Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa

Abstract

Type Three Secretion Systems (T3SS) from many gram‐negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically, and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with Vmax of 3.9 ± 0.2 μmol/min/mg and K m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni‐NTA pull‐down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN–PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN–PscL heterotrimeric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN–PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN–PscL complex in T3SS mediated pathogenic pathway.

Pseudomonas aeruginosa Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections

“…The structure of the T3SS comprises a macromolecular complex spanning the bacterium's internal membrane, periplasmic space, peptidoglycan layer, outer membrane, then the extracellular space and the target cell membrane [71,72]. Upon contact with the target cell, a translocation pore can form across the host cell membrane, allowing the T3SS injectisome to actively secrete the exotoxins from the bacterial cytosol into the host cytosol under the influence of ATPases [14,[73][74][75]. The T3SS injectisome has a narrow lumen of approximately 25-40 Å diameter, formed by an assembly of helical PscF proteins [72,76,77]; thus, the exotoxins have to unfold to pass through the channel.…”

The PopN gate-keeper complex acts on the ATPase PscN to regulate the T3SS secretion switch from early to middle substrates inPseudomonas aeruginosa

“…This indicates that although SctN shares 40-50% of sequence identity among the Gramnegative pathogenic bacteria (Zarivach et al, 2007), the T3SS ATPase of P. aeruginosa is more difficult to produce in E. coli. Indeed, a recent study of PscN required refolding after solubilisation of inclusion bodies (Halder et al, 2018). Therefore, in order to obtain the purified protein, we fused PscN to a triple tag and achieved efficient production and purification under native conditions from an engineered P. aeruginosa strain (Arora et al, 1997;Brunschwig and Darzins, 1992).…”

“…The interaction of PscN with its negative regulator PscL was recently described using both proteins refolded from inclusion bodies (Halder et al, 2018). Using the construct employed for the pull-down study, several attempts failed to provide enough material for biochemical characterization.…”

“…PscN was shown to interact with the stator PscL (SctL) (Halder et al, 2018) while SctN from other species was shown to interact with several classes of exported substrates, such as effector, translocator and gate-keeper proteins (Akeda and Galan, 2005;Akeda and Galán, 2004;Allison et al, 2014;Botteaux et al, 2009;Cooper et al, 2010;Gauthier, A. and Finlay, 2003;Lorenz and Buttner, 2009;Zarivach et al, 2007). To further explore the binding spectrum and specificities of PscN, an ELISAbased assay was performed with purified FSS-PscN and purified partner proteins including effector, translocator, needle and gate-keeper proteins (the cargos) and their chaperones in complex or alone.…”

Hyperpiliation reducesPseudomonas aeruginosapathogenicity by impeding type III secretion

“…We tested the contribution of T3S to slow killing of C. elegans by strain PAK by generating a pscN deletion mutant lacking the T3S system ATPase, which prevents the secretion of toxic effectors as shown for Yersinia enterocolitica (50) and a number of other T3SS-expressing pathogens (51)(52)(53)(54). Loss of pscN significantly reduced the pathogenicity of PAK in the slow killing assay (Figure 4A).…”