A multi-pronged search for a common structural motif in the secretion signal of Salmonella enterica serovar Typhimurium type III effector proteins

Many pathogenic Gram-negative bacteria use a type III secretion system (T3SS) to deliver effector proteins into the host cell where they reprogram host defenses and facilitate pathogenesis. The first 20-30 N-terminal residues usually contain the 'secretion signal' that targets effector proteins for translocation, however, a consensus sequence motif has never been discerned. Recent machine-learning approaches, such as support vector machine (SVM)-based Identification and Evaluation of Virulence Effectors (SIEVE), have improved the ability to identify effector proteins from genomics sequence information. While these methods all suggest that the T3SS secretion signal has a characteristic amino acid composition bias, it is still unclear if the amino acid pattern is important and if there are any unifying structural properties that direct recognition. To address these issues a peptide corresponding to the secretion signal for Salmonella enterica serovar Typhimurium effector SseJ was synthesized (residues 1-30, SseJ) along with scrambled peptides of the same amino acid composition that produced high (SseJ-H) and low (SseJ-L) SIEVE scores. The secretion properties of these three peptides were tested using a secretion signal-CyaA fusion assay and their structural properties probed using circular dichroism, nuclear magnetic resonance, and ion mobility spectrometry-mass spectrometry. The secretion predictions from SIEVE matched signal-CyaA fusion experimental results with J774 macrophages suggesting that the SseJ secretion signal has some sequence order dependence. The structural studies showed that the SseJ, SseJ-H, and SseJ-L peptides were intrinsically disordered in aqueous solution with a small predisposition to adopt nascent helical structure only in the presence of structure stabilizing agents such as 1,1,1,3,3,3- hexafluoroisopropanol. Intrinsic disorder may be a universal feature of effector secretion signals as similar conclusions were reached following structural characterization of peptides corresponding to the N-terminal regions of the S. Typhimurium effectors SptP, SopD-2, GtgE, and the Yersinia pestis effector YopH.