Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.

Perforin‐2 (P‐2) is a recently described antimicrobial protein with unique properties to kill intracellular bacteria. We investigated P‐2 expression pattern and cellular distribution in human skin and its importance in restoration of barrier function during wound healing process and infection with the common wound pathogen Staphylococcus aureus. We describe a novel approach for the measurement of P‐2 mRNA within individual skin cells using an amplified fluorescence in situ hybridization (FISH) technique. The unique aspect of this approach is simultaneous detection of P‐2 mRNA in combination with immune‐phenotyping for cell surface proteins using fluorochrome‐conjugated antibodies. We detected P‐2 transcript in both hematopoietic (CD45+) and non‐hematopoietic (CD45−) cutaneous cell populations, confirming the P‐2 expression in both professional and non‐professional phagocytes. Furthermore, we found an induction of P‐2 during wound healing. P‐2 overexpression resulted in a reduction of intracellular S. aureus, while infection of human wounds by this pathogen resulted in P‐2 suppression, revealing a novel mechanism by which S. aureus may escape cutaneous immunity to cause persistent wound infections.

Inducible expression of major cytosolic and nuclear chaperone proteins is mediated by the heat-shock transcription factor HSF1 that is activated by derepressive mechanisms triggered by transient heat stress and sustained proteotoxicity. Despite progress in defining essential aspects of HSF1 regulation, little is known about the cellular dynamics enabling this factor to mediate gene responses to cytosolic stress signals. We report that the inactive, stress-responsive form of HSF1 accumulates in the nucleus due to a relatively potent import signal, which can be recognized by importin-a/b, and simultaneously undergoes continuous nucleocytoplasmic shuttling due to a comparatively weak, nonetheless efficient, export activity not involving the classical exportin-1 pathway. Strikingly, experimental stresses at physiological or elevated temperature reversibly inactivate the export competence of HSF1. Likewise, mutations mimicking stress-induced derepression impair export but not import. These findings are consistent with a dynamic process whereby exported molecules that are derepressed in an inductive cytosolic environment are recollected and pause in the nucleoplasm, replacing progressively the inactive pool. While steady-state nuclear distribution of the bulk of HSF1 ensures a rapid gene response to acute heat stress, our results suggest that the capture in the nucleus of molecules primed for activation in the cytosol may underlie responses to sustained proteotoxicity.

Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH-YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called -motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the -motif. Biochemical studies establish that the -motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed.

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