Mycobacterium tuberculosis

Tese de mestrado em Biologia Molecular e Genética apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007The Beijing/W family of Mycobacterium tuberculosis represents a global threat in tuberculosis control. It has been frequently associated with drug resistance and its wide distribution suggests that these strains may have selective advantages over others. On the present study a collection of Beijing/W strains isolated in different countries was analyzed under the genetic markers: Large Sequence Polymorphisms (LSPs) and Single Nucleotide Polymorphisms (SNPs). Through a Multiplex-PCR isolates were tested for the presence or absence of the LSPs, described by a previous study for the Beijing/W family (RD105, RD181, RD150, RD142). The analysis of these regions proved to be very useful for defining and broadly subdivide the Beijing/W family in four groups. Furthermore, it provided a support for selection of representative isolates for the further study of other markers like SNPs in recently identified polymorphic DNA repair, recombination and replication (3R) genes. Selected 3R genes were sequenced for representative Beijing/W isolates to search for variations and comparative analysis with M.tuberculosis H37Rv identified SNPs for all genes. Almost half corresponded to not previously described SNPs and a large proportion seemed specific to Beijing/W strains. SNPs were much more informative than LSPs distinguishing twenty-four groups for which a hypothetic phylogenetic network was constructed. Phylogenetic relations originated by SNPs were found to be congruent with the ones originated by LSPs and also with previous studies on mutT2, muT4 and ogt putative DNA repair genes. Analysis according to geographic origin of isolates suggested that the collection used might be representative of the Beijing/W family. Certain genes were found to be more polymorphic than others with the accumulation of several non-synonymous SNPs (nsSNPs) that may potentially affect the function of the protein. Altered function in proteins involved in such important mechanisms in genome dynamics and stability might be associated with mutator phenotypes strains having high mutation rates in Beijing/W strains, as previously suggested. This might explain their higher adaptability and frequent association with resistance to antibiotics. Overall, this study provided useful information that, although may need to be validated using a larger set of isolates, it could be the start point for several future studies. It may contribute to clarify the role of the Beijing/W family in the tuberculosis pandemic.Resumo alargado disponível em portuguê

Mycobacterium tuberculosis host adaptation and evolution reflected by defense mechanisms against oxidative stress

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2012Part of the success of Mycobacterium tuberculosis lies in its ability to thrive inside macrophages, where it is exposed to strong antimicrobials molecules, such as reactive oxygen species (ROS). However, the role of defense mechanisms against ROS in M. tuberculosis pathogenesis remains unclear. We used, for the first time, a functional genomic approach to investigate M. tuberculosis responses against ROS. By screening a transposon mutant library we identified a high number of mutants, with increased susceptibility to H2O2, in genes related to cell envelope functions, including mmpL9. This revealed the importance of M. tuberculosis cell envelope against oxidative stress. However, we have also identified genes implicated in other kind of defense mechanism against ROS, such as moaD1. Infection of human macrophages has shown that both mmpL9 and moaD1 have a role in M. tuberculosis intracellular lifestyle, and previous studies suggested the same for several other genes identified during our screening. Therefore, these represent potential virulence factors useful for the development of future anti-tuberculosis strategies. DNA repair, recombination and replication (3R) are important mechanisms in maintaining genome stability by repairing DNA damages, such as those induced by ROS, however, variations on 3R genes potentially increase genomic variability, due to an increase in mutation rates. Thus, analysis of polymorphisms in 3R genes could indicate which strains are more prone to adapt and evolve. We have analyzed polymorphisms in 3R genes in a collection of strains of a successful family of M. tuberculosis strains, the Beijing/W family. Specific 3R polymorphisms were found to characterize particular groups of Beijing/W strains for which a phylogeny was constructed. Certain groups were found to be predominant, suggesting that strains of these genotypes might have some selective advantage. Therefore, particular 3R SNPs may define pathogenic features that have contributed to the evolution of the Beijing/W family.Mycobacterium tuberculosis, a bactéria responsável pela tuberculose, infecta cerca de 1/3 da população mundial e é responsável pela morte de aproximadamente 1,5 milhões de pessoas por ano. Estes números podem ser explicados, em parte, pela capacidade desta bactéria persistir no interior de macrófagos, num ambiente extremamente agressivo, no qual é exposta a diferentes moléculas antimicrobianas tais como as espécies reactivas de oxigénio. Estas, sendo altamente reativas, causam danos em todo o tipo de moléculas incluindo lípidos, proteínas e DNA. Desta forma, os mecanismos de defesa utilizados pelo M. tuberculosis contra as espécies reativas de oxigénio são sem dúvida importantes para este, porém, o seu papel na virulência e patogénese desta bactéria ainda é pouco claro. O trabalho desenvolvido nesta tese consistiu na investigação dos mecanismos de defesa utilizados pelo M. tuberculosis contra as espécies reactivas de oxigénio. Com este objectivo, realizou-se pela primeira vez, um screening de uma biblioteca de mutantes construída por transposição, na estirpe clinica da família Beijing/W - GC1237, de forma a selecionar mutantes sensíveis a espécies reativas de oxigénio. A análise de cerca de 6000 mutantes permitiu a selecção de 18, sensíveis a peróxido de hidrogénio, para os quais a amplificação e posterior sequenciação do sítio de inserção do transposão levaram à identificação de 11 genes. Verificou-se que grande parte dos genes apresenta funções associadas ao invólucro celular bacteriano, o que revela que o invólucro do M. tuberculosis representa a primeira barreira de defesa contra as espécies reactivas de oxigénio nesta bactéria. O gene mmpL9 foi um dos identificados, cuja proteína se encontra possivelmente envolvida no transporte de lípidos para o invólucro celular. Curiosamente, três mutantes com diferentes mutações neste gene foram seleccionados. Identificaram-se também alguns genes envolvidos noutro tipo de mecanismos tais como, moaD1, envolvido na síntese do cofactor molibdénio. Uma vez que o M. tuberculosis tem de fazer face às espécies reativas de oxigénio no interior de macrófagos, o passo seguinte consistiu em analisar o fenótipo de alguns destes mutantes nestas células. Para o efeito, macrófagos humanos foram infectados com um dos mutantes no gene mmpL9 ou com o mutante no gene moaD1, uma vez que estudos anteriores demonstraram o papel de diferentes MmpLs na virulência desta bactéria e que outros genes envolvidos na síntese do cofactor molibdénio são importantes para a sobrevivência do M. tuberculosis no interior de macrófagos. Em ambos os casos observou-se uma diminuição na capacidade de crescimento destas estirpes no interior destas células, em comparação com a estirpe selvagem. A complementação destas estirpes mutantes restaurou o fenótipo da estirpe selvagem, o que demonstra o papel destes genes no crescimento de M. tuberculosis em macrófagos humanos. Porém, a complementação restaurou apenas parcialmente a sensibilidade destes mutantes às espécies reactivas de oxigénio in vitro, em comparação com a estirpe selvagem. No entanto, mmpL9 e moaD1 deverão ter um papel na resposta às espécies reativas de oxigénio em M. tuberculosis, caso contrário, a complementação não teria restaurado de todo o fenótipo selvagem, nas condições referidas. A comparação da sequência do gene mmpL9 da nossa estirpe selvagem, GC1237, com a estirpe de referência laboratorial, H37Rv, revelou a presença de uma deleção de um nucleótido na posição 218. Contudo, os nossos resultados obtidos a partir da infecção de macrófagos com o mutante mmpL9 e com a mesma estirpe após complementação da mutação sugerem que o gene mmpL9 deverá ser expresso apesar desta deleção. É possível que ocorra uma reniniação da tradução a partir de um codão de iniciação situado a montante da deleção, na posição 258, no gene mmpL9. Para além disso, a complementação do nosso mutante mmpL9 com o respectivo gene da estirpe H37Rv, restaurou apenas parcialmente o fenótipo da estirpe selvagem, GC1237, durante a infecção de macrófagos. Estas observações sugerem que a proteína MmpL9 na estirpe GC1237 poderá ser diferente da existente na estirpe H37Rv. A análise da sequência de todos os genes mmpL existentes no genoma de M. tuberculosis, numa coleção de estirpes que representam diferentes famílias, sugeriu que a deleção identificada poderá realmente ter um efeito na proteína. Verificou-se ainda que esta deleção é aparentemente específica para todas as estirpes modernas da família Beijing. Isto indica que a deleção do nucleótido 218 poderá ter conferido alguma vantagem selectiva a estas estirpes que contribuiu para a sua evolução. Para além de moaD1 e mmpL9, alguns dos outros genes identificados durante o screening como importantes para a resposta de M. tuberculosis às espécies reativas de oxigénio, foram igualmente descritos, em estudos anteriores, como importantes para a capacidade desta bactéria sobreviver intracelularmente. Isto indica uma correlação entre sensibilidade ao stress oxidativo e capacidade de sobreviver ou replicar-se no interior de macrófagos. No entanto, a infecção de macrófagos tratados com inibidores da NADPH oxidase, a enzima que produz as espécies reativas de oxigénio nestas células, com os mutantes nos genes mmpL9 e moaD1, não permitiu confirmar esta hipótese. É possível que estes mutantes sejam sensíveis a outras moléculas antimicrobianas encontradas no interior do macrófago, tais como as espécies reativas de azoto, que, em conjunto com as espécies reativas de oxigénio, são responsáveis pelo crescimento atenuado observado em macrófagos. Em conclusão, este trabalho permitiu a identificação de genes envolvidos na defesa do M. tuberculosis contra as espécies reactivas de oxigénio, os quais aparentam ter igualmente um papel na sobrevivência desta bactéria nas suas células hospedeiras, os macrófagos. Estes genes representam potenciais factores de virulência que poderão ser úteis para o desenvolvimento de futuros antibióticos ou vacinas. Numa segunda parte desta tese, realizou-se uma análise dos polimorfismos existentes em genes de reparação, recombinação e replicação (3R) de DNA numa família específica de estirpes de M. tuberculosis, a família Beijing. Os mecanismos de reparação, recombinação e replicação são extremamente importantes na manutenção da estabilidade genómica, através da reparação de danos causados no DNA, tais como os induzidos pelas espécies reativas de oxigénio. Por outro lado, estes mecanismos podem ser igualmente responsáveis pela introdução de variabilidade genética. Polimorfismos nos genes que codificam para as proteínas envolvidas nestes mecanismos poderão induzir um aumento na taxa de mutação, e consequentemente, a acumulação de mutações vantajosas para as estirpes, como por exemplo, mutações responsáveis pela resistência a antibióticos. Deste modo, a análise de polimorfismos nos genes 3R poderá indicar que estirpes estão mais predispostas a adaptar-se ou evoluir. A família Beijing é uma família de M. tuberculosis que apresenta uma maior virulência e que tem sido identificada um pouco por todo o mundo como estando frequentemente associada a resistência a antibióticos. Deste modo, analisaram-se polimorfismos em genes 3R numa coleção de estirpes Beijing, isoladas em várias partes do mundo. Diferentes polimorfismos foram identificados que permitiram a discriminação de vários grupos, para os quais foi construída uma árvore filogenética. Estes resultados foram congruentes com os resultados obtidos utilizando outros marcadores genéticos, o que significa que as mesmas associações filogenéticas foram obtidas, para a colecção de estirpes utilizada neste estudo, usando polimorfismos nos genes 3R ou outro tipo de marcadores genéticos. Uma grande percentagem das estirpes foi agrupada num dos grupos, Bmyc10, que incluiu estirpes isoladas em diferentes partes do mundo. Um estudo anterior demonstrou que estirpes com características genéticas que definem as deste grupo são igualmente predominantes numa outra região geográfica, não representada no nosso estudo. Em conjunto, estes resultados sugerem que o genótipo das estirpes do grupo Bmyc10 poderá ter-lhes conferido alguma vantagem selectiva que permitiu a sua evolução. Isto é igualmente sugerido num estudo anterior, no qual a caracterização do efeito da mutação no gene mutT2, que caracteriza todas as estirpes do grupo Bmyc10, indica que esta induz uma alteração na função da proteína que pode ser vantajosa para as estirpes que a possuem. Portanto, alguns dos polimorfismos identificados no nosso estudo poderão ter um efeito na função das respectivas proteínas, o que significa que este tipo de análise é importante para compreender o significado dos mesmos. Em conclusão, a análise de polimorfismos em genes 3R demonstrou que diferentes grupos podem ser identificados na família Beijing caracterizados por polimorfismos específicos. Estes podem ter conferido alguma vantagem que permitiu a expansão de certos grupos e contribuiu para a evolução desta família. A análise da variabilidade genética entre estirpes é importante uma vez que estas podem reflectir diferenças patogénicas. Este estudo poderá contribuir para aprofundar os nossos conhecimentos acerca dos mecanismos que determinam o êxito das estirpes da família Beijing.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/39079/2007); projeto TB-ADAPT (LSHP-CT-2006-037919) - European Commission baseado na Health Cooperation Work Programme of the 6th Framework Programme; outros projetos TB-VIR (Grant agreement n° 200973) e NEWTBVAC (HEALTH-F3-2009-241745) do 7th Framework Programme

Genomic organization, gene expression and activity profile of Marinobacter hydrocarbonoclasticus denitrification enzymes

POCI-01-0145-FEDER-007728Background. Denitrification is one of the main pathways of the N-cycle, during which nitrate is converted to dinitrogen gas, in four consecutive reactions that are each catalyzed by a different metalloenzyme. One of the intermediate metabolites is nitrous oxide, which has a global warming impact greater then carbon dioxide and which atmospheric concentration has been increasing in the last years. The four denitrification enzymes have been isolated and biochemically characterized from Marinobacter hydrocarbonoclasticus in our lab. Methods. Bioinformatic analysis of the M. hydrocarbonoclasticus genome to identify the genes involved in the denitrification pathway. The relative gene expression of the gene encoding the catalytic subunits of those enzymes was analyzed during the growth under microoxic conditions. The consumption of nitrate and nitrite, and the reduction of nitric oxide and nitrous oxide by whole-cells was monitored during anoxic and microoxic growth in the presence of 10 mM sodium nitrate at pH 7.5. Results. The bioinformatic analysis shows that genes encoding the enzymes and accessory factors required for each step of the denitrification pathway are clustered together. An unusual feature is the co-existence of genes encoding a q- and a c-type nitric oxide reductase, with only the latter being transcribed at similar levels as the ones encoding the catalytic subunits of the other denitrifying enzymes, when cells are grown in the presence of nitrate under microoxic conditions. Using either a batch- or a closed system, nitrate is completely consumed in the beginning of the growth, with transient formation of nitrite, and whole-cells can reduce nitric oxide and nitrous oxide from mid-exponential phase until being collected (time-point 50 h). Discussion. M. hydrocarbonoclasticus cells can reduce nitric and nitrous oxide in vivo, indicating that the four denitrification steps are active. Gene expression profile together with promoter regions analysis indicates the involvement of a cascade regulatory mechanism triggered by FNR-type in response to low oxygen tension, with nitric oxide and nitrate as secondary effectors, through DNR and NarXL, respectively. This global characterization of the denitrification pathway of a strict marine bacterium, contributes to the understanding of the N-cycle and nitrous oxide release in marine environments.publishersversionpublishe

The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase

PTDC/BBB-BQB/0129/2014 (IM). This work was supported by the Applied Molecular Biosciences Unit-UCIBIO, and Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT (UIDB/04378/2020 and UIDB/50006/2020, respectively).Abstract: Increasing atmospheric concentration of N2O has been a concern, as it is a potent greenhouse gas and promotes ozone layer destruction. In the N-cycle, release of N2O is boosted upon a drop of pH in the environment. Here, Marinobacter hydrocarbonoclasticus was grown in batch mode in the presence of nitrate, to study the effect of pH in the denitrification pathway by gene expression profiling, quantification of nitrate and nitrite, and evaluating the ability of whole cells to reduce NO and N2O. At pH 6.5, accumulation of nitrite in the medium occurs and the cells were unable to reduce N2O. In addition, the biochemical properties of N2O reductase isolated from cells grown at pH 6.5, 7.5 and 8.5 were compared for the first time. The amount of this enzyme at acidic pH was lower than that at pH 7.5 and 8.5, pinpointing to a post-transcriptional regulation, though pH did not affect gene expression of N2O reductase accessory genes. N2O reductase isolated from cells grown at pH 6.5 has its catalytic center mainly as CuZ(4Cu1S), while that from cells grown at pH 7.5 or 8.5 has it as CuZ(4Cu2S). This study evidences that an in vivo secondary level of regulation is required to maintain N2O reductase in an active state. Graphic abstract: [Figure not available: see fulltext.].preprintpublishe

Innate Immune Response to Mycobacterium tuberculosis Beijing and Other Genotypes

Contains fulltext : 124335.pdf (publisher's version ) (Open Access)BACKGROUND: As a species, Mycobacterium tuberculosis is more diverse than previously thought. In particular, the Beijing family of M. tuberculosis strains is spreading and evaluating throughout the world and this is giving rise to public health concerns. Genetic diversity within this family has recently been delineated further and a specific genotype, called Bmyc10, has been shown to represent over 60% of all Beijing clinical isolates in several parts of the world. How the host immune system senses and responds to various M. tuberculosis strains may profoundly influence clinical outcome and the relative epidemiological success of the different mycobacterial lineages. We hypothesised that the success of the Bmyc10 group may, at least in part, rely upon its ability to alter innate immune responses and the secretion of cytokines and chemokines by host phagocytes. METHODOLOGY/PRINCIPAL FINDINGS: We infected human macrophages and dendritic cells with a collection of genetically well-defined M. tuberculosis clinical isolates belonging to various mycobacterial families, including Beijing. We analyzed cytokine and chemokine secretion on a semi-global level using antibody arrays allowing the detection of sixty-five immunity-related soluble molecules. Our data indicate that Beijing strains induce significantly less interleukin (IL)-6, tumor necrosis factor (TNF), IL-10 and GRO-alpha than the H37Rv reference strain, a feature that is variously shared by other modern and ancient M. tuberculosis families and which constitutes a signature of the Beijing family as a whole. However, Beijing strains did not differ relative to each other in their ability to modulate cytokine secretion. CONCLUSIONS/SIGNIFICANCE: Our results confirm and expand upon previous reports showing that M. tuberculosis Beijing strains in general are poor in vitro cytokine inducers in human phagocytes. The results suggest that the epidemiological success of the Beijing Bmyc10 is unlikely to rely upon any specific ability of this group of strains to impair anti-mycobacterial innate immunity

The Tryptophan System in Cocaine-Induced Depression

Major depression disorder (MDD) is the most prevalent psychiatric comorbid condition in cocaine use disorder (CUD). The comorbid MDD might be primary-MDD (CUD-primary-MDD) or cocaine-induced MDD (CUD-induced-MDD), and their accurate diagnoses and treatment is a challenge for improving prognoses. This study aimed to assess the tryptophan/serotonin (Trp/5-HT) system with the acute tryptophan depletion test (ATD), and the kynurenine pathway in subjects with CUD-primary-MDD, CUD-induced-MDD, MDD and healthy controls. The ATD was performed with a randomized, double-blind, crossover, and placebo-controlled design. Markers of enzymatic activity of indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase, kynurenine aminotransferase (KAT) and kynureninase were also established. Following ATD, we observed a decrease in Trp levels in all groups. Comparison between CUD-induced-MDD and MDD revealed significant differences in 5-HT plasma concentrations (512 + 332 ng/mL vs. 107 + 127 ng/mL, p = 0.039) and the Kyn/5-HT ratio (11 + 15 vs. 112 + 136; p = 0.012), whereas there were no differences between CUD-primary-MDD and MDD. Effect size coefficients show a gradient for all targeted markers (d range 0.72-1.67). Results suggest different pathogenesis for CUD-induced-MDD, with lower participation of the tryptophan system, probably more related to other neurotransmitter pathways and accordingly suggesting the need for a different pharmacological treatment approach

Caracterización de membranas cerámicas de bajo coste para procesos avanzados de tratamientos de aguas

En el presente trabajo se ha realizado la caracterización estructural de una línea de membranas de bajo coste, que se está desarrollando para su uso en tratamientos de aguas residuales, en reactores biológicos de membrana (MBR) y/o en tratamientos terciarios. Se comparan los resultados de tamaño de poro obtenidos en membranas cerámicas simétricas según dos métodos de caracterizacion: punto de burbuja y porosimetría de mercurio

Methylprednisolone Pulses Plus Tacrolimus in Addition to Standard of Care vs. Standard of Care Alone in Patients With Severe COVID-19. A Randomized Controlled Trial

Introduction: Severe lung injury is triggered by both the SARS-CoV-2 infection and the subsequent host-immune response in some COVID-19 patients. Methods: We conducted a randomized, single-center, open-label, phase II trial with the aim to evaluate the efficacy and safety of methylprednisolone pulses and tacrolimus plus standard of care (SoC) vs. SoC alone, in hospitalized patients with severe COVID-19. The primary outcome was time to clinical stability within 56 days after randomization. Results: From April 1 to May 2, 2020, 55 patients were prospectively included for subsequent randomization; 27 were assigned to the experimental group and 28 to the control group. The experimental treatment was not associated with a difference in time to clinical stability (hazard ratio 0.73 [95% CI 0.39-1.37]) nor most secondary outcomes. Median methylprednisolone cumulative doses were significantly lower (360 mg [IQR 360-842] vs. 870 mg [IQR 364-1451]; p = 0.007), and administered for a shorter time (median of 4.00 days [3.00-17.5] vs. 18.5 days [3.00-53.2]; p = 0.011) in the experimental group than in the control group. Although not statistically significant, those receiving the experimental therapy showed a numerically lower all-cause mortality than those receiving SoC, especially at day 10 [2 (7.41%) vs. 5 (17.9%); OR 0.39 (95% CI 0.05-2.1); p = 0.282]. The total number of non-serious adverse events was 42 in each the two groups. Those receiving experimental treatment had a numerically higher rate of non-serious infectious adverse events [16 (38%) vs. 10 (24%)] and serious infectious adverse events [7 (35%) vs. 3 (23%)] than those receiving SoC. Conclusions: The combined use of methylprednisolone pulses plus tacrolimus, in addition to the SoC, did not significantly improve the time to clinical stability or other secondary outcomes compared with the SoC alone in severe COVID-19. Although not statistically significant, patients receiving the experimental therapy had numerically lower all-cause mortality than those receiving SoC, supporting recent non-randomized studies with calcineurin inhibitors. It is noteworthy that the present trial had a limited sample size and several other limitations. Therefore, further RCTs should be done to assess the efficacy and safety of tacrolimus to tackle the inflammatory stages of COVID-19

Phylogeny of Mycobacterium tuberculosis Beijing Strains Constructed from Polymorphisms in Genes Involved in DNA Replication, Recombination and Repair

The original publication is available at http:/www.plosone.orgBackground: The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R) genes. It was shown that evolution of M. tuberculosis complex strains can be studied using 3R SNPs and a high-resolution tool for strain discrimination was developed. Here we investigated the genetic diversity and propose a phylogeny for Beijing strains by analyzing polymorphisms in 3R genes. Methodology/Principal Findings: A group of 3R genes was sequenced in a collection of Beijing strains from different geographic origins. Sequence analysis and comparison with the ones of non-Beijing strains identified several SNPs. These SNPs were used to type a larger collection of Beijing strains and allowed identification of 26 different sequence types for which a phylogeny was constructed. Phylogenetic relationships established by sequence types were in agreement with evolutionary pathways suggested by other genetic markers, such as Large Sequence Polymorphisms (LSPs). A recent Beijing genotype (Bmyc10), which included 60% of strains from distinct parts of the world, appeared to be predominant. Conclusions/Significance: We found SNPs in 3R genes associated with the Beijing family, which enabled discrimination of different groups and the proposal of a phylogeny. The Beijing family can be divided into different groups characterized by particular genetic polymorphisms that may reflect pathogenic features. These SNPs are new, potential genetic markers that may contribute to better understand the success of the Beijing family. © 2011 Mestre et al.Publishers' Versio