Final Report Summary - BACTINSECT (Molecular mechanisms in the establishment of disease transmission by invertebrate vectors)
Bacterial symbionts have a profound effect in the lifestyle of many insects playing major roles in adaptation, nutrient acquisition, protection against predators and infections. Some symbiotic interactions are complex and dynamic as those found in enteric consortia whilst others arise from an extreme level of specialization. In this project we aimed to investigate one of these rare interactions where the plant pathogen Pectobacterium carotovorum ECC15 has acquired the means to colonize and survive in the midgut of the fruit fly Drosophila melanogaster (Basset et al., 2003). Given its low spread amongst P. carotovorum strains, this novel symbiosis constitutes an extraordinary opportunity for the study of the origin of novel symbioses. The goal of my project was to investigate the molecular mechanisms behind monospecific bacterial-host interactions and to study the role that bacterial cell-cell communication (quorum sensing) plays in it. P. carotovorum ECC15 possesses a rare gene, named Erwinia virulence factor (EVF) which provides the bacterium with the ability to survive inside the midgut of D. melanogaster (Basset et al., 2003). Although the crystal structure of EVF has been solved and this gene has been carefully analyzed (Quevillon-Cheruel et al., 2009; Rigden, 2009) the lack of close homologues in any phyla together with the lack of biochemical characterization have hampered the elucidation of the EVF function. EVF has been shown to be controlled by Hor (Basset et al., 2003), a common bacterial regulator controlled by quorum sensing (Sjöblom et al., 2008) and a second regulator termed Erwinia virulence Regulator (EVR) (Lemaitre, personal communications, (Williamson et al., 2010)). Full genome sequencing in our lab revealed that P. carotovorum ECC15 is indeed a P. carotovorum strain closely related to strains PC1 and PCC21, discarding the possibility of a species misclassification. Evf originates from a recombination event in the P. carotovorum chromosome but the source of this gene remains unknown. Following a novel approach to study the impact of EVF in D. melanogaster larvae, we observed how larvae fed on P. carotovorum ECC15 WT displayed a delay in development when comparing to larvae fed on P. carotovorum ∆evf. This novel phenotype allowed us to screen multiple cell-cell signaling mutants that we constructed in P. carotovorum and led us to the discovery that EVF is controlled by quorum sensing. Where the AHL quorum sensing system has a major role followed by the Gac/Rsm cell-cell signaling system. Together these two cell-cell signaling systems control the expression of the HOR regulator that in turn regulates EVF expression. EVR, an additional regulator that had been postulated to be involved in EVF production has a minor role that seems insignificant.
In parallel, we aimed to dissect the quorum-sensing network of Pectobacterium spp. This was essential since previous data on the quorum-sensing network of this group of bacteria is scarce and has been diluted in the last three decades providing an unclear picture. This hampers our aim of elucidating the role of quorum sensing in EVF regulation. To answer this question, we used P. wasabiae SCC3193 (formerly misclassified as P. carotovorum) to dissect the network in Pectobacteria since we had in our lab a series of single knockouts in the quorum-sensing genes in this strain.
In the plant pathogen P. wasabiae, two cell-to-cell signaling networks - the N-acyl-homoserine lactone (AHL) quorum-sensing network and the Gac/Rsm signal transduction network- control the expression of plant cell-wall degrading enzymes (PCWDEs), its major virulence determinants. We revealed that the AHL system controls the Gac/Rsm system, by regulating the RNA, rsmB, providing evidence for a previously unreported link between these two cell-to-cell signaling networks. Next, using gacA and gacS mutants, we sought to investigate how much do AHLs modulate the levels of RsmB. This provided us with a surprising result: addition of exogenous AHLs could override the requirement of the Gac/Rsm system and bring in these mutants the level of RsmB to wt levels.
We investigated the potential advantages that having rsmB under control of the AHL-system could provide to pectobacteria. The fact that AHLs were sufficient to restore virulence induction in a gacA and a gacS mutant lead us to hypothesize that cross-species activation by AHL-producing bacteria could override the need for the Gac/Rsm system, enabling virulence activation even at low P. wasabiae cell density. To test this hypothesis, we examined the expression of rsmB and pehA (one of the PCWDEs responsible for virulence) in various P. wasabiae SCC3193 strains when mixed with a higher cell density of two P. carotovorum strains (Ecc71 and Ecc15). Our results revealed that in mixed communities, P. wasabiae can solely rely in the AHLs produced by other species to produce RsmB and consequently virulence. The work described here was performed in collaboration with Rita Valente and Filipe Vieira under the supervision of Karina Xavier.
In parallel, we aimed to dissect the quorum-sensing network of Pectobacterium spp. This was essential since previous data on the quorum-sensing network of this group of bacteria is scarce and has been diluted in the last three decades providing an unclear picture. This hampers our aim of elucidating the role of quorum sensing in EVF regulation. To answer this question, we used P. wasabiae SCC3193 (formerly misclassified as P. carotovorum) to dissect the network in Pectobacteria since we had in our lab a series of single knockouts in the quorum-sensing genes in this strain.
In the plant pathogen P. wasabiae, two cell-to-cell signaling networks - the N-acyl-homoserine lactone (AHL) quorum-sensing network and the Gac/Rsm signal transduction network- control the expression of plant cell-wall degrading enzymes (PCWDEs), its major virulence determinants. We revealed that the AHL system controls the Gac/Rsm system, by regulating the RNA, rsmB, providing evidence for a previously unreported link between these two cell-to-cell signaling networks. Next, using gacA and gacS mutants, we sought to investigate how much do AHLs modulate the levels of RsmB. This provided us with a surprising result: addition of exogenous AHLs could override the requirement of the Gac/Rsm system and bring in these mutants the level of RsmB to wt levels.
We investigated the potential advantages that having rsmB under control of the AHL-system could provide to pectobacteria. The fact that AHLs were sufficient to restore virulence induction in a gacA and a gacS mutant lead us to hypothesize that cross-species activation by AHL-producing bacteria could override the need for the Gac/Rsm system, enabling virulence activation even at low P. wasabiae cell density. To test this hypothesis, we examined the expression of rsmB and pehA (one of the PCWDEs responsible for virulence) in various P. wasabiae SCC3193 strains when mixed with a higher cell density of two P. carotovorum strains (Ecc71 and Ecc15). Our results revealed that in mixed communities, P. wasabiae can solely rely in the AHLs produced by other species to produce RsmB and consequently virulence. The work described here was performed in collaboration with Rita Valente and Filipe Vieira under the supervision of Karina Xavier.