Identity Determinants of the Translocation Signal for a Type 1 Secretion System.

Olivia Spitz,Ben F Luisi,Miao Ma,Lutz Schmitt,Sander H J Smits,Sandra Peherstorfer,Kerstin Kanonenberg,Jens Reiners,Michael H H Lenders,Isabelle N Erenburg

doi:10.3389/fphys.2021.804646

Olivia Spitz, Ben F Luisi + Show 8 more

Open Access

https://doi.org/10.3389/fphys.2021.804646

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Abstract

The toxin hemolysin A was first identified in uropathogenic E. coli strains and shown to be secreted in a one-step mechanism by a dedicated secretion machinery. This machinery, which belongs to the Type I secretion system family of the Gram-negative bacteria, is composed of the outer membrane protein TolC, the membrane fusion protein HlyD and the ABC transporter HlyB. The N-terminal domain of HlyA represents the toxin which is followed by a RTX (Repeats in Toxins) domain harboring nonapeptide repeat sequences and the secretion signal at the extreme C-terminus. This secretion signal, which is necessary and sufficient for secretion, does not appear to require a defined sequence, and the nature of the encoded signal remains unknown. Here, we have combined structure prediction based on the AlphaFold algorithm together with functional and in silico data to examine the role of secondary structure in secretion. Based on the presented data, a C-terminal, amphipathic helix is proposed between residues 975 and 987 that plays an essential role in the early steps of the secretion process.

Highlights

Type 1 secretion systems (T1SS) are widespread in Gram-negative bacteria and translocate a large variety of mainly proteinaceous substrates (Holland et al, 2016)
A well-known member of sub-family 2 of T1SS is the hemolysin A (HlyA) T1SS, which is composed of the ABC transporter HlyB, the membrane fusion protein HlyD and the outer membrane protein TolC [for recent reviews see Kanonenberg et al (2013)], which was first identified in uropathogenic E. coli strains (Felmlee et al, 1985)
Mutations were introduced in the pro-HlyA plasmid pSU-HlyA (Thomas et al, 2014a) by applying the quick-change PCR method using primers listed in Table 1 and following the protocol of the manufacturer (New England Biolabs)

Summary

Introduction

Type 1 secretion systems (T1SS) are widespread in Gram-negative bacteria and translocate a large variety of mainly proteinaceous substrates (Holland et al, 2016). The general blueprint of such a nanomachinery consists of an ABC transporter, a membrane fusion protein (MFP) and an outer membrane protein (OMP). A well-known member of sub-family 2 of T1SS is the hemolysin A (HlyA) T1SS, which is composed of the ABC transporter HlyB, the membrane fusion protein HlyD and the outer membrane protein TolC [for recent reviews see Kanonenberg et al (2013)], which was first identified in uropathogenic E. coli strains (Felmlee et al, 1985). The secretion signal of the substrate is located at the extreme C-terminus and is not cleaved prior, during or after transport (Gray et al, 1986). These substrates are characterized by Gly- and Asp-rich nonapeptide

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