An Atlas of Genetic Variation Linking Pathogen-Induced Cellular Traits to Human Disease.

Pathogens have been a strong driving force for natural selection. Therefore, understanding how human genetic differences impact infection-related cellular traits can mechanistically link genetic variation to disease susceptibility. Here we report the Hi-HOST Phenome Project (H2P2): a catalog of cellular genome-wide association studies (GWAS) comprising 79 infection-related phenotypes in response to 8 pathogens in 528 lymphoblastoid cell lines. Seventeen loci surpass genome-wide significance for infection-associated phenotypes ranging from pathogen replication to cytokine production. We combined H2P2 with clinical association data from patients to identify a SNP near CXCL10 as a risk factor for inflammatory bowel disease. A SNP in the transcriptional repressor ZBTB20 demonstrated pleiotropy, likely through suppression of multiple target genes, and was associated with viral hepatitis. These data are available on a web portal to facilitate interpreting human genome variation through the lens of cell biology and should serve as a rich resource for the research community.

Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis.

Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.

Intracellular Concentrations of Borrelia burgdorferi Cyclic Di-AMP Are Not Changed by Altered Expression of the CdaA Synthase.

The second messenger nucleotide cyclic diadenylate monophosphate (c-di-AMP) has been identified in several species of Gram positive bacteria and Chlamydia trachomatis. This molecule has been associated with bacterial cell division, cell wall biosynthesis and phosphate metabolism, and with induction of type I interferon responses by host cells. We demonstrate that B. burgdorferi produces a c-di-AMP synthase, which we designated CdaA. Both CdaA and c-di-AMP levels are very low in cultured B. burgdorferi, and no conditions were identified under which cdaA mRNA was differentially expressed. A mutant B. burgdorferi was produced that expresses high levels of CdaA, yet steady state borrelial c-di-AMP levels did not change, apparently due to degradation by the native DhhP phosphodiesterase. The function(s) of c-di-AMP in the Lyme disease spirochete remains enigmatic.

Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia.

Gene inactivation by transposon insertion or allelic exchange is a powerful approach to probe gene function. Unfortunately, many microbes, including Chlamydia, are not amenable to routine molecular genetic manipulations. Here we describe an arrayed library of chemically induced mutants of the genetically intransigent pathogen Chlamydia trachomatis, in which all mutations have been identified by whole-genome sequencing, providing a platform for reverse genetic applications. An analysis of possible loss-of-function mutations in the collection uncovered plasticity in the central metabolic properties of this obligate intracellular pathogen. We also describe the use of the library in a forward genetic screen that identified InaC as a bacterial factor that binds host ARF and 14-3-3 proteins and modulates F-actin assembly and Golgi redistribution around the pathogenic vacuole. This work provides a robust platform for reverse and forward genetic approaches in Chlamydia and should serve as a valuable resource to the community.

Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates.

Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform.

Chlamydia trachomatis-induced alterations in the host cell proteome are required for intracellular growth.

Intracellular pathogens directly alter host cells in order to replicate and survive. While infection-induced changes in host transcription can be readily assessed, posttranscriptional alterations are more difficult to catalog. We applied the global protein stability (GPS) platform, which assesses protein stability based on relative changes in an adjoining fluorescent tag, to identify changes in the host proteome following infection with the obligate intracellular bacteria Chlamydia trachomatis. Our results indicate that C. trachomatis profoundly remodels the host proteome independently of changes in transcription. Additionally, C. trachomatis replication depends on a subset of altered proteins, such as Pin1 and Men1, that regulate the host transcription factor AP-1 controlling host inflammation, stress, and cell survival. Furthermore, AP-1-dependent transcription is activated during infection and required for efficient Chlamydia growth. In summary, this experimental approach revealed that C. trachomatis broadly alters host proteins and can be applied to examine host-pathogen interactions and develop host-based therapeutics.

STING-dependent recognition of cyclic di-AMP mediates type I interferon responses during Chlamydia trachomatis infection.

UNLABELLED: STING (stimulator of interferon [IFN] genes) initiates type I IFN responses in mammalian cells through the detection of microbial nucleic acids. The membrane-bound obligate intracellular bacterium Chlamydia trachomatis induces a STING-dependent type I IFN response in infected cells, yet the IFN-inducing ligand remains unknown. In this report, we provide evidence that Chlamydia synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite not previously identified in Gram-negative bacteria, and that this metabolite is a prominent ligand for STING-mediated activation of IFN responses during infection. We used primary mouse lung fibroblasts and HEK293T cells to compare IFN-ß responses to Chlamydia infection, c-di-AMP, and other type I IFN-inducing stimuli. Chlamydia infection and c-di-AMP treatment induced type I IFN responses in cells expressing STING but not in cells expressing STING variants that cannot sense cyclic dinucleotides but still respond to cytoplasmic DNA. The failure to induce a type I IFN response to Chlamydia and c-di-AMP correlated with the inability of STING to relocalize from the endoplasmic reticulum to cytoplasmic punctate signaling complexes required for IFN activation. We conclude that Chlamydia induces STING-mediated IFN responses through the detection of c-di-AMP in the host cell cytosol and propose that c-di-AMP is the ligand predominantly responsible for inducing such a response in Chlamydia-infected cells. IMPORTANCE: This study shows that the Gram-negative obligate pathogen Chlamydia trachomatis, a major cause of pelvic inflammatory disease and infertility, synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite that thus far has been described only in Gram-positive bacteria. We further provide evidence that the host cell employs an endoplasmic reticulum (ER)-localized cytoplasmic sensor, STING (stimulator of interferon [IFN] genes), to detect c-di-AMP synthesized by Chlamydia and induce a protective IFN response. This detection occurs even though Chlamydia is confined to a membrane-bound vacuole. This raises the possibility that the ER, an organelle that innervates the entire cytoplasm, is equipped with pattern recognition receptors that can directly survey membrane-bound pathogen-containing vacuoles for leaking microbe-specific metabolites to mount type I IFN responses required to control microbial infections.

A bacterial two-hybrid system that utilizes Gateway cloning for rapid screening of protein-protein interactions.

Comprehensive clone sets representing the entire genome now exist for a large number of organisms. The Gateway entry clone sets are a particularly useful means to study gene function, given the ease of introduction into any Gateway-suitable destination vector. We have adapted a bacterial two-hybrid system for use with Gateway entry clone sets, such that potential interactions between proteins encoded within these clone sets can be determined by new destination vectors. We show that utilizing the Gateway clone sets for Francisella tularensis and Vibrio cholerae, known interactions between F. tularensis IglA and IglB and V. cholerae VipA and VipB could be confirmed with these destination vectors. Moreover, the introduction of unique tags into each vector allowed for visualization of the expressed hybrid proteins via Western immunoblot. This Gateway-suitable bacterial two-hybrid system provides a new tool for rapid screening of protein-protein interactions.

Oral live vaccine strain-induced protective immunity against pulmonary Francisella tularensis challenge is mediated by CD4+ T cells and antibodies, including immunoglobulin A.

Francisella tularensis is an intracellular gram-negative bacterium and the etiological agent of pulmonary tularemia. Given the high degrees of infectivity in the host and of dissemination of bacteria following respiratory infection, immunization strategies that target mucosal surfaces are critical for the development of effective vaccines against this organism. In this study, we have characterized the efficacy of protective immunity against pneumonic tularemia following oral vaccination with F. tularensis LVS (live vaccine strain). Mice vaccinated orally with LVS displayed colocalization of LVS with intestinal M cells, with subsequent enhanced production of splenic antigen-specific gamma interferon and of systemic and mucosal antibodies, including immunoglobulin A (IgA). LVS-vaccinated BALB/c mice were highly protected against intranasal (i.n.) SCHU S4 challenge and exhibited significantly less bacterial replication in the lungs, liver, and spleen than mock-immunized animals. Depletion of CD4(+) T cells significantly abrogated the protective immunity, and mice deficient in B cells or IgA displayed partial protection against SCHU S4 challenge. These results suggest that oral vaccination with LVS induces protective immunity against i.n. challenge with F. tularensis SCHU S4 by a process mediated cooperatively by CD4(+) T cells and antibodies, including IgA.

The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.

Francisella tularensis causes the human disease tularemia. F. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. F. tularensis virulence is dependent upon the Francisella pathogenicity island (FPI), a cluster of genes that we show here shares homology with type VI secretion gene clusters in Vibrio cholerae and Pseudomonas aeruginosa. We demonstrate that two FPI proteins, VgrG and IglI, are secreted into the cytosol of infected macrophages. VgrG and IglI are required for F. tularensis phagosomal escape, intramacrophage growth, inflammasome activation and virulence in mice. Interestingly, VgrG secretion does not require the other FPI genes. However, VgrG and other FPI genes, including PdpB (an IcmF homologue), are required for the secretion of IglI into the macrophage cytosol, suggesting that VgrG and other FPI factors are components of a secretion system. This is the first report of F. tularensis FPI virulence proteins required for intramacrophage growth that are translocated into the macrophage.

The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression.

Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.

The C-terminus of IpaC is required for effector activities related to Shigella invasion of host cells.

Invasion plasmid antigen C (IpaC) is secreted by the Shigella flexneri type III secretion system (TTSS) as an essential trigger of epithelial cell invasion. At the molecular level, IpaC possesses a distinct functional organization. The IpaC C-terminal region between amino acids 319 and 345 is predicted to form a coiled-coil structure. Such alpha-helical motifs appear to be a recurring structural theme among TTSS components. Together with IpaB, this IpaC region is also required for the formation of translocon pores in target cell membranes. In contrast, mutations within the C-terminal tail of IpaC (defined by residues 345-363) have no effect on contact hemolysis (a putative measure of translocon pore formation), but they can contribute significantly to IpaC's ability to trigger S. flexneri entry into cultured cells. Here we describe the molecular dissection of the IpaC C-terminus and how changes in this region affect selected virulence-related activities. IpaC invasion function requires its immediate C-terminus and this general region may be involved in its ability to trigger actin nucleation. In contrast, IpaC could not be shown to interact directly with Cdc42, a host GTPase closely tied to Shigella invasion.

Lipidation of an FlrC-dependent protein is required for enhanced intestinal colonization by Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, has a sheathed, polar flagellum, and motility has been linked to virulence. An operon with two genes, flgO and flgP (VC2207 and VC2206), is positively regulated by FlrC, the activator of class III flagellar genes. Deletion of flgP results in a nonmotile phenotype, demonstrating the requirement of this gene for V. cholerae motility. V. cholerae delta flgP cells synthesize fragile and defective flagella but transcribe flagellar genes similar to the wild-type strain. PhoA fusion analysis indicated that the putative lipoprotein FlgP is localized external to the cytoplasm, and fractionation demonstrated that it was localized to the outer membrane. Mutagenesis of the site of lipidation of FlgP (C18G) prevented [3H]palmitate incorporation and outer membrane localization. Interestingly, FlgP with the mutation C18G [FlgP(C18G)] could complement the delta flgP mutant for motility, and the cells synthesized wild-type flagella. The delta flgP mutant strain was defective for intestinal colonization (approximately 20-fold), but FlgP(C18G) was unable to complement this defect, demonstrating that lipidation of FlgP is essential for its role in intestinal colonization but not flagellar synthesis. FlgP thus represents a novel V. cholerae intestinal colonization factor that is regulated by the flagellar transcription hierarchy.

Construction of targeted insertion mutations in Francisella tularensis subsp. novicida.

Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species.

A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol.

Francisella tularensis is an intracellular bacterial pathogen, and is a category A bioterrorism agent. Within quiescent human macrophages, the F. tularensis pathogenicity island (FPI) is essential for bacterial growth within quiescent macrophages. The F. tularensis-containing phagosome matures to a late endosome-like stage that does not fuse to lysosomes for 1-8 h, followed by gradual bacterial escape into the macrophage cytosol. Here we show that the FPI protein IglD is essential for intracellular replication in primary human monocyte-derived macrophages (hMDMs). While the parental strain replicates robustly in pulmonary, hepatic and splenic tissues of BALB/c mice associated with severe immunopathologies, the isogenic iglD mutant is severely defective. Within hMDMs, the iglD mutant-containing phagosomes mature to either a late endosome-like phagosome, similar to the parental strain, or to a phagolysosome, similar to phagosomes harbouring the iglC mutant control. Despite heterogeneity and alterations in phagosome biogenesis, the iglD mutant bacteria escape into the cytosol faster than the parental strain within hMDMs and pulmonary cells of BALB/c mice. Co-infections of hMDMs with the wild-type strain and the iglD mutant, or super-infection of iglD mutant-infected hMDMs with the wild-type strain show that the mutant strain replicates robustly within the cytosol of hMDMs coinhabited by the wild strain. However, when the wild-type strain-infected hMDMs are super-infected by the iglD mutant, the mutant fails to replicate in the cytosol of communal macrophages. This is the first demonstration of a F. tularensis novel protein essential for proliferation in the macrophage cytosol. Our data indicate that F. tularensis transduces signals to the macrophage cytosol to remodel it into a proliferative niche, and IglD is essential for transduction of these signals.

Molecular and genetic basis of pathogenesis in Francisella tularensis.

The genetic means of pathogenesis of Francisella tularensis are poorly understood. F. tularensis is able to survive and replicate within macrophages, and a virulence factor, MglA, has been identified that is essential for this ability. MglA regulates the transcription of genes necessary for intracellular growth, including those located within the Francisella Pathogenicity Island (FPI). The FPI genes are required for intramacrophage growth and virulence, and appear to encode a protein secretion system, but the exact function of individual FPI proteins remains to be determined. Additional regulatory factors required for virulence have recently been discovered, as well a number of virulence genes identified through transposon mutant screens. The role of surface components, including LPS, pili, and capsule, in F. tularensis virulence is also beginning to be illuminated. The identification of virulence genes has enabled the study of defined attenuated mutants as live vaccine strains against tularemia. As new components of the F. tularensis virulence gene repertoire are discovered, we will achieve a better understanding of how this bacterium interacts with the host and evades immune function, which will facilitate the development of therapeutic and preventive measures against tularemia.

The Vibrio cholerae FlgM homologue is an anti-sigma28 factor that is secreted through the sheathed polar flagellum.

Vibrio cholerae has a single polar sheathed flagellum that propels the cells of this bacterium. Flagellar synthesis, motility, and chemotaxis have all been linked to virulence in this human pathogen. V. cholerae expresses flagellar genes in a hierarchy consisting of sigma54- and sigma28-dependent transcription. In other bacteria, sigma28 transcriptional activity is controlled by an anti-sigma28 factor, FlgM. We demonstrate that the V. cholerae FlgM homologue (i) physically interacts with sigma28, (ii) has a repressive effect on some V. cholerae sigma28-dependent flagellar promoters, and (iii) is secreted through the polar sheathed flagellum, consistent with anti-sigma28 activity. Interestingly, FlgM does not have a uniform repressive effect on all sigma28-dependent promoters, as determined by measurement of sigma28-dependent transcription in cells either lacking FlgM (DeltaflgM) or incapable of secretion (DeltafliF). Further analysis of a DeltafliF strain revealed that this flagellar assembly block causes a decrease in class III (FlrC- and sigma54-dependent) and class IV (sigma28-dependent), but not class II (FlrA- and sigma54-dependent), flagellar transcription. V. cholerae flgM and fliA (encodes sigma28) mutants were only modestly affected in their ability to colonize the infant mouse intestine, a measure of virulence. Our results demonstrate that V. cholerae FlgM functions as an anti-sigma28 factor and that the sheathed flagellum is competent for secretion of nonstructural proteins.

MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival.

Francisella tularensis is able to survive and grow within macrophages, a trait that contributes to pathogenesis. Several genes have been identified that are important for intramacrophage survival, including mglA and iglC. F. tularensis is also able to survive within amoebae. It is shown here that F. tularensis mglA and iglC mutant strains are not only defective for survival and replication within the macrophage-like cell line J774, but also within Acanthamoebae castellanii. Moreover, these strains are highly attenuated for virulence in mice, suggesting that a common mechanism underlies intramacrophage and intraamoebae survival and virulence. A 2D gel analysis of cell extracts of wild-type and mglA mutant strains revealed that at least seven prominent proteins were at low levels in the mglA mutant, and one MglA-regulated protein was identified as the IglC protein. RT-PCR analysis demonstrated reduced transcription of iglC and several other known and suspected virulence genes in the mglA mutant. Thus, MglA regulates the transcription of virulence factors of F. tularensis that contribute to intramacrophage and intraamoebae survival.

Allelic exchange in Francisella tularensis using PCR products.

We describe here a technique for allelic exchange in Francisella tularensis subsp. novicida utilizing polymerase chain reaction (PCR) products. Linear PCR fragments containing gene deletions with an erythromycin resistance cassette insertion were transformed into F. tularensis. The subsequent ErmR progeny were found to have undergone allelic exchange at the correct location in the genome; the minimum flanking homology necessary was 500 bp. This technique was used to create mglA, iglC, bla, and tul4 mutants in F. tularensis subsp. novicida strains. The mglA and iglC mutants were defective for intramacrophage growth, and the tul4 mutant lacked detectable Tul4 by Western immunoblot, as expected. Interestingly, the bla mutant maintained resistance to ampicillin, indicating the presence of multiple ampicillin resistance genes in F. tularensis.

Structural characterization of the N terminus of IpaC from Shigella flexneri.

The primary effector for Shigella invasion of epithelial cells is IpaC, which is secreted via a type III secretion system. We recently reported that the IpaC N terminus is required for type III secretion and possibly other functions. In this study, mutagenesis was used to identify an N-terminal secretion signal and to determine the functional importance of the rest of the IpaC N terminus. The 15 N-terminal amino acids target IpaC for secretion by Shigella flexneri, and placing additional amino acids at the N terminus does not interfere with IpaC secretion. Furthermore, amino acid sequences with no relationship to the native IpaC secretion signal can also direct its secretion. Deletions introduced beyond amino acid 20 have no effect on secretion and do not adversely affect IpaC function in vivo until they extend beyond residue 50, at which point invasion function is completely eliminated. Deletions introduced at amino acid 100 and extending toward the N terminus reduce IpaC's invasion function but do not eliminate it until they extend to the N-terminal side of residue 80, indicating that a region from amino acid 50 to 80 is critical for IpaC invasion function. To explore this further, the ability of an IpaC N-terminal peptide to associate in vitro with its translocon partner IpaB and its chaperone IpgC was studied. The N-terminal peptide binds tightly to IpaB, but the IpaC central hydrophobic region also appears to participate in this binding. The N-terminal peptide also associates with the chaperone IpgC and IpaB is competitive for this interaction. Based on additional biophysical data, we propose that a region between amino acids 50 and 80 is required for chaperone binding, and that the IpaB binding domain is located downstream from, and possibly overlapping, this region. From these data, we propose that the secretion signal, chaperone binding region, and IpaB binding domain are located at the IpaC N terminus and are essential for presentation of IpaC to host cells during bacterial entry; however, IpaC effector activity may be located elsewhere.