Mutagenesis of the<i>Shigella flexneri</i>Autotransporter IcsA Reveals Novel Functional Regions Involved in IcsA Biogenesis and Recruitment of Host Neural Wiscott-Aldrich Syndrome Protein

May, Kerrie L.; Morona, Renato

doi:10.1128/jb.00093-08

Cited by 40 publications

(87 citation statements)

References 56 publications

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“…Consistent with this hypothesis, large endogenous AT passengers appear to have stricter requirements for efficient secretion than small heterologous passengers (6,51). For example, secretion of IcsA is compromised by mutations in the C-terminal end of the endogenous passenger (52), whereas substituting the entire passenger for the smaller heterologous MalE protein results in efficient secretion (43).…”

Section: Discussionmentioning

confidence: 82%

Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins

Drobnak¹,

Braselmann²,

Clark

2015

Journal of Biological Chemistry

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Background: Many virulence proteins are secreted using the autotransporter system. Results: Autotransporter proteins do not fold until secreted and are secreted poorly in the absence of folding. Conclusion: Folding helps drive autotransporter secretion, but another energy source is required for its initiation. Significance: Our conclusions reconcile apparent contradictions in the literature and importantly contribute to understanding and manipulating autotransporter secretion.

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Section: Discussionmentioning

confidence: 82%

Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins

Drobnak¹,

Braselmann²,

Clark

2015

Journal of Biological Chemistry

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“…This AC domain is conserved among many ATs, including members of the SPATE (for serine protease autotransporters of Enterobacteriaceae) subfamily to which Hbp belongs (22). Deletion and mutagenesis experiments have shown that this region is crucial for efficient folding of several passenger domains (23)(24)(25)(26). Recent evidence indicates that translocation of the passenger across the OM occurs in an C-to-N direction, possibly following the formation of a hairpin structure by the C terminus of the passenger in the ␤-domain channel (27,28).…”

mentioning

confidence: 99%

A Conserved Aromatic Residue in the Autochaperone Domain of the Autotransporter Hbp Is Critical for Initiation of Outer Membrane Translocation

Soprova

Saurı́

Ulsen

et al. 2010

Journal of Biological Chemistry

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Autotransporters are bacterial virulence factors that share a common mechanism by which they are transported to the cell surface. They consist of an N-terminal passenger domain and a C-terminal ␤-barrel, which has been implicated in translocation of the passenger across the outer membrane (OM). The mechanism of passenger translocation and folding is still unclear but involves a conserved region at the C terminus of the passenger domain, the so-called autochaperone domain. This domain functions in the stepwise translocation process and in the folding of the passenger domain after translocation. In the autotransporter hemoglobin protease (Hbp), the autochaperone domain consists of the last rung of the ␤-helix and a capping domain. To examine the role of this region, we have mutated several conserved aromatic residues that are oriented toward the core of the ␤-helix. We found that non-conservative mutations affected secretion with Trp 1015 in the cap region as the most critical residue. Substitution at this position yielded a DegP-sensitive intermediate that is located at the periplasmic side of the OM. Further analysis revealed that Trp 1015 is most likely required for initiation of processive folding of the ␤-helix at the cell surface, which drives sequential translocation of the Hbp passenger across the OM.

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“…Indeed, heterologous expression of IcsA506-620-GFP fusion proteins has frequently been used to study the nature of spatial protein targeting in bacteria (Fixen et al, 2012;Janakiraman et al, 2009). However, in previous mutagenesis studies of full-length IcsA, we have found that insertions (5 aa linkers) within the nPT region (positions i56, i81 and i87) were unable to perturb PT, whilst insertions within the cPT region (positions i532 and i563) dramatically perturbed PT, resulting in circumferential distribution on the surface of bacteria (May & Morona, 2008). These data indicate that the cPT regions plays a more significant role in IcsA PT.…”

Section: Introductionmentioning

confidence: 78%

“…Polyclonal rabbit anti-N-WASP and polyclonal rabbit anti-IcsA were produced and validated as described previously (May & Morona, 2008;Van den Bosch et al, 1997). Monoclonal mouse anti-DnaK antibody was from Enzo Life Sciences.…”

Section: R564amentioning

confidence: 99%

A small conserved motif supports polarity augmentation of Shigella flexneri IcsA

2015

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The rod-shaped enteric intracellular pathogen Shigella flexneri and other Shigella species are the causative agents of bacillary dysentery. S. flexneri are able to spread within the epithelial lining of the gut, resulting in lesion formation, cramps and bloody stools. The outer membrane protein IcsA is essential for this spreading process. IcsA is the initiator of an actin-based form of motility whereby it allows the formation of a filamentous actin 'tail' at the bacterial pole. Importantly, IcsA is specifically positioned at the bacterial pole such that this process occurs asymmetrically. The mechanism of IcsA polarity is not completely understood, but it appears to be a multifactorial process involving factors intrinsic to IcsA and other regulating factors. In this study, we further investigated IcsA polarization by its intramolecular N-terminal and central polar-targeting (PT) regions (nPT and cPT regions, respectively). The results obtained support a role in polar localization for the cPT region and contend the role of the nPT region. We identified single IcsA residues that have measurable impacts on IcsA polarity augmentation, resulting in decreased S. flexneri sprading efficiency. Intriguingly, regions and residues involved in PT clustered around a highly conserved motif which may provide a functional scaffold for polarity-augmenting residues. How these results fit with the current model of IcsA polarity determination is discussed.

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Mutagenesis of theShigella flexneriAutotransporter IcsA Reveals Novel Functional Regions Involved in IcsA Biogenesis and Recruitment of Host Neural Wiscott-Aldrich Syndrome Protein

Cited by 40 publications

References 56 publications

Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins

Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins

A Conserved Aromatic Residue in the Autochaperone Domain of the Autotransporter Hbp Is Critical for Initiation of Outer Membrane Translocation

A small conserved motif supports polarity augmentation of Shigella flexneri IcsA

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