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The development of new tuberculosis vaccines remains a global priority, and recombinant vaccines are a frequently investigated option. These vaccines follow a molecular strategy that may enhance protective efficacy. However, their functional differences, particularly with respect to glycosylation, remain unknown. Recent studies have shown that glycosylation plays a key role in the host-pathogen interactions during immune recognition. The aim of this study was to determine the differences in the glycosylation profiles of two recombinant strains of Mycobacterium microti, overexpressing Ag85B (Rv1886c) and PstS-1 (Rv0934) antigens of M. tuberculosis . For each strain, the glycosylation profile was determined by Western blotting with lectins. The results showed the presence of mannosylated proteins and evidence of linked sialic acid proteins. Interestingly, different proteome and glycoproteome profiles were observed between the two recombinant strains and the wild-type strain. We have shown here that the construction of the recombinant strains of M. microti has altered the proteome and glycosylation profiles of these strains, leading us to ask what impact these changes might have on the immune response.

Currently, the only available vaccine against tuberculosis is Mycobacterium bovis Bacille Calmette-Guérin (BCG). Pulmonary tuberculosis protection provided by the vaccine varies depending on the strain, the patient's age and the evaluated population. Although the adaptive immune responses induced by different BCG strains have been widely studied, little conclusive data is available regarding innate immune responses, especially in macrophages. Here, we aimed to characterize the innate immune responses of human THP-1-derived macrophages at the transcriptional level following a challenge with either the BCG Mexico (M.BCG) or Phipps (P.BCG) strains. After a brief in vitro characterization of the bacterial strains and the innate immune responses, including nitric oxide production and cytokine profiles, we analyzed the mRNA expression patterns and performed pathway enrichment analysis using RNA microarrays. Our results showed that multiple biological processes were enriched, especially those associated with innate inflammatory and antimicrobial responses, including tumor necrosis factor (TNF)-α, type I interferon (IFN-I) and IFN-γ. However, four DEGs were identified in macrophages infected with M.BCG compared to P. BCG. These findings indicated the proinflammatory stimulation of macrophages induced by both BCG strains, at the cytokine level and in terms of gene expression, suggesting a differential expression pattern of innate immune transcripts depending on the mycobacterial strain.

Introduction: The growing incidence of non-tuberculous mycobacteria (NTM) and changes in epidemiological factors have indicated that immune dysregulation may be associated with the emergence of NTM. Minireview entails to acknowledge complex interaction and new ways NTM are evolving around diverse immune status., Methods: In order to perform this review, we selected peer reviewed, NLM database articles under the terms NTM, mycobacterium complex 'AND' -Host- immune response, immunity regulation, Disease, Single Nucleotide Polymorphism (SNP´s), and -pathogen- followed by a snow ball rolling basis search on immune components and NTM related with diseases distribution., Results: The universal exposure and diversity of NTM are well-documented; however, hospitals seldom establish vigilant control of water quality or immunodeficiencies for patients with NTM infections. Depending on the chemical structures and immune mechanisms presented by various NTM varieties, they can trigger different effects in dendritic and natural killer cells, which release interleukin (IL)-17, tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and rIL-1B. The T helper (Th)2-acquired immune response is responsible for autoimmune responses in patients with NTM infections, and, quite disturbingly, immunocompetent patients have been reported to suffer from NTM infections., Conclusion: New technologies and a comprehensive view has taught us; to acknowledge metabolic/immune determinants and trade-offs along transit through mutualism-parasite continuous., (© 2021 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The ScandinavianFoundation for Immunology.)

Background: In contrast to the well-characterized Celiac Disease (CD), the clinical scenarios encompassed by the non-celiac self-reported wheat sensitivity (NCSRWS) might be related to different antigens that trigger distinct immune-inflammatory reactions. Although an increased number of intestinal intraepithelial lymphocytes is observed at the inception of both diseases, the subsequent immunopathogenic pathways seem to be different. We aimed to describe the cytokine profile observed in the duodenal mucosa of patients with NCSRWS., Methods: In a blind, cross-sectional study, we included duodenal biopsies from 15 consecutive untreated patients with active CD, 9 individuals with NCSRWS and 10 subjects with dyspepsia without CD and food intolerances. Immunohistochemistry and flow-cytometry were used to determine the presence of pro-inflammatory cytokine expressing monocytes and monocyte-derived dendritic cells involved in innate immune activation, cytokine-driven polarization and maintenance of Th1 and Th17/Th 22, and anti-inflammatory/profibrogenic cytokines., Results: The percentage of cells expressing all tested cytokines in the lamina propria and the epithelium was higher in CD patients than in the control group. Cytokines that induce and maintain Th1 and Th17 polarization were higher in CD than in NCSRWS and controls, also were higher in NCSRWS compared to controls. Similar differences were detected in the expression of IL-4 and TGF-1, while IL-10-expressing cells were lower in NCSRWS patients than in controls and CD subjects., Conclusions: NCSRWS patients exhibit components of both, innate and adaptive immune mechanisms but to a lesser extent compared to CD.

Background: Pichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein., Results: The recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb., Conclusions: rPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.

Nontuberculous mycobacteria (NTM) are recognized as emerging pathogens and their immune regulatory mechanisms are not well described yet. From them, Mycobacterium avium is known to be a weak activator of dendritic cells (DCs) that impairs the response induced by BCG vaccine. However, whether other NTM such as Mycobacterium scrofulaceum may modulate the activation of DCs, has not been extensively studied. Here, we exposed bone marrow-derived DCs (BMDCs) to M. scrofulaceum and we analyzed the effect on the activation of DCs. We found that M. scrofulaceum has a comparable ability to induce a semi-mature DC phenotype, which was produced by its interaction with DC-SIGN and TLR4 receptors in a synergic effect. BMDCs exposed to M. scrofulaceum showed high expression of PD-L2 and production of IL-10, as well as low levels of co-stimulatory molecules and pro-inflammatory cytokines. In addition to immunophenotype induced on DCs, changes in morphology, re-organization of cytoskeleton and decreased migratory capacity are consistent with a semi-mature phenotype. However, unlike other pathogenic mycobacteria, the DC-semi-mature phenotype induced by M. scrofulaceum was reversed after re-exposure to BCG, suggesting that modulation mechanisms of DC-activation used by M. scrofulaceum are different to other known pathogenic mycobacteria. This is the first report about the immunophenotypic characterization of DC stimulated by M. scrofulaceum., (Copyright © 2016. Published by Elsevier Ltd.)

Nontuberculous mycobacteria (NTM) have been isolated from water, soil, air, food, protozoa, plants, animals, and humans. Although most NTM are saprophytes, approximately one-third of NTM have been associated with human diseases. In this study, we did a comparative proteomic analysis among five NTM strains isolated from several sources. There were different numbers of protein spots from M. gordonae (1,264), M. nonchromogenicum type I (894), M. nonchromogenicum type II (935), M. peregrinum (806), and M. scrofulaceum/Mycobacterium mantenii (1,486) strains, respectively. We identified 141 proteins common to all strains and specific proteins to each NTM strain. A total of 23 proteins were selected for its identification. Two of the common proteins identified (short-chain dehydrogenase/reductase SDR and diguanylate cyclase) did not align with M. tuberculosis complex protein sequences, which suggest that these proteins are found only in the NTM strains. Some of the proteins identified as common to all strains can be used as markers of NTM exposure and for the development of new diagnostic tools. Additionally, the specific proteins to NTM strains identified may represent potential candidates for the diagnosis of diseases caused by these mycobacteria.

Tuberculosis is a disease caused by the Mycobacterium tuberculosis complex (MTb). In 2011, global mortality due to tuberculosis was 1·4 million individuals. The only available vaccine is the attenuated M. bovis [bacillus Calmette-Guérin (BCG)] strain, which confers variable protection against pulmonary tuberculosis. Some widely distributed non-tuberculous mycobacteria (NTM), such as M. avium and M. arupense, are also potential pathogens for humans. This work aimed to produce and characterize monoclonal antibodies against the M. bovis BCG Mexico strain of the MTb, M. avium subs. hominissuis and the M. arupense strain from NTM. Hybridomas were produced from splenocytes of BALB/c female mice immunized with radiation-inactivated mycobacteria, and the immunoglobulin (Ig)G2a antibody-producing clones with the highest antigenic recognition were selected. The selected clones, Mbv 2A10 for M. bovis BCG Mexico, Mav 3H1 for M. avium and Mar 2D10 for M. arupense, were used in further studies. Enzyme-linked immunosorbent assay (ELISA) and immune proteomics analyses characterized the clones as having the highest cross-reactivity with mycobacteria. Using mass spectrometry, a number of proteins recognized by the monoclonal antibody (mAb) clones were identified. These proteins had roles in metabolic processes, hypoxia, cell cycle and dormancy. In addition, a Clustal W and Immune Epitope Database (IEDB) in-silico analysis was performed in protein sequences that result in the conserved regions within probability epitopes that could be recognized for Mbv2A10 and Mav3H1 clones., (© 2014 British Society for Immunology.)

Systemic release of norepinephrine (NE) is a component of the acute host response to infection, and studies in the field of microbial endocrinology indicate generally that NE increases the bacterial growth rate and promotes invasive disease. However, NE attenuates experimental invasive pneumococcal disease. We determined that NE promoted pneumococcal growth but paradoxically decreased pneumococcal adhesion to host cells. This effect was independent of the classical adhesin CbpA. Microarray analysis indicated that the effect of NE involved two two-component regulatory systems that both regulate expression of the Piu iron uptake ABC transport operon. We propose that NE, a known siderophore, enhances iron availability to the bacteria, resulting in greater bacterial replication and decreased expression of Piu operon products. Downregulation of the operon includes decreased expression of the Piu-associated adhesin PiuD. Our results suggested that the iron-dependent inhibitory effect of NE on pneumococcal adherence is a mechanism underlying the amelioration of pneumococcal disease by NE.

We investigated the presence of nontuberculous mycobacteria (NTM) in three Mexican aquatic systems to evaluate the prevalence with the distribution of NTM species. Key physicochemical parameters of the water samples were determined to find correlations with the species' distributions. We used multilocus sequence analysis (MLSA) based on hsp65, rpoB, and 16S rRNA fragments to determine their taxonomic affiliations. NTM were recovered from water distribution systems and reclaimed water from the Mexico City Metropolitan Area (MCMA). The isolated species were associated with a temperature of 21°C and pH >7.7. The phylogenetic analysis showed that eight of the 14 different NTM strains were unambiguously classifiable: Mycobacterium peregrinum, M. nonchromogenicum (2), M. smegmatis (2), M. fortuitum, M. avium ssp. hominissuis, M. arupense, M. gordonae, and M. chitae. One strain was tentatively identified as M. mantenni/ scrofulaceum and another strain was related to M. porcinum/M. septicum. All NTM species identified in the water distribution system were also detected in the reclaimed water, but some species from the reclaimed water were not found in the water distribution systems. Two of the identified species found in the reclaimed water, M. avium and M. fortuitum, are considered important human opportunistic pathogens.

Background: Intraepithelial lymphocytes (IELs) phenotyping has emerged as a useful test in intestinal pathology. In celiac disease (CD), a permanent and marked increase of gammadelta+ IELs has been described. However, there is a lack of knowledge about this peculiar IELs population in other intestinal pathologies., Aim: To analyze the percentage of IELs, specifically gammadelta+ IELs subset, present in duodenal mucosa biopsies from patients with CD and compare it with those obtained from patients with small intestinal bacterial overgrowth (SIBO) or irritable bowel syndrome (IBS)., Methods: Twelve patients with untreated CD, 8 patients with SIBO, and 10 patients with diarrhea-predominant IBS were evaluated. All subjects underwent upper endoscopy for mucosal biopsy and jejunal aspirate. From 2 small bowel biopsies, intraepithelial cells were isolated and labeled with the following monoclonal antibodies CD103-PE (phycoerythrin), CD3-FITC (fluoresecein isothio-cynate), CD-7R-PE, CD45RO-APC (allophycocyanin), and TcR gammadelta-FITC. Flow cytometry analysis was performed on a standard FACScan. Total and IELs subset counts were expressed as percentage., Results: Mean total IELs percentage was 16.7+/-6% in IBS, 25.4+/-17% in SIBO, and 26+/-13% in CD patients (P=0.2). CD and SIBO patients, had significantly higher percentages of gammadelta+ IELs (15.7+/-13% and 14.6+/-8%) than IBS subjects (4.1+/-2.5%, P<0.05). There was no difference between CD and SIBO (P=0.6)., Conclusions: An increased density of gammadelta+ IELs is typical, but not specific for CD. A similar increase was observed in subjects with SIBO. Our findings suggest that this unique T-cell population might have a key role against intestinal bacterial infections.

Mycobacterium bovis BCG is the only available vaccine against tuberculosis. Reasons for why diverse BCG substrains induce different levels of protection in clinical trials remain unclear. The aim of this study was to compare the effectiveness of 10 BCG substrains in a mouse model of pulmonary tuberculosis. BALB/c mice were subcutaneously vaccinated and 2 months later were challenged with Mycobacterium tuberculosis H37Rv by intratracheal injection. Two and 4 months after challenge, delayed-type hypersensitivity (DTH) response, lung tissue affected by pneumonia, CFU, T-cell counts, and cytokine expression (interleukin-2 [IL-2], IL-4, IL-10, and gamma interferon) were determined. A differential protective effect of the diverse BCG substrains was found. BCG Phipps led to the largest and most persistent reduction of CFU counts and of the area of pneumonia at 2 and 4 months after challenge. This protection was accompanied by reduced IL-10-producing T cells. Contemporary BCG substrains induce a wide range of protection in this animal model. These data can help in the selection of the best vaccine for human immunization and for the development of novel recombinant BCG-based vaccine.