Genetic Dissection of the Signaling Cascade that Controls Activation of the Shigella Type III Secretion System from the Needle Tip.

I Murillo,I Martinez-Argudo,A J Blocker

doi:10.1038/srep27649

Abstract

Many Gram-negative bacterial pathogens use type III secretion systems (T3SSs) for virulence. The Shigella T3SS consists of a hollow needle, made of MxiH and protruding from the bacterial surface, anchored in both bacterial membranes by multimeric protein rings. Atop the needle lies the tip complex (TC), formed by IpaD and IpaB. Upon physical contact with eukaryotic host cells, T3S is initiated leading to formation of a pore in the eukaryotic cell membrane, which is made of IpaB and IpaC. Through the needle and pore channels, further bacterial proteins are translocated inside the host cell to meditate its invasion. IpaD and the needle are implicated in transduction of the host cell-sensing signal to the T3S apparatus. Furthermore, the sensing-competent TC seems formed of 4 IpaDs topped by 1 IpaB. However, nothing further is known about the activation process. To investigate IpaB’s role during T3SS activation, we isolated secretion-deregulated IpaB mutants using random mutagenesis and a genetic screen. We found ipaB point mutations in leading to defects in secretion activation, which sometimes diminished pore insertion and host cell invasion. We also demonstrated IpaB communicates intramolecularly and intermolecularly with IpaD and MxiH within the TC because mutations affecting these interactions impair signal transduction.

Highlights

Type III secretion systems (T3SSs) are macromolecular structures used by many Gram-negative bacteria
The Shigella T3SS basal body is anchored in both bacterial membranes and followed by a hollow needle, formed of MxiH, that protrudes from the bacterial surface and acts as the secretion channel[5,6,7,8]
Was the decrease in hemolytic activity of some ipaB*mutants due to a problem in membrane-insertion of mutant IpaB? For those mutants with reduced hemolytic activity, we examined the composition of the lysed Red Blood Cells (RBCs) membranes isolated by floatation in a sucrose density gradient

Summary

Introduction

Type III secretion systems (T3SSs) are macromolecular structures used by many Gram-negative bacteria. Single amino acid mutations in needle proteins alter secretion regulation, host cell sensing and TC composition[13,17,18]. Point mutations in the upper part of IpaD’s C-terminal helix render the T3SS unresponsive to an artificial inducer of secretion, the small amphipathic dye Congo red (CR20) or to host cells[21]. This and its position atop needles indicate it is involved in sensing host cells. IpaB deletion mutants pleiotrophically affect T3SS regulation and host cell invasion[23,24], a direct role for IpaB in host cell sensing remains uninvestigated

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Conclusion