Translational regulation in mycobacteria and its implications for pathogenicity.

Protein synthesis is a fundamental requirement of all cells for survival and replication. To date, vast numbers of genetic and biochemical studies have been performed to address the mechanisms of translation and its regulation in Escherichia coli, but only a limited number of studies have investigated these processes in other bacteria, particularly in slow growing bacteria like Mycobacterium tuberculosis, the causative agent of human tuberculosis. In this Review, we highlight important differences in the translational machinery of M. tuberculosis compared with E. coli, specifically the presence of two additional proteins and subunit stabilizing elements such as the B9 bridge. We also consider the role of leaderless translation in the ability of M. tuberculosis to establish latent infection and look at the experimental evidence that translational regulatory mechanisms operate in mycobacteria during stress adaptation, particularly focussing on differences in toxin-antitoxin systems between E. coli and M. tuberculosis and on the role of tuneable translational fidelity in conferring phenotypic antibiotic resistance. Finally, we consider the implications of these differences in the context of the biological adaptation of M. tuberculosis and discuss how these regulatory mechanisms could aid in the development of novel therapeutics for tuberculosis

Translation of a Leaderless Reporter Is Robust During Exponential Growth and Well Sustained During Stress Conditions in Mycobacterium tuberculosis.

Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5' leader region, which usually contains the Shine-Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins like toxin-antitoxin systems are predominantly leaderless and the overall ratio of leaderless to Shine-Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during optimal growth conditions and during stress conditions that induce growth arrest, is unclear. Here, we have used the desA1 (Rv0824c) and desA2 (Rv1094) gene pair as representative for Shine-Dalgarno and leaderless transcripts in M. tuberculosis respectively; and used them to construct bioluminescent reporter strains. We detect robust leaderless translation during exponential in vitro growth, and we show that leaderless translation is more stable than Shine-Dalgarno translation during adaptation to stress conditions. These changes are independent from transcription, as transcription levels did not significantly change following quantitative real-time PCR analysis. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine-Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are transiently more stable than those of Shine-Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection

The SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) in myalgic encephalomyelitis/chronic fatigue syndrome: A meta-analysis of public DNA methylation and gene expression data

Expresión génica; Encefalomielitis miálgica/síndrome de fatiga crónica; Coronavirus SARS-CoV-2; COVID-19; 2019-nCoVExpressió gènica; Encefalomielitis miàlgica/síndrome de fatiga crònica; Coronavirus SARS-CoV-2; COVID-19; 2019-nCoVGene expression; Myalgic encephalomyelitis/chronic fatigue syndrome; Coronavirus SARS-CoV-2; COVID-19; 2019-nCoVPeople with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) often report a high frequency of viral infections and flu-like symptoms during their disease course. Given that this reporting agrees with different immunological abnormalities and altered gene expression profiles observed in the disease, we aimed at answering whether the expression of the human angiotensin-converting enzyme 2 (ACE2), the major cell entry receptor for SARS-CoV-2, is also altered in these patients. In particular, a low expression of ACE2 could be indicative of a high risk of developing COVID-19. We then performed a meta-analysis of public data on CpG DNA methylation and gene expression of this enzyme and its homologous ACE protein in peripheral blood mononuclear cells and related subsets. We found that patients with ME/CFS have decreased methylation levels of four CpG probes in the ACE locus (cg09920557, cg19802564, cg21094739, and cg10468385) and of another probe in the promoter region of the ACE2 gene (cg08559914). We also found a decreased expression of ACE2 but not of ACE in patients when compared to healthy controls. Accordingly, in newly collected data, there was evidence for a significant higher proportion of samples with an ACE2 expression below the limit of detection in patients than healthy controls. Altogether, patients with ME/CFS can be at a higher COVID-19 risk and, if so, they should be considered a priority group for vaccination by public health authorities. To further support this conclusion, similar research is recommended for other human cell entry receptors and cell types, namely, those cells targeted by the virus.João Malato and André Fonseca were fully funded by FCT – Fundação para a Ciência e Tecnologia, Portugal (ref.grant: SFRH/BD/149758/2019 and SFRH/BD/147629/2019, respectively). Nuno Sepúlveda and Clara Cordeiro were partially funded by FCT – Fundação para a Ciência e a Tecnologia, Portugal (ref. grant: UIDB/00006/2020). Luís Nacul and Eliana M Lacerda acknowledge the funding from the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH -Award Number: R01AI103629), and from the ME Association (Award number: PF8947) for their studies on ME/CFS

Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism

<p>Abstract</p> <p>Background</p> <p>Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in <it>Campylobacter jejuni </it>is more complex than the one in the laboratory <it>E. coli </it>K-12 strain.</p> <p>Results</p> <p>In the <it>C. jejuni </it>81-176 genome, the <it>dsb </it>genes of the oxidative pathway are arranged in three transcriptional units: <it>dsbA2</it>-<it>dsbB</it>-<it>astA, dsbA1 </it>and <it>dba</it>-<it>dsbI</it>. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in <it>dsb </it>gene regulation was proven by a reporter gene study in a <it>C. jejuni </it>wild type strain and its isogenic <it>fur </it>mutant. An electrophoretic mobility shift assay (EMSA) confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind <it>in vitro </it>to the <it>C. jejuni </it>promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives <it>dsbA1 </it>gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary <it>dba-dsbI </it>mRNA structure for <it>dsbI </it>mRNA translation was verified by estimating individual <it>dsbI </it>gene expression from its own promoter.</p> <p>Conclusions</p> <p>The present work shows that iron concentration is a significant factor in <it>dsb </it>gene transcription. These results support the concept that iron concentration - also through its influence on <it>dsb </it>gene expression - might control the abundance of extracytoplasmic proteins during different stages of infection. Our work further shows that synthesis of the DsbI membrane oxidoreductase is controlled by a translational coupling mechanism. The <it>dba </it>expression is not only essential for the translation of the downstream <it>dsbI </it>gene, but also Dba protein that is produced might regulate the activity and/or stability of DsbI.</p

Functional and bioinformatics analysis of two Campylobacter jejuni homologs of the thiol-disulfide oxidoreductase, DsbA.

BACKGROUND: Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. METHODS AND RESULTS: Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. CONCLUSIONS: Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far

3D hydrogels reveal medulloblastoma subgroup differences and identify extracellular matrix subtypes that predict patient outcome

© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, SHH, Group 3, and Group 4. These molecular subgroups differ in their metastasis patterns and related prognosis rates. Conventional 2D cell culture methods fail to recapitulate these clinical differences. Realistic 3D models of the cerebellum are therefore necessary to investigate subgroup-specific functional differences and their role in metastasis and chemoresistance. A major component of the brain extracellular matrix (ECM) is the glycosaminoglycan hyaluronan. MB cell lines encapsulated in hyaluronan hydrogels grew as tumour nodules, with Group 3 and Group 4 cell lines displaying clinically characteristic laminar metastatic patterns and levels of chemoresistance. The glycoproteins, laminin and vitronectin, were identified as subgroup-specific, tumour-secreted ECM factors. Gels of higher complexity, formed by incorporation of laminin or vitronectin, revealed subgroup-specific adhesion and growth patterns closely mimicking clinical phenotypes. ECM subtypes, defined by relative levels of laminin and vitronectin expression in patient tissue microarrays and gene expression data sets, were able to identify novel high-risk MB patient subgroups and predict overall survival. Our hyaluronan model system has therefore allowed us to functionally characterize the interaction between different MB subtypes and their environment. It highlights the prognostic and pathological role of specific ECM factors and enables preclinical development of subgroup-specific therapies. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland

Functional genetic variation in pe/ppe genes contributes to diversity in Mycobacterium tuberculosis lineages and potential interactions with the human host.

INTRODUCTION: Around 10% of the coding potential of Mycobacterium tuberculosisis constituted by two poorly understood gene families, the pe and ppe loci, thought to be involved in host-pathogen interactions. Their repetitive nature and high GC content have hindered sequence analysis, leading to exclusion from whole-genome studies. Understanding the genetic diversity of pe/ppe families is essential to facilitate their potential translation into tools for tuberculosis prevention and treatment. METHODS: To investigate the genetic diversity of the 169 pe/ppe genes, we performed a sequence analysis across 73 long-read assemblies representing seven different lineages of M. tuberculosis and M. bovis BCG. Individual pe/ppe gene alignments were extracted and diversity and conservation across the different lineages studied. RESULTS: The pe/ppe genes were classified into three groups based on the level of protein sequence conservation relative to H37Rv, finding that >50% were conserved, with indels in pe_pgrs and ppe_mptr sub-families being major drivers of structural variation. Gene rearrangements, such as duplications and gene fusions, were observed between pe and pe_pgrs genes. Inter-lineage diversity revealed lineage-specific SNPs and indels. DISCUSSION: The high level of pe/ppe genes conservation, together with the lineage-specific findings, suggest their phylogenetic informativeness. However, structural variants and gene rearrangements differing from the reference were also identified, with potential implications for pathogenicity. Overall, improving our knowledge of these complex gene families may have insights into pathogenicity and inform the development of much-needed tools for tuberculosis control

Comparison of whole genome expression profile between preterm and full-term newborns

Objectives: Evaluate the time dependent expression of genes in preterm neonates and verify the influence of ontogenic maturation and the environmental factors on the gene expression after birth. Material and methods: The study was carried out on 20 full-term newborns and 62 preterm newborns (mean birth weight = 1002 [g] (SD: 247), mean gestational age = 27.2 weeks (SD: 1.9)). Blood samples were drawn from all the study participants at birth and at the 36th week postmenstrual age from the preterm group to assess whole genome expression in umbilical cord blood and in peripheral blood leukocytes, respectively. (SurePrint G3 Human Gene Expression v3, 8x60K Microarrays (Agilent)). Results: A substantial number of genes was found to be expressed differentially at the time of birth and at 36 PMA in comparison to the term babies with more genes being down-regulated than up-regulated. However, the fold change in the majority of cases was &lt; 2.0. Extremely preterm and very preterm infants were characterized by significantly down-regulated cytokine and chemokine related pathways. The number of down-regulated genes decreased and number of up-regulated genes increased at 36 PMA vs. cord blood. There were no specific gene expression pathway profiles found within the groups of different gestational ages. Conclusions: Preterm delivery is associated with a different gene expression profile in comparison to term delivery. The gene expression profile changes with the maturity of a newborn measured by the gestational age