Shutting Down Shigella Secretion: Characterizing Small Molecule Type Three Secretion System ATPase Inhibitors

Abstract

Many important human pathogens rely on one or more type three secretion systems (T3SS) to inject bacterial effector proteins directly into the host cell cytoplasm. Protein secretion through the needle-like type three secretion apparatus (T3SA) is essential for pathogen virulence and relies on a highly-conserved ATPase at the base of the apparatus, making it an attractive target for anti-infective therapeutics. Here, we leveraged the ability to purify an active oligomeric Shigella T3SS ATPase to provide kinetic analyses of three T3SS ATPase inhibitors of Spa47. In agreement with in silico docking simulations, the inhibitors displayed non-competitive inhibition profiles and efficiently reduced Spa47 ATPase activity with IC50s as low as 52 ± 3 μM. Two of the inhibitors functioned well in vivo, nearly abolishing effector protein secretion without significantly affecting Shigella growth phenotype or HeLa cell viability. Furthermore, characterization of Spa47 complexes in vitro and Shigella T3SA formation in vivo showed that the inhibitors do not function through disruption of Spa47 oligomers or by preventing T3SA formation. Together, these findings suggest that inhibitors targeting Spa47 may be an effective means of combating Shigella infection by shutting down type three secretion without preventing presentation of the highly antigenic T3SA tip proteins that aid in clearing the infection and developing pan-Shigella immunological memory. In summary, this is the first report of Shigella T3SS ATPase inhibitors and one of only a small number of studies characterizing T3SS ATPase inhibition in general. The work presented here provides much-needed insight into T3SS ATPase inhibition mechanisms and provides a strong platform for developing and evaluating non-antibiotic therapeutics targeting Spa47 and other T3SS ATPases.

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence

“…Each of the Spa47 constructs encoded in pTYB21 were transformed into E. coli Tuner (DE3) cells and were expressed and purified as previously described [22,29,30]. Briefly, the expression strains were grown to an OD 600 of approximately 0.8 in Terrific Broth medium containing 0.1 mg/ml ampicillin at 37˚C and 200 RPM.…”

On the road to structure-based development of anti-virulence therapeutics targeting the type III secretion system injectisome

“…103 Case et al (2018) identified 3 non-competitive inhibitors of Spa47 (SctN of Shigella flexneri) through virtual screening techniques and were predicted to bind at the interface between protomers based on modelling the oligomeric structure of Spa47 after a heterohexameric F-type ATPase. 105 Two inhibitors functioned in vivo, exhibiting almost no secretion without compromising S. flexneri or HeLa viability. All three compounds exhibited inhibition of the ATPase.…”

“…The authors note that IpaD is still present in needle complexes, suggesting that the inhibitors are inhibiting secretion, potentially via an ATP dependent manner. 105 New knowledge from the recent cryo-EM structure of homohexameric EscN (SctN of EPEC discussed above) may assist with a more targeted approach to drug design against the ATPase. 18 Phenoxyacetamide inhibitors have previously exhibited inhibitory properties towards the T3SS, with a few compounds displaying high nanomolar IC 50 values.…”

“…18 Previously, inhibitors against the ATPase of several T3SS's have been designed, based off of high throughput or virtual screening using homology modelling and notably in the absence of the central stalk component, which defined the observed asymmetry. [103][104][105]127 As the active site(s) sits between adjacent protomers, the precise architecture provided by the novel atomic model of EscN/O may assist with optimization of previously designed inhibitors through SAR techniques.…”

Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation

“…We have previously demonstrated that the Shigella protein Spa47 is a T3SS ATPase whose activity is essential for protein secretion through the apparatus and overall Shigella virulence . Furthermore, Spa47 oligomerization is required for ATPase activity, as residues from opposite sides of adjacent Spa47 protomers contribute to interfacial active sites within the complex, making Spa47 oligomerization a likely means of regulating T3SS activity in vivo and an attractive target for nonantibiotic therapeutics against Shigella infections …”