Tuberculosis caused by Mycobacterium africanum: Knowns and unknowns.

Tuberculosis (TB), one of the deadliest threats to human health, is mainly caused by 2 highly related and human-adapted bacteria broadly known as Mycobacterium tuberculosis and Mycobacterium africanum. Whereas M. tuberculosis is widely spread, M. africanum is restricted to West Africa, where it remains a significant cause of tuberculosis. Although several differences have been identified between these 2 pathogens, M. africanum remains a lot less studied than M. tuberculosis. Here, we discuss the genetic, phenotypic, and clinical similarities and differences between strains of M. tuberculosis and M. africanum. We also discuss our current knowledge on the immune response to M. africanum and how it possibly articulates with distinct disease progression and with the geographical restriction attributed to this pathogen. Understanding the functional impact of the diversity existing in TB-causing bacteria, as well as incorporating this diversity in TB research, will contribute to the development of better, more specific approaches to tackle TB.

Characterization of Erythroferrone in a Teleost Fish (Dicentrarchus labrax) With Two Functional Hepcidin Types: More Than an Erythroid Regulator.

Erythroferrone is a recently identified erythroid regulator produced by erythroblasts in the mammalian bone marrow and extramedullary sites, known to be induced in conditions of anemia or blood loss. Iron metabolism is affected by erythroferrone through its capacity to inhibit hepcidin production, leading to the increase of iron availability required for erythropoiesis. However, little is known about erythroferrone function in other vertebrates, in particular teleost fish, that unlike mammals, present two different functional types of hepcidin, one type mostly involved in iron metabolism and the other in antimicrobial response. The study of erythroferrone evolution and its biological role in teleost fish can give us valuably new insights into its function. To address these questions, we characterized erythroferrone in the European sea bass (Dicentrarchus labrax), a species presenting two hepcidin types, and evaluated variations in its expression levels in response to different experimental conditions. During experimental anemia, erythroferrone responds by increasing its expression and suppressing hepcidin production, following the pattern observed in mammals, but it is not influenced by iron overload. However, during bacterial infection, erythroferrone is downregulated and hepcidin levels increase. Furthermore, administration of Hamp1 but not of Hamp2 peptides suppresses erythroferrone expression. In conclusion, in dual hepcidin teleost fish erythroferrone seems to only interact with type 1 hepcidin, known to be involved in iron homeostasis, but not with type 2, which has an almost exclusive antimicrobial role.

Incidence of SARS-CoV-2 infection in a cohort of workers from the University of Porto, Portugal.

BACKGROUND: Repeated serosurveys in the same population provide more accurate estimates of the frequency of SARS-CoV-2 infection and more comparable data over time than notified cases. We aimed to estimate the incidence of SARS-CoV-2 infection, identify associated factors, and assess time trends in the ratio of serological/molecular diagnosis in a cohort of university workers. METHODS: Participants had a serological rapid test for SARS-CoV-2 immunoglobulins M and G, and completed a questionnaire, in May-July 2020 (n = 3628) and November 2020-January 2021 (n = 2661); 1960 participated in both evaluations and provided data to compute the incidence proportion and the incidence rate. Crude and adjusted incidence rate ratios (aIRR) and 95% confidence intervals (CI) were computed using generalized linear models with Poisson regression. RESULTS: The incidence rate was 1.8/100 person-months (95% CI: 1.5-2.0), and the 6 months' cumulative incidence was 10.7%. The serological/molecular diagnosis ratio was 10:1 in the first evaluation and 3:1 in the second. Considering newly identified seropositive cases at the first (n = 69) and second evaluation (n = 202), 29.0% and 9.4% never reported symptoms, respectively, 14.5% and 33.3% reported contact with a confirmed case and 82.6%, and 46.0% never had a molecular test. Males (aIRR: 0.61; 95% CI: 0.44-0.85) and 'high-skilled white-collar' workers (aIRR: 0.74, 95% CI: 0.53-1.04) had lower risk of infection. CONCLUSION: University workers presented a high SARS-CoV-2 incidence while restrictive measures were in place. The time decrease in the proportion of undiagnosed cases reflected the increased access and awareness to testing, but opportunities continued to be missed, even in the presence of COVID-19-like symptoms.

The Era of Antimicrobial Peptides: Use of Hepcidins to Prevent or Treat Bacterial Infections and Iron Disorders.

The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass (Dicentrarchus labrax), two different hepcidin types are found, with specialized roles: the single type 1 hepcidin is involved in iron homeostasis trough the regulation of ferroportin, the only known iron exporter; and the various type 2 hepcidins present antimicrobial activity against a number of different pathogens. In this study, we tested the administration of sea bass derived hepcidins in models of infection and iron overload. Administration with hamp2 substantially reduced fish mortalities and bacterial loads, presenting itself as a viable alternative to the use of antibiotics. On the other hand, hamp1 seems to attenuate the effects of iron overload. Further studies are necessary to test the potential protective effects of hamp2 against other pathogens, as well as to understand how hamp2 stimulate the inflammatory responses, leading to an increased fish survival upon infection.

Antimicrobial Peptides: Identification of two Beta-Defensins in a Teleost Fish, the European Sea Bass (Dicentrarchus labrax).

Beta-defensins consist in a group of cysteine-rich antimicrobial peptides (AMPs), widely found throughout vertebrate species, including teleost fish, with antimicrobial and immunomodulatory activities. However, although the European sea bass (Dicentrarchus labrax) is one of the most commercially important farmed fish species in the Mediterranean area, the characterization of its beta-defensins and its potential applications are still missing. In this study, we characterized two members of the beta-defensin family in this species. Phylogenetic and synteny analysis places sea bass peptides in the beta-defensin subfamilies 1 and 2, sharing similar features with the other members, including the six cysteines and the tertiary structure, that consists in three antiparallel beta-sheets, with beta-defensin 1 presenting an extra alpha-helix at the N-terminal. Further studies are necessary to uncover the functions of sea bass beta-defensins, particularly their antimicrobial and immunomodulatory properties, in order to develop novel prophylactic or therapeutic compounds to be used in aquaculture production.

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.

A role for hepcidin in the anemia caused by Trypanosoma brucei infection.

Trypanosomiasis is a parasitic disease affecting both humans and animals in the form of Human African Trypanosomiasis and Nagana disease, respectively. Anemia is one of the most common symptoms of trypanosomiasis, and if left unchecked can cause severe complications and even death. Several factors have been associated with the development of this anemia, including dysregulation of iron homeostasis, but little is known about the molecular mechanisms involved. Here, using murine models, we study the involvement of hepcidin, the key regulator of iron metabolism and an important player in the development of anemia of inflammation. Our data show two stages for the progression of anemia, to which hepcidin contributes a first stage when anemia develops, with a likely cytokine-mediated stimulation of hepcidin and subsequent limitation in iron availability and erythropoiesis, and a second stage of recovery, where the increase in hepcidin then declines due to the reduced inflammatory signal and increased production of erythroid regulators by the kidney, spleen and bone marrow, thus leading to an increase in iron release and availability, and enhanced erythropoiesis. In agreement with this, in hepcidin knockout mice, anemia is much milder and its recovery is complete, in contrast to wild-type animals which have not fully recovered from anemia after 21 days. Besides all other factors known to be involved in the development of anemia during trypanosomiasis, hepcidin clearly makes an important contribution to both its development and recovery.

The Diverse Piscidin Repertoire of the European Sea Bass (Dicentrarchus labrax): Molecular Characterization and Antimicrobial Activities.

Fish rely on their innate immune responses to cope with the challenging aquatic environment, with antimicrobial peptides (AMPs) being one of the first line of defenses. Piscidins are a group of fish specific AMPs isolated in several species. However, in the European sea bass (Dicentrarchus labrax), the piscidin family remains poorly understood. We identified six different piscidins in sea bass, performed an in-depth molecular characterization and evaluated their antimicrobial activities against several bacterial and parasitic pathogens. Sea bass piscidins present variable amino acid sequences and antimicrobial activities, and can be divided in different sub groups: group 1, formed by piscidins 1 and 4; group 2, constituted by piscidins 2 and 5, and group 3, formed by piscidins 6 and 7. Additionally, we demonstrate that piscidins 1 to 5 possess a broad effect on multiple microorganisms, including mammalian parasites, while piscidins 6 and 7 have poor antibacterial and antiparasitic activities. These results raise questions on the functions of these peptides, particularly piscidins 6 and 7. Considering their limited antimicrobial activity, these piscidins might have other functional roles, but further studies are necessary to better understand what roles might those be.

Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1ß 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-1ß 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-1ß 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.

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.

Experimental Evidence for Limited in vivo Virulence of Mycobacterium africanum.

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.

IFN-γ-Dependent Reduction of Erythrocyte Life Span Leads to Anemia during Mycobacterial Infection.

Anemia is a frequent and challenging complication of mycobacterial infections. We used a model of disseminated Mycobacterium avium infection in mice to investigate the mechanisms of mycobacteria-induced anemia. We found increased formation of RBC in the bone marrow and spleen of infected mice. Infection induced reticulocytosis and the premature egress of immature progenitors to the systemic circulation in an IFN-γ (IFNG)-dependent way. The newly formed RBC had reduced CD47 surface expression and a reduced life span and were phagocytosed in the liver of infected mice, increasing iron recycling in this organ. The increased engulfment and degradation of RBC was independent of IFNG sensing by macrophages. Together, our findings demonstrate that mycobacterial infection alters the formation of erythrocytes, leading to their accelerated removal from circulation and hemolytic anemia. This comprehensive elucidation of the mechanisms underlying mycobacteria-induced anemia has important implications for its efficient clinical management.

Hamp1 but not Hamp2 regulates ferroportin in fish with two functionally distinct hepcidin types.

Hepcidin is a small cysteine rich peptide that regulates the sole known cellular iron exporter, ferroportin, effectively controlling iron metabolism. Contrary to humans, where a single hepcidin exists, many fish have two functionally distinct hepcidin types, despite having a single ferroportin gene. This raises the question of whether ferroportin is similarly regulated by the iron regulator Hamp1 and the antimicrobial Hamp2. In sea bass (Dicentrarchus labrax), iron overload prompted a downregulation of ferroportin, associated with an upregulation of hamp1, whereas an opposite response was observed during anemia, with no changes in hamp2 in either situation. During infection, ferroportin expression decreased, indicating iron withholding to avoid microbial proliferation. In vivo administration of Hamp1 but not Hamp2 synthetic peptides caused significant reduction in ferroportin expression, indicating that in teleost fish with two hepcidin types, ferroportin activity is mediated through the iron-regulator Hamp1, and not through the dedicated antimicrobial Hamp2. Additionally, in vitro treatment of mouse macrophages with fish Hamp1 but not Hamp2 caused a decrease in ferroportin levels. These results raise questions on the evolution of hepcidin and ferroportin functional partnership and open new possibilities for the pharmaceutical use of selected fish Hamp2 hepcidins during infections, with no impact on iron homeostasis.

Experimental study of tuberculosis: From animal models to complex cell systems and organoids.

Tuberculosis (TB) is a devastating disease to mankind that has killed more people than any other infectious disease. Despite many efforts and successes from the scientific and health communities, the prospect of TB elimination remains distant. On the one hand, sustainable public health programs with affordable and broad implementation of anti-TB measures are needed. On the other hand, achieving TB elimination requires critical advances in three areas: vaccination, diagnosis, and treatment. It is also well accepted that succeeding in advancing these areas requires a deeper knowledge of host-pathogen interactions during infection, and for that, better experimental models are needed. Here, we review the potential and limitations of different experimental approaches used in TB research, focusing on animal and human-based cell culture models. We highlight the most recent advances in developing in vitro 3D models and introduce the potential of lung organoids as a new tool to study Mycobacterium tuberculosis infection.

Hepcidin-Dependent Regulation of Erythropoiesis during Anemia in a Teleost Fish, Dicentrarchus labrax.

Anemia is a common disorder, characterized by abnormally low levels of red blood cells or hemoglobin. The mechanisms of anemia development and response have been thoroughly studied in mammals, but little is known in other vertebrates, particularly teleost fish. In this study, different degrees of anemia were induced in healthy European sea bass specimens (Dicentrarchus labrax) and at pre-determined time points hematological parameters, liver iron content and the expression of genes involved in iron homeostasis and hematopoiesis, with particular attention on hepcidins, were evaluated. The experimental anemia prompted a decrease in hamp1 expression in all tested organs, in accordance to an increased need for iron absorption and mobilization, with slight increases in hamp2 in the kidney and intestine. The liver was clearly the major organ involved in iron homeostasis, decreasing its iron content and showing a gene expression profile consistent with an increased iron release and mobilization. Although both the spleen and head kidney are involved in erythropoiesis, the spleen was found to assume a more preponderant role in the recovery of erythrocyte levels. The intestine was also involved in the response to anemia, through the increase of iron transporting genes. Administration of Hamp1 or Hamp2 mature peptides showed that only Hamp1 affects hematological parameters and liver iron content. In conclusion, the molecular mechanisms of response to anemia present in sea bass are similar to the ones described for mammals, with these results indicating that the two hepcidin types from teleosts assume different roles during anemia.

Multiple Hepcidins in a Teleost Fish, Dicentrarchus labrax: Different Hepcidins for Different Roles.

Teleost fish rely heavily on their innate immunity for an adequate response against pathogens and environmental challenges, with the production of antimicrobial peptides being one of their first lines of defense. Among those is hepcidin, a small cysteine-rich antimicrobial peptide that is also the key regulator of iron metabolism. Although most mammals possess a single hepcidin gene, with a dual role in both iron metabolism regulation and antimicrobial response, many teleost fish present multiple copies of hepcidin, most likely because of genome duplications and positive Darwinian selection, suggesting that different hepcidins may perform different functions. To study the roles of hepcidin in teleost fish, we have isolated and characterized several genes in the European sea bass (Dicentrarchus labrax) and evaluated variations in their expression levels in response to different experimental conditions. Although several hepcidin genes were found, after phylogenetic analysis they could be clustered in two groups: hamp1-like, with a single isoform similar to mammalian hepcidins, and hamp2-like, with several isoforms. Under experimental conditions, hamp1 was upregulated in response to iron overload and infection and downregulated during anemia and hypoxic conditions. Hamp2 did not respond to either iron overload or anemia but was highly upregulated during infection and hypoxia. In addition, Hamp2 synthetic peptides exhibited a clear antimicrobial activity against several bacterial strains in vitro. In conclusion, teleost fish that present two hepcidin types show a degree of subfunctionalization of its functions, with hamp1 more involved in the regulation of iron metabolism and hamp2 mostly performing an antimicrobial role.

Natural history of SLC11 genes in vertebrates: tales from the fish world.

BACKGROUND: The SLC11A1/Nramp1 and SLC11A2/Nramp2 genes belong to the SLC11/Nramp family of transmembrane divalent metal transporters, with SLC11A1 being associated with resistance to pathogens and SLC11A2 involved in intestinal iron uptake and transferrin-bound iron transport. Both members of the SLC11 gene family have been clearly identified in tetrapods; however SLC11A1 has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. In the present work we characterized the SLC11 genes in teleosts and evaluated if the roles attributed to mammalian SLC11 genes are assured by other fish specific SLC11 gene members. RESULTS: Two different SLC11 genes were isolated in the European sea bass (Dicentrarchus. labrax), and named slc11a2-α and slc11a2-ß, since both were found to be evolutionary closer to tetrapods SLC11A2, through phylogenetic analysis and comparative genomics. Induction of slc11a2-α and slc11a2-ß in sea bass, upon iron modulation or exposure to Photobacterium damselae spp. piscicida, was evaluated in in vivo or in vitro experimental models. Overall, slc11a2-α was found to respond only to iron deficiency in the intestine, whereas slc11a2-ß was found to respond to iron overload and bacterial infection in several tissues and also in the leukocytes. CONCLUSIONS: Our data suggests that despite the absence of slc11a1, its functions have been undertaken by one of the slc11a2 duplicated paralogs in teleost fish in a case of synfunctionalization, being involved in both iron metabolism and response to bacterial infection. This study provides, to our knowledge, the first example of this type of sub-functionalization in iron metabolism genes, illustrating how conserving the various functions of the SLC11 gene family is of crucial evolutionary importance.

Transferrin and ferritin response to bacterial infection: the role of the liver and brain in fish.

Iron is essential for growth and survival, but it is also toxic when in excess. Thus, there is a tight regulation of iron that is accomplished by the interaction of several genes including the iron transporter transferrin and iron storage protein ferritin. These genes are also known to be involved in response to infection. The aim of this study was to understand the role of transferrin and ferritin in infection and iron metabolism in fish. Thus, sea bass transferrin and ferritin H cDNAs were isolated from liver, cloned and characterized. Transferrin constitutive expression was found to be highest in the liver, but also with significant expression in the brain, particularly in the highly vascularized region connecting the inferior lobe of the hypothalamus and the saccus vasculosus. Ferritin, on the other hand, was expressed in all tested organs, but also significantly higher in the liver. Fish were subjected to either experimental bacterial infection or iron modulation and transferrin and ferritin mRNA expression levels were analyzed, along with several iron regulatory parameters. Transferrin expression was found to decrease in the liver and increase in the brain in response to infection and to increase in the liver in iron deficiency. Ferritin expression was found to inversely reflect transferrin in the liver, increasing in infection and iron overload and decreasing in iron deficiency, whereas in the brain, ferritin expression was also increased in infection. These findings demonstrate the evolutionary conservation of transferrin and ferritin dual functions in vertebrates, being involved in both the immune response and iron metabolism.

Dual function of fish hepcidin: response to experimental iron overload and bacterial infection in sea bass (Dicentrarchus labrax).

The role of hepcidin in iron metabolism regulation and bacterial infection has been the focus of recent attention. However, in spite of the growing number of hepcidin genes known from different organisms, little is known about its putative dual function in fish. The aim of this study was to characterize the sea bass hepcidin gene and to study its role in iron metabolism and infection. The novel sea bass hepcidin gene was found to be organized into two introns and three exons with several copies present in the genome. The transcript showed a constitutive low basal expression being mainly expressed in liver and encoding a putative 85 residues long peptide. Fish were submitted either to iron status modulation or bacterial infection and the hepcidin transcript levels were analysed along with a number of other parameters. Liver hepcidin expression was found to increase in both the iron-overloaded and infected fish, while in the iron-deficient fish no alteration in expression levels was detected. These results point to the evolutionary conservation of hepcidin's dual functions.