Role of AIP56 disulphide bond and its reduction by cytosolic redox systems for efficient intoxication.

Apoptosis-inducing protein of 56 kDa (AIP56) is a major virulence factor of Photobacterium damselae subsp. piscicida, a gram-negative pathogen that infects warm water fish species worldwide and causes serious economic losses in aquacultures. AIP56 is a single-chain AB toxin composed by two domains connected by an unstructured linker peptide flanked by two cysteine residues that form a disulphide bond. The A domain comprises a zinc-metalloprotease moiety that cleaves the NF-kB p65, and the B domain is involved in binding and internalisation of the toxin into susceptible cells. Previous experiments suggested that disruption of AIP56 disulphide bond partially compromised toxicity, but conclusive evidences supporting the importance of that bond in intoxication were lacking. Here, we show that although the disulphide bond of AIP56 is dispensable for receptor recognition, endocytosis, and membrane interaction, it needs to be intact for efficient translocation of the toxin into the cytosol. We also show that the host cell thioredoxin reductase-thioredoxin system is involved in AIP56 intoxication by reducing the disulphide bond of the toxin at the cytosol. The present study contributes to a better understanding of the molecular mechanisms operating during AIP56 intoxication and reveals common features shared with other AB toxins.

Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56.

Bacterial AB toxins are secreted key virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, bacterial AB toxins evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way. In this work, the determination of the three-dimensional structure of AIP56 shows that, instead of a two-domain organization suggested by previous studies, AIP56 has three-domains: a non-LEE encoded effector C (NleC)-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only upon endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation.

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-containing 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 ( PhotobacteriumNlpC-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.

Bearing My Heart: The Role of Extracellular Matrix on Cardiac Development, Homeostasis, and Injury Response.

The extracellular matrix (ECM) is an essential component of the heart that imparts fundamental cellular processes during organ development and homeostasis. Most cardiovascular diseases involve severe remodeling of the ECM, culminating in the formation of fibrotic tissue that is deleterious to organ function. Treatment schemes effective at managing fibrosis and promoting physiological ECM repair are not yet in reach. Of note, the composition of the cardiac ECM changes significantly in a short period after birth, concurrent with the loss of the regenerative capacity of the heart. This highlights the importance of understanding ECM composition and function headed for the development of more efficient therapies. In this review, we explore the impact of ECM alterations, throughout heart ontogeny and disease, on cardiac cells and debate available approaches to deeper insights on cell-ECM interactions, toward the design of new regenerative therapies.

Author Correction: Involvement of Hsp90 and cyclophilins in intoxication by AIP56, a metalloprotease toxin from Photobacterium damselae subsp. piscicida.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Involvement of Hsp90 and cyclophilins in intoxication by AIP56, a metalloprotease toxin from Photobacterium damselae subsp. piscicida.

AIP56 (apoptosis inducing protein of 56 kDa) is a key virulence factor secreted by virulent strains of Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes septicemic infections in several warm water marine fish species. AIP56 is systemically disseminated during infection and induces massive apoptosis of host macrophages and neutrophils, playing a decisive role in the disease outcome. AIP56 is a single-chain AB-type toxin, being composed by a metalloprotease A domain located at the N-terminal region connected to a C-terminal B domain, required for internalization of the toxin into susceptible cells. After binding to a still unidentified surface receptor, AIP56 is internalised through clathrin-mediated endocytosis, reaches early endosomes and translocates into the cytosol through a mechanism requiring endosomal acidification and involving low pH-induced unfolding of the toxin. At the cytosol, the catalytic domain of AIP56 cleaves NF-κB p65, leading to the apoptotic death of the intoxicated cells. It has been reported that host cytosolic factors, including host cell chaperones such as heat shock protein 90 (Hsp90) and peptidyl-prolyl cis/trans isomerases (PPIases), namely cyclophilin A/D (Cyp) and FK506-binding proteins (FKBP) are involved in the uptake of several bacterial AB toxins with ADP-ribosylating activity, but are dispensable for the uptake of other AB toxins with different enzymatic activities, such as Bacillus anthracis lethal toxin (a metalloprotease) or the large glycosylating toxins A and B of Clostridium difficile. Based on these findings, it has been proposed that the requirement for Hsp90/PPIases is a common and specific characteristic of ADP-ribosylating toxins. In the present work, we demonstrate that Hsp90 and the PPIases cyclophilin A/D are required for efficient intoxication by the metalloprotease toxin AIP56. We further show that those host cell factors interact with AIP56 in vitro and that the interactions increase when AIP56 is unfolded. The interaction with Hsp90 was also demonstrated in intact cells, at 30 min post-treatment with AIP56, suggesting that it occurs during or shortly after translocation of the toxin from endosomes into the cytosol. Based on these findings, we propose that the participation of Hsp90 and Cyp in bacterial toxin entry may be more disseminated than initially expected, and may include toxins with different catalytic activities.

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.

Dietary nitrite induces nitrosation of the gastric mucosa: the protective action of the mucus and the modulatory effect of red wine.

The stomach chemical environment promotes the production of new molecules that can induce post-translational modifications of endogenous proteins with physiological impact. The nitrate-nitrite-nitric oxide pathway is relevant in this process via production of nitric oxide ((•)NO) and nitric oxide-derived nitrogen oxides (NOx) at high concentrations. Using a highly sensitive and selective chemiluminescence approach, we found that exposure the stomach of rats to nitrite yielded S- and N-nitroso derivatives in gastric mucus cysteine-rich glycoproteins (mucins). To lesser extent, the underlying epithelial cell layers also suffered nitrite-driven S- and N-nitroso modifications which increased upon mucus removal, indicating that, under normal nitrite load, (•)NO and NOx can reach inner layers of the stomach wall and locally modify proteins. S-nitrosation was by large the predominant modification. In vitro and ex vivo experiments indicated that the gastric nitrosation pattern is triggered by dietary nitrite in a concentration dependent manner, encompassing the intermediary formation of (•)NO and is susceptible to modulation by dietary reductants, notably red wine polyphenols. Collectively, these results suggest a protective action of the mucus and potential (•)NO-dependent biochemical effects at deeper cells layers of the mucosa.

A shortcut to wide-ranging biological actions of dietary polyphenols: modulation of the nitrate-nitrite-nitric oxide pathway in the gut.

Dietary polyphenols are complex, natural compounds with recognized health benefits. Initially attractive to the biomedical area due to their in vitro antioxidant properties, the biological implications of polyphenols are now known to be far from their acute ability to scavenge free radicals but rather to modulate redox signaling pathways. Actually, it is now recognized that dietary polyphenols are extensively metabolized in vivo and that the chemical, biophysical and biological properties of their metabolites are, in most cases, quite different from the ones of the parent molecules. Hence, the study of the metabolic, absorptive and signaling pathways of both phenolics and derivatives has become a major issue. In this paper we propose a short-cut for the systemic effects of polyphenols in connection with nitric oxide (˙NO) biology. This free radical is a ubiquitous signaling molecule with pivotal functions in vivo. It is produced through an enzymatic pathway and also through the reduction of dietary nitrate and nitrite in the human stomach. At acidic gastric pH, dietary polyphenols, in the form they are conveyed in foods and at high concentration, not only promote nitrite reduction to ˙NO but also embark in a complex network of chemical reactions to produce higher nitrogen oxides with signaling functions, namely by inducing post-translational modifications. Modified endogenous molecules, such as nitrated proteins and lipids, acquire important physiological functions. Thus, local and systemic effects of ˙NO such as modulation of vascular tone, mucus production in the gut and protection against ischemia-reperfusion injury are, in this sense, triggered by dietary polyphenols. Evidence to support the signaling and biological effects of polyphenols by modulation of the nitrate-nitrite-NO pathway will be herein provided and discussed. General actions of polyphenols encompassing absorption and metabolism in the intestine/liver are short-cut via the production of diffusible species in the stomach that have not only a local but also a general impact.

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.

Dietary nitrite in nitric oxide biology: a redox interplay with implications for pathophysiology and therapeutics.

Until recently, nitrite has been considered a stable oxidation inert metabolite of nitric oxide ((∙)NO) metabolism. This view is now changing as it has been shown that nitrite can be reduced back to (∙)NO and thus one may consider a reversible interaction regarding (∙)NO:nitrite couple. Not only physiological regulatory actions have been assigned to nitrite but also may represent, in addition to nitrate, the largest (∙)NO reservoir in the body. This notion has obvious importance when considering that (∙)NO is a ubiquitous regulator of cell functions, ranging from neuromodulation to the regulation of vascular tone. Particularly in the stomach, following ingestion of nitrate and food or beverages-containing polyphenols, a rich chemistry occurs in which (∙)NO, (∙)NO-derived species and nitroso or nitrated derivatives may be formed. Most of these molecules may play an important role in vivo. For instance, it has been shown that polyphenol-catalyzed nitrite reduction to (∙)NO may induce local vasodilation and that ethanol (from wine) reacts with (∙)NO-derived species yielding nitroso derivatives endowed with (∙)NO-donating properties. Thus, this review reveals new pathways for the biological effects of dietary nitrite encompassing its interaction with dietary components (polyphenols, red wine, lipids), yielding products with impact on human physiology and pathology, namely cardiovascular, urinary and gastrointestinal systems. Novel therapeutic strategies are therefore expected to follow the elucidation of the mechanisms of nitrite biology.