6 research outputs found
A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. piscicida Cleaves the Peptidoglycan of Potentially Competing Bacteria
Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-contain-ing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (Photobacterium NlpC-like protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-D-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior and Fundação para a Ciência e a Tecnologia (FCT), I.P., within the scope of the Norma Transitória - DL57/2016/CP1355/CT0010. This work had also support from the State Agency for Research (AEI) of Spain cofunded by the FEDER Program from the European Union (grants AGL2016-79738-R and
BIO2016-77639-P
The role of nitrite-derived nitric oxide in gastric physiology: biochemical mechanisms, molecular targets and the modulatory effect of red wine
Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Bioquímica, apresentada à Faculdade de Farmácia da Universidade de CoimbraO óxido nítrico é um mediador celular ubíquo com papel relevante nos sistemas cardiovascular, imunitário e nervoso. As propriedades físico-químicas do, nomeadamente o facto de ser um gás, difusível e hidrofóbico tornam-no distinto de outros mensageiros celulares, uma vez que permeia membranas biológicas, não sendo, portanto armazenado em vesículas. A dinâmica da concentração do, ou seja, o equilíbrio entre a sua síntese (via óxido nítrico sintases (NOS) ou por redução química do nitrito) e a sua inativação (por reação com heme proteínas ou via oxidação a nitrito e nitrato) determina a sua bioatividade.
O nitrito foi considerado por muitos e durante muito tempo como um produto do metabolismo do cujo destino é a excreção, e como um tóxico existente em determinados alimentos causador de cancro gástrico pela formação de N-nitrosaminas e envolvido em casos de meta-hemoglobinemia infantil. A biologia do nitrito chamou a atenção da comunidade científica quando a produção de a partir de nitrito inorgânico no estômago foi reportada. O consumo de alimentos como alface, beterraba, espinafres, brócolos e outros vegetais de folhas verdes ricos em nitrato (e algum nitrito) levam a um aumento de nitrato e nitrito no plasma. Na cavidade oral, bactérias comensais reduzem nitrato a nitrito que misturado com a saliva chega ao estômago onde o pH ácido promove a redução do nitrito a. Esta sequência de eventos designada por Nitrate-Nitrite- pathway origina no estômago a maior concentração de formada in vivo. O nitrito representa um vasto reservatório de no organismo e tem também sido implicado na modulação de funções celulares de uma forma independente do. Várias enzimas mostram atividade de nitrito reductases em condições de baixa tensão de oxigénio implicando o envolvimento do nitrito na sinalização em hipoxia.
No trato gastrointestinal, o derivado do nitrito demonstra propriedades antimicrobianas e modula o fluxo sanguíneo, a produção de muco, a motilidade gástrica e está envolvido na prevenção ulcerogénica. No meio acídico do estômago, o nitrito da dieta leva à formação de diversos óxidos de nitrogénio (RNOS) além do, que podem induzir modificações pós-tradução como a nitrosação e a nitração em proteínas com impacto biológico. A ingestão concomitante de alimentos cujos componentes detém potencial de oxidação-redução tais como os polifenóis do vinho tinto potencia a formação de por redução univalente do nitrito e consequente oxidação do polifenol ao seu radical semiquinónico.
O trabalho apresentado nesta tese mostra que o nitrito derivado da dieta tem a capacidade de induzir nitrosação, especialmente S-nitrosação, em proteínas constituintes da camada de muco (mucinas) que recobre a superfície gástrica. Além disso, foi observado que proteínas do epitélio gástrica são alvos para nitrosação por nitrito acidificado. Estes resultados apontam para o muco como filtro ativo ao estresse nitrosativo e para potencias efeitos celulares mediados pelo. O perfil de nitrosação é modulado pela presença de vinho tinto o que sugere uma nova atividade para os polifenóis do vinho tinto no que toca à formação de compostos S- e N-nitrosados no compartimento gástrico. A quantificação de nitrosação na mucosa gástrica foi conseguida recorrendo a uma metodologia de quimiluminescência de elevada sensibilidade e seletividade. O S-nitrosotióis são compostos relativamente estáveis e que podem funcionar como transportadores e dadores de, com efeitos locais e sistémicos. De facto, foi observado que o muco de estômago de rato nitrosado com nitrito acidificado liberta a pH fisiológico. Em condições inflamatórias in vivo a extensão de nitrosação por nitrito é aumentada, particularmente na fração correspondente aos S-nitrosotióis.
Além dos conhecidos efeitos do nitrito derivado da dieta relativamente à produção de muco gástrico, neste trabalho foi observado que o nitrito estimula a produção de TFF1 (trefoil factor 1), um importante peptídeo para proteção e regeneração da mucosa e com propriedades anti-tumorogénicas, contribuindo para a manutenção da integridade mucosal. Também aqui a modulação redox pelo vinho tinto tem impacto, aumentando a expressão de TFF1.
As elevadas concentrações de nitrito e •NO atingidas no estômago derivadas da dieta, levantam a questão sobre o seu impacto na função mitocondrial da mucosa. A mitocôndria é um conhecido alvo para o •NO e mais recentemente foi também reconhecida como alvo para o nitrito. Nos resultados apresentados, pode observar-se que além da capacidade para lidar com elevadas concentrações de •NO e nitrito, a função mitocondrial da mucosa gástrica surge melhorada por efeito do nitrito in vivo. A análise da função mitocondrial foi efetuada utilizando a respirometria de alta-resolução. Considerando que a respiração mitocondrial é essencial no funcionamento celular e em vias de sinalização, a modulação da função mitocondrial por constituintes da dieta como nitrito inorgânico pode ter implicação na fisiologia e patologia gástrica.
No seu conjunto, estes resultados destacam a importância de compostos da dieta diária com atividade biológica, tais como o nitrato e o nitrito, da sua interação com outros componentes da dieta como o vinho tinto e o seu impacto coletivo na fisiologia e patologia gástrica.Nitric oxide is a ubiquitous messenger implicated in several important signalling pathways. Critical physiological functions such as regulation of the vascular tone, immune response and neuromodulation depend on dynamics. In between its synthesis (by synthases or by chemical reduction of nitrite) and its inactivation (by heme globins or oxidation to nitrite and nitrate), diffuses trough biological milieu reaching its molecular targets.
Considered for long as waste product, capable of induce gastric cancer via the formation of carcinogenic nitrosamines, nitrite is now proving that is more than a stable NO metabolite. Nitrite represents a vast reservoir in the body and has been implicated in many modulatory pathways itself. The nitrite biology gained attention upon the report of production in the stomach from inorganic nitrite. Nitrate from diet is reduced to nitrite in the saliva that reaches the gastric lumen where the acidic pH promotes the univalent reduction to, in the so-called nitrate-nitrite- pathway. This pathway originates the highest yield of in vivo. Several enzymes have been shown to acquire nitrite reductase properties at low oxygen tensions, suggesting a role for nitrite in the hypoxic signalling.
In the gastrointestinal tract, nitrite-derived has been shown to modulate host defence, blood flow, mucus production and gastric motility and protection. At the acidic pH, nitrite generates several nitrogen oxides (RNOS) beside such as nitrogen dioxide and dinitrogen trioxide (N2O3) that can induce post-translational modifications of endogenous proteins with physiological impact. Other dietary components with redox potential such as red wine polyphenols are known to be implicated in the nitrite chemistry in the gastric lumen, enhancing production by univalent reduction of nitrite and consequent oxidation of the polyphenols to its o-semiquinone radical.
The work presented in this thesis shows that dietary nitrite is able to induce nitrosation (mostly S-nitrosation) of mucus glycoproteins (mucins) and in the gastric mucosa cells, pointing towards -mediated actions in the mucosa and to the filter effect of the mucus. This pattern is redox-modulated by red wine, suggesting novel actions for wine polyphenols in vivo via the balance of S- an N-nitroso compounds in the gastric wall. A highly sensitive chemiluminescence methodology was used to quantify the formation of nitroso compounds. S-nitrosothiols are fairly stable compounds that may act as carriers and exert both local and systemic impact. In fact, nitrosated mucus from rat stomach with acidified nitrite is shown to release at physiological pH. The alteration of the gastric environment by inflammation in vivo shows to increase nitrite induced nitrosation, particularly the S-nitrosothiols fraction.
Alongside with mucus production and blood flow regulation, dietary nitrite and its derivatives demonstrated to contribute for the maintenance of gastric mucosal integrity via the stimulation of the expression of an important signaling peptide, the trefoil factor 1 (TFF1) involved mucosal protection and anti-tumorigenesis. Again, the redox modulation of the nitrite chemistry by red wine plays an important role, particularly under inflammatory conditions, by increasing TFF1 expression.
The high concentration of nitrite and achieved in the stomach raised the question of how can gastric mitochondria cope with such challenge. Mitochondrial is a known target for and more recently was identified as a target for nitrite also. In fact, it was observed in these results that not only gastric mitochondria can deal with both and nitrite amounts easily achieved by a vegetables rich diet, but also mitochondrial function is improved with the nitrate-nitrite pathway. The analysis of mitochondrial function was achieved recurring to high-resolution oxygraphy. Mitochondria are key in cell function and signaling, and the modulation of their functionality by dietary derived inorganic molecules such as nitrite and can have major effects in gastric physiology and disease.
Taken together, these results highlight the relevance of bioactive compounds in everyday diet such as nitrate and nitrite, their interaction with other diet components as red wine and their impact in the gastric physiopathology.FCT - SFRH/BD/62265/200
The Apoptogenic Toxin AIP56 Is Secreted by the Type II Secretion System of Photobacterium damselae subsp. piscicida
AIP56 (apoptosis-inducing protein of 56 kDa) is a key virulence factor of Photobacterium damselae subsp. piscicida (Phdp), the causative agent of a septicaemia affecting warm water marine fish species. Phdp-associated pathology is triggered by AIP56, a short trip AB toxin with a metalloprotease A domain that cleaves the p65 subunit of NF-κB, an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes and plays a central role in host responses to infection. During infection by Phdp, AIP56 is systemically disseminated and induces apoptosis of macrophages and neutrophils, compromising the host phagocytic defence and contributing to the genesis of pathology. Although it is well established that the secretion of AIP56 is crucial for Phdp pathogenicity, the protein secretion systems operating in Phdp and the mechanism responsible for the extracellular release of the toxin remain unknown. Here, we report that Phdp encodes a type II secretion system (T2SS) and show that mutation of the EpsL component of this system impairs AIP56 secretion. This work demonstrates that Phdp has a functional T2SS that mediates secretion of its key virulence factor AIP56
The redox interplay between nitrite and nitric oxide: From the gut to the brain
The reversible redox conversion of nitrite and nitric oxide (·NO) in a physiological setting is now widely accepted. Nitrite has long been identified as a stable intermediate of ·NO oxidation but several lines of evidence support the reduction of nitrite to nitric oxide in vivo. In the gut, this notion implies that nitrate from dietary sources fuels the longstanding production of nitrite in the oral cavity followed by univalent reduction to ·NO in the stomach. Once formed, ·NO boosts a network of reactions, including the production of higher nitrogen oxides that may have a physiological impact via the post-translational modification of proteins and lipids. Dietary compounds, such as polyphenols, and different prandial states (secreting specific gastric mediators) modulate the outcome of these reactions. The gut has unusual characteristics that modulate nitrite and ·NO redox interplay: (1) wide range of pH (neutral vs acidic) and oxygen tension (c.a. 70 Torr in the stomach and nearly anoxic in the colon), (2) variable lumen content and (3) highly developed enteric nervous system (sensitive to ·NO and dietary compounds, such as glutamate). The redox interplay of nitrite and ·NO might also participate in the regulation of brain homeostasis upon neuronal glutamatergic stimulation in a process facilitated by ascorbate and a localized and transient decrease of oxygen tension. In a way reminiscent of that occurring in the stomach, a nitrite/·NO/ascorbate redox interplay in the brain at glutamatergic synapses, contributing to local ·NO increase, may impact on ·NO-mediated process.
We here discuss the implications of the redox conversion of nitrite to ·NO in the gut, how nitrite-derived ·NO may signal from the digestive to the central nervous system, influencing brain function, as well as a putative ascorbate-driven nitrite/NO pathway occurring in the brain
The Apoptogenic Toxin AIP56 Is Secreted by the Type II Secretion System of Photobacterium damselae subsp. piscicida
Characterisation of microbial attack on archaeological bone
As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved