Come out and play:Exploring bacterial cell wall synthesis and cell division

Bacteriën zijn eencellige organismen die over de hele wereld zijn verspreid. Normaal gesproken neemt de celgrootte van de bacterie toe voordat de bacteriecel opsplitst in twee identieke cellen. Voor deze bacteriële levenscyclus zijn twee verschillende, maar gerelateerde cellulaire processen nodig: de synthese en de deling van de celwand. Het celwandsynthese proces is erg belangrijk om er voor te zorgen dat de celwand goed gesynthetiseerd en georganiseerd wordt, aangezien het betrokken is bij de uiteindelijk grens en vorm van de bacterie. De synthese van de nieuwe celwand heeft verschillende eiwitten nodig die samenwerken om peptidoglycaan te maken, de belangrijkste component voor de celwand. Celwandsynthese is niet alleen nodig om de cel te laten groeien, maar ook om de cel zich goed te laten delen zonder dat de celwand breekt. Ook bij celdeling werken veel verschillende eiwitten samen, zodat uiteindelijk de cel gedeeld wordt in twee dochtercellen. Zowel de celwandsynthese als de celdeling zijn uitermate belangrijk en om die reden uitgebreid bestudeerd in de afgelopen decennia. Echter veel specifieke details zijn nog onbekend. Daarom wordt er in dit proefschrift een aantal verschillende manieren gebruikt om de complexiteit van de celwandsynthese en celdeling verder te ontrafelen. Verschillende aspecten zijn hierbij onderzocht: zowel de interne membraanorganisatie, als de chaperonne activiteit van het eiwit YidC en het antibacteriële effect van alkylgallaten. Deze studie onderstreept daarmee de nauw verweven relatie tussen de bacteriële celwandsynthese en de celdeling

Fas Ligando como indicador de apoptose na doença coronária

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2009A progressão da aterosclerose leva ao desenvolvimento do ateroma na parede arterial, o qual por erosão e/ou ruptura súbita pode conduzir à formação de trombo com oclusão total ou parcial das artérias coronárias e consequente enfarte do miocárdio. Estudos in vitro revelaram que a apoptose das células constituintes da parede vascular está associada à expressão do receptor Fas e do seu ligando FasL assim como da interacção entre eles. Admite-se que a expressão do FasL pelas células endoteliais possa contribuir para os níveis de concentração da forma solúvel de FasL (sFasL) em circulação. Com este trabalho pretendeu-se relacionar a concentração do sFasL, determinada pelo método de ELISA, com as características da placa de ateroma identificadas em pacientes com doença coronária, confirmada por angioplastia e caracterizados por VH IVUS (do inglês, Virtual Histology Intravascular Ultrasound), juntamente com os parâmetros antropomórficos, bioquímicos e fisiológicos dos doentes. Embora não se tenham encontrado correlações significativas entre o sFasL e as características biológicas da placa de ateroma, o aumento de sFasL plasmático relacionou-se com a terapêutica com estatinas. Por outro lado, factores de risco como a hipertensão, glucose e idade associaram-se à diminuição de sFasL. Relativamente à placa, incrementos do seu conteúdo em tecidos fibrótico e fibrolipídico associaram-se a um menor número de leucócitos e plaquetas em circulação. O tecido necrótico correlacionou-se com o perímetro abdominal e o tecido calcificado com o peso. A estenose arterial associou-se à concentração de pro-BNP e ao tabagismo. O aumento de sFasL pode ter uma consequência benéfica devido à sua capacidade de induzir apoptose nos leucócitos em circulação que tentam atravessar o endotélio. A associação do aumento de sFasL à toma regular de estatinas sugere um papel do sFasL na ateroprotecção e na regulação do endotélio vascular. Estes resultados permitiram avaliar a potencialidade do sFasL como indicador da DC, melhorar o protocolo de selecção de doentes e identificar factores a ter em consideração na avaliação dos doentes e na actividade da placa de ateroma. É possível que a ausência de correlações entre o sFasL e os componentes da placa esteja condicionada pelo tamanho reduzido da amostra, pelo que serão necessários estudos adicionais para confirmar os resultados obtidos.The atherosclerosis progression leads to the development of an atheroma in the arterial wall, whereby erosion and/or sudden rupture can lead to the formation of a thrombus with partial or total occlusion of coronary arteries and consequently can lead to myocardial infarction. In vitro studies revealed that the constituent cells of vascular wall apoptosis is linked to Fas receptor and its ligand FasL expression and the interaction between them. It is assumed FasL expression by endothelial cells may contribute to the soluble form of FasL (sFasL) plasma concentration. The aim of this work was to correlate sFasL concentration, by ELISA method, with plaque characteristics identified in patients with coronary heart disease, confirmed by angioplasty and characterized by VH IVUS (Virtual Histology Intravascular Ultrasound), in addition to patients anthropomorphic, biochemical and physiological parameters. Although no significant correlations were found between sFasL and biological plaque characteristics, the increase of plasmatic sFasL was associated with the therapy with statins. On the other hand, risk factors such as hypertension, glucose, and age were related with sFasL decrease. Relatively to atheromatous plaque, an increase in its fibrous and fibrolipidic tissues was related to a lower number of leukocytes and platelets in circulation. Necrotic tissue was linked with abdominal perimeter and calcified tissue with weight. Arterial stenosis was associated with pro-BNP concentration and smoke. The increase in sFasL concentration may have a beneficial outcome due to its capacity to induce apoptosis in circulating leukocytes trying to go through the endothelium. The association between sFasL increase and statins consume seem to suggest an atheroprotective and vascular endothelium regulation effect of sFasL. These results allowed the assessment of sFasL as a potential coronary artery disease indicator, improving the selection protocol of patients and identifying aspects to take into consideration in patients assessment and atheroma plaque activity. It is possible that the absence of sFasL associations with any of the plaque components might be influenced by the small data; therefore, future studies will be needed in order to overcome these limitations

Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles

The purpose of this work was to assess the ability of recombinant hepatitis B vaccine, encapsulated in alginate-coated chitosan nanoparticles, to induce local and systemic immune responses following oral vaccination. The antigen was administered either alone or in combination with the immunopotentiator, synthetic oligodeoxynucleotide containing immunostimulatory CpG motif (CpG ODN) as adjuvant, and associated or not with the alginate-coated chitosan nanoparticles. After two immunizations the group I (HBsAg associated with nanoparticles) and the group VI (HBsAg and CpG, both associated with nanoparticles) showed enhanced immune responses. Both groups showed significant higher values of the CD69 expression in CD4+ and CD8+ T-lymphocytes and lower values of this marker in B lymphocytes. Moreover, a strongest proliferative response of the splenocytes, ex vivo stimulated with concanavalin A, was observed in the same groups. Although with a presence of non-responder mice within the groups, only mice of the groups I and VI elicited the generation of anti-HBsAg antibodies detected in serum (IgG) and in the intestinal washings (sIgA). The results demonstrated that coated chitosan nanoparticles might have potential for being used as a deliver system for oral vaccination with the recombinant hepatitis B surface antigen.http://www.sciencedirect.com/science/article/B6T25-4PF1WCM-2/1/3fe2a6633c054a684fa0fafa7bb4a8b

a clinical perspective

This article was funded by the project NORTE-08-5369-FSE-000018, supported by Norte Portugal Regional Programme (Norte2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by FCT—Fundação para a Ciência e a Tecnologia [UID/ BIM/04293/2013].Background: In phenylketonuria (PKU), modified casein glycomacropeptide supplements (CGMP-AA) are used as an alternative to the traditional phenylalanine (Phe)-free L-amino acid supplements (L-AA). However, studies focusing on the long-term nutritional status of CGMP-AA are lacking. This retrospective study evaluated the long-term impact of CGMP-AA over a mean of 29 months in 11 patients with a mean age at CGMP-AA onset of 28 years (range 15–43) [8 females; 2 hyperphenylalaninaemia (HPA), 3 mild PKU, 3 classical PKU and 3 late-diagnosed]. Outcome measures included metabolic control, anthropometry, body composition and biochemical parameters. Results: CGMP-AA, providing 66% of protein equivalent intake from protein substitute, was associated with no significant change in blood Phe with CGMP-AA compared with baseline (562 ± 289 µmol/L vs 628 ± 317 µmol/L; p = 0.065). In contrast, blood tyrosine significantly increased on CGMP-AA (52.0 ± 19.2 μmol/L vs 61.4 ± 23.8 μmol/L; p = 0.027). Conclusions: Biochemical nutritional markers remained unchanged which is an encouraging finding in adults with PKU, many of whom are unable to maintain full adherence with nutritionally fortified protein substitutes. Longitudinal, prospective studies with larger sample sizes are necessary to fully understand the metabolic impact of using CGMP-AA in PKU.publishersversionpublishe

Characterization of ftsZ Mutations that Render Bacillus subtilis Resistant to MinC

Background: Cell division in Bacillus subtilis occurs precisely at midcell. Positional control of cell division is exerted by two mechanisms: nucleoid occlusion, through Noc, which prevents division through nucleoids, and the Min system, where the combined action of the MinC, D and J proteins prevents formation of the FtsZ ring at cell poles or recently completed division sites. Methodology/Principal Findings: We used a genetic screen to identify mutations in ftsZ that confer resistance to the lethal overexpression of the MinC/MinD division inhibitor. The FtsZ mutants were purified and found to polymerize to a similar or lesser extent as wild type FtsZ, and all mutants displayed reduced GTP hydrolysis activity indicative of a reduced polymerization turnover. We found that even though the mutations conferred in vivo resistance to MinC/D, the purified FtsZ mutants did not display strong resistance to MinC in vitro. Conclusions/Significance: Our results show that in B. subtilis, overproduction of MinC can be countered by mutations that alter FtsZ polymerization dynamics. Even though it would be very likely that the FtsZ mutants found depend on other Z-ring stabilizing proteins such as ZapA, FtsA or SepF, we found this not to be the case. This indicates that the cell division process in B. subtilis is extremely robust.

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Bacterial dynamin as a membrane puncture repair kit

Dynamin(-like) proteins (DLPs) are widely distributed inbacteria, and both structural and biochemical data haveclearly demonstrated that, like their eukaryotic counter-parts, these proteins can function in membrane modelling.Although various functions for bacterial DLPs have beenhypothesized, a clear function for these proteins withinbacteria remained an enigma. A new study by Bramkampand colleagues (Sawantet al., 2016) provides the firstglimpse for a role of bacterial dynamins as a surveillancesystem that monitors membrane integrity and that coun-teracts pore formation by phages and antibiotics

Metal-dependent SpoIIE oligomerization stabilizes FtsZ during asymmetric division in Bacillus subtilis

SpoIIE is a bifunctional protein involved in asymmetric septum formation and in activation of the forespore compartment-specific transcription factor σF through dephosphorylation of SpoIIAA-P. The phosphatase activity of SpoIIE requires Mn2+ as a metal cofactor. Here, we show that the presence of a metal cofactor also influences SpoIIE oligomerization and asymmetric septum formation. Absence of Mn2+ from sporulation medium results in a delay of the formation of polar FtsZ-rings, similar to a spoIIE null mutant. We purified the entire cytoplasmic part of the SpoIIE protein, and show that the protein copurifies with bound metals. Metal binding both stimulates SpoIIE oligomerization, and results in the formation of larger oligomeric structures. The presence of SpoIIE oligomers reduces FtsZ GTP hydrolysis activity and stabilizes FtsZ polymers in a light scattering assay. Combined, these results indicate that metal binding is not just required for SpoIIE phosphatase activity but also is important for SpoIIE's role in asymmetric septum formation

The Escherichia coli membrane protein insertase YidC assists in the biogenesis of Penicillin Binding Proteins

Membrane proteins need to be properly inserted and folded in the membrane in order to perform a range of activities that are essential for the survival of bacteria. The Sec translocon and the YidC insertase are responsible for the insertion of the majority of proteins into the cytoplasmic membrane. YidC can act in combination with the Sec translocon in the insertion and folding of membrane proteins. However, YidC also functions as an insertase independently of the Sec translocon for so-called YidC-only substrates. In addition, YidC can act as a foldase and promote the proper assembly of membrane protein complexes. Here, we investigate the effect of Escherichia coli YidC depletion on the assembly of Penicillin Binding Proteins (PBPs), that are involved in cell wall synthesis. YidC depletion does not affect the total amount of the specific cell division PBP3 (FtsI) in the membrane, but the amount of active PBP3, as assessed by substrate binding, is reduced two-fold. A similar reduction in the amount of active PBP2 was observed, while the levels of active PBP1A/1B and PBP5 were essentially similar. PBP1B and PBP3 disappeared from higher Mw bands upon YidC depletion, indicating that YidC might play a role in PBP complex formation. Taken together, our results suggest that the foldase activity of YidC can extend to the periplasmic domains of membrane proteins. IMPORTANCE: This work addresses the role of the membrane protein insertase YidC in the biogenesis of Penicillin Binding Proteins (PBPs). PBPs are proteins containing one transmembrane segment and a large periplasmic or extracellular domain, which are involved in peptidoglycan synthesis. We observe that in the absence of YidC, two critical PBPs are not correctly folded even though the total amount of protein in the membrane is not affected. Our findings extend the function of YidC as a foldase for membrane protein (complexes) to periplasmic domains of membrane proteins