Preparation and characterization of translocator/chaperone complexes and their component proteins from Shigella flexneri

Shigella flexneri causes a severe form of bacillary dysentery also known as shigellosis. Onset of shigellosis requires bacterial invasion of colonic epithelial cells which is initiated by the delivery of translocator and effector proteins to the host cell membrane and cytoplasm, respectively, by the Shigella type III secretion system (TTSS). The Shigella translocator proteins, IpaB and IpaC, form a pore complex in the host cell membrane to facilitate effector delivery; however, prior to their secretion IpaB and IpaC are partitioned in the bacterial cytoplasm by association with the cytoplasmic chaperone IpgC. To determine their structural and biophysical properties, recombinant IpaB/IpgC and IpaC/IpgC complexes were prepared for their first detailed in vitro analysis. Both IpaB/IpgC and IpaC/IpgC complexes are highly stable and soluble heterodimers whose formation prevents IpaB-IpaC interaction as well as Ipa-dependent disruption of phospholipid membranes. Circular dichroism spectroscopy shows that IpgC binding has a detectable influence on IpaC secondary/tertiary structure and stability. In contrast, IpaB structure is not as dramatically affected by chaperone binding. To more precisely ascertain the influence of chaperone binding on IpaC structure and stability, single tryptophan mutants were generated for detailed fluorescence spectroscopy analysis. These mutants provide a low-resolution picture of how IpaC exists in the Shigella cytoplasm with chaperone binding possibly involving distinct regions within the N- and C-terminal halves of IpaC. This preliminary assessment of the IpaC-IpgC interaction is supported by initial deletion mutagenesis studies. The data provide the first structural analysis of IpgC association with IpaB and IpaC.     

IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells.

Shigella proteins that are targeted to host cells by a type III secretion apparatus are essential for reorganization of the cytoskeleton during cell invasion. We have developed a semi-permeabilized cell assay that tests the effects of bacterial proteins on the actin cytoskeleton. The Shigella IpaC protein was found to induce the formation of filopodial and lamellipodial extensions in these semi-permeabilized cells. Microinjection of IpaC into cells, or cellular expression of IpaC also led to the formation of filopodial structures. Monoclonal antibodies (mAbs) directed against the C-terminus of IpaC inhibited the IpaC-induced extensions, whereas an anti-N-terminal IpaC mAb stimulated extensive lamellae formation. Shigella induced foci of actin polymerization in the permeabilized cells and these were inhibited by anti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induced cytoskeletal rearrangements during entry, stable transfectants expressing IpaC challenged with Shigella showed increased bacterial internalization. IpaC-induced extensions were inhibited by a dominant-interfering form of Cdc42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering form of Rac resulted in inhibition of lamellae formation. We conclude that IpaC leads to activation of Cdc42 which in turn, causes activation of Rac, both GTPases being required for Shigella entry.     

Bacterial signals and cell responses during Shigella entry into epithelial cells

Shigella invades epithelial cells by inducing cytoskeletal reorganization localized at the site of bacterial–host cell interaction. During entry, the Shigella type III secretion apparatus allows the insertion of a pore that contains the IpaB and IpaC proteins into cell membranes. Insertion of this complex is thought to allow translocation of the carboxy-terminus moiety of IpaC, but also of other Shigella effectors, such as IpaA, into the cell cytosol. IpaC triggers actin polymerization and the formation of filopodial and lamellipodial extensions dependent on the Cdc42 and Rac GTPases. IpaA, on the other hand, binds to the focal adhesion protein vinculin and induces depolymerization of actin filaments. IpaA and the GTPase Rho are not required for actin polymerization at the site of bacterial contact with the cell membrane, but allow the transformation of the IpaC-induced extensions into a structure that is productive for bacterial entry. Rho is required for the recruitment at entry foci of ezrin, a cytoskeletal linker required for Shigella entry, and also of the Src tyrosine kinase. The Src tyrosine kinase activity, which is required for Shigella -induced actin polymerization, also appears to be involved in a negative regulatory loop that downregulates Rho at the site of entry.     

Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes

Shigella flexneri causes dysentery after invading the epithelial cells of the human colon. Enterocyte invasion is induced by the bacterial effector IpaC (invasion plasmid antigen C), which triggers Shigella entry into epithelial cells by a rather poorly understood mechanism. IpaC is also involved in pathogen escape into the host cell cytoplasm following uptake, and this property may be reflected in its ability to disrupt phospholipid vesicles in vitro. Purified recombinant IpaC interacts with liposome vesicles to cause the release of small molecules trapped inside. This interaction requires that the liposomes possess an acidic phospholipid component. To better understand the events involved in the disruption of liposomes by IpaC, single tryptophan mutants were generated to permit the use of intrinsic fluorescence, circular dichroism, and ultraviolet absorption spectroscopies to examine the effect that phospholipid membrane association has on IpaC structure and stability. These mutants were also used to determine how amino acid substitutions within specific regions of IpaC influence its activity in vivo. The outcomes of this study include findings that cholesterol greatly impacts IpaC association with phospholipid membranes, tryptophan incorporation into specific regions of IpaC (especially near the C-terminus) can greatly impact its in vivo activity, and interaction with phospholipid membranes causes differing degrees of change in the fluorescence of tryptophan residues introduced at specific sites within IpaC. These data, together with fluorescence quenching analyses, provide new functional and structural information concerning IpaC and its insertion into phospholipid membranes.     

Shigella invasion of macrophage requires the insertion of IpaC into the host plasma membrane. Functional analysis of IpaC.

Shigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death. The bacterial strategy of macrophage infection, however, remains largely speculative. Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria. When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000. J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester. Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake of Escherichia coli. The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining. The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117-169, which contains a putative membrane-spanning sequence. Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial.     

Cdc42 facilitates invasion but not the actin-based motility of Shigella

The enteric pathogen Shigella utilizes host-encoded proteins to invade the gastrointestinal tract. Efficient invasion of host cells requires the stimulation of Rho-family GTPases and cytoskeletal alterations by Shigella-encoded IpaC. Following invasion and lysis of the phagosome, Shigella exploits the host's actin-based polymerization machinery to assemble an actin tail that serves as the propulsive force required for spreading within and between cells. The Shigella surface protein IcsA stimulates actin-tail formation by recruiting host-encoded N-WASP to drive Arp2/3-mediated actin assembly. N-WASP is absolutely required for Shigella motility, but not for Shigella invasion. Although Rho-family GTPases have been implicated in both the invasion and motility of Shigella, the role of Cdc42, an N-WASP activator, in this process has been controversial. In these studies, we have examined the role of Cdc42 in Shigella invasion and actin-based motility using Cdc42-deficient cells. We demonstrate that Cdc42 is required for efficient Shigella invasion but reveal a minor Cdc42-independent pathway that can permit Shigella invasion. However, the actin-based motility of Shigella, as well as vaccinia, proceeds unperturbed in the absence of Cdc42. These data further support the involvement of distinct host-encoded proteins in the steps regulating invasion and intercellular spread of Shigella.     

IpaC from Shigella and SipC from Salmonella possess similar biochemical properties but are functionally distinct

Invasion plasmid antigen C (IpaC) is secreted via the type III secretion system (TTSS) of Shigella flexneri and serves as an essential effector molecule for epithelial cell invasion. The only homologue of IpaC identified thus far is Salmonella invasion protein C (SipC/SspC), which is essential for enterocyte invasion by Salmonella typhimurium. To explore the biochemical and functional relatedness of IpaC and SipC, recombinant derivatives of both proteins were purified so that their in vitro biochemical properties could be compared. Both proteins were found to: (i) enhance the entry of wild-type S. flexneri and S. typhimurium into cultured cells; (ii) interact with phospholipid membranes; and (iii) oligomerize in solution; however, IpaC appeared to be more efficient in carrying out several of the biochemical properties examined. Overall, the data indicate that purified IpaC and SipC are biochemically similar, although not identical with respect to their in vitro activities. To extend these observations, complementation analyses were conducted using S. flexneri SF621 and S. typhimurium SB220, neither of which is capable of invading epithelial cells because of non-polar null mutations in ipaC and sipC respectively. Interestingly, both ipaC and sipC restored invasiveness to SB220 whereas only ipaC restored invasiveness to SF621, suggesting that SipC lacks an activity possessed by IpaC. This functional difference is not at the level of secretion because IpaC and SipC are both secreted by SF621 and it does not appear to be because of SipC dependency on this native chaperone as coexpression of sipC and sicA in SF621 still failed to restore detectable invasiveness. Taken together, the data suggest that IpaC and SipC differ in either their ability to be translocated into host cells or in their function as effectors of host cell invasion. Because IpaB shares significant sequence homology with the YopB translocator of Yersinia species, the ability for IpaC and SipC to associate with this protein was explored as a potential indicator of translocation function. Both proteins were found to bind to purified IpaB with an apparent dissociation constant in the nanomolar range, suggesting that they may differ with respect to effector function. Interestingly, whereas SB220 expressing sipC behaved like wild-type Salmonella, in that it remained within its membrane-bound vacuole following entry into host cells, SB220 expressing ipaC was found in the cytoplasm of host cells. This observation indicates that IpaC and SipC are responsible for a major difference in the invasion strategies of Shigella and Salmonella, that is, they escape into the host cell cytoplasm. The implications of the role of each protein's biochemistry relative to its in vivo function is discussed.     

A system for identifying post-invasion functions of invasion genes: requirements for the Mxi-Spa type III secretion pathway of Shigella flexneri in intercellular dissemination

Invasion and intercellular spread are hallmarks of Shigella pathogenicity. Invasion of the eukaryotic cell cytosol requires a type III secretion system (Mxi-Spa) and its cognate set of secreted Ipa invasins. Once intracellular, the IcsA protein directs a form of actin-based motility that helps to drive intracellular bacterial movement, formation of cellular protrusions and cell-to-cell spread. Work in our laboratory has focused on identifying additional factors required for this intercellular form of dissemination. In this study, we sought to identify novel contributions of the type III secretion pathway to post-invasion-specific processes, distinct from its previously characterized roles in invasion. Studies of post-invasion Ipa and Mxi-Spa functions are complicated by an absolute requirement for these virulence proteins in invasion. To circumvent this problem, we developed a system called TIER (for test of intracellular expression requirements), whereby specific ipa, mxi or spa loci are transiently expressed before infection of tissue culture cell monolayers (thus supporting invasion), but then repressed after invasion in the intracellular environment. Such invasive type III secretion mutants (called TIER mutants) were severely restricted in their ability to spread intercellularly and form plaques in confluent tissue culture cell monolayers. Intercellular spread defects were associated with the repression of most type III pathway components examined, including structural (MxiM and Spa33), secreted effector (IpaB, IpaC and IpaD) and regulatory elements (VirF and VirB). A kinetic analysis of bacterial growth in L2 cell monolayers showed that each of the TIER mutants was defective with respect to long-term intracellular proliferation and viability. Examination of TIER mutant-infected monolayers by electron microscopy revealed that the type III pathway was required for a late step in intercellular spread - bacterial escape from protrusion-derived, double-membrane-bound vacuoles. The TIER mutants were eventually degraded in a process involving vacuolar acidification. Based on these findings, we propose that Ipa secretion via Mxi-Spa is required in the protrusion vacuole for double-membrane lysis.     

Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells.

Entry into host cells is an essential feature in the pathogenicity of Salmonella spp. The inv locus of Salmonella typhimurium encodes several proteins which are components of a type III protein secretion system required for these organisms to gain access to host cells. We report here the identification of several proteins whose secretion into the culture supernatant of S. typhimurium is dependent on the function of the inv-encoded translocation apparatus. Nucleotide sequence analysis of the genes encoding two of these secreted proteins, SipB and SipC, indicated that they are homologous to the Shigella sp. invasins IpaB and IpaC, respectively. An additional gene was identified, sicA, which encodes a protein homologous to IpgC, a Shigella protein that serves as a molecular chaperone for the invasins IpaB and IpaC. Nonpolar mutations in sicA, sipB, and sipC rendered S. typhimurium unable to enter cultured epithelial cells, indicating that these genes are required for bacterial internalization.     

The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri

Invasion of epithelial cells by Shigella flexneri involves entry and dissemination. The main effectors of entry, IpaB and IpaC, are also required for contact haemolytic activity and escape from the phagosome in infected macrophages. These proteins are stored in the cytoplasm in association with the chaperone IpgC, before their secretion by a type III secretion apparatus is activated by host cells. We used a His-tagged IpgC protein to purify IpgC-containing complexes and showed that only IpaB and IpaC are associated with IpgC. Plasmids expressing His6-IpgC either alone or together with IpaB or IpaC under the control of an IPTG-inducible lac promoter were introduced into ipgC , ipaB or ipaC mutants. Induction of expression of the recombinant plasmid-encoded proteins by IPTG allowed bacteria to enter epithelial cells, and the role of these proteins in dissemination was investigated by incubating infected cells in either the absence or the presence of IPTG. The size of plaques produced by recombinant strains on cell monolayers was regulated by IPTG, indicating that IpgC, IpaB and IpaC were each required for efficient dissemination. Electron microscopy analysis of infected cells indicated that these proteins were necessary for lysis of the membrane of the protrusions during cell-to-cell spread.     

The IpaC Carboxyterminal Effector Domain Mediates Src-Dependent Actin Polymerization during Shigella Invasion of Epithelial Cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells by locally reorganizing the actin cytoskeleton. Shigella invasion requires actin polymerization dependent on the Src tyrosine kinase and a functional bacterial type III secretion (T3S) apparatus. Using dynamic as well as immunofluorescence microscopy, we show that the T3S translocon component IpaC allows the recruitment of the Src kinase required for actin polymerization at bacterial entry sites during the initial stages of Shigella entry. Src recruitment occurred at bacterial-cell contact sites independent of actin polymerization at the onset of the invasive process and was still observed in Shigella strains mutated for translocated T3S effectors of invasion. A Shigella strain with a polar mutation that expressed low levels of the translocator components IpaB and IpaC was fully proficient for Src recruitment and bacterial invasion. In contrast, a Shigella strain mutated in the IpaC carboxyterminal effector domain that was proficient for T3S effector translocation did not induce Src recruitment. Consistent with a direct role for IpaC in Src activation, cell incubation with the IpaC last 72 carboxyterminal residues fused to the Iota toxin Ia (IaC) component that translocates into the cell cytosol upon binding to the Ib component led to Src-dependent ruffle formation. Strikingly, IaC also induced actin structures resembling bacterial entry foci that were enriched in activated Src and were inhibited by the Src inhibitor PP2. These results indicate that the IpaC effector domain determines Src-dependent actin polymerization and ruffle formation during bacterial invasion.     

Molecular basis of actin nucleation factor cooperativity: crystal structure of the Spir-1 kinase non-catalytic C-lobe domain (KIND)•formin-2 formin SPIR interaction motif (FSI) complex

The distinct actin nucleation factors of the Spir and formin subgroup families cooperate in actin nucleation. The Spir/formin cooperativity has been identified to direct two essential steps in mammalian oocyte maturation, the asymmetric spindle positioning and polar body extrusion during meiosis. Understanding the nature and regulation of the Spir/Fmn cooperation is an important requirement to comprehend mammalian reproduction. Recently we dissected the structural elements of the Spir and Fmn family proteins, which physically link the two actin nucleation factors. The trans-regulatory interaction is mediated by the Spir kinase non-catalytic C-lobe domain (KIND) and the C-terminal formin Spir interaction motif (FSI). The interaction inhibits formin nucleation activity and enhances the Spir activity. To get insights into the molecular mechanism of the Spir/Fmn interaction, we determined the crystal structure of the KIND domain alone and in complex with the C-terminal Fmn-2 FSI peptide. Together they confirm the proposed structural homology of the KIND domain to the protein kinase fold and reveal the basis of the Spir/formin interaction. The complex structure showed a large interface with conserved and positively charged residues of the Fmn FSI peptide mediating major contacts to an acidic groove on the surface of KIND. Protein interaction studies verified the electrostatic nature of the interaction. The data presented here provide the molecular basis of the Spir/formin interaction and give a first structural view into the mechanisms of actin nucleation factor cooperativity.     

Delineation and characterization of the actin nucleation and effector translocation activities of Salmonella SipC

Salmonella type III secreted SipC possesses dual functions: translocation of effectors and actin modulation. The biological significance of SipC's actin nucleation activity in Salmonella-induced actin cytoskeleton rearrangements has not been studied. We report here the delineation of the actin nucleation activity from the effector translocation activity of SipC. Our data show that the central amino acid region (residues: 201-220) is essential for its actin nucleation activity and the C-terminal amino acid region (321-409) is required for translocation of effectors. A SipC nucleation-deficient mutant, which maintained its effector translocation activity, was obtained. This nucleation-deficient mutant had significantly reduced ability to induce actin cytoskeleton rearrangements, resulting in lower bacterial invasion into HeLa cells. Contrary to a previous report, we found that the purified recombinant wild-type SipC(199-409) protein is monomeric in solution by size exclusion chromatography coupled with multiangle laser light scattering assays (SEC-LS). Our data established that the actin nucleation activity of SipC plays a vital role in Salmonella-induced membrane ruffles and subsequent bacteria invasion.     

Significance of the secreted form of IpaC, a 45 kDa protein of Shigella dysenteriae 1, in the invasive process as determined by monoclonal antibodies

Abstract Invasion plasmid antigen C (IpaC), a 45 kDa plasmid encoded protein, is associated with the virulence of virulent Shigella spp. In S. dysenteriae type 1 the 45 kDa IpaC protein is secreted to a greater extent into the surrounding medium in comparison to other Shigella spp. Monoclonal antibodies (mAbs) to the secreted form of IpaC protein were raised in this study. Of the four secretory hybrid cells, one (3G4) was found to have a very high antibody titre as determined by ELISA. The specificity of 3G4 was confirmed by immunoblotting of whole cell extract of Escherichia coli strain MC1061 carrying the plasmid pHW756 which synthesizes both the IpaB and C proteins. The effect of the mAbs on plaque formation by virulent Shigella dysenteriae 1 was determined and it was found that the clone 3G4 substantially (55%) reduced plaque formation on HeLa cell monolayer. The epitope specificity of the mAb 3G4 was competitively inhibited by the convalescent phase sera from human, suggesting that the epitope recognized by clone 3G4 was expressed during the natural course of infection and also indicating that the 45 kDa (IpaC) protein in secreted form has a definite role in the invasive process.     

Capping of actin filaments by vinculin activated by the Shigella IpaA carboxyl-terminal domain

Shigella, the causative agent of bacillary dysentery, invades epithelial cells. Upon bacterial-cell contact, the type III bacterial effector IpaA binds to the cytoskeletal protein vinculin to promote actin reorganization required for efficient bacterial uptake. We show that the last 74 C-terminal residues of IpaA (A559) bind to human vinculin (HV) and promotes its association with actin filaments. Polymerisation experiments demonstrated that A559 was sufficient to induce HV-dependent partial capping of the barbed ends of actin filaments. These results suggest that IpaA regulates actin polymerisation/depolymerisation at sites of Shigella invasion by modulating the barbed end capping activity of vinculin.     

Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function

Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.     

Cytoplasmic targeting of IpaC to the bacterial pole directs polar type III secretion in Shigella

Type III secretion (T3S) systems are largely used by pathogenic gram-negative bacteria to inject multiple effectors into eukaryotic cells. Upon cell contact, these bacterial microinjection devices insert two T3S substrates into host cell membranes, forming a so-called 'translocon' that is required for targeting of type III effectors in the cell cytosol. Here, we show that secretion of the translocon component IpaC of invasive Shigella occurs at the level of one bacterial pole during cell invasion. Using IpaC fusions with green fluorescent protein variants (IpaCi), we show that the IpaC cytoplasmic pool localizes at an old or new bacterial pole, where secretion occurs upon T3S activation. Deletions in ipaC identified domains implicated in polar localization. Only polar IpaCi derivatives inhibited T3S, while IpaCi fusions with diffuse cytoplasmic localization had no detectable effect on T3S. Moreover, the deletions that abolished polar localization led to secretion defects when introduced in ipaC. These results indicate that cytoplasmic polar localization directs secretion of IpaC at the pole of Shigella, and may represent a mandatory step for T3S.     

Influence of the C-terminal residues on oligomerization of alpha A-crystallin

Earlier studies have shown that the chaperone activity of alpha-crystallin is significantly affected in diabetic rat and human lenses. Subsequently, mass spectrometric analysis showed diabetic lenses having high levels of the alphaA-crystallins in which different numbers of C-terminal residues were deleted. The present study was aimed to show whether cleavage of these residues influences protein structure, oligomerization, and chaperone function. For generation of various mutants, a stop codon was introduced at the positions of interest, proteins were expressed in BL21(DE3)pLys S E. coli, and the truncated alphaA-crystallins were purified by size-exclusion chromatography. The molecular masses, as determined by molecular sieve HPLC, of mutants with deletions of 1, 5, and 10 C-terminal residues (group-1) were 519-602 kDa, and those of mutants with deletions of 11, 16, and 22 C-terminal residues (group-2) were 148-152 kDa, as compared to 607 kDa for alphaA-wild type. On the basis of circular dichroism measurements, the alpha helix content was 2-fold higher and the tertiary structure was significantly altered in the group-2 mutants. Chaperoning abilities, as determined by the ADH assay and the betaL-crystallin heat denaturation assay, of the group-1 mutants, with the exception of alphaA(1-163), were slightly improved or unchanged, that of alphaA(1-163) was moderately affected, and those of the group-2 mutants were severely affected. Most strikingly, cleavage of 11 C-terminal residues including Arg-163 showed a substantial decrease in oligomeric size and chaperone function and significant changes in protein structure whereas cleavage of 10 residues had either a small effect or no effect at all. This points to an important role for the C-terminal extension, Arg-163 in particular, and no significant role for the C-terminal flexible tail in the oligomer assembly of alphaA-crystallin.     

Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure

Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nanomachine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.     

Identification of functional regions within invasion plasmid antigen C (IpaC) of Shigella flexneri

Shigella flexneri causes bacillary dysentery with symptoms resulting from the inflammation that accompanies bacterial entry into the cells of the colonic epithelium. The effectors of S. flexneri invasion are the Ipa proteins, particularly IpaB and IpaC, which are secreted at the host-pathogen interface following bacterial contact with a host cell. Of the purified Ipa proteins, only IpaC has been shown to possess quantifiable in vitro activities that are related to cellular invasion. In this study, ipaC deletion mutants were generated to identify functional regions within the IpaC protein. From these data, we now know that the N-terminus and an immunogenic central region are not required for IpaC-dependent enhancement of cellular invasion by S. flexneri. However, to restore invasiveness to an ipaC null mutant of S. flexneri, the N-terminus is essential, because IpaC mutants lacking the N-terminus are not secreted by the bacterium. Deletion of the central hydrophobic region eliminates IpaC's ability to interact with phospholipid membranes, and fusion of this region to a modified form of green fluorescent protein converts it into an efficient membrane-associating protein. Meanwhile, deletion of the C-terminus eliminates the mutant protein's ability to establish protein-protein contacts with full-length IpaC. Interestingly, the mutant form of ipaC that restores partial invasiveness to the S. flexneri ipaC null mutant also restores full contact-mediated haemolysis activity to this bacterium. These data support a model in which IpaC possesses a distinct functional organization that is important for bacterial invasion. This information will be important in defining the precise role of IpaC in S. flexneri pathogenesis and in exploring the potential effects of purified IpaC at mucosal surfaces.