On the role of a 5'-leader region in controlling the levels of the aromatic-responsive transcriptional activator DmpR

Dissertação de mestrado em Genética MolecularMetabolically versatile bacteria play an important role in recycling carbon in the environment. For certain bacteria this metabolic versatility extends to seeming obnoxious toxic carbon sources such as aromatic compounds that can cause environmental pollution. One such example is Pseudomonas putida CF600 that carries the dmp-system for catabolism of dimethylphenols, mono-methylated phenols, and phenol on a catabolic plasmid. The dmpsystem consists of the dmp-operon encoding the specialized catabolic enzymes divergently transcribed from the dmpR gene. The dmpR gene encodes the aromatic-responsive transcriptional activator DmpR whose activity is strictly required for transcription of the dmpoperon. Because DmpR is a sensor-regulator that is activated upon binding substrates of the dmp-pathway enzymes, the dmp-system is always silent unless substrates are available. However, like other auxiliary catabolic pathways, regulation of expression of the Dmp-enzymes is also highly integrated within the host global regulatory network such that the system is also silent if more energetically favourable carbon sources are present. Failure to engineer such integration within host physiology has lead to unpredictable performance of artificial constructed catabolic pathways under field conditions. This provides a practical impetus to gain a greater understanding of the mechanisms involved. Much previous work had focused on the multiple roles of a bacterial alarmone that converge to stimulate activity of the promoter that drives transcription of dmpR to maximize performance of the dmp-system under lowenergy / stress conditions. However, the 5‟-leader region of the dmpR mRNA has also been implicated in playing a regulatory role. In this work, it is presented evidence, from in vivo and in vitro assays, that the DNA encoding the 5‟-leader region and the cognate region of the resulting mRNA exert control of the levels of DmpR by at least three different mechanisms: I) at the level of transcription through a ATAAATA motif within the 5‟-leader region DNA, II) at the level of translation by binding of Crc to the 5‟-leader region RNA, and III) by a less well defined, Crcindependent mechanism, that likely involved coupling of translation between a small openreading frame with the 5‟-leader region and that of the downstream dmpR gene. The results of these analysis and their physiological and mechanistic implications are discussed.Bactérias metabolicamente versáteis são importantes na reciclagem de carbono no ambiente. Em algumas delas, a sua versatilidade abrange fontes de carbono aparentemente tóxicas, como compostos aromáticos, e causadoras de poluição ambiental. Um exemplo é a espécie Pseudomonas putida CF600 que possui o sistema dmp que permite o catabolismo de dimetil-fenois, metil-fenois e fenol. O sistema dmp consiste no operão dmp, que codifica enzimas catabolicas especializadas, e o gene dmpR divergentemente transcrito. Este último codifica o ativador transcriptional DmpR cuja atividade é estritamente necessária para ocorrer transcrição do operão. Sendo o DmpR um sensor / regulador apenas ativo após a ligação a substractos da via metabólica, o sistema dmp encontra-se sempre silenciado, exceto, quando substratos estão presentes. No entanto, como qualquer outra via metabólica auxiliar, a regulação da expressão das enzimas Dmp está também integrada nas vias regulatórias globais da célula; desta forma, o sistema é silenciado quando fontes de carbono mais favoráveis estão presentes. A falha em construir esta integração com a fisiologia do hospedeiro tem levado a resultados imprevistos por parte de vias catabólicas artificialmente construídas quando submetidas a condições de campo. Este facto impulsiona a obtenção de um melhor entendimento dos mecanismos envolvidos. Uma grande parte do trabalho previamente efetuado focou-se nos múltiplos papéis de uma alarmona bacterial, os quais convergem para estimular a atividade do promotor do gene dmpR, de modo a maximizar a performance do sistema em condições de baixa energia / stress. No entanto, a região 5‟ líder do mRNA do gene dmpR parece também estar implicada na regulação dos níveis da proteína. Neste trabalho, são apresentadas evidências, de ensaios realizados in vivo e in vitro, em como o DNA codificante desta região e a correspondente região do mRNA controlam os níveis de DmpR através de pelo menos 3 mecanismos: ao nível da transcrição através do motivo ATAAATA presente no DNA; ao nível da tradução através da ligação da proteína Crc ao mRNA e através de um mecanismo pouco definido mas que parece envolver a tradução acoplada entre uma pequena ORF (dentro da região 5‟ líder) e o gene dmpR. A discussão dos resultados desta análise, as implicações fisiológicas e os mecanismos associados são apresentados

Infection with hypervirulent Mycobacterium tuberculosis triggers emergency myelopoiesis but not trained immunity

IntroductionDuring infection, bone marrow (BM) hematopoiesis is reprogrammed toward myeloid cell production, a mechanism named emergency myelopoiesis. In addition to replenishing myeloid cells, emergency myelopoiesis has been linked to trained immunity, a process that allows enhanced innate immune responses to secondary challenges. Although hematopoietic alterations during tuberculosis (TB) have been described and Mycobacterium tuberculosis may colonize the BM, studies using the mouse model of infection and the laboratory reference strain M. tuberculosis H37Rv have demonstrated limited emergency myelopoiesis and trained immunity.MethodsTo further address this issue, we aerosol- infected C57BL/6 mice with high doses of the hypervirulent M. tuberculosis isolate HN878 and monitored alterations to the BM. This experimental model better resembles the human blood immune signature of TB.Results and discussionWe found increased frequencies of lineage-Sca-1+cKit+ (LSK) cells and the granulocyte/macrophage progenitor (GMP) population. At the mature cell level, we observed an increase of monocytes and neutrophils in the blood and lung, likely reflecting the increased BM myeloid output. Monocytes or monocyte-derived macrophages recovered from the BM of M. tuberculosis HN878-infected mice did not show signs of trained immunity, suggesting an uncoupling of emergency myelopoiesis and trained immunity in the BM. Surprisingly, M. tuberculosis HN878-induced emergency myelopoiesis was not fully dependent on IFNγ, as mice lacking this cytokine and infected under the same conditions as wild-type mice still presented BM alterations. These data expand our understanding of the immune response to M. tuberculosis and raise awareness of pathogen strain-imposed differences to host responses

Mycobacterium tuberculosis Infection Up-Regulates Sialyl Lewis X Expression in the Lung Epithelium

Glycans display increasingly recognized roles in pathological contexts, however, their impact in the host-pathogen interplay in many infectious diseases remains largely unknown. This is the case for tuberculosis (TB), one of the ten most fatal diseases worldwide, caused by infection of the bacteria Mycobacterium tuberculosis. We have recently reported that perturbing the core-2 O -glycans biosynthetic pathway increases the host susceptibility to M. tuberculosis infection, by disrupting the neutrophil homeostasis and enhancing lung pathology. In the present study, we show an increased expression of the sialylated glycan structure Sialyl-Lewis X (SLeX) in the lung epithelium upon M. tuberculosis infection. This increase in SLeX glycan epitope is accompanied by an altered lung tissue transcriptomic signature, with up-regulation of genes codifying enzymes that are involved in the SLeX core-2 O -glycans biosynthetic pathway. This study provides novel insights into previously unappreciated molecular mechanisms involving glycosylation, which modulate the host response to M. tuberculosis infection, possibly contributing to shape TB disease outcome

Experimental evidence for limited in vivo virulence of Mycobacterium africanum

Funding Information: The authors thank the excellent support from the i3S scientific platforms, namely Animal Facility and Translational Cytometry. Funding. This work was supported by Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 ? Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Funda??o para a Ci?ncia e a Tecnologia, Minist?rio da Ci?ncia, Tecnologia e Inova??o in the framework of the project ?Institute for Research and Innovation in Health Sciences? (POCI-01-0145-FEDER-007274), and by grants FCT ? Aga Khan Development Network (ref 333197025), POCI-01-0145-FEDER-028955 (to MS), PTDC/BIA-MIC/30692/2017, and UID/Multi/04413/2013 (to DM and MV). BC and KF were funded by FCT Ph.D. scholarships SFRH/BD/114403/2016 and SFRH/BD/114405/2016, respectively. The Gulbenkian Foundation is acknowledged for a field work research grant to BC, Bolsas de apoio ? investiga??o para estudantes de doutoramento dos PALOP, Ref. P-146397. DM and MS were supported by FCT through Estimulo Individual ao Emprego Cient?fico. Publisher Copyright: © Copyright © 2019 Cá, Fonseca, Sousa, Maceiras, Machado, Sanca, Rabna, Rodrigues, Viveiros and Saraiva.Tuberculosis remains a public health problem and a main cause of death to humans. Both Mycobacterium tuberculosis and Mycobacterium africanum cause tuberculosis. In contrast to M. tuberculosis, which is geographically spread, M. africanum is restricted to West Africa. Differences have also been found in the growth rate and type of disease caused by M. africanum, globally suggesting an attenuation of this bacteria. In this study, we used the mouse model of infection to follow the dynamics of M. africanum infection in terms of bacterial burdens and tissue pathology, as well as the immune response triggered. Our findings support a lower virulence of M. africanum as compared to M. tuberculosis, including in mice lacking IFN-γ, a major protective cytokine in tuberculosis. Furthermore, the lung immune response triggered by M. africanum infection in wild-type animals was characterized by a discrete influx of leukocytes and a modest transcriptional upregulation of inflammatory mediators. Our findings contribute to elucidate the pathogenesis of M. africanum, supporting the hypothesis that this is an attenuated member of the tuberculosis-causing bacteria. Understanding the biology of M. africanum and how it interacts with the host to establish infection will have implications for our knowledge of TB and for the development of novel and better tools to control this devastating disease.publishersversionpublishe

Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1β production

Genetic diversity of Mycobacterium tuberculosis affects immune responses and clinical outcomes of tuberculosis (TB). However, how bacterial diversity orchestrates immune responses to direct distinct TB severities is unknown. Here we study 681 patients with pulmonary TB and show that M. tuberculosis isolates from cases with mild disease consistently induce robust cytokine responses in macrophages across multiple donors. By contrast, bacteria from patients with severe TB do not do so. Secretion of IL-1β is a good surrogate of the differences observed, and thus to classify strains as probable drivers of different TB severities. Furthermore, we demonstrate that M. tuberculosis isolates that induce low levels of IL-1β production can evade macrophage cytosolic surveillance systems, including cGAS and the inflammasome. Isolates exhibiting this evasion strategy carry candidate mutations, generating sigA recognition boxes or affecting components of the ESX-1 secretion system. Therefore, we provide evidence that M. tuberculosis strains manipulate host-pathogen interactions to drive variable TB severities

Interleukin-10 induces interferon-γ-dependent emergency myelopoiesis

International audienceIn emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production are observed and often mediated by the pro-inflammatory cytokine interferon gamma (IFN-γ). Interleukin-10 (IL-10) inhibits IFN-γ secretion, but paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work, we use different in vivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also find that IL-10, unexpectedly, reprograms CD4 and CD8 T cells toward an activation state that includes IFN-γ production by these T cell subsets in vivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening additional perspectives for the design of IL-10-based immunotherapies

Human blood Tfr cells are indicators of ongoing humoral activity not fully licensed with suppressive function

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government WorksGerminal center (GC) responses are controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells and are crucial for the generation of high-affinity antibodies. Although the biology of human circulating and tissue Tfh cells has been established, the relationship between blood and tissue Tfr cells defined as CXCR5+Foxp3+T cells remains elusive. We found that blood Tfr cells are increased in Sjögren syndrome, an autoimmune disease with ongoing GC reactions, especially in patients with high autoantibody titers, as well as in healthy individuals upon influenza vaccination. Although blood Tfr cells correlated with humoral responses, they lack full B cell–suppressive capacity, despite being able to suppress T cell proliferation. Blood Tfr cells have a naïve-like phenotype, although they are absent from human thymus or cord blood. We found that these cells were generated in peripheral lymphoid tissues before T-B interaction, as they are maintained in B cell–deficient patients. Therefore, blood CXCR5+Foxp3+ T cells in human pathology indicate ongoing humoral activity but are not fully competent circulating Tfr cells.This study was funded by HMSP-ICT/0034/2013, FAPESP/19906/2014, PTDC/IMI-IMU/7038/2014 research grants, and LISBOA-01-0145-FEDER-007391, projeto cofinanciado pelo FEDER através POR Lisboa 2020–Programa Operacional Regional de Lisboa, do PORTUGAL 2020, e pela Fundação para a Ciência e a Tecnologia. The vaccination study was funded by the European Research Council Starting Grant TWILIGHT (to M.A.L.). W.P. was funded by a Newton International Fellowship from the Royal Society. M.A.L. was funded by the Bioscience and Biotechnology Research Council.info:eu-repo/semantics/publishedVersio

Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils

Modulation of immunity and disease by glycans is increasingly recognized. However, how host glycosylation shapes and is shaped by tuberculosis remains poorly understood. We show that deficiency in the glucosaminyl (N-acetyl) transferase 1 (Gcnt1), a key enzyme for core-2 O -glycans biosynthesis, drives susceptibility to Mycobacterium tuberculosis infection. The increased susceptibility of Gcnt1 deficient mice was characterized by extensive lung immune pathology, mechanistically related to neutrophils. Uninfected Gcnt1 deficient mice presented bone marrow, blood and lung neutrophilia, which further increased with infection. Blood neutrophilia required Gcnt1 deficiency in the hematopoietic compartment, relating with enhanced granulopoiesis, but normal cellular egress from the bone marrow. Interestingly, for the blood neutrophilia to translate into susceptibility to M. tuberculosis infection, Gnct1 deficiency in the stroma was also necessary. Complete Gcnt1 deficiency associated with increased lung expression of the neutrophil chemoattractant CXCL2. Lastly, we demonstrate that the transcript levels of various glycosyltransferase-encoding genes were altered in whole blood of active tuberculosis patients and that sialyl Lewis x, a glycan widely present in human neutrophils, was detected in the lung of tuberculosis patients. Our findings reveal a previously unappreciated link between Gcnt1, neutrophilia and susceptibility to M. tuberculosis infection, uncovering new players balancing the immune response in tuberculosis