0.009 and P=0.020). Kaplan-Meier survival model data showed that the patients with low counting of NPL+ macrophages and high counting of MR+ macrophages significantly led to worse disease progression and poorer prognosis (P=0.008). Cox regression analysis further demonstrated that high expression of MR+ macrophages was an independent predictor of poor prognosis in patients with gastric cancer (P=0.033).
Conclusions:
Occurrence of mannose and MR in tumor tissues of gastric cancer might be prognostic factors for estimating risk of gastric cancer patients.
... Murine macrophage RAW264.7 cells were purchased from China Center for Type Culture Collection (CCTCC). To prepare murine peritoneal macrophages, thioglycolate-elicited macrophages were prepared by injecting mice with 3.5 mL of 3% sterile thioglycolate media (BD Biosciences, NJ, USA) [12,13]. After 5 days, peritoneal cells were collected by lavage. ...
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb) bacteria, is a leading infectious cause of mortality worldwide. The emergence of drug-resistant M. tb has made control of TB more difficult. The selective optimization of side activities (SOSA) approach uses old drugs for new pharmacological targets. In the present study by using SOSA approach, we have successfully identified pyrvinium pamoate (PP) which is capable of inhibiting the growth of mycobacteria, including M. tb H37Rv, Mycobacterium smegmatis, Bacille Calmette-Guérin (BCG), M. tb H37Ra, and drug-resistant M. tb clinical isolates in vitro from 1280 known drugs library. The MIC99 of PP, the minimum inhibitory concentration that inhibits more than 99% of M. tb H37Rv and the drug-resistant M. tb clinical isolates, ranges from 1.55 to 4.8 µg/mL. Importantly, PP could reduce the bacterial colony-forming units (CFUs) in lung, spleen and liver tissues, and effectively inhibit inflammatory response in M. tb H37Rv, multidrug-resistant (MDR) M. tb and extensively drug-resistant (XDR) M.tb-infected mice. Our results clearly show that the PP has the potential application for treatment of TB.
... After mycobacteria are inhaled by the host, they are engulfed by the alveolar macrophages, which become infected, pathogenesis of human and BTB occurs in a similar way, beginning with bacterial entry to host lungs by inhalation and bacteria phagocytosis by alveolar macrophages. Establishment of a chronic infection status is accomplished due to mycobacterial virulence factors that allow it to enter and survive within the host phagocytic cells [2], higher production of nitric oxide by bovine macrophages infected with M. avium subsp paratuberculosis, macrophages involved in M. tuberculosis infection by collection and identification of specific glycoproteins and glycogen biomarkers [3,4]. The macrophages play an important role in tuberculosis pathogenesis, being the first defense line, the niche for the bacteria and the leading control mechanism. ...
The immune signalling genes during challenge of bovine macrophages with bacterial products derived from disease causing bacteria in cattle were investigated. An in-vitro cell culture model were challenged to Mycobacterium bovis of bovine monocyte derived macrophages. Macrophages from healthy and already infected animals can both be fully activated during M. bovis infection. Analysis of mRNA abundance in peripheral blood mononuclear cells from M. bovis infected and non-infected cattle were performed as a controls. Cells of treatment were challenged after six days for six hours incubation at 37 °C, with 5% CO2, to total RNA was extracted then cDNA labelling, hybridization and scanning for microarray methods have been developed for microarray based immune related gene expression analysis. The differential expressions twenty genes (IL1, CCL3, CXCR4, TNF, TLR2, IL12, CSF3, CCR5, CCR3, MAPT, NFKB1, CCL4, IL6, IL2, IL23A, CCL20, IL8, CXCL8, TRIP10, CXCL2 and IL1B) implicated in M. bovis response were examined Agilent Bovine_GXP_8 × 60 K microarray platform. The outcomes provided confirmation of a pro-inflammatory gene expression profile in PBMC from Bovine tuberculosis infected animals in response to antigen stimulation. Cells of treatment were challenged after six days for six hours incubation then pathways analysis of Toll like receptor and Chemokine signalling pathway study of responsible genes in bovine tuberculosis. The PBMC from M. bovis infected cattle exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to M. bovis antigen stimulation, providing evidence of a novel genes expression program due to M. bovis exposure. It will guide future studies, regarding the complex macrophage specific signalling pathways stimulated upon phagocytosis of M. bovis and role of signalling pathways in creating the host immune response to cattle tuberculosis.
Macrophages derived from human monocytic leukemia THP-1 cell line are often used as the alternative of human primary macrophage. However, the polarization method of THP-1 to macrophages varies between different laboratories, which may unknowingly affect the relevance of research output across research groups. In this regard, a systematic search was developed in Pubmed, BioOne, Scopus, and Science Direct to identify articles focusing on THP-1 polarization into M1 and M2 macrophage. All selected articles were read and discussed by two independent reviewers. The selection process was based on selected keywords on the title, abstract and full-text level. A total of 85 articles were selected and categorized based on the field of studies, method of THP-1 differentiation, and markers or genes expressed upon differentiation. THP-1 derived macrophages were mainly used together with primary monocyte-derived macrophages in cellular inflammation studies, while it was commonly employed alone in cancer research. THP-1 derived macrophages are also of paramount importance in biomaterials studies to prevent unfavorable immune responses in-vivo. We explored various methods of THP-1 differentiation and suggested several common genes encountered to characterize M1 and M2 macrophages differentiated from THP-1. The systematic review highlights the relevance of using THP-1 derived macrophage as a useful alternative to primary macrophage. Although it is not possible to derive a standard method of THP-1 polarization into M1 and M2 macrophage from this review, it may lead researchers to obtain reproducible polarization protocol based on commonly used stimulants and markers of differentiation.
Objectives:
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a widespread infectious disease around the world. Early diagnosis is always important in order to avoid spreading. At present, many studies have confirmed that microRNA (miRNA) could be a useful tool for diagnosis. This study aimed to evaluate whether miRNAs could be regarded as a noninvasive diagnosis biomarker from sputum for pulmonary tuberculosis (PTB).
Materials and methods:
The M. tuberculosis strain H37Rv was incubated and cultured with human macrophage line THP-1. The total RNA was extracted from the THP-1 cells for detection. Six increased expressions of miRNAs were selected by miRNA microarray chips and the miRNAs were confirmed by qRT-PCR in the M. tuberculosis infection cell model. At last, the efficiency of other methods was compared with using miRNA.
Results:
Only miR-155 showed a better diagnostic value for PTB than the other five miRNAs to distinguish PTB from non-PTB, including pneumonia, lung cancer, and unexplained pulmonary nodules. Next, we detected and analyzed the results of 68 PTB patients and 122 non-PTB, the sensitivity and specificity of miR-155 detection was 94.1% and 87.7%, respectively. It was higher than sputum smear detection and anti-TB antibody detection. But slightly lower than ELISpot (97%, P=0.404). Interestingly, the ranking of sputum smear by Ziehl-Neelsen staining had positive correlation with the expression level of miR-155 in smear-positive sputum (R2=0.8443, P<0.05).
Conclusion:
Our research suggested that miR-155 may be an efficiency biomarker for active PTB diagnosis and bacteria-loads evaluation.
Introduction: Glycoproteomics is an important subdiscipline of proteomics, focusing on the role of protein glycosylation in various biological processes. Protein glycosylation is the enzymatic addition of sugars or oligosaccharides to proteins. Altered glycosylation often occurs in the early stages of disease development, for example, certain tumor-associated glycans have been shown to be expressed in precursor lesions of different types of cancer, making them powerful early diagnostic markers. Lectin microarrays have become a powerful tool for both the study of glycosylation and the diagnosis of various diseases including cancer.
Areas covered: This review will discuss the most useful features of lectin microarrays, such as their technological advances, their capability for parallel/high-throughput analysis for the important glycopatterns of glycoprotein, and an overview of their use for glycosylation analysis of various complex protein samples, as well as their diagnostic potential in various diseases.
Expert opinion: Lectin microarrays have proved to be useful in studying multiple lectin–glycan interactions in a single experiment and, with the advances made in the field, hold a promise of enabling glycopatterns of diseases in a fast and efficient manner. Lectin microarrays will become increasingly powerful early diagnostic tool for a variety of conditions.
Mycobacterium tuberculosis is a successful intracellular pathogen. Numerous host innate immune responses signaling pathways are induced upon mycobacterium invasion, however their impact on M. tuberculosis replication is not fully understood. Here we reinvestigate the role of STAT3 specifically inside human macrophages shortly after M. tuberculosis uptake. We first show that STAT3 activation is mediated by IL-10 and occurs in M. tuberculosis infected cells as well as in bystander non-colonized cells. STAT3 activation results in the inhibition of IL-6, TNF-α, IFN-γ and MIP-1β. We further demonstrate that STAT3 represses iNOS expression and NO synthesis. Accordingly, the inhibition of STAT3 is detrimental for M. tuberculosis intracellular replication. Our study thus points out STAT3 as a key host factor for M. tuberculosis intracellular establishment in the early stages of macrophage infection.
Exposure to silver nanoparticles (AgNP) used in consumer products carries potential health risks including increased susceptibility to infectious pathogens. Systematic assessments of antimicrobial macrophage immune responses in the context of AgNP exposure are important because uptake of AgNP by macrophages may lead to alterations of innate immune cell functions. In this study we examined the effects of exposure to AgNP with different particle sizes (20 and 110 nm diameters) and surface chemistry (citrate or polyvinlypyrrolidone capping) on cellular toxicity and innate immune responses against Mycobacterium tuberculosis (M.tb) by human monocyte-derived macrophages (MDM). Exposures of MDM to AgNP significantly reduced cellular viability, increased IL8 and decreased IL10 mRNA expression. Exposure of M.tb-infected MDM to AgNP suppressed M.tb-induced expression of IL1B, IL10, and TNFA mRNA. Furthermore, M.tb-induced IL-1β, a cytokine critical for host resistance to M.tb, was inhibited by AgNP but not by carbon black particles indicating that the observed immunosuppressive effects of AgNP are particle specific. Suppressive effects of AgNP on the M.tb-induced host immune responses were in part due to AgNP-mediated interferences with the TLR signaling pathways that culminate in the activation of the transcription factor NF-κB. AgNP exposure suppressed M.tb-induced expression of a subset of NF-κB mediated genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). In addition, AgNP exposure increased the expression of HSPA1A mRNA and the corresponding stress-induced Hsp72 protein. Up-regulation of Hsp72 by AgNP can suppress M.tb-induced NF-κB activation and host immune responses. The observed ability of AgNP to modulate infectious pathogen-induced immune responses has important public health implications.
Cystic fibrosis (CF) patients battle life-long pulmonary infections with the respiratory pathogen Pseudomonas aeruginosa (PA). An overabundance of mucus in CF airways provides a favorable niche for PA growth. When compared with that of non-CF individuals, mucus of CF airways is enriched in sialyl-Lewis(x), a preferred binding receptor for PA. Notably, the levels of sialyl-Lewis(x) directly correlate with infection severity in CF patients. However, the mechanism by which PA causes increased sialylation remains uncharacterized. In this study, we examined the ability of PA virulence factors to modulate sialyl-Lewis(x) modification in airway mucins. We found pyocyanin (PCN) to be a potent inducer of sialyl-Lewis(x) in both mouse airways and in primary and immortalized CF and non-CF human airway epithelial cells. PCN increased the expression of C2/4GnT and ST3Gal-IV, two of the glycosyltransferases responsible for the stepwise biosynthesis of sialyl-Lewis(x), through a tumor necrosis factor (TNF)-α-mediated phosphoinositol-specific phospholipase C (PI-PLC)-dependent pathway. Furthermore, PA bound more efficiently to airway epithelial cells pre-exposed to PCN in a flagellar cap-dependent manner. Importantly, antibodies against sialyl-Lewis(x) and anti-TNF-α attenuated PA binding. These results indicate that PA secretes PCN to induce a favorable environment for chronic colonization of CF lungs by increasing the glycosylation of airway mucins with sialyl-Lewis(x).Mucosal Immunology advance online publication 11 November 2015. doi:10.1038/mi.2015.119.
Matricellular proteins such as osteopontin (OPN), galectin-9 (Gal-9), and tenascin-C (TN-C) are expressed not only under normal physiological conditions, but also during infection, inflammation, or tumorigenesis. We studied the levels of matricellular proteins in plasma to determine their diagnostic value as potential markers of tuberculosis (TB) activity. We found that levels of OPN and TN-C were higher in active TB patients compared to healthy individuals (HC) and latently infected (LTBI) individuals. Moreover, LTBI patients had higher OPN levels compared to HC. The Gal-9 levels did not differ significantly between groups. The levels of the matricellular proteins were higher in pleural fluid compared to their levels in the plasma of TB patient. Expression of matricellular proteins was also investigated in TB granulomas and in other granulomatous diseases. Positive OPN and Gal-9 staining was observed in TB and sarcoidosis granulomas, but not in Crohn's disease granuloma. The fibrotic ring around granulomas stained positive for TN-C in TB and sarcoidosis, but not in Crohn's disease. Of the three matricellular proteins studied, OPN and TN-C may serve as reliable plasma markers to monitor TB activity, whereas Gal-9 seems to be expressed more at the site of infection rather than in systemic circulation.
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The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of cross-contamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine>small intestine>caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1-2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sd(a)-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection.
Mycobacterium bovis bacille Calmette-Gué rin (BCG) is the most widely used live attenuated vaccine. However, the correlates of protection and waning of its immunity against tuberculosis is poorly understood. In this study, we correlated the longitudinal changes in the magnitude and functional quality of CD4 + and CD8 + T-cell response over a period of two years after mucosal or parenteral BCG vaccination with the strength of protection against Mycobacterium tuberculosis in mice. The BCG vaccination-induced CD4 + and CD8 + T cells exhibited comparable response kinetics but distinct functional attributes in-terms of IFN-c, IL-2 and TNF-a co-production and CD62L memory marker expression. Despite a near life-long BCG persistence and the induction of enduring CD4 + T-cell responses characterized by IFN-c and/or TNF-a production with comparable protection, the protective efficacy waned regardless of the route of vaccination. The progressive decline in the multifactorial functional abilities of CD4 + and CD8 + T cells in-terms of type-1 cytokine production, proliferation and cytolytic potential corresponded with the waning of protection against M. tuberculosis infection. In addition, simultaneous increase in the dysfunctional and terminally-differentiated T cells expressing CTLA-4, KLRG-1 and IL-10 during the contraction phase of BCG-induced response coincided with the loss of protection. Our results question the empirical development of BCG-booster vaccines and emphasize the pursuit of strategies that maintain superior T-cell functional capacity. Furthermore, our results underscore the importance of understanding the comprehensive functional dynamics of OPEN ACCESS
The development of effective Mycobacterial antigen diagnostic reagents remains a high priority. The 6-kDa early secreted antigenic target (ESAT6) and 10-kDa culture filtrate protein (CFP10) are secreted early by virulent Mycobacterium tuberculosis (M.tb) and are not present in the non-virulent Bacillus Calmette-Guerin (BCG). In this study, we used a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique to screen for a functional ssDNA aptamer “antibody” that specifically bound to ESAT6-CFP10 (CE) protein. The selected single ssDNA aptamers (CE24 and CE15) demonstrated the highest specificity and binding affinity to CFP10 (CE24: Kd=3.75×10-7 M) and ESAT6 (CE15: Kd=1.6×10-7 M). We further detected CFP10 and ESAT6 proteins in serum samples from active pulmonary tuberculosis (TB) patients, extrapulmonary TB patients and healthy donors by using an enzyme-linked oligonucleotide assay (ELONA). The results showed that the sensitivity and specificity were 100% and 94.1% (using CE24 aptamer-based ELONA) and 89.6% and 94.1% (using CE15 aptamer-based ELONA), respectively. A good correlation was observed between aptamer-based ELONA and T-SPOT TB assay. Thus, our study suggests that CE24 and CE15 have potentially broad applications as early antigen diagnostic agents not only for active pulmonary TB, extrapulmonary TB, but also possibly for latent TB infection and TB with immune-deficiency.
Adhesion of Helicobacter pylori to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylori–related diseases. In this study, we investigated the GalNAcβ1-4GlcNAc motif (also known as N,N’-diacetyllactosediamine [lacdiNAc])
carried by MUC5AC gastric mucins as the target for bacterial binding to the human gastric mucosa. The expression of LacdiNAc
carried by gastric mucins was correlated with H. pylori localization, and all strains tested adhered significantly to this motif. Proteomic analysis and mutant construction allowed
the identification of a yet uncharacterized bacterial adhesin, LabA, which specifically recognizes lacdiNAc. These findings
unravel a target of adhesion for H. pylori in addition to moieties recognized by the well-characterized adhesins BabA and SabA. Localization of the LabA target, restricted
to the gastric mucosa, suggests a plausible explanation for the tissue tropism of these bacteria. These results pave the way
for the development of alternative strategies against H. pylori infection, using adherence inhibitors.
The major surface lipoglycan of Mycobacterium tuberculosis (M. tb), mannose-capped lipoarabinomannan (ManLAM) is an immunosuppressive epitope of M. tb. We used Systematic Evolution of Ligands by EXponential enrichment (SELEX) to generate an aptamer (ZXL1) that specifically bound to ManLAM from the virulent M. tb strain H37Rv. Aptamer ZXL1 had the highest binding affinity with equilibrium dissociation constant-Kd of 436.3±37.84 nM and competed with the mannose receptor for binding to ManLAM and M. tb H37Rv. ZXL1 significantly inhibited the ManLAM-induced immunosuppression of CD11c(+) dendritic cells (DCs) and enhanced the M. tb-antigen-presenting activity of DCs for naïve CD4(+) Th1 cell activation. More importantly, we demonstrated that injection of aptamer ZXL1 significantly reduced the progression of M. tb H37Rv infections and bacterial loads in lungs in mice and rhesus monkeys. These results suggest that aptamer ZXL1 is a new potential antimycobacterial agent and TB vaccine immune-adjuvant.Molecular Therapy (2014); doi:10.1038/mt.2014.31.
Alveolar macrophages exist in a unique microenvironment and, despite historical evidence showing that they are in close contact with the respiratory epithelium, have until recently been investigated in isolation. The microenvironment of the airway lumen has a considerable influence on many aspects of alveolar macrophage phenotype, function and turnover. As the lungs adapt to environmental challenges, so too do alveolar macrophages adapt to accommodate the ever-changing needs of the tissue. In this Review, we discuss the unique characteristics of alveolar macrophages, the mechanisms that drive their adaptation and the direct and indirect influences of epithelial cells on them. We also highlight how airway luminal macrophages function as sentinels of a healthy state and how they do not respond in a pro-inflammatory manner to antigens that do not disrupt lung structure. The unique tissue location and function of alveolar macrophages distinguish them from other macrophage populations and suggest that it is important to classify macrophages according to the site that they occupy.
Tumorigenesis is characterised by changes in transcriptional regulation and the androgen receptor (AR) has been identified as a key driver in prostate cancer. In this study, we show that the hexosamine biosynthetic pathway (HBP) genes are overexpressed in clinical prostate cancer and androgen-regulated in cell-lines. HBP senses metabolic status of the cell and produces an essential substrate for O-GlcNAc transferase (OGT), which regulates target proteins via glycosylation. Using immunohistochemistry, we found that OGT is up-regulated in the protein level in prostate cancer (n=1987) and its expression correlates with high Gleason Score (GS), pT and pN stages and biochemical recurrence (for all, p<0.0001). Both a small molecule inhibitor and siRNAs targeting OGT decreased prostate cancer cell growth. Microarray profiling revealed that the principal effects of the OGT inhibitor in prostate cancer cells are on cell cycle progression and DNA replication. We identified MYC as a candidate upstream regulator of these genes and found that OGT inhibitor caused a dose-dependent loss of c-MYC protein but not mRNA in cell lines. Finally, we observed a statistically significant co-expression between c-MYC and OGT in human prostate cancer samples (n=1306, p=0.0012).
Microenvironmental interactions are crucial for the survival and proliferation of chronic lymphocytic leukemia (CLL) cells. CD4+ T cells that express CD40 ligand (CD40L), along with other accessory immune and stromal cells within CLL lymph nodes (LNs) provide signals needed for activation and outgrowth of the tumor clone. Further, correct positioning of CLL cells within lymphoid subcompartments is essential for the transmission of these supportive signals. Thereby, interstitial cell migration and adhesion events, influenced by activational stimuli, determine CLL cell localization. CD44 has been implicated in cell activation, migration and tissue retention via binding to its extracellular matrix ligand hyaluronan (HA). In this study, we investigated the role of CD44-HA interactions for CLL positioning and interaction with supportive microenvironments in peripheral LNs, focusing on its regulation via CD40L-dependent, T cell-mediated activation of CLL cells. We found that HA triggered a robust CCL21-induced motility of resting CLL cells. However, CD40L-stimulation promoted the firm, CD44-mediated adhesion of CLL cells to HA, antagonizing their motile behavior. N-linked glycosylations of CD44, particularly associated with the variant isoform CD44v6 after CD40L-activation, appeared to facilitate HA recognition by CD44. We propose that the CD40L-CD40 signaling axis provides a stop signal to motile CLL cells within LN compartments by inducing high avidity CD44-HA adhesion. This might retain CLL cells close to T cell stimuli and facilitate essential interactions with HA bearing stromal cells, collectively promoting CLL cell proliferation and survival.
Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease taking one human life every 15 s globally. The two well-characterized strains H37Rv and H37Ra, derived from the same parental strain M. tuberculosis H37, show dramatically different pathogenic phenotypes. PE/PPE gene family, comprising of 176 open reading frames and present
exclusively in genus Mycobacterium, accounts for ∼10% of the M. tuberculosis genome. Our comprehensive in silico analyses of PE/PPE family of H37Ra and virulent H37Rv strains revealed genetic differences between these strains in terms of several single nucleotide variations and InDels
and these manifested in changes in physico-chemical properties, phosphorylation sites, and protein: protein interacting domains
of the corresponding proteomes. Similar comparisons using the 13 sigma factor genes, 36 members of the mammalian cell entry
family, 13 mycobacterial membrane protein large family members and 11 two-component signal transduction systems along with
5 orphaned response regulators and 2 orphaned sensor kinases failed to reveal very significant difference between H37Rv and H37Ra, reinforcing the importance of PE/PPE genes. Many of these changes between H37Rv and H37Ra can be correlated to differences in pathogenesis and virulence of the two strains.
Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines.
Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response-altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)-mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40-producing cells from the draining lymph nodes of treated Leishmania major-infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar-treated and control groups was not observed in draining lymph node cells from TLR2(-/-) mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections.
Macrophage cell death following infection with Mycobacterium tuberculosis plays a central role in tuberculosis disease pathogenesis. Certain attenuated strains induce extrinsic apoptosis of infected macrophages but virulent strains of M. tuberculosis suppress this host response. We previously reported that virulent M. tuberculosis induces cell death when bacillary load exceeds ∼20 per macrophage but the precise nature of this demise has not been defined.
We analyzed the characteristics of cell death in primary murine macrophages challenged with virulent or attenuated M. tuberculosis complex strains. We report that high intracellular bacillary burden causes rapid and primarily necrotic death via lysosomal permeabilization, releasing hydrolases that promote Bax/Bak-independent mitochondrial damage and necrosis. Cell death was independent of cathepsins B or L and notable for ultrastructural evidence of damage to lipid bilayers throughout host cells with depletion of several host phospholipid species. These events require viable bacteria that can respond to intracellular cues via the PhoPR sensor kinase system but are independent of the ESX1 system.
Cell death caused by virulent M. tuberculosis is distinct from classical apoptosis, pyroptosis or pyronecrosis. Mycobacterial genes essential for cytotoxicity are regulated by the PhoPR two-component system. This atypical death mode provides a mechanism for viable bacilli to exit host macrophages for spreading infection and the eventual transition to extracellular persistence that characterizes advanced pulmonary tuberculosis.
Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.
The number of glycan determinants that comprise the human glycome is not known. This uncertainty arises from limited knowledge of the total number of distinct glycans and glycan structures in the human glycome, as well as limited information about the glycan determinants recognized by glycan-binding proteins (GBPs), which include lectins, receptors, toxins, microbial adhesins, antibodies, and enzymes. Available evidence indicates that GBP binding sites may accommodate glycan determinants made up of 2 to 6 linear monosaccharides, together with their potential side chains containing other sugars and modifications, such as sulfation, phosphorylation, and acetylation. Glycosaminoglycans, including heparin and heparan sulfate, comprise repeating disaccharide motifs, where a linear sequence of 5 to 6 monosaccharides may be required for recognition. Based on our current knowledge of the composition of the glycome and the size of GBP binding sites, glycoproteins and glycolipids may contain approximately 3000 glycan determinants with an additional approximately 4000 theoretical pentasaccharide sequences in glycosaminoglycans. These numbers provide an achievable target for new chemical and/or enzymatic syntheses, and raise new challenges for defining the total glycome and the determinants recognized by GBPs.
CD44 designates a large family of proteins with a considerable structural and functional diversity, which are generated from one gene by alternative splicing. As such, the overexpression of CD44 variant isoform (CD44v) has been causally related to the metastatic spread of cancer cells. To study the underlying mechanism, stable knockdown clones with deletion of exon v7 containing CD44 isoforms (CD44v(kd)) of the highly metastatic rat adenocarcinoma line BSp73ASML (ASML(wt)) were established. ASML-CD44v(kd) clones hardly form lung metastases after intrafootpad application and the metastatic load in lymph nodes is significantly reduced. Rescuing, albeit at a reduced level, CD44v expression in ASML-CD44v(kd) cells (ASML-CD44v(rsc)) restores the metastatic potential. The following major differences in ASML(wt), ASML-CD44v(kd), and ASML-CD44v(rsc) clones were observed: (a) ASML(wt) cells produce and assemble a matrix in a CD44v-dependent manner, which supports integrin-mediated adhesion and favors survival. This feature is lost in the ASML-CD44v(kd) cells. (b) CD44v cross-linking initiates phosphatidylinositol 3-kinase/Akt activation in ASML(wt) cells. Accordingly, apoptosis resistance is strikingly reduced in ASML-CD44v(kd) cells. The capacity to generate an adhesive matrix but not apoptosis resistance is restored in ASML-CD44v(rsc) cells. These data argue for a 2-fold effect of CD44v on metastasis formation: CD44v-mediated matrix formation is crucial for the settlement and growth at a secondary site, whereas apoptosis resistance supports the efficacy of metastasis formation.
Galactosyl beta-1,3-N-acetyl galactosamine (Gal beta-1,3-GalNAc) (Thomsen Friedenreich antigen), the Class I core sequence in O-linked oligosaccharide chains, behaves as an oncofetal antigen showing increased expression in many epithelial malignancies. Previous work has shown that peanut agglutinin (PNA), a lectin that binds Gal beta-1,3-GalNAc, stimulates proliferation in HT-29 (human colon cancer) cells and normal human colonic epithelium and this implies that cell surface glycoproteins which express Gal beta-1,3-GalNAc may play an important role in the regulation of epithelial cell proliferation. We have now studied the effect on epithelial cells of another dietary Gal beta-1,3-GalNAc-binding lectin, the edible mushroom Agaricus bisporus lectin (ABL). This differs from PNA in its ability to bind also to sialylated Gal beta-1,3-GalNAc. In contrast to PNA, ABL (25 micrograms/ml) inhibited incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87% (95% confidence limit, 85-89%), Caco-2 colon cancer cells by 16% (95% confidence limit, 12-20%), MCF-7 breast cancer cells by 50% (95% confidence limit, 47-52%), and Rama-27 rat mammary fibroblasts by 55% (95% confidence limit, 51-60%) when these cells were grown for 24 h in serum-free medium. When assessed by cell count, similar inhibition of proliferation of HT29 cells by ABL was found. In the presence of 2% fetal calf serum (which contains the ABL-binding glycoprotein fetuin), the inhibitory effect of ABL on cell proliferation was still demonstrable but at increased ABL concentration (60 micrograms/ml for 49% inhibition). Ten micrograms/ml ABL completely abolished the stimulatory effect on [3H]thymidine incorporation of epidermal growth factor (100 pg/ml) and PNA (25 micrograms/ml) and markedly inhibited the stimulatory effect of insulin (50 ng/ml). ABL (0.2 mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50 micrograms/ml ABL was reversible after removal of the lectin. Binding studies with 125I-labeled ABL suggested a single class of binding site with an apparent Kd value of (4.12 +/- 0.29) x 10(-7) M with (3.6 +/- 0.3) x 10(7) binding sites/cell. A. bisporus lectin is a reversible noncytotoxic inhibitor of epithelial cell proliferation which deserves study as a potential agent for cancer therapy.
The Galbeta1-3GalNAcalpha (TF antigen)-binding lectin (ABL) from the common edible mushroom (Agaricus bisporus) has a potent anti-proliferative effect without any apparent cytotoxicity. This unusual combination of properties prompted investigation of its mechanism of action. In contrast to soluble lectin, agarose-immobilized, and hence noninternalizable ABL had no effect on proliferation of HT29 colon cancer cells. Electron microscopy of HT29 cells incubated with fluorescein- and gold-conjugated ABL showed internalization of the lectin into endocytotic vesicles and multivesicular bodies. Confocal microscopy showed perinuclear accumulation of fluorescein isothiocyanate-conjugated lectin, which also inhibits HT29 cell proliferation, raising the possibility that the lectin might interfere with nuclear pore function. Transport of heat shock protein 70 into the nucleus in response to heat shock was blocked by preincubation of HT29 cells for 6 h with 40 micrograms/ml ABL. In digitonin-permeabilized cells, nuclear uptake of bovine albumin conjugated to a nuclear localization sequence (NLS)-containing peptide was also inhibited by a 15-min preincubation with 40-100 micrograms/ml ABL. In contrast, serum-stimulated nuclear translocation of mitogen-activated protein kinase, which is NLS-independent, was not affected by pretreatment of cells with the lectin. These results suggest that the anti-proliferative effect of ABL is likely to be a consequence of the lectin trafficking to the nuclear periphery, where it blocks NLS-dependent protein uptake into the nucleus.
The massive T cell death that occurs in HIV type 1 (HIV-1) infection contributes profoundly to the pathophysiology associated with AIDS. The mechanisms controlling cell death of both infected and uninfected T cells ("bystander" death) are not completely understood. We have shown that HIV-1 infection of T cells results in altered glycosylation of cell surface glycoproteins; specifically, it decreased sialylation and increased expression of core 2 O-glycans. Galectin-1 is an endogenous human lectin that recognizes these types of glycosylation changes and induces cell death of activated lymphocytes. Therefore we studied the possible contribution of galectin-1 in the pathophysiology of AIDS. O-glycan modifications were investigated on peripheral lymphocytes from AIDS patients. Oligosaccharides from CD43 and CD45 of CEM cells latently infected with HIV-1 were chemically analyzed. Consistent with our previous results, we show that HIV-1 infection results in accumulation of exposed lactosamine residues, oligosaccharides recognized by galectin-1 on cell surface glycoproteins. Both latently HIV-1-infected T cell lines and peripheral CD4 and CD8 T cells from AIDS patients exhibited exposed lactosamine residues and demonstrated marked susceptibility to galectin-1-induced cell death, in contrast to control cultures or cells from uninfected donors. The fraction of cells that died in response to galectin-1 exceeded the fraction of infected cells, indicating that death of uninfected cells occurred. Altered cell surface glycosylation of T cells during HIV-1 infection increases the susceptibility to galectin-1-induced cell death, and this death pathway can contribute to loss of both infected and uninfected T cells in AIDS.
CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, has additional functions in inflammatory and immune responses, contributing to the ingestion and clearance of particles and apoptotic cells. Our goal was to determine the specific role of CD44 in phagocytosis and whether it functions as a primary or accessory phagocytic receptor. Using hyaluronan-coated beads and erythrocytes coated with antiCD44 antibodies as the phagocytic prey, we determined that CD44 mediates efficient phagocytosis in primary murine peritoneal macrophages and in the murine macrophage cell line RAW 264.7. In RAW cells, the phagocytic index for anti-CD44-coated erythrocytes was 25 +/- 3 (mean +/- SEM) compared with less than 1 for erythrocytes coated with isotype-matched control antibodies. Uptake of anti-CD44-coated erythrocytes was abrogated by pretreatment with a blocking antibody to CD44 and was absent in primary cultures of CD44-deficient murine macrophages. Down-regulation of Fc receptors by aggregated IgG-induced internalization, which blocks uptake of IgG-coated particles, had no effect on CD44-mediated particle engulfment. Using a combination of immunoprecipitation, pharmacologic inhibition, and genetic deletion, we determined that CD44-mediated phagocytosis involves Syk, Rac1, and phosphatidylinositol 3-kinase and induced activation of the phagocyte oxidase. We conclude that CD44 is a competent phagocytic receptor that efficiently mediates internalization of large particles.
Infection of human monocyte-derived macrophages with Mycobacterium tuberculosis at low multiplicities of infection leads 48-72 h after the infection to cell death with the characteristics of apoptosis or necrosis. Predominant induction of one or the other cell death modality depends on differences in mitochondrial membrane perturbation induced by attenuated and virulent strains. Infection of macrophages with the attenuated H37Ra or the virulent H37Rv causes mitochondrial outer membrane permeabilization characterized by cytochrome c release from the mitochondrial intermembrane space and apoptosis. Mitochondrial outer membrane permeabilization is transient, peaks 6 h after infection, and requires Ca(2+) flux and B cell chronic lymphocytic leukemia/lymphoma 2-associated protein X translocation into mitochondria. In contrast, only the virulent H37Rv induces significant mitochondrial transmembrane potential (Deltapsi(m)) loss caused by mitochondrial permeability transition. Dissipation of Deltapsi(m) also peaks at 6 h after infection, is transient, is inhibited by the classical mitochondrial permeability transition inhibitor cyclosporine A, has a requirement for mitochondrial Ca(2+) loading, and is independent of B cell chronic lymphocytic leukemia/lymphoma translocation into the mitochondria. Transient dissipation of Deltapsi(m) 6 h after infection is essential for the induction of macrophage necrosis by Mtb, a mechanism that allows further dissemination of the pathogen and development of the disease.
Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents.
Tuberculosis (TB) remains a prevalent and lethal infectious disease. The glycobiology associated with Mycobacterium tuberculosis infection of front-line alveolar macrophages is still unresolved. Herein, we investigated the regulation of protein N-glycosylation in human macrophages and their secreted microparticles (MPs) used for intercellular communication upon M. tb infection. LC-MS/MS-based proteomics and glycomics were performed to monitor the regulation of glycosylation enzymes and receptors and the N-glycome in in vitro-differentiated macrophages and in isolated MPs upon M. tb infection. Infection promoted a dramatic regulation of the macrophage proteome. Most notably, significant infection-dependent down-regulation (4-26 fold) of 11 lysosomal exoglycosidases e.g. β-galactosidase, β-hexosaminidases and α-/β-mannosidases was observed. Relative weak infection-driven transcriptional regulation of these exoglycosidases and a stronger augmentation of the extracellular hexosaminidase activity demonstrated that the lysosome-centric changes may originate predominantly from infection-induced secretion of the lysosomal content. The macrophages showed heterogeneous N-glycan profiles and displayed significant up-regulation of complex-type glycosylation and concomitant down-regulation of paucimannosylation upon infection. Complementary intact N-glycopeptide analysis supported a subcellular-specific manipulation of the glycosylation machinery and altered glycosylation patterns of lysosomal N-glycoproteins within infected macrophages. Interestingly, the corresponding macrophage-derived MPs displayed unique N-glycome and proteome signatures supporting a preferential packaging from plasma membranes. The MPs were devoid of infection-dependent N-glycosylation signatures, but interestingly displayed increased levels of the glyco-initiating oligosaccharyltransferase complex and associated α-glucosidases that correlated with increased formation, N-glycan precursor levels and N-glycan density of infected MPs. In conclusion, this system-wide study provides new insight into the host- and pathogen-driven N-glycoproteome manipulation of macrophages in TB.
Because of the Mycobacterium bovis, termed Bacillus Calmette–Guérin (BCG), the only available used Tuberculosis (TB) vaccine, retaining immunomodulatory properties that limit its protective immunogenicity, there are continuous efforts to identify the immunosuppression mechanism as well as new strategies for improving immunogenicity of BCG. Here, an ssDNA aptamer “antibody” BM2 that specifically bound to Mannose-capped lipoarabinomannan (ManLAM) of BCG was selected. BM2 significantly blocked ManLAM-Mannose Receptor (MR) binding and triggered ManLAM-CD44 signaling, and enhanced M1 macrophage and Th1 activation via cellular surface CD44 in vitro and in vivo. BM2 enhanced immunoprotective effects of BCG against virulent Mycobacterium tuberculosis (M. tb) H37Rv infection in mice and monkeys models. Thus, we report a new mechanism of the interaction between ManLAM and CD44 on macrophages and CD4+ T cells, and reveal that ManLAM-binding membrane molecule CD44 is a novel target for the enhancement of BCG immunogenicity and BM2 has strong potential as an immune enhancer for BCG.
Protein glycosylation processes play a crucial role in most physiological functions, including cell signaling, cellular differentiation and adhesion. We previously demonstrated that rapid deglycosylation of membrane proteins was specifically triggered after infection of human macrophages by the bacterial pathogen Francisella tularensis. Using a glycan processing gene microarray, we found here that Francisella infection modulated expression of numerous glycosidase and glycosyltransferase genes. Furthermore, analysis of cell extracts from infected macrophages by Lectin and Western blotting revealed an important increase of N- and O-protein glycosylation. We chose to focus in the present work on one of the O-glycosylated proteins identified by mass spectrometry, the multifunctional endoplasmic reticulum chaperone BiP (HSPA5/GRP78). We demonstrate that BiP expression is modulated upon Francisella infection and is required to support its intracellular multiplication. Moreover, we show that Francisella differentially modulates the BiP-dependent activation of three key proteins of the unfolded protein response (UPR), IRE1, PERK and ATF6. The effects exerted on human cells by Francisella may thus constitute a novel example of UPR manipulation contributing to intracellular bacterial adaptation.
In recent years, a critical role for β-galactoside-binding protein, Galectin-9 (Gal-9) has emerged in infectious disease, autoimmunity, and cancer. It is a ligand for T cell immunoglobulin mucin domain 3 (Tim-3), a type-I glycoprotein that is persistently expressed on dysfunctional T cells during chronic viral infections. Gal-9 exerts its pivotal immunomodulatory effects by inducing apoptosis or suppressing effector functions via engagement with its receptor, Tim-3. Recent studies report elevation of circulating Gal-9 in humans infected with different viral infections. Interaction of soluble Gal-9 with Tim-3 expressed on the surface of activated CD4+ T cells renders them less susceptible to HIV-1 infection, while enhanced HIV infection occurs when Gal-9 interacts with a different receptor than Tim-3. This indicates the versatile role of Gal-9 in viral pathogenesis. For instance, higher expression of Tim-3 during chronic viral infection and elevation of plasma Gal-9 may have evolved to limit persistent immune activation and pathogenic T cells activity. In contrast, Gal-9 can suppress the effectiveness of immunity against viral infections. In agreement, Gal-9 knockout mice mount a more robust and vigorous virus-specific immune response in acute and chronic viral infections resulting in rapid viral clearance. In line with this observation, blocking Gal-9 signals to Tim-3-expressing T cells result in improved immune responses. Here we review the biological and immunological properties of Gal-9 in viral infections (HIV, HCV, HBV, HSV, CMV, influenza, and dengue virus). Manipulating Gal-9 signals may have immunotherapeutic potential and could represent an alternative approach for improving immune responses to viral infections/vaccines. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
Epithelial-to-mesenchymal transition (EMT) is an essential biological process that occurs in embryonic development, metastatic diseases, and cancer progression. Altered expression of glycans is known to be associated with cancer progression. No studies to date have presented global analysis of the precise variation of N-glycans in EMT. We describe here the profile of N-glycans and glycogene expression in the EMT process induced by transforming growth factor-β1 (TGFβ1) in a normal mouse mammary gland epithelial (NMuMG) cell model. An integrated strategy with a combination of mass spectrometry, glycogene microarray analysis, and lectin microarray analysis was applied, and results were confirmed by lectin histochemistry and quantitative real-time PCR. In TGFβ-induced EMT, levels of high-mannose-type N-glycans were enhanced, antennary N-glycans and fucosylation were suppressed, and bisecting GlcNAc N-glycans were greatly suppressed. The expression of 7 N-glycan-related genes was significantly changed. The products of glycogenes ALG9, MGAT3, and MGAT4B appeared to contribute to the observed alteration of N-glycans. The findings indicate that dysregulation of N-glycan synthesis plays a role in the EMT process. Systematic glycomic analysis based on the combination of techniques described here is expected to facilitate the discovery of the aberrant N-glycosylation in tumor progression and provide essential information in systems glycobiology.
Glycosylation of proteins is one of the most important post-translational modifications, with more than half of all human proteins estimated to be glycosylated. It is widely known that aberrant glycosylation has been implicated in many different diseases due to changes associated with biological function and protein folding. In cancer, there is increasing evidence pertaining to the role of glycosylation in tumour formation and metastasis. Alterations in cell surface glycosylation, particularly terminal motifs, can promote invasive behaviour of tumour cells that ultimately lead to the progression of cancer. While a majority of studies have investigated protein glycosylation changes in cancer cell lines and tumour tissue for individual cancers, the review presented here represents a comprehensive, in-depth overview of literature on the structural changes of glycosylation and their associated synthetic enzymes in five different cancer types originating from the breast, colon, liver, skin and ovary. More importantly, this review focuses on key similarities and differences between these cancers which reflect the importance of structural changes of cell surface N- and O-glycans, such as sialylation, fucosylation, degree of branching and the expression of specific glycosyltransferases for each cancer. It is envisioned that the understanding of these biologically relevant glycan alterations on cellular proteins will facilitate the discovery of novel glycan-based biomarkers which could potentially serve as diagnostic and prognostic indicators of cancer. This article is protected by copyright. All rights reserved.
We have previously shown that the pro-inflammatory cytokine TNF (tumour necrosis factor) could drive sLex (sialyl-Lewisx) biosynthesis through the up-regulation of the BX transcript isoform of ST3GAL4 sialyltransferase gene in lung epithelial cells and human bronchial mucosa. Here, we show that the TNF-induced up-regulation of ST3GAL4 BX transcript is mediated by the mitogen and stress activated protein kinases MSK1/2 through Erk (extracellular signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and increases sLex expression on high molecular weight glycoproteins in the inflamed airway epithelium. We also show that the TNF-induced sLex expression increases the adhesion of Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells in a FliD-dependent manner. These results suggest that Erk and p38 MAPK, and the downstream kinase MSK1/2 should be considered as potential targets to hamper inflammation, bronchial mucins glycosylation changes and P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.
Tuberculosis (TB) remains one of the greatest threats to human health. The causative bacterium, Mycobacterium tuberculosis (Mtb), is acquired by the respiratory route. It is exquisitely human adapted and a prototypic intracellular pathogen of macrophages, with alveolar macrophages (AMs) being the primary conduit of infection and disease. The outcome of primary infection is most often a latently infected healthy human host, in whom the bacteria are held in check by the host immune response. Such individuals can develop active TB later in life with impairment in the immune system. In contrast, in a minority of infected individuals, the host immune response fails to control the growth of bacilli, and progressive granulomatous disease develops, facilitating spread of the bacilli via infectious aerosols coughed out into the environment and inhaled by new hosts. The molecular details of the Mtb-macrophage interaction continue to be elucidated. However, it is clear that a number of complex processes are involved at the different stages of infection that may benefit either the bacterium or the host. Macrophages demonstrate tremendous phenotypic heterogeneity and functional plasticity which, depending on the site and stage of infection, facilitate the diverse outcomes. Moreover, host responses vary depending on the specific characteristics of the infecting Mtb strain. In this chapter, we describe a contemporary view of the behavior of AMs and their interaction with various Mtb strains in generating unique immunologic lung-specific responses.
Recent studies have elucidated that expression of certain glycoproteins in human saliva are increased or decreased according to age, meanwhile, human saliva may inhibit viral infection and prevent viral transmission. However, little is known about the age- and sex-associated differences in the glycopatterns of human salivary glycoproteins and their significant roles against influenza A virus (IVA). Here we investigate the glycopatterns of human salivary glycoproteins with 180 healthy saliva samples divided into six age/sex groups using lectin microarrays and fabricate saliva microarrays to validate the terminal carbohydrate moieties of glycoproteins in individual saliva samples. Furthermore, we assess the inhibiting and neutralizing activity of saliva against two strains of influenza A (H9N2) virus. We find that seven lectins (e.g., MAL-II and SNA) show significant age differences in both females and males, and seven lectins (e.g., WFA and STL) show significant sex differences in children, adults and elderly people. Interestingly, we observe that elderly individuals have strongest resistance to IVA partly by presenting more terminal α2-3/6-linked sialic acid residues in their saliva, which bind with the influenza viral hemagglutinations. We conclude that age- and sex- associated differences in the glycopatterns of human salivary glycoproteins may provide pivotal information to help understand some age related diseases and physiological phenomenon.
Sixty four sequences containing lectin domains with homologues of known three-dimensional structure were identified through a search of mycobacterial genomes. They appear to belong to the β-prism II, the C-type, the Microcystis virdis (MV), and the β-trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI-PLC and the β-grasp domains respectively while mycobacterial β-trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologues of known three dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and non-pathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one or the other of the four lectin domains can be gleaned through the examination of homologous proteins, although the structure of none of them is available. Their biological role is also yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins. Proteins 2012. © 2012 Wiley Periodicals, Inc.
Francisella tularensis, a Gram-negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP-1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post-translational modification of a eukaryotic amino acid transporter.
The interfacial surface tension of the lung is regulated by phospholipid-rich pulmonary surfactant films. Small changes in temperature affect surfactant structure and function in vitro. We compared the compositional, thermodynamic and functional properties of surfactant from hibernating and summer-active 13-lined ground squirrels (Ictidomys tridecemlineatus) with porcine surfactant to understand structure-function relationships in surfactant membranes and films. Hibernating squirrels had more surfactant large aggregates with more fluid monounsaturated molecular species than summer-active animals. The latter had more unsaturated species than porcine surfactant. Cold-adapted surfactant membranes displayed gel-to-fluid transitions at lower phase transition temperatures with reduced enthalpy. Both hibernating and summer-active squirrel surfactants exhibited lower enthalpy than porcine surfactant. LAURDAN fluorescence and DPH anisotropy revealed that surfactant bilayers from both groups of squirrels possessed similar ordered phase characteristics at low temperatures. While ground squirrel surfactants functioned well during dynamic cycling at 3, 25, and 37°C, porcine surfactant demonstrated poorer activity at 3°C but was superior at 37°C. Consequently the surfactant composition of ground squirrels confers a greater thermal flexibility relative to homeothermic mammals, while retaining tight lipid packing at low body temperatures. This may represent the most critical feature contributing to sustained stability of the respiratory interface at low lung volumes. Thus, while less effective than porcine surfactant at 37°C, summer-active surfactant functions adequately at both 37°C and 3°C allowing these animals to enter hibernation. Here further compositional alterations occur which improve function at low temperatures by maintaining adequate stability at low lung volumes and when temperature increases during arousal from hibernation.
CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis-induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis-induced CD44 surface expression in THP-1 human monocytes.
Interleukin-10 (IL-10), a cytokine with anti-inflammatory properties, has a central role in infection by limiting the immune response to pathogens and thereby preventing damage to the host. Recently, an increasing interest in how IL10 expression is regulated in different immune cells has revealed some of the molecular mechanisms involved at the levels of signal transduction, epigenetics, transcription factor binding and gene activation. Understanding the specific molecular events that regulate the production of IL-10 will help to answer the remaining questions that are important for the design of new strategies of immune intervention.
In this study, we have identified a secreted 13 kDa lectin from Mtb (Mtb, Mycobacterium tuberculosis; sMTL-13) by homology search of a non-redundant lectin database. Bioinformatic analysis revealed that sMTL-13 belongs to the ricin-type beta-trefoil family of proteins containing a Sec-type signal peptide present in Mtb complex species, but not in non-tuberculous mycobacteria. Following heterologous expression of sMTL-13 and generation of an mAb (clone 276.B7/IgG1kappa), we confirmed that this lectin is present in culture filtrate proteins from Mtb H37Rv, but not in non-tuberculous mycobacteria-derived culture filtrate proteins. In addition, sMTL-13 leads to an increased IFN-gamma production by PBMC from active tuberculosis (ATB) patients. Furthermore, sera from ATB patients displayed high titers of IgG Ab against sMTL-13, a response found to be decreased following successful anti-tuberculosis therapy. Together, our findings reveal a secreted 13 kDa ricin-like lectin from Mtb, which is immunologically recognized during ATB and could serve as a biomarker of disease treatment.
Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-kappaB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb.
Macrophages are not only host cells for many pathogens, but also fulfill several key functions in the innate and adaptive immune response, including the release of pro- and anti-inflammatory cytokines, the generation of organic and inorganic autacoids, the phagocytosis and killing of intracellular microorganisms or tumor cells, and the degradation and presentation of antigens. Several of these functions are shared by other immune cells, including dendritic cells, granulocytes, NK cells, and/or T lymphocytes. Thus, the analysis of macrophage functions in vitro using primary mouse cell populations requires standardized methods for the generation and culture of macrophages that guarantee high cell purity as well as the absence of stimulatory microbial contaminants. This chapter presents methodology to achieve these aims.
CD44 alternative splicing patterns differ between normal and malignant tissue, and accordingly, modulation of CD44 splicing has received the most attention in studies that have examined the role of CD44 in tumor progression. Many investigators have examined functional differences between individual CD44 alternative splice variants. However, specific CD44 isoforms function uniquely depending on the type of cell on which they are expressed, thereby suggesting that additional tissue-specific mechanisms regulate CD44 function. In the present study we have demonstrated that colon carcinoma cells modify CD44 with O-linked glycosyl groups, and blockade of this glycosylation enhances their CD44-mediated adhesion to hyaluronate. This enhancement is attributable principally to CD44H (CD44s) rather than high molecular weight CD44 variants. Use of site-directed mutant CD44H cDNA transfectants demonstrated that CD44 O-linked glycosylation modulates interaction between hyaluronate and the B loop domain of CD44. The influence of glycosylation on CD44 function in colon carcinoma cells is specific to the presence of O-linked sugars; inhibition of N-linked glycosylation had minimal influence on CD44 function. These findings indicate that O-linked glycosylation may be as important as alternative splicing in the regulation of CD44 function and the broad spectrum of biological processes attributed to it, including normal development, tumor metastases, and lymphocyte function.
Tumor necrosis factor (TNF)-alpha-dependent apoptosis of alveolar macrophages (AM) after infection with avirulent Mycobacterium tuberculosis (Mtb) results in bacillary death and the destruction of a growth niche for the pathogen. This response is minimized after infection with virulent strains of Mtb. To study the genetic control of Mtb-induced apoptosis, we used microarrays to interrogate the expression profile of infected human AM. Although we found variation in gene expression between different donors of AM, a set of genes were constant for each condition. A group of proapoptotic genes were downregulated after infection by virulent Mtb strain H37Rv, whereas infection with avirulent Mtb H37Ra led to a gene expression profile that would favor macrophage apoptosis. Neutralizing TNF in macrophage cultures infected with H37Ra changed the gene expression profile to one that resembled the profile of macrophages infected with H37Rv. These data reveal that apoptosis-related genes are regulated differently by virulent or attenuated Mtb strains, and are consistent with the hypothesis that virulent Mtb interfere with TNF death signaling. Given the importance of TNF in host defense against tuberculosis, the ability to repress the expression of genes activated by TNF may constitute a bacillary virulence mechanism.
Macrophage apoptosis plays a role in mycobacterial infection. To define the mechanism by which virulent Mycobacterium tuberculosis escapes apoptosis and killing in macrophages, J774 macrophages were infected with virulent M. tuberculosis H37Rv and attenuated H37Ra strains. H37Rv induced less apoptosis than H37Ra, and caspase 3 was activated in H37Ra- and H37Rv-infected macrophages. Intracellular H37Rv bacilli were released at a higher rate into the supernatant than were H37Ra by the sixth day of infection, and this was simultaneously accompanied by the increased necrosis of infected cells showing lactate dehydrogenase (LDH) release. Fas mRNA expression was downregulated and FasL was upregulated in H37Ra- and H37Rv-infected macrophages, while Bcl-2 was upregulated in H37Rv-infected macrophages but downregulated in H37Ra-infected macrophages as seen by real-time PCR. These results indicate that M. tuberculosis H37Ra and H37Rv proliferate in macrophages by preventing them from inducing apoptosis during the early phase of infection, and that M. tuberculosis H37Rv-infected macrophages are found to express Bcl-2 mRNA, which leads to anti-apoptotic activity, and that relatively distinct necrosis might occur during the later phase of infection.
World Health Organization. The WHO 2014 global tuberculosis report-further to go
World Health Organization. The WHO 2014 global tuberculosis report-further
to go. Lancet Glob Health; 2015.