ExlA Pore-Forming Toxin: Localization at the Bacterial Membrane, Regulation of Secretion by Cyclic-Di-GMP, and Detection In Vivo

Abstract

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.

ExlA: A New Contributor to Pseudomonas aeruginosa Virulence

“…The current hypothesis is thus that the active form of the toxin is that anchored by ExlB in the outer membrane, rather than the secreted protein (Figure 1C). Proteinase digestion experiments showed that a part of ExlA is pointing out of the bacterial membrane and further supports a model in which ExlA, entrapped in the outer membrane, could interact with a potential receptor located at the host cell surface (Deruelle et al, 2021). This secretion model is analogous to that described for contact-dependent inhibition (CDI) toxins, belonging to the TPS family, for which passenger translocation across the outer membrane stalls at mid-length in the TPS transporter and resumes upon interaction with a target receptor (Ruhe et al, 2018).…”

“…Both ExlA and ExlB are predicted to be secreted into the periplasm by the sec pathway. ExlA accumulates at the outer membrane as shown by cellular fractionation experiments ( Deruelle et al., 2021 ) ( Figure 1C ). Microscopy immunolocalization identified ExlA spots at the bacterial periphery.…”

“…ExlA secretion from the outer membrane into the extracellular milieu is controlled by cyclic-di-GMP ( Figure 1C ), a second messenger known to induce the production of biofilm components by P. aeruginosa ( Hall and Lee, 2018 ). High bacterial concentrations of c-di-GMP have no effect on exlB–exlA transcription but retain ExlA at the membrane, which is, as previously mentioned, predicted to increase bacterial virulence ( Deruelle et al., 2021 ). The signal transduction pathway transferring the signal from cytosolic c-di-GMP to ExlA and ExlB, both located at the outer membrane, remains unknown and probably involves shuttle proteins in the periplasm.…”

“…In erfA mutant bacteria, the proportion of ExlA-positive cells increased to ~10%, confirming that ExlAB synthesis repression is induced by ErfA. In the erfA mutant, the signal intensities increased rather than the number of spots, suggesting that multiple ExlB proteins aggregate in the same membrane location when they are overproduced ( Deruelle et al., 2021 ).…”

“…Only very low amounts of ExlA are detected in PA7-like strain secretomes ( Reboud et al., 2016 ), and they are at the limit of detection by ELISA ( Deruelle et al., 2021 ). Furthermore, secretomes do not induce the cytolytic activity observed when cells are in direct contact with bacteria, leading to the hypothesis that ExlA toxicity is contact-dependent ( Basso et al., 2017a ).…”