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. 2010 Dec;6(12):2448-58.
doi: 10.1039/c0mb00097c. Epub 2010 Sep 29.

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

Garry W Buchko  1 , George Niemann, Erin S Baker, Mikhail E Belov, Richard D Smith, Fred Heffron, Joshua N Adkins, Jason E McDermott
Affiliations

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

Garry W Buchko et al. Mol Biosyst. 2010 Dec.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Levels of cAMP generated by the secretion signal–CyaA fusion assay using fusions with the peptides SseJ (black), SseJ-L (red), SseJ-H (blue), and SrfH (green). The solid colored columns are with wildtype S. Typhimurium and the white columns with SPI-2 inactivated mutant S. Typhimurium. All experiments were performed with J774 macrophage in triplicate with the error in these measurements indicated by the black error bars.
Fig. 2
Fig. 2
(A) Circular dichroism spectra for the peptides SseJ (black solid line), SseJ-L (red dotted line), and SseJ-H (blue dashed line) at 0, 20, and 100% HFIP (v/v). (B) Dependency of [Θ]220 on the concentration of HFIP (volume percent) determined for the peptides SseJ (black solid line), SseJ-L (red dotted line), and SseJ-H (blue dashed line). Titration data were collected at 25 °C with an approximately equal concentration of each peptide (0.08 mM).
Fig. 3
Fig. 3
Overlay of the one-dimensional proton spectra for the peptides SseJ, SseJ-L, and SseJ-H in water (black spectrum) and in 25% HFIP (blue spectrum), collected at a 1H resonance frequency of 750 MHz, 20 °C.
Fig. 4
Fig. 4
Amide (1HN) and alpha proton (1Hα) region of the two-dimensional 1H–1H NOESY spectrum for the peptide SseJ in 25% HFIP collected at a 1H resonance frequency of 750 MHz, 20 °C. Sequential walk through the amide and alpha proton assignments are shown in red.
Fig. 5
Fig. 5
The nested IMS-MS spectra over a 5 ms drift time (x-axis) for the (A) triply protonated plus-three charge state of SseJ, SseJ-L, and SseJ-H in 25% HFIP and (B) the plus-three charge state of SseJ in 0, 25, and 100% HFIP. An example of a two-dimensional slice through the mass spectrum of SseJ is shown on the left. In addition to the triply protonated state, the (2H+ + Na+)3+ and (2H+ + K+)3+ species are also shown in (B). At the top of each spectrum is shown the ion arrival time distribution (ATD) for each peptide. All peptides exhibit a range of multiple conformations that have been grouped into three distinct groups: purple = compact; yellow = intermediate; blue = extended.
Fig. 6
Fig. 6
Circular dichroism dependency of [Θ]220 on the concentration of HFIP (volume percent) determined for the peptides corresponding to the N-terminal regions of the S. Typhimurium effectors SptP (red), SopD-2 (purple), GtgE (cyan), and SseJ (black) and Y. pestis effector YopH (green). The data were corrected for concentration differences between the peptides SptP (0.06 mM), SopD-2 (0.12 mM), GtgE (0.13 mM), and YopH (0.07 mM), and SseJ (0.08 mM) and have been normalized to SseJ.
Fig. 7
Fig. 7
Graphical output of PONDR predictions using the VL-XT algorithm for (A) SseJ (black solid), SseJ-L (red dots), and SseJ-H (blue dashes) and (B) the N-terminal regions of the S. Typhimurium effectors SptP (red), SopD-2 (purple), GtgE (cyan), and SseJ (black) and Y. pestis effector YopH (green). Consecutive values above and below 0.5 predict disordered and ordered regions, respectively, within the protein.
Fig. 8
Fig. 8
Structure of the first 35 residues in the only effector structures containing an intact N-terminal region, Y. pestis YopH (residues 1–129). (A) Crystal structure of a domain-swapped dimer, 1K46. (B) The solution structure of a monomer bound to N-aceyl-DEpYDDPF-NH2, 1M0V. The α-helix, shown in gold, comprises most of the residues (1–17) necessary for secretion. The β-strands are colored blue.
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References

    1. Tindall BJ, Grimont PA, Garrity GM, Euzeby JP. Int. J. Syst. Evol. Microbiol. 2005;55:521–524. - PubMed
    1. Bagger-Skjot L, Nielsen EM, Sandvang D, Ethelberg S, Monnet DL, Hammerum AM. J. Antimicrob. Chemother. 2007;59:814–815. - PubMed
    1. Crump JA, Luby SP, Mintz ED. Bull. W. H. O. 2004;82:346–353. - PMC - PubMed
    1. Chen S, Zhoa C, White DG, Schroeder CM, Lu R, Yang H, McDermott PF, Ayers S, Meng J. Appl. Environ. Microbiol. 2003;2004:1–7. - PMC - PubMed
    1. Glynn MK, Bopp C, Dewitt W, Dabney P, Mokhtar M, Angulo FJ. N. Engl. J. Med. 1998;338:1333–1338. - PubMed

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