Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / JOB Viviana
Contact person(s) related to this article / JOB Viviana
Contact person(s) related to this article / JOB Viviana

Exolysin and new virulence strategy of clinical strains

Until 2014, it was widely admitted in the Pseudomonas community, that the main pathogenic mechanism of P. aeruginosa requires T3SS. This dogma was challenged when we discovered a novel toxin, named Exolysin (ExlA) required for cell and tissue destruction [1]. We demonstrated that ExlA possesses a pore-forming activity, leading to cell death [2,3]. In collaboration with Dessen’s team (PATBAC) we gained knowledge on the ExlA structure-function relationship [4].

https://youtu.be/gpVClMUGmoM

Fig. 1 : Partial structural model of ExlA based on homologous sequence elements present in the Protein Data Bank. (Reboud et al., Toxins 2017)

References
[1] Elsen et al (2014) Cell host & microbe 15(2):164-176.,
[2] Basso et al (2017) mBio 8(1).
[3] Reboud et al (2017) Toxins 9 ;9(11):364. doi : 10.3390/toxins9110364.
[4] Bertrand Q et al (2020) J Mol Biol. 24 ;432(16):4466-4480. doi : 10.1016/j.jmb.2020.05.025.

ExlA induces disruption of cell-cell junctions

ExlA+ strains induce a disruption of cell-cell junctions.

Furthermore, E-cadherin at epithelial junctions and VE-cadherin at endothelial junctions, were missing after incubation with bacteria. We found that ADAM10, an eukaryotic transmembrane protease, whose natural substrates are the cadherins, is activated by ExlA. ExlA pore formation induces a calcium influx that triggers ADAM10 activation and export to the plasma membrane, where it cleaves the cadherins.
Furthermore, our work demonstrates that ADAM10 is not the cellular receptor for ExlA and that calcium influx is also responsible for the cell death program induced by ExlA. In addition, we showed that the mechanism of ADAM10 activation/cadherin cleavage was similarly induced by ShlA, a pore-forming toxin secreted by Serratia marcescens, suggesting that this mechanism might be used by several pore-forming toxins [5].


Figure from Reboud E. et al (2017) PLoS Pathogens

Reference
[5] Reboud et al (2017) PLoS pathogens 13(8):e1006579.,

ExlA toxicity in vivo

We first phenotypically analyzed ExlA-positive cohort collected worldwide revealing differential levels of expression of virulence factors and different motility patterns [6].
In the mouse, ExlA promotes bacterial growth in infected lungs and dissemination in several organs. It induces major lesions in lung alveoli, owing to its capacity to trigger necrosis of epithelial and endothelial cells, hence provoking hemorrhages by rupture of the alveolo-capillary barrier [7]. P. aeruginosa-induced hyperinflammation is known to be deleterious for the host. Inflammation is orchestrated by inflammasome activation within resident macrophages, leading to caspase-1-dependent IL-1β maturation and pyroptosis. IHMA87, the ExlA-positive strain that was particularly studied in the lab, was able of inflammasome activation in bone marrow-derived macrophages, leading to pyroptosis and IL-1β secretion [8]. To understand the role of inflammasome in IHMA87 infection in vivo, caspase-I-deficient and wild-type mice were infected using an acute pneumonia model. Infected mice survived better when they lacked caspase-1, indicating that the hyperinflammation induced by the presence of IHMA87 is detrimental for the animals. Some pro-inflammatory cytokines (IL-6, IL-17a and KC) are less produced and less neutrophils are recruited in caspase-I-deficient lungs. Thus, as for T3SS-positive strains, IHMA87 triggers an excessive inflammasome-dependent inflammatory response that is deleterious for the host.

Fig 2. : Macrophage damage induced by Pseudomonas aeruginosa (ExlA+) strain.

Références
[6] Reboud et al (2016) Environmental microbiology 18(10):3425-3439.,
[7] Bouillot et al (2017) Scientific reports 7(1):2120.,
[8] Basso et al (2017) Environmental microbiology 19(10):4045-4064.