• Título:  CHARACTERIZATION OF THE HIGH-PATHOGENICITY ISLAND ENCODING THE SIDEROPHORE PISCIBACTIN IN VIBRIO ANGUILLARUM: EFFECTS ON BACTERIAL VIRULENCE AND IMPLICATIONS FOR THE CONTROL OF VIBRIOSIS IN FISH
• Autor:  Afonso Lages, Marta Carolina
• Universidad:  Universidad de Santiago de Compostela
• Departamento:  Escuela de Doctorado Internacional (EDIUS)
• Fecha de Lectura:  18/03/2022
• Mención Europeo / Mención Internacional: concedido
• Programa de doctorado:  Programa de Doctorado en Avances en Biología Microbiana y Parasitaria por la Universidad de Santiago de Compostela
• Dirección: 
  • Lemos Ramos, Manuel Luis (DIRECTOR)
  • Balado Dacosta, Miguel (CODIRECTOR)
• Tribunal: 
  • López Romalde, Jesús Angel (PRESIDENTE)
  • Amaro González, Carmen (SECRETARIO)
  • Rivas Fontenla, Amable José (VOCAL)
• Descriptores: 
  • BIOLOGIA MOLECULAR DE MICROORGANISMOS
• Localización:  BIBLIOTECA XERAL USC
• 624360_1418682.pdf
• Resumen:
  Vibrio anguillarum is one of the most important pathogens for aquacultured fish, causing vibriosis, a haemorrhagic septicaemia that affects fish species living at different temperatures. Besides, V. anguillarum is an inhabitant of marine and estuarine environments, so it must be able to adapt its physiology to the changing levels of salinity, nutrients, and temperature. Although water temperature raising is usually associated with a higher incidence of vibriosis, 15 °C is considered enough for the occurrence of vibriosis outbreaks. In this work, we analyzed the physiological adaptations of V. anguillarum to low-iron availability and temperature changes that enable it to cause vibriosis in a wide diversity of fish species. Some of these adaptations include the expression of virulence factors that will led to the disease. We used the serotype O2a pathogenic strain RV22 as a model of study. The faster growth shown by V. anguillarum RV22 at 25 ºC compared to growth at 15 ºC, contrasts with its higher virulence at 15 ºC. Temperature and iron levels usually act as signals during host colonization. Therefore, first we analyzed by RNAseq the effect of iron levels and temperature changes on the transcriptome of V. anguillarum RV22. The comparative analysis revealed that under iron deficiency deep changes in the metabolism appear and virulence factors are induced. Metabolic routes requiring iron such as TCA cycle and cytochromes were down-regulated, while genes related to the glycolytic and pentose phosphate pathways were up-regulated. Some components of amino acids and carbohydrate metabolism were down-regulated at 15 ºC, which suggests a slower nutrient import rate at 15 ºC. Interestingly, V. anguillarum expresses different virulence factors at 15 ºC or 25 ºC. At warm temperature, V. anguillarum induces chemotaxis, motility, exopolysaccharide biosynthesis, haemolysin Vah1, T6SS, and the outer membrane components OmpA and OmpV. Conversely, the haemolysin RTX, T6SS2, OmpC, and genes related to exopolysaccharide transport and assembly, were induced preferentially at 15 ºC. Regarding iron uptake systems, ferrous iron uptake feoB, the vanchrobactin siderophore system and haem utilization were preferentially expressed at 25 ºC whereas the piscibactin siderophore system was strongly up-regulated at 15 ºC. The differential expression of virulence factors in relation to temperature indicates that their relevance may vary between hosts, enabling V. anguillarum to infect fish species living at cold and warm waters. Siderophores are key virulence factors in most pathogens. In V. anguillarum, two siderophore systems have been described: the pJM1 plasmid-encoded anguibactin, which is restricted to serotype O1 strains, and the chromosomally encoded vanchrobactin, which is widespread among pathogenic and environmental isolates. In addition, the genome of V. anguillarum RV22 contains a genomic island that is homologous to the pathogenicity island irp-HPI harbored in plasmid pPHDP70 of Photobacterium damselae subsp. piscicida (Pdp). This island encodes the siderophore piscibactin and it is likely spread among bacteria through HGT. An in silico analysis revealed that irp-HPI is present in several highly virulent V. anguillarum strains that have an active vanchrobactin system. In fact, the 4¿-phosphopantetheinyl transferase activity needed for piscibactin synthesis is only encoded by vabD, which is part of the vanchrobactin gene cluster. The chemical analysis clearly demonstrated that V. anguillarum synthesizes piscibactin in addition to vanchrobactin. In vitro growth ability and virulence assays performed with single and combined mutants, suggest that vanchrobactin could be more related to environmental survival, while piscibactin is a key virulence factor for V. anguillarum pathogenesis. The irp-HPI island putatively encodes synthesis and transport of piscibactin and their regulation. The construction of mutants by allelic exchange allowed the elucidation of the role of Irp1, Irp4, Irp8 and FrpABC in piscibactin biosynthesis and utilization. Irp1 is the main NRPS/PKS essential for piscibactin synthesis. The type II thioesterase Irp4 is required for the biosynthesis final steps. Piscibactin is secreted out of the cell through the MFS protein Irp8 located at the plasma membrane. The ferri-piscibactin complex reaches the periplasm through FrpA, a TonB-dependent outer membrane transporter, and it is transported through the inner membrane by the ABC-type transporter FrpBC. To identify the key structural elements of piscibactin necessary for its transport through FrpA, piscibactin analogues were synthesized and their ability to act as iron source was investigated. The growth promotion assays identified the configuration of C-13 as crucial for iron chelation and recognition by FrpA. The widespread distribution of FrpA suggests that piscibactin analogues carrying antimicrobials could be used in novel therapies against a wide range of pathogens. The transcriptional analysis of irp-HPI showed that piscibactin promoters PpbtA, PfrpA and PfrpBC respond to iron levels and temperature. The measure of transcriptional activity revealed that piscibactin regulatory (PpbtA) and transport genes (PfrpA and PfrpBC) are preferentially expressed under iron deficiency at 15 ºC. irp-HPI putatively encodes two AraC-like transcriptional regulators, PbtA and PbtB. In addition, the global regulators H-NS and ToxR-S are also present in V. anguillarum. To analyze the regulation of irp-HPI expression, in-frame deletion mutants of pbtA, pbtB and the global regulators h-ns and toxR-S were constructed and their relevance for growth under iron deficiency and irp-HPI gene expression was analyzed at 15 and 25 ºC. The results showed that whereas pbtA is required for the expression of piscibactin biosynthetic and transport genes, pbtB is not. The inactivation of H-NS resulted in a decreased growth under iron deficiency and a low activity of PfrpA at 15 and 25 ºC. Inactivation of ToxR-S increased the transcriptional activity of PfrpA and PpbtA at 25 ºC. Notably, the inactivation of PbtA and H-NS resulted in a marked decrease of virulence for fish, which corroborates the importance of piscibactin for the virulence of V. anguillarum. The results suggest that PbtA is the main modulator of irp-HPI gene expression, and that H-NS is indirectly required for the maximum production of piscibactin. ToxR-S could be involved in the repression of piscibactin genes at 25 ºC. Several versions of the promoters PpbtA and PfrpA were identified in different bacteria harbouring irp-HPI. PfrpA showed the highest variability, which indicates that different expression patterns might be present. For instance, the shorter version of PfrpA present in Pdp does not act as transcription start and, in contrast to V. anguillarum, irp-HPI from Pdp did not show a temperature dependent expression. However, when PfrpA and PpbtA promoters of irp-HPI from Pdp were cloned into V. anguillarum, they also showed a temperature-dependent expression pattern. This indicates that the temperature-dependent expression of irp-HPI depends on activator(s) present in V. anguillarum and absent in Pdp. Since siderophore systems are expressed during the infection process, we finally evaluated the use of the siderophore outer membrane transporters FrpA and FvtA as subunit vaccines against vibriosis. Fish were immunized with the recombinant proteins rFrpA or rFvtA, a RV22 or Pdp bacterin and the antibody levels were measured by ELISA. Both FrpA and FvtA are immunogenic proteins, but they confer limited protection against V. anguillarum infection. However, it is noteworthy that V. anguillarum FrpA conferred cross protection against Pdp. This result indicates that the widespread distribution of FrpA could be used to formulate subunit vaccines against irp-HPI-harbouring pathogens. In summary, in this thesis we demonstrated that V. anguillarum expresses a specific cocktail of virulence factors depending on the environmental temperature, which enables it to infect fish living at cold and warm waters. In addition, V. anguillarum produces the siderophore piscibactin as a key virulence factor, whose expression is regulated by iron levels and favoured at low temperatures.