Tropism based analysis of Chlamydia trachomatis chromosome

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2014Alguns microrganismos possuem vários fatores de virulência que lhes conferem a capacidade de infetar de forma específica nichos biológicos distintos. Chlamydia é um excelente exemplo para representar esta competência, uma vez que evoluiu de forma a poder colonizar diversos grupos de animais vertebrados. Trata-se de um género bacteriano gram-negativo e intracelular obrigatório, com um ciclo de vida bifásico único entre os procariotas, de 30 a 72 horas. Apresenta duas formas celulares morfologicamente distintas: forma extracelular infeciosa, o corpo elementar e uma forma replicativa não-infeciosa, o corpo reticulado. Ao longo do desenvolvimento, a bactéria reside e multiplica-se dentro de um vacúolo - a inclusão, e manipula a célula hospedeira através de um sistema de secreção do tipo III (T3SS) pela translocação de proteínas efetoras para o interior da célula hospedeira. Embora a história taxonómica deste género bacteriano tenha sido controversa, propôs-se recentemente que a família das Chlamydiaceae fosse agregada num único género, Chlamydia, incluindo nove espécies com um largo espetro de hospedeiros e de patologias: C. muridarum, C. suis, C. pecorum, C. caviae, C. psittaci, C. abortum, C. felis, C. pneumoniae e C. trachomatis, sendo esta última o foco do nosso estudo, dado tratar-se da única espécie que infecta estritamente o homem e cujas infeções constituem um sério problema de saúde pública. Chlamydia trachomatis é classificada em 15 serovars principais, de acordo com o serotipo diferencial da MOMP (proteína principal da membrana externa). Os serovars de A a C infetam a conjuntiva ocular, provocando o tracoma, que é a principal causa de cegueira susceptível de prevenção em todo o mundo; os serovars D a K estão associados às infeções ano-urogenitais não invasivas, constituindo a primeira causa de doenças bacterianas sexualmente transmissíveis a nível mundial; finalmente, os serovars L1 a L3 são responsáveis por doenças mais invasivas e sistémicas, tais como o linfogranuloma venéreo (LGV), através da infeção do tecido genital e proliferação para os nódulos linfáticos inguinais. O genoma de C. trachomatis, o qual foi sequenciado em 1998, tem aproximadamente 1-Mpb, o que é considerado pequeno para uma bactéria, resultando dum processo de redução evolutiva aquando da transição deste patogéneo para o meio intracelular. Esta bactéria contém também um plasmídeo altamente conservado com ~7,5 kb, que codifica para 8 genes, dois dos quais (pgp3 a pgp4) já tendo sido implicados em funções de virulência. Apesar das diferenças biológicas dos vários serovars de C. trachomatis em relação ao seu tropismo, virulência e sucesso ecológico, estes apresentam um elevado grau de similaridade genómica (>98%). Assim, pensa-se que estas discrepâncias fenotípicas tenham origem em polimorfismos genéticos específicos (mutações pontuais ou pequenos eventos de inserção/deleção) circunscritos aos restantes 2% do genoma. Neste âmbito, este estudo visa avaliar quais os genes que possam estar envolvidos nas diferenças de tropismo, virulência e sucesso ecológico entre os diversos serovars de C. trachomatis, sendo dividido em duas partes. Na primeira parte estudámos todos os ~900 genes de C. trachomatis, tanto a nível filogenético, como evolutivo, para avaliar a sua possível associação com apetência celular e sucesso ecológico, tendo como base cerca de 50 genomas totalmente sequenciados que estão disponíveis no GenBank. Vimos que apenas ~1% dos genes mostram ter uma segregação filogenética dos três grupos de doença (agrupando diferencialmente estirpes que infetam a conjuntiva ocular, o epitélio genital e os nódulos linfáticos). Por outro lado, aproximadamente 80% dos genes segregam as estirpes de LGV, e 28% de todos os genes, incluindo a maioria dos efetores do T3SS e proteínas da inclusão de membrana, as agrupam de forma exclusiva. Metade dos genes estão envolvidos na segregação das estirpes dos serovars genitais mais prevalentes, mas apenas 61 proteínas exibem este padrão mutacional de forma exclusiva. Notavelmente, estas últimas mostraram ser co-segregadas com as estirpes LGV por ~20% dos genes, o que não deixa de ser curioso, tendo em conta o carácter mais invasivo destas últimas. Identificámos também alguns pseudogenes, especificamente associados a estirpes com determinado tipo de tropismo. Aproximadamente 3.5% dos genes mostraram uma sobre representação de mutações não-sinónimas, onde a maioria codifica proteínas que interagem diretamente com o hospedeiro. Globalmente, esta previsão in silico dos genes de C. trachomatis associados a um fenótipo específico pode constituir uma importante base de dados, abrindo portas para futuros estudos cujo objetivo seja o desenvolvimento de medidas profiláticas para o combate às infeções por esta bactéria. A segunda parte deste trabalho foca-se na avaliação da dinâmica genómica de C. trachomatis, subjacente à adaptação ao meio laboratorial. Este tipo de estudos de evolução adaptativa in vitro têm permitido recolher conhecimento importante relativo à base molecular subjacente a processos de evolução microbiana, podendo posteriormente relacioná-lo com dinâmicas adaptativas ocorrentes em populações naturais. Demonstram normalmente a existência de alterações fenotípicas tipicamente observadas em populações propagadas em laboratório, nomeadamente o desenvolvimento de novas capacidades metabólicas, o desenvolvimento de resistência/sensibilidade a antibióticos e perda geral de virulência. Estas características têm sido exploradas pelos investigadores, de forma a esclarecer mecanismos subjacentes ao processo de infeção, e descobrir novos genes de virulência, uma vez que a sua perda de função poderá estar possivelmente relacionada com a sua dispensabilidade no meio in vitro, corroborando o facto de que estes genes possam ter um papel essencial in vivo. Foram utilizadas neste estudo de propagação laboratorial intensiva e de análise genómica comparativa (sequenciação genómica total das estirpes antes e após a sua propagação laboratorial), estirpes com características de tropismo diferentes, nomeadamente seis estirpes representativas dos três grupos de doença (4 urogenitais não-invasivas, uma ocular e uma LGV). Propagámos as estirpes através de 30 passagens in vitro de forma a perceber quais os mecanismos que levam à acumulação de mutações ao longo da passagem laboratorial. Detetámos a emergência de clones com mutações inativantes no gene CT135 (previamente descrito como potencial fator de virulência), para todas as estirpes urogenitais, mas não para as restantes estirpes (oculares e LGV), sendo que os mutantes CT135-nulos subiram rapidamente a sua frequência na população. Ocorreu um cenário semelhante para o gene CT713/porB, em que 3 das 4 estirpes urogenitais não invasivas foram alvo de possíveis mutações inativantes, um panorama que possivelmente reflete um processo de adaptação metabólica, dado que este gene está associado ao transporte de dicarboxilatos. Detetámos também dois genes que foram alvos de mutações inativantes: CT257 e CT645, indicando que estes genes não são essenciais para o crescimento in vitro de C. trachomatis. Curiosamente, a estirpe ocular C/TW-3 evoluiu de forma a reter um gene CT135 funcional, opostamente à estirpe LGV, que não mostrou o aparecimento de qualquer mutação. Também verificámos que a taxa de crescimento para as estirpes propagadas in vitro aumentou relativamente às populações ancestrais, refletindo uma melhoria gradual do fitness ao longo do tempo. Globalmente, esta segunda parte do trabalho contribui para a compreensão de alguns mecanismos subjacentes à adaptação laboratorial de C. trachomatis e corrobora o papel do gene CT135 como um importante fator de virulência. Também identifica possíveis problemas em relação à interpretação de resultados de estudos in vivo que usam estirpes propagadas em laboratório. De uma forma geral, pensamos que este trabalho possa fornecer novo conhecimento em relação aos genes que possam estar envolvidos nos processos de tropismo diferencial, sucesso ecológico e virulência dos diversos serovars de C. trachomatis, abrindo novos caminhos de pesquisa para estabelecer associações entre genótipo e fenótipo

Redirected Stress Responses in a Genome-Minimized 'midiBacillus' Strain with Enhanced Capacity for Protein Secretion

Genome engineering offers the possibility to create completely novel cell factories with enhanced properties for biotechnological applications. In recent years, genome minimization was extensively explored in the Gram-positive bacterial cell factory Bacillus subtilis, where up to 42% of the genome encoding dispensable functions was removed. Such studies showed that some strains with minimized genomes gained beneficial features, especially for secretory protein production. However, strains with the most minimal genomes displayed growth defects. This focused our attention on strains with less extensive genomic deletions that display close-to-wild-type growth properties while retaining the acquired beneficial traits in secretory protein production. A strain of this category is B. subtilis IIG-Bs27-47-24, here referred to as midiBacillus, which lacks 30.95% of the parental genome. To date, it was unknown how the altered genomic configuration of midiBacillus impacts cell physiology in general, and protein secretion in particular. The present study bridges this knowledge gap through comparative quantitative proteome analyses with focus on protein secretion. Interestingly, the results show that the secretion stress responses of midiBacillus, as elicited by high-level expression of the immunodominant staphylococcal antigen A, are completely different from secretion stress responses that occur in the parental strain 168. We further show that midiBacillus has an increased capacity for translation and that a variety of critical Sec secretion machinery components is present at elevated levels. Altogether, our observations demonstrate that high-level protein secretion has different consequences for wild-type and genome-engineered Bacillus strains, dictated by the altered genomic and proteomic configurations. IMPORTANCE Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midiBacillus, as a chassis for the development of future industrial strains that serve in the production of high-value difficult-to-produce proteins. In particular, we explain how midiBacillus, which lacks about one-third of the original genome, effectively secretes a protein of the major human pathogen Staphylococcus aureus that cannot be produced by the parental Bacillus subtilis strain. This is important, because the secreted S. aureus protein is exemplary for a range of targets that can be implemented in future antistaphylococcal immunotherapies. Accordingly, we anticipate that midiBacillus chassis will contribute to the development of vaccines that protect both humans and livestock against diseases caused by S. aureus, a bacterial pathogen that is increasingly difficult to fight with antibiotics, because it has accumulated resistances to essentially all antibiotics that are currently in clinical practice

Escherichia coli Can Adapt Its Protein Translocation Machinery for Enhanced Periplasmic Recombinant Protein Production

Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm

Sppl Forms a Membrane Protein Complex with SppA and Inhibits Its Protease Activity in Bacillus subtilis

The membrane protease SppA of Bacillus subtilis was first described as a signal peptide peptidase and later shown to confer resistance to lantibiotics. Here, we report that SppA forms octameric complexes with YteJ, a membrane protein of thus-far-unknown function. Interestingly, sppA and yid deletion mutants exhibited no protein secretion defects. However, these mutant strains differed significantly in their resistance to antimicrobial peptides. In particular, sppA mutant cells displayed increased sensitivity to the lantibiotics nisin and subtilin and the human lysozyme-derived cationic antimicrobial peptide LP9. Importantly, YteJ was shown to antagonize SppA activity both in vivo and in vitro, and this SppA-inhibitory activity involved the C-terminal domain of YteJ, which was therefore renamed Sppl. Most likely, Sppl-mediated control is needed to protect B. subtilis against the potentially detrimental protease activity of SppA since a mutant overexpressing sppA by itself displayed defects in cell division. Altogether, we conclude that the SppA-Sppl complex of B. subtills has a major role in protection against antimicrobial peptides. IMPORTANCE Our study presents new insights into the molecular mechanism that regulates the activity of SppA, a widely conserved bacterial membrane protease. We show that the membrane proteins SppA and Sppl form a complex in the Gram-positive model bacterium B. subtilis and that Sppl inhibits SppA protease activity in vitro and in vivo. Furthermore, we demonstrate that the C-terminal domain of Sppl is involved in SppA inhibition. Since SppA, through its protease activity, contributes directly to resistance to lantibiotic peptides and cationic antibacterial peptides, we propose that the conserved SppA-Sppl complex could play a major role in the evasion of bactericidal peptides, including those produced as part of human innate immune defenses

Novel genomic approach to decode syphilis

A sífilis é uma doença sexualmente transmissível causada pela bactéria Treponema pallidum e constitui um problema de saúde pública mundial, em parte devido à ausência de uma vacina para prevenção da sua transmissão. A investigação desta doença tem sido atrasada pela incapacidade histórica de cultivar T. pallidum in vitro, dificultando por exemplo o desenvolvimento de estudos genómicos. De facto, há uma grande lacuna no conhecimento da epidemiologia molecular deste agente patogénico, assim como da base molecular que medeia a patogénese da sífilis. No estudo aqui apresentado, foi possível implementar uma abordagem inovadora para capturar o genoma de T. pallidum no contexto de infeção humana, evitando-se, assim, a necessidade da cultura da bactéria em modelo animal. Esta estratégia permitiu estudar, pela primeira vez, como é que este agente patogénico vai alterando o seu genoma para se adaptar e sobreviver como agente infecioso humano. Nomeadamente permitiu descodificar os principais mecanismos genéticos pelos quais a bactéria T. pallidum evade o sistema imunitário e se adapta ao Homem nesta complexa e multifásica doença. A aplicação desta estratégia inovadora de monitorização da interação Homem-bactéria poderá ser importante para o desenvolvimento de novas medidas profiláticas e/ou terapêuticas. Acresce que esta abordagem constitui também um ponto de viragem para o aperfeiçoamento de metodologias de diagnóstico e de epidemiologia molecular, o que permitirá aumentar o conhecimento da distribuição geográfica, das vias de transmissão e das propriedades de virulência deste agente patogénico para bem da saúde pública.Syphilis, a sexually transmitted disease caused by the bacterium T. pallidum, remains a global problem with an estimated 6 million people infected each year, which is in part attributed to the absence of a vaccine to prevent infection and transmission. Despite its tremendous public health impact, research in syphilis has been considerable hampered due to the historical inability to culture T. pallidum in vitro, which has hampered, for instance, the acquisition of consistent genomic data. As such, there is a strong lack of knowledge on the molecular epidemiology of this important human pathogen as well as on the molecular mechanisms underlying syphilis pathogenesis. In the work presented here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the contex t of human infection. This strategy allowed, for the first time, to understand how this pathogen shapes its genome towards adaptation and sur vival during syphilis. While this work demonstrates the exceptional power of monitoring the pathogen adaptability during human infection, it also provides critical data that may guide the development of novel treatments and prophylactic measures, such as a vaccine. In other perspective, it is anticipated that the implemented methodological approach constitutes a disruptive step towards the improvement of the current diagnostics and typing methodologies, which will enhance the knowledge on the geographic distribution of strains, its transmissibility and propensity to cause disease.Este estudo foi financiado pela Fundação para a Ciência e Tecnologia (EXPL/BIA-MIC/0309/2013).info:eu-repo/semantics/publishedVersio

Membrane Modulation of Super-Secreting “midiBacillus” Expressing the Major Staphylococcus aureus Antigen – A Mass-Spectrometry-Based Absolute Quantification Approach

Bacillus subtilis has been extensively used as a microbial cell factory for industrial enzymes due to its excellent capacities for protein secretion and large-scale fermentation. This bacterium is also an attractive host for biopharmaceutical production. However, the secretion potential of this organism is not fully utilized yet, mostly due to a limited understanding of critical rearrangements in the membrane proteome upon high-level protein secretion. Recently, it was shown that bottlenecks in heterologous protein secretion can be resolved by genome minimization. Here, we present for the first time absolute membrane protein concentrations of a genome-reduced B. subtilis strain (“midiBacillus”) expressing the immunodominant Staphylococcus aureus antigen A (IsaA). We quantitatively characterize the membrane proteome adaptation of midiBacillus during production stress on the level of molecules per cell for more than 400 membrane proteins, including determination of protein concentrations for ∼61% of the predicted transporters. We demonstrate that ∼30% of proteins with unknown functions display a significant increase in abundance, confirming the crucial role of membrane proteins in vital biological processes. In addition, our results show an increase of proteins dedicated to translational processes in response to IsaA induction. For the first time reported, we provide accumulation rates of a heterologous protein, demonstrating that midiBacillus secretes 2.41 molecules of IsaA per minute. Despite the successful secretion of this protein, it was found that there is still some IsaA accumulation occurring in the cytosol and membrane fraction, leading to a severe secretion stress response, and a clear adjustment of the cell’s array of transporters. This quantitative dataset offers unprecedented insights into bioproduction stress responses in a synthetic microbial cell

Functional association of the stress-responsive LiaH protein and the minimal TatAyCy protein translocase in Bacillus subtilis

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound co-factors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat

The Emerging Potential of Advanced Targeted Mass Spectrometry to Become a Routine Tool for Protein Quantification in Biomedical Research

Mass spectrometry-based proteomics has become an indispensable tool for system-wide protein quantification in systems biology, biomedical research, and increasing for clinical applications. In particular, targeted mass spectrometry offers the most sensitive and reproducible quantitative detection of proteins, peptides and post-translational modifications of any currently applied mass spectrometry technique and is therefore ideally suited to generate high quality quantitative datasets. Despite these apparent advantages, targeted mass spectrometry is only slowly gaining popularity in academia and pharmaceutical industries, mainly due to the additional efforts in assay generation and manual data validation. However, with the increasing accumulation of mass spectrometry data, advances in deep learning spectral prediction for automated assay development, these obstacles can and will be considerably reduced in the near future. Here, we describe the latest technological developments in this field and discuss the emerging importance of targeted mass spectrometry for systems biology research and potential key roles in bridging biomedical discovery and clinical implementation.  &nbsp

Absolute & relative membrane protein quantification - a mass spectrometry-based approach

Microbial cell factories have been largely exploited for the controlled production of recombinant proteins, including industrial enzymes and biopharmaceuticals. The advent of high-throughput ‘-omics’ techniques have boosted the design of these production systems due to their valuable contribution to the field of systems metabolic engineering, a discipline integrating metabolic engineering with systems and synthetic biology. In order to thrive, the field of systems metabolic engineering needs absolute proteomics data to be generated, as proteins are the central players in the complex metabolic and adaptational networks. Due to advent of mass spectrometry-based proteomics, a substantial amount of absolute proteomic data became available in the past decade. However, membrane proteins remained inaccessible to these efforts. Nonetheless, comparative studies targeting the membrane proteome have been quite successful in characterizing physiological processes. Hence, label-free proteomics was used in a study (Quesada-Ganuza et al, 2019 – Article I) to identify and optimize PrsA in Bacillus subtilis, for improved yield of amylase. Amylase is one of the most relevant enzymes in the biotechnological sector. By employing a label-free mass spectrometry approach targeting the membrane proteome of this bacterium, relative changes in heterologous and native levels of PrsA could be quantified. The results of this study evidenced that each PrsA shows different relative abundancies, but with no relevant impact in the yield of amylase. Even though relative protein quantification can already provide a good visualization of the physiological changes occurring between different conditions, they are not sufficient to understand how resources are allocated in the cell under certain physiological conditions. Therefore, a global method for absolute membrane protein quantification remains the biggest requirement for systems metabolic engineering. Hence, with this work, we successfully developed a mass spectrometry-based approach enabling the absolute quantification of membrane proteins (Antelo-Varela et al, 2019 – Article II). This study was also performed in the Gram-positive model organism Bacillus subtilis, regarded as a prolific microbial cell factory. The method developed in this work combines the comprehensiveness of shotgun proteomics with the sensitivity and accuracy of targeted mass spectrometry. Fundamental to the method is that it relies on the application of a correction and an enrichment factor to calibrate absolute membrane protein abundances derived from shotgun mass spectrometry. This has permitted, for the first time reported, the calculation of absolute membrane protein abundances in a living organism. The newly developed approach enabled to accurately quantify ~40% of the predicted proteome of this bacterium, offering a clear visualization of the physiological rearrangements occurring upon the onset of osmotic stress. In addition, this work also provides evidence for new membrane protein stoichiometries. Overall, this study enabled the development of a straightforward methodology long-needed in the scientific and biotechnological community and, for the first time reported, providing absolute abundances of one of the most puzzling fractions of the cell – the membrane proteome. The next step of the work summarized here was to implement the afore described method to a biotechnological relevant strain, as absolute membrane protein abundances are essential to understand the fundamental principles of protein secretion and production stress. Hence, this work was applied in a genome-reduced B. subtilis strain, ‘midiBacillus’, expressing the major staphylococcal antigen IsaA (Antelo-Varela et al, submitted – Article III). The employed absolute membrane protein quantification methodology enabled the analysis of physiological rearrangements occurring upon the induction of heterologous protein production. This work showed that, even though IsaA was successfully secreted into the growth medium, one of the main requirements for the biotechnological sector, it was still partly accumulated in the cell membrane of this bacterium. This led to an exacerbated physiological response where membrane proteins involved in the management of secretion stress were activated. In addition, this study also showed that a rearrangement of the cell’s translocation machinery occurs upon induction of production, where a ‘game’ of in- and decrease of transporters takes place. Anticipating the impact of genetic and environmental insults, such as the ones caused by production stress, is essential for the field of systems metabolic engineering. Thus, the highly accurate and comprehensive dataset generated during this work can be implemented in predictive mathematical models, thereby contributing in the rational design of next-generation secretion systems.Mikrobielle Zellfabriken wurden weitgehend für die kontrollierte Produktion rekombinanter Proteine genutzt, einschließlich industrieller Enzyme und Biopharmazeutika. Das Aufkommen von Hochdurchsatz-"Omics"-Techniken hat das Design dieser Produktionssysteme aufgrund ihres wertvollen Beitrags zum Bereich der System-Metabolic-Engineering, einer Disziplin, die das Metabolic-Engineering mit Systembiologie und synthetischer Biologie verbindet, gefördert. Damit der Bereich System-Metabolic-Engineering florieren kann, müssen absolute Proteomicsdaten generiert werden, da Proteine die zentralen Akteure in den komplexen Stoffwechsel- und Anpassungsnetzwerken sind. Aufgrund des Aufkommens der massenspektrometrie-basierten Proteomik wurde in den letzten zehn Jahren eine beträchtliche Menge an absoluten Proteomdaten verfügbar. Membranproteine blieben für diese Bemühungen jedoch weiterhin kaum zugänglich. Nichtsdestotrotz waren vergleichende Studien, die auf das Membranproteom abzielten, bei der Charakterisierung physiologischer Prozesse recht erfolgreich. Daher wurde in einer Studie (Quesada-Ganuza et al., 2019 - Artikel I) die markierungsfreie Proteomanalyse verwendet, um PrsA in Bacillus subtilis zu identifizieren und zu optimieren, mit dem Ziel die Ausbeute an Amylase zu verbessern. Amylase ist eines der relevantesten Enzyme im biotechnologische Sektor. Durch Verwendung eines markierungsfreien Massenspektrometrie-Ansatzes, der auf das Membranproteom dieses Bakteriums abzielt, konnten relative Änderungen der heterologen und nativen PrsA-Spiegel quantifiziert werden. Die Ergebnisse dieser Studie zeigten, dass jedes PrsA unterschiedliche relative Häufigkeiten aufweist, jedoch keine relevanten Auswirkungen auf die Ausbeute an Amylase hat. Obwohl die relative Proteinquantifizierung bereits eine gute Darstellung der physiologischen Veränderungen, die zwischen verschiedenen Zuständen auftreten, liefern kann, reichen sie nicht aus, um zu verstehen, wie Ressourcen unter bestimmten physiologischen Bedingungen in der Zelle zugewiesen werden. Daher bleibt eine globale Methode zur absoluten Quantifizierung von Membranproteinen nach wie vor die größte Herausforderung für das System-Metabolic-Engineering. In dieser Arbeit wurde erfolgreich ein massenspektrometrischer Ansatz entwickelt, der die absolute Quantifizierung von Membranproteinen ermöglicht (Antelo-Varela et al., 2019 - Artikel II). Diese Studie wurde ebenfalls am grampositiven Modellorganismus Bacillus subtilis durchgeführt, der als eine produktive mikrobielle Zellfabrik angesehen wird. Die in dieser Arbeit entwickelte Methode kombiniert die Vollständigkeit der Shotgun-Proteomik mit der Empfindlichkeit und Genauigkeit der gezielten Massenspektrometrie. Grundlegend für das Verfahren ist, dass es auf der Anwendung eines Korrektur- und eines Anreicherungsfaktors beruht, um absolute Membranproteinhäufigkeiten zu kalibrieren, die aus der Shotgun-Massenspektrometrie stammen. Dies ermöglichte erstmals die Berechnung der absoluten Membranproteinhäufigkeit in einem lebenden Organismus. Der neu entwickelte Ansatz ermöglichte die genaue Quantifizierung von ~ 40% des vorhergesagten Proteoms dieses Bakteriums und ermöglichte die klare Darstellung der physiologischen Anpassungen, die beim Einsetzen von osmotischem Stress auftreten. Darüber hinaus liefert diese Arbeit Hinweise auf neue Membranproteinstöchiometrien. Insgesamt ermöglichte diese Studie die Entwicklung einer einfachen Methodik, die in der wissenschaftlichen und biotechnologischen Gemeinschaft seit langem benötigt wird und lieferte zum ersten Mal absolute Häufigkeiten einer der rätselhaftesten Fraktionen der Zelle - des Membranproteoms. Der nächste Schritt der hier zusammengefassten Arbeit war die Implementierung der oben beschriebenen Methode bei einem biotechnologisch relevanten Stamm, da die Bestimmung der absoluten Membranproteinhäufigkeiten enorm hilfreich für das Verständnis der Grundprinzipien der Proteinsekretion und des Produktionsstresses sein kann. Diese Methode wurde in einem genomreduzierten B. subtilis-Stamm, „midiBacillus“, angewendet, der das Hauptstaphylokokken-Antigen IsaA exprimiert (Antelo-Varela et al., Eingereicht - Artikel III). Die angewandte Methode zur absoluten Quantifizierung der Membranproteine ermöglichte die Analyse physiologischer Anpassungen, die nach Induktion der heterologen Proteinproduktion auftraten. Diese Arbeit zeigte, dass IsaA, obwohl es erfolgreich in das Wachstumsmedium sezerniert wurde, eine der Hauptanforderungen für den biotechnologischen Sektor, teilweise noch in der Zellmembran dieses Bakteriums akkumuliert war. Dies führte zu einer verstärkten physiologischen Reaktion, bei der Proteine aktiviert wurden, die am Management von Sekretionsstress beteiligt sind. Darüber hinaus zeigt diese Studie auch eine Neuordnung der Translokationsmaschinerie der Zelle, bei der ein "Spiel" des Ein- und Abbaus von Transportern stattfindet. Die Vorhersage der Auswirkungen genetischer und umweltbedingter Belastungen, wie sie beispielsweise durch Produktionsstress verursacht werden, ist für den Bereich des Metabolic-Engineering von wesentlicher Bedeutung. Somit kann der während dieser Arbeit erzeugte hochgenaue und umfassende Datensatz in prädiktiven mathematischen Modellen implementiert werden, wodurch ein Beitrag zum rationalen Design von Sekretionssystemen der nächsten Generation geleistet wird

A retrospective cross-sectional quantitative molecular approach in biological samples from patients with syphilis

Syphilis is the sexually transmitted disease caused by Treponema pallidum, a pathogen highly adapted to the human host. As a multistage disease, syphilis presents distinct clinical manifestations that pose different implications for diagnosis. Nevertheless, the inherent factors leading to diverse disease progressions are still unknown. We aimed to assess the association between treponemal loads and dissimilar disease outcomes, to better understand syphilis. We retrospectively analyzed 309 DNA samples distinct anatomic sites associated with particular syphilis manifestations. All samples had previously tested positive by a PCR-based diagnostic kit. An absolute quantitative real-time PCR procedure was used to precisely quantify the number of treponemal and human cells to determine T. pallidum loads in each sample. In general, lesion exudates presented the highest T. pallidum loads in contrast with blood-derived samples. Within the latter, a higher dispersion of T. pallidum quantities was observed for secondary syphilis. T. pallidum was detected in substantial amounts in 37 samples of seronegative individuals and in 13 cases considered as syphilis-treated. No association was found between treponemal loads and serological results or HIV status. This study suggests a scenario where syphilis may be characterized by: i) heterogeneous and high treponemal loads in primary syphilis, regardless of the anatomic site, reflecting dissimilar duration of chancres development and resolution; ii) high dispersion of bacterial concentrations in secondary syphilis, potentially suggesting replication capability of T. pallidum while in the bloodstream; and iii) bacterial evasiveness, either to the host immune system or antibiotic treatment, while remaining hidden in privileged niches. This work highlights the importance of using molecular approaches to study uncultivable human pathogens, such as T. pallidum, in the infection process.This work was supported by the Portuguese Foundation for Science and Technology (EXPL/BIA-MIC/0309/2013).info:eu-repo/semantics/publishedVersio