Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo Cross-linking

Zoufaly, Stefan; Fröbel, Julia; Rose, Patrick P.; Flecken, Tobias; Maurer, H. Carlo; Moser, Michael J.; Müller, Matthias

doi:10.1074/jbc.m112.343798

Cited by 64 publications

(130 citation statements)

References 49 publications

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“…Our finding is in line with the findings of previous mutant screening studies where sufI mutants of Y. pseudotuberculosis YPIII were found to be avirulent (37,38). SufI has been used in mechanistic studies of Tat structure and function in E. coli (27,39,40), and we confirmed that Tat also promotes SufI translocation to the periplasm in Y. pseudotuberculosis. This highlights that the periplasmic localization of SufI is essential for its function.…”

Section: Discussionsupporting

confidence: 92%

The Tat Substrate SufI Is Critical for the Ability of Yersinia pseudotuberculosis To Cause Systemic Infection

et al. 2017

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The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important humanpathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a ΔsufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosis. In vivo bioluminescent imaging of orally infected mice revealed that both the ΔsufI and ΔtatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the ΔtatC mutant nor the ΔsufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the ΔtatC and ΔsufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the ΔtatC mutant.KEYWORDS Yersinia pseudotuberculosis, Tat pathway, virulence, SufI, mesenteric lymph nodes, neutrophils T he genus Yersinia includes three species that are pathogenic to humans: Yersinia pestis, the causative agent of plague, and the two enteropathogens Y. enterocolitica and Y. pseudotuberculosis, which normally cause a self-limiting disease with symptoms ranging from mild diarrhea, enterocolitis, septicemia, and mesenteric lymphadenitis to reactive arthritis in humans after ingestion of contaminated food or water (1). Once it is ingested, Y. pseudotuberculosis traverses the epithelial barrier through M cells and infects the associated lymphoid tissues, such as Peyer's patches (PPs) and cecal patches, and later spreads to the mesenteric lymph nodes (MLNs). Although the infection is usually self-limited in humans, the infection caused by the two enteropathogenic Yersinia species in mice readily progresses to become systemic and disseminates to the spleen and liver (2). The main virulence arsenal of Yersinia is the type III secretion system (T3SS), which is encoded by an ϳ70-kb virulence plasmid. The T3SS enables the translocation of virulence effector proteins directly into the cytosol of the target host cell, which results in the disruption of host signaling and early immune

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Section: Discussionsupporting

confidence: 92%

The Tat Substrate SufI Is Critical for the Ability of Yersinia pseudotuberculosis To Cause Systemic Infection

et al. 2017

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“…Thus, our results provide a map of the domains and residues involved in signal peptide binding. As such, they are consistent with and extend previous photo-cross-linking studies (Alami et al, 2003;Gérard and Cline, 2006;Zoufaly et al, 2012) as well as recent isothermal calorimetric analysis (Rollauer et al, 2012).…”

Section: Discussionsupporting

confidence: 90%

“…Thus, it is possible that these three regions, containing mutants F200AF201A, D276, and T272AP273A, define a hotspot for Tat component interactions. Consistent with this general conclusion is the fact that the L3 residue Thr-275 directed strong cpTatC-cpTatC cross-linking (Figure 6), the E. coli TatC P3 residue Asp-211 directed photo-cross-linking to TatA (Tha4 ortholog) (Zoufaly et al, 2012), and the TM3-proximal P2 region residue 150 of E. coli TatC directed cross-linking to TatB and TatA (Zoufaly et al, 2012) Mutations in L1 resulted in complete loss of receptor complex and absence of any endogenous cpTatC in the purified mutant cpTatC ( Figure 5). The E. coli TatC periplasmic loop P1 (comparable to L1) is also hypersensitive to mutation (Kneuper et al, 2012) and at least one mutant in P1, P48A, resulted in loss of receptor complex (Barrett et al, 2005).…”

Section: Discussionsupporting

confidence: 59%

“…Zoufaly et al (2012), using site-directed photo-cross-linking, recently showed that the E. coli TatC N terminus and first cytoplasmic loop are in close contact with bound substrate. They suggested but did not demonstrate that the observed interactions were with the twin Arg motif of signal peptide.…”

Section: Discussionmentioning

confidence: 99%

“…The E. coli TatC periplasmic loop P1 (comparable to L1) is also hypersensitive to mutation (Kneuper et al, 2012) and at least one mutant in P1, P48A, resulted in loss of receptor complex (Barrett et al, 2005). Also of interest is that L1 Cys substitution directed strong cpTatC-cpTatC cross-linking (Figure 6), and a P1 residue of E. coli TatC directed photo-crosslinking to adjacent TatC (Zoufaly et al, 2012). It is tempting to conclude that L1 (P1) is involved cpTatC-cpTatC interactions.…”

Section: Discussionmentioning

confidence: 99%

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Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

Cline

2013

The Plant Cell

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Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

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Twin-Arginine Protein Translocation

Goosens

Dijl

2016

Current Topics in Microbiology and Immunology

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Twin-arginine protein translocation systems (Tat) translocate fully folded and co-factor-containing proteins across biological membranes. In this review, we focus on the Tat pathway of Gram-positive bacteria. The minimal Tat pathway is composed of two components, namely a TatA and TatC pair, which are often complemented with additional TatA-like proteins. We provide overviews of our current understanding of Tat pathway composition and mechanistic aspects related to Tat-dependent cargo protein translocation. This includes Tat pathway flexibility, requirements for the correct folding and incorporation of co-factors in cargo proteins and the functions of known cargo proteins. Tat pathways of several Gram-positive bacteria are discussed in detail, with emphasis on the Tat pathway of Bacillus subtilis. We discuss both shared and unique features of the different Gram-positive bacterial Tat pathways. Lastly, we highlight topics for future research on Tat, including the development of this protein transport pathway for the biotechnological secretion of high-value proteins and its potential applicability as an antimicrobial drug target in pathogens.

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Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo Cross-linking

Cited by 64 publications

References 49 publications

The Tat Substrate SufI Is Critical for the Ability of Yersinia pseudotuberculosis To Cause Systemic Infection

The Tat Substrate SufI Is Critical for the Ability of Yersinia pseudotuberculosis To Cause Systemic Infection

Mapping the Signal Peptide Binding and Oligomer Contact Sites of the Core Subunit of the Pea Twin Arginine Protein Translocase

Twin-Arginine Protein Translocation

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