Projecto nº:016608

Referência do Projecto:PTDC/BIA-MIC/2007/2014 (POCI-01-0145-FEDER-016608)

Título:Host cell factors and molecular mechanisms implicated in intoxication by AIP56, a bacterial

toxin targeting NF-kB

Montante envolvidos:

Investimento total: 198.048,00 €

IBMC-Instituto de Biologia Molecular e Celular - 198.048,00€

Apoio FEDER: 168.340,80€

Apoio OE: 29.707,20€

Localização do projecto: Porto, Portugal

Sintese do projecto:

AB toxins are potent proteins secreted by bacteria into the extracellular medium that are able to reach the cytosol of eukaryotic cells. For many pathogens, they are the main virulence determinants and elicit most of the symptoms and lesions associated to infection. AB toxins possess multi-domain structures adapted to their mode of action [1], being composed of an enzymatically active A subunit and a B subunit responsible for delivering the A subunit into the cytosol. By modifying key cytosolic factors, AB toxins modulate host cell functions in favour of microbial infection. Depending on their specific target, they can interfere with a variety of key cellular processes such as protein synthesis (e.g. diphtheria and shiga toxins), cAMP levels (e.g. cholera and adenylate cyclase toxins), cytoskeleton structure (e.g. Clostridium difficile toxins A and B) and neurotransmitter release (tetanus and botulinum toxins) [2]. Studies of AB toxins have been fundamental to the understanding of many infectious diseases and also to elucidate fundamental issues in cell biology.

Amongst the most recently discovered bacterial AB toxins is AIP56 (apoptosis-inducing protein of 56 kDa), a key virulence factor of Photobacterium damselae piscicida, a Gram-negative bacterial pathogen that infects economically important marine fish [3]. AIP56 is the founding and only characterized member of a recently uncovered family of bacterial proteins (Annex 1) that includes members found mainly in marine Vibrio species. Interestingly, AIP56 seems to have originated from a fusion of two components: its A domain is related to NleC, a type III secreted effector present in several enteric pathogenic bacteria associated with human illness and death worldwide while its B domain is related to a protein of unknown function from the bacteriophage APSE2. Our previous work showed that AIP56 is a zinc metalloprotease that acts by cleaving NF-kB p65 in target cells, leading to apoptosis [4, 5]. We further showed that AIP56 reaches its intracellular target using a mechanism that is conserved between fish and mammalian cells [6]. After binding to a still unidentified cell surface receptor, the toxin undergoes clathrin-dependent endocytosis and low pH-triggered translocation across the endosomal membrane into the cytosol [6]. Remarkably, a significant pool of endocytosed AIP56 is recycled back to the extracellular medium, representing the first example of an AB toxin following the endocytic recycling pathway [6].Although our recent work allowed uncovering structural determinants of AIP56 required for different steps of intoxication and defining the intracellular trafficking routes followed by the toxin [6], the molecular and cellular mechanisms that confer to AIP56 its specificities remain to be defined. In this project, we propose to address this issue, using a combination of hypothesis-driven research based on previous results of our lab and global proteomic analysis of intoxicated cells. We anticipate that the results of this project will allow a more comprehensive view of the mechanisms involved in AIP56 toxicity. This will not only broaden our understanding of AB toxin biology and microbial pathogenicity but may contribute to disclose new molecular mechanisms of endocytosis and intracellular transport in eukaryotic cells. Additionally, considering that uncontrolled activation of NF κB is associated with multiple human pathologies, including inflammatory diseases and cancers [7], a deeper understanding of the intoxication mechanism used by AIP56 may foster its use as anticancer or anti-inflammatory therapeutics. Finally, since the delivery of proteins directly into viable cells has a huge biotechnological interest, a fine knowledge of the molecular mechanism dictating the uptake and intracellular routes followed by AIP56 may support the use of engineered versions of the toxin as delivery

systems able to translocate different cargos into eukaryotic cells. We strongly feel that our research team is well qualified for this work and that our skills, together with the excellent human resources and high-quality equipment/services of the Proponent Institution will allow this project to be carried out successfully.

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