MARTX Toxin in the Zoonotic Serovar of Vibrio vulnificus Triggers an Early Cytokine Storm in Mice

Abstract

Vibrio vulnificus biotype 2-serovar E is a zoonotic clonal complex that can cause death by sepsis in humans and fish. Unlike other biotypes, Bt2 produces a unique type of MARTXVv (Multifunctional-Autoprocessive-Repeats-in-Toxin; RtxA13), which is encoded by a gene duplicated in the pVvBt2 plasmid and chromosome II. In this work, we analyzed the activity of this toxin and its role in human sepsis by performing in vitro, ex vivo, and in vivo assays. First, we demonstrated that the ACD domain, present exclusively in this toxin variant, effectively has an actin-cross-linking activity. Second, we determined that the whole toxin caused death of human endotheliocytes and monocytes by lysis and apoptosis, respectively. Finally, we tested the hypothesis that RtxA13 contributes to human death caused by this zoonotic serovar by triggering an early cytokine storm in blood. To this end, we used a Bt2-SerE strain (R99) together with its rtxA13 deficient mutant, and a Bt1 strain (YJ016) producing RtxA11 (the most studied MARTXVv) together with its rtxA11 deficient mutant, as controls. Our results showed that RtxA13 was essential for virulence, as R99ΔΔrtxA13 was completely avirulent in our murine model of infection, and that R99, but not strain YJ016, induced an early, strong and dysregulated immune response involving the up-regulation of a high number of genes. This dysregulated immune response was directly linked to RtxA13. Based on these results and those obtained ex vivo (human blood), we propose a model of infection for the zoonotic serovar of V. vulnificus, in which RtxA13 would act as a sepsis-inducing toxin.

Adaptation to host in Vibrio vulnificus, a zoonotic pathogen that causes septicemia in fish and humans

“…When RtxA1 interacts with the appropriate immune cell, it triggers a cytokine storm by inducing the overexpression of inflammation mediators as described by Murciano et al . () [Figure modified from the work of Murciano et al . ()].…”

“…() [Figure modified from the work of Murciano et al . ()]. Bacterial and eukaryotic cells are not drawn to scale.…”

“…The protected bacteria multiply and produce both VvhA (yellow ball) and RtxA1 (grey bars), which collaborate in cellular destruction (3). When RtxA1 interacts with the appropriate immune cell, it triggers a cytokine storm by inducing the overexpression of inflammation mediators as described by Murciano et al (2017) were transcribed in parallel and exponentially in artificial iron-overloaded human blood during the 6 h of incubation we analysed. A role for this additive function in blood invasion from the intestine has already been suggested for V. vulnificus strains, other than the zoonotic ones (Jeong and Satchell, 2012).…”

“…Bacterial strains (Supporting Information Table S1) were grown in TSA‐1 (TSA, 1% NaCl), CM9 (Miller, ), CM9+Tf [CM9 + 10 μM human transferrin (Sigma‐Aldrich)]), CM9‐Fe [CM9 + 200 or 400 μM of the iron chelator 2,2′‐bipyridyl (Sigma‐Aldrich)], CM9+Fe (CM9 + 100 μM FeCl 3 ), eel serum [obtained as previously described (Lee et al ., )], iron‐overloaded eel serum (eel serum + 2.5, 5 or 100 μM FeCl 3 ), human serum (Sigma‐Aldrich), and iron‐overloaded human serum [human serum +100 μM FeCl 3 (Murciano et al ., )]. Unless clearly specified, 2.5 ml of each serum/broth were inoculated with washed bacteria (10 5 CFU ml −1 ) and incubated with agitation (50 rpm) at 28°C (eels) or 37°C (humans).…”

“…Currently, the zoonotic clonal-complex is present worldwide and includes strains isolated over a 40-year period from humans, diseased/healthy animals and water-samples (Roig et al, 2018). The most relevant virulence factors described in the zoonotic clonal-complex are the RtxA1 toxin, involved in invasion and death by sepsis (Jeong and Satchell, 2012;Lee et al, 2013;Callol et al, 2015;Murciano et al, 2017), a capsule, involved in resistance to human serum, and the O-antigen, conferring resistance to eel serum (Amaro et al, 1994;Amaro et al, 1997). No role has been assigned to VvhA, an hemolysin that seems to have an additive function to RtxA1 in strains from which it has been analysed, all of which belong to lineage 1 (Jeong and Satchell, 2012).…”