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-¿B 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.This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/BIA-MIC/2007/2014 (POCI-01-0145-FEDER-016608). Ana do Vale was supported by the FCT fellowship SFRH/BPD/95777/2013. We thank Steve Leppla (Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, USA) for providing PA and Alexander E. Lang (Institute for Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Germany) for providing His-TccC3hvr. We are grateful to Paula Sampaio for assistance in the fluorescence microscopy and acknowledge the support of the ALM i3S Scientific Platform, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122) and to André Maia of the BioSciences Screening i3S Scientific Platform for assistance in image acquisition with IN Cell analyzer. We also acknowledge the support of the Biochemical and Biophysical Technologies i3S Scientific Platform in protein quantification and circular dichroism

Characterization and Vaccine Potential of Outer Membrane Vesicles from Photobacterium damselae subsp. piscicida

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative fish pathogen with worldwide distribution and broad host specificity that causes heavy economic losses in aquaculture. Although Phdp was first identified more than 50 years ago, its pathogenicity mechanisms are not completely understood. In this work, we report that Phdp secretes large amounts of outer membrane vesicles (OMVs) when cultured in vitro and during in vivo infection. These OMVs were morphologically characterized and the most abundant vesicle-associated proteins were identified. We also demonstrate that Phdp OMVs protect Phdp cells from the bactericidal activity of fish antimicrobial peptides, suggesting that secretion of OMVs is part of the strategy used by Phdp to evade host defense mechanisms. Importantly, the vaccination of sea bass (Dicentrarchus labrax) with adjuvant-free crude OMVs induced the production of anti-Phdp antibodies and resulted in partial protection against Phdp infection. These findings reveal new aspects of Phdp biology and may provide a basis for developing new vaccines against this pathogen.This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundacao para a Ciencia e a Tecnologia/Ministerio da Ciencia, Tecnologia e Ensino Superior (FCT) in the framework of the project POCI-01-0145-FEDER-030018 (PTDC/CVT-CVT/30018/2017). This work also received funding from the European Union's Horizon Europe research and innovation program under grant agreement No. 101084651 (project IGNITION). Views and opinions expressed are those of the authors only and do not necessarily reflect those of the European Union. The European Union cannot be held responsible for them. AdV was funded by Portuguese national funds through the FCT-Fundacao para a Ciencia e a Tecnologia, I.P. and, when eligible, by COMPETE 2020 FEDER funds, under the Scientific Employment Stimulus-Individual Call 2021.02251.CEECIND/CP1663/CT0016

The RstAB system impacts virulence, motility, cell morphology, penicillin tolerance and production of type II secretion system-dependent factors in the fish and human pathogen photobacterium damselae subsp. damselae

The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC) cytotoxins in the fish and human pathogen Photobacterium damselae subsp. damselae, a marine bacterium of the family Vibrionaceae. However, the function of the predicted cognate response regulator RstA has not been studied so far, and the role of the RstAB system in other cell functions and phenotypes remain uninvestigated. Here, we analyzed the effect of rstA and rstB mutations in cell fitness and in diverse virulence-related features. Both rstA and rstB mutants were severely impaired in virulence for sea bream and sea bass fish. Mutants in rstA and rstB genes were impaired in hemolysis and in Dly-dependent phospholipase activity but had intact PlpV-dependent phospholipase and ColP-dependent gelatinase activities. rstA and rstB mutants grown at 0.5% NaCl exhibited impaired swimming motility, enlarged cell size and impaired ability to separate after cell division, whereas at 1% NaCl the mutants exhibited normal phenotypes. Mutation of any of the two genes also impacted tolerance to benzylpenicillin. Notably, rstA and rstB mutants showed impaired secretion of a number of type II secretion system (T2SS)-dependent proteins, which included the three major cytotoxins Dly, PhlyP and PhlyC, as well as a putative delta-endotoxin and three additional uncharacterized proteins which might constitute novel virulence factors of this pathogenic bacterium. The analysis of the T2SS-dependent secretome of P. damselae subsp. damselae also led to the identification of RstAB-independent potential virulence factors as lipoproteins, sialidases and proteases. The RstAB regulon included plasmid, chromosome I and chromosome II-encoded genes that showed a differential distribution among isolates of this subspecies. This study establishes RstAB as a major regulator of virulence and diverse cellular functions in P. damselae subsp. damselae.This work has been supported by grant AGL2016-79738-R (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union. The support of Xunta de Galicia (Spain) with grant ED431C 2018/18 is also acknowledged. MT thanks the Brazilian Ministry of Education and CAPES (Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior) for a predoctoral fellowship. XM thanks Xunta de Galicia for a predoctoral fellowship. AdV was supported by the FCT fellowship SFRH/BPD/95777/2013. The mass spectrometry technique was performed at the Proteomics i3S Scientific Platform with the assistance of Hugo Osório. This work had support from the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125)

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 ¿-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.We are grateful for access to the HTX crystallization facility (Proposal ID: BIOSTRUCTX_8167). The support of the X-ray Crystallography Scientific Platform of i3S (Porto, Portugal) is also acknowledged. This work was financed by Fundo 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 (FCT) in the framework of the project POCI-01-0145-FEDER-030018 M8(PTDC/CVT-CVT/30018/2017). A.D.V. was supported by national funds from 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) and from the French Government’s Investissement d’Avenir program, Laboratoire d´Excellence “Integrative Biology of Emerging Infectious Diseases” (grant ANR-10-LABX-62-IBEID; http://www.agence-nationale-recherche.fr/investissements-d-avenir/). AR. was supported by a postdoctoral fellowship from the Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” and from an Infec-ERA grant (INTRABACWALL- 16-IFEC-0004-03)

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. © 2023, The Author(s).This work was supported by National funds through FCT under the project UIDB/04293/2020 and by FEDER funds through Programa Operacional Factores de Competitividade – COMPETE and by national funds through FCT – Fundação para a Ciência e a Tecnologia under the project PTDC/BIA-MIC/29910/2017 to N.M.S.S. A.d.V. was funded by Portuguese national funds through the FCT and, when eligible, by COMPETE 2020 FEDER funds, under the Scientific Employment Stimulus–Individual Call 2021.02251.CEECIND/CP1663/CT0016. We acknowledge access to the HTX crystallization facility (Proposal ID: BIOSTRUCTX_8167) and SOLEIL, ESRF and ALBA synchrotrons for provision of measurement time and thank their staff for help with data collection. The authors acknowledge the support of i3S Scientific Platforms (https://www.i3s.up.pt/scientific-platforms.php) Advanced Light Microscopy, member of the national infrastructure PPBI-Portuguese Platform of BioImaging (supported by POCI-01-0145-FEDER-022122), Animal Facility, Biochemical and Biophysical Technologies and X-ray Crystallography. A special thanks to Dr. Marc Graille and Dr. João Morais Cabral for constructive discussions in structural biology and Dr. Dimitri Panagiotis Papatheodorou for providing plasmid p327

Peritonitis and catheter exit-site infection in patients on peritoneal dialysis at home

Objective: to analyze the complications related to peritonitis and catheter exit-site infections, in patients on peritoneal dialysis at home.Method: quantitative and cross-sectional study, carried out with 90 patients on peritoneal dialysis at home, in a municipality in the Northeast region of Brazil. For data collection, it was used two structured scripts and consultation on medical records. Descriptive analysis and comparison tests among independent groups were used, considering p<0.05 as level of statistical significance.Results: by comparing the frequency of peritonitis and the length of treatment, it was found that patients over two years of peritoneal dialysis were more likely to develop peritonitis (X²=6.39; p=0.01). The number of episodes of peritoneal catheter exit-site infection showed association with the length of treatment (U=224,000; p=0.015).Conclusion: peritonitis and catheter exit-site infection are associated with the length of treatment

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.This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/BIA-MIC/2007/2014 (POCI-01-0145-FEDER-016608). Ana do Vale was supported by the FCT fellowship SFRH/BPD/95777/2013. We thank Steve Leppla (Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, USA) for providing PA. The authors acknowledge the support of the BioSciences Screening i3S Scientific Platform and the assistance of André Maia for the image acquisition with IN CELL analyzer. The authors also acknowledge the support of the Biochemical and Biophysical Technologies i3S Scientific Platform in protein quantification and circular dichroism