Contribution of Noncanonical Antigens to Virulence and Adaptive Immunity in Human Infection with Enterotoxigenic E. coli.

Enterotoxigenic Escherichia coli (ETEC) contributes significantly to the substantial burden of infectious diarrhea among children living in low- and middle-income countries. In the absence of a vaccine for ETEC, children succumb to acute dehydration as well as nondiarrheal sequelae related to these infections, including malnutrition. The considerable diversity of ETEC genomes has complicated canonical vaccine development approaches defined by a subset of ETEC pathovar-specific antigens known as colonization factors (CFs). To identify additional conserved immunogens unique to this pathovar, we employed an "open-aperture" approach to capture all potential conserved ETEC surface antigens, in which we mined the genomic sequences of 89 ETEC isolates, bioinformatically selected potential surface-exposed pathovar-specific antigens conserved in more than 40% of the genomes (n = 118), and assembled the representative proteins onto microarrays, complemented with known or putative colonization factor subunit molecules (n = 52) and toxin subunits. These arrays were then used to interrogate samples from individuals with acute symptomatic ETEC infections. Surprisingly, in this approach, we found that immune responses were largely constrained to a small number of antigens, including individual colonization factor antigens and EtpA, an extracellular adhesin. In a Bangladeshi cohort of naturally infected children <2 years of age, both EtpA and a second antigen, EatA, elicited significant serologic responses that were associated with protection from symptomatic illness. In addition, children infected with ETEC isolates bearing either etpA or eatA genes were significantly more likely to develop symptomatic disease. These studies support a role for antigens not presently targeted by vaccines (noncanonical) in virulence and the development of adaptive immune responses during ETEC infections. These findings may inform vaccine design efforts to complement existing approaches.

Vinculin proteolysis unmasks an ActA homolog for actin-based Shigella motility.

To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1-3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 +/- 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that supplementing cells with p90 stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878-21885) and to vasodilator-stimulated phosphoprotein (VASP) (Brindle, N.P.J., M. R. Hold, J.E. Davies, C.J. Price, and D.R. Critchley. 1996. Biochem. J. 318:753-757). We now offer a working model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and profilin.

Eukaryotic gene expression: metabolite control by amino acids.

Our understanding of the metabolite control in mammalian cells lags far behind that in prokaryotes. This is particularly true for amino-acid-dependent gene expression. Few proteins have been identified for which synthesis is selectively regulated by amino-acid availability, and the mechanisms for control of transcription and translation in response to changes in amino-acid availability have not yet been elucidated. The intimate relationship between amino-acid supply and the fundamental cellular process of protein synthesis makes amino-acid-dependent control of gene expression particularly important. Future studies should provide important insight into amino-acid and other nutrient signaling pathways, and their impact on cellular growth and metabolism.

Identification of the discontinuous epitope in human complement protein C9 recognized by anti-melittin antibodies.

Polyclonal rabbit antibodies against melittin recognize human C protein C9 and retard C9-mediated hemolysis. Human C9 contains a tetrameric and a pentameric sequence (amino acids 293-296 and 528-532, respectively) that together match a continuous segment in the melittin sequence, i.e., residues 8-16. It has been suggested that the tetrameric and the pentameric regions on C9 form a discontinuous epitope on folded C9 that mimics the structure of melittin. To further test this hypothesis, antibodies to C9-sequence-specific peptides were prepared. Peptides containing either the homologous tetrameric or the homologous pentameric sequence together with short stretches of the respective amino- and carboxyl-terminal flanking regions were synthesized, as well as a composite peptide predicted to resemble the discontinuous epitope as a linear, nine-amino acid sequence. Direct and competitive binding assays demonstrated that the tetrameric and the pentameric sequences are part of the epitope on human C9 that is recognized by anti-melittin IgG. However, only antibodies directed against the complete epitope are capable of inhibiting hemolysis. Because neither anti-tetramer nor anti-pentamer antibodies affect hemolysis whereas anti-melittin and anti-composite antibodies do, we propose that human C9 changes conformation around a hinge located between residues 296 and 528 and that the latter two antibodies inhibit unfolding required for membrane insertion and subsequent hemolysis.

Detection of refolding conformers of complement protein C9 during insertion into membranes.

Human complement protein C9 is a hydrophilic serum glycoprotein responsible for efficient expression of the cytotoxic and cytolytic functions of complement. It assembles on the surface of a target cell together with C5, C6, C7 and C8 to form the membrane attack complex (MAC) and therefore has to change structure to become an integral membrane protein. As the protein assumes a stable structure in an aqueous environment, the question arises as to how it can enter the hydrophobic interior of a membrane. During MAC assembly C9 polymerizes into a circular structure, termed poly(C9) (ref. 8), which is responsible for the cylindrical electron microscopic appearance of the MAC. The suggestion has been made that C9 must at least partly unfold in order to enter a membrane and also that polymerization of the molecule is intimately linked to insertion and cytotoxicity. The extent of unfolding and the mechanism of polymerization are not understood, nor is it known precisely which parts of the molecule participate in the proposed structural changes. We have been able to capture refolding C9 conformers during membrane insertion with the help of sequence-specific anti-peptide antibodies. Some of these antibodies inhibit C9-mediated haemolysis but not C9 polymerization, while others have the opposite effect. This suggests that the two processes are independent.

Enhanced xenobiotic transporter expression in normal teleost hepatocytes: response to environmental and chemotherapeutic toxins.

Many aquatic organisms are resistant to environmental pollutants, probably because their inherent multi-drug-resistant protein extrusion pump (pgp) can be co-opted to handle man-made pollutants. This mechanism of multixenobiotic resistance is similar to the mechanism of multidrug resistance exhibited in chemotherapy-resistant human tumor cells. In the present study, a variety of techniques were used to characterize this toxin defense system in killifish (Fundulus heteroclitus) hepatocytes. The cellular localization and activity of the putative drug efflux system were evaluated. In addition, in vitro and in vivo studies were used to examine the range of expression of this putative drug transporter in the presence of environmental and chemotherapeutic toxins. The broad range of pgp expression generally observed in transformed mammalian cells was found in normal cells of our teleost model. Our findings suggest that the expression of the pgp gene in the killifish could be an excellent indicator of toxin levels or stressors in the environment.

Comparison between complement and melittin hemolysis: anti-melittin antibodies inhibit complement lysis.

A comparison is made between the hemolytic actions of melittin and the ninth component of complement (C9). Melittin and C9 produce "pores" of similar effective radius in erythrocytes under standardized conditions, and their hemolytic action is suppressed by metal ions at similar concentrations, suggesting a common mechanism. Polyclonal anti-melittin immunoglobulin G (IgG) produced in rabbits retards hemolysis mediated by human C9 in a specific manner. Such antibodies react in several immunoassays with human and monkey C9 but not with C9 from lower animals, and no inhibition of lysis mediated by C9 molecules from these animals is observed. Thus, it is unlikely that anti-melittin IgG reacts with a structural element, such as an amphipathic helix, on human C9 since such structures are also predicted to exist in other C9 molecules. Human C9 and melittin block cross-reactivity in a dose-dependent manner, and anti-melittin IgG recognizes an epitope located between amino acid residues 245 and 390 of human C9 on "Western" blots. Comparison of the melittin and human C9 sequences indicates two regions of complete homology, a tetrapeptide at positions 292-295, and a pentapeptide at positions 527-531 in human C9, corresponding to residues 8-16 in melittin. Inhibition of hemolysis is not caused by blocking of C9 binding to the C5b-8 complex; rather the antibody must dissociate from the bound C9 before lysis ensues, indicating that it interferes with a postbinding event. It is proposed that anti-melittin binds to a conformational epitope on native, folded human C9 and thereby retards unfolding of the molecule, which is required for membrane insertion and hemolysis.

Nuclear retention of the induced mRNA following amino acid-dependent transcriptional regulation of mammalian ribosomal proteins L17 and S25.

An amino acid starvation-induced mRNA, increased up to 4-fold in the absence of amino acids, was identified previously as rat 60 S subunit ribosomal protein L17. The data presented here demonstrate that among ribosomal proteins, L17, as well as the smaller subunit ribosomal protein S25, are uniquely regulated by amino acid deprivation of cells; the increase in L17 and S25 mRNA content in response to substrate starvation is not shared by the 11 other ribosomal proteins tested. When rat Fao hepatoma cells were incubated in the presence of actinomycin D, the L17 mRNA level decayed below the basal level, regardless of medium amino acid content, and nuclear run-off assays confirmed that nutrient starvation leads to enhanced transcription of the L17 ribosomal protein gene. Likewise, the induction of S25 mRNA also was prevented in the presence of actinomycin D. Furthermore, the induction of L17 and S25 mRNA content was blocked by cycloheximide, demonstrating the requirement for a newly synthesized protein in the signaling pathway. Northern analysis with RNA isolated from cytoplasmic, polysomal, and nuclear enriched fractions indicated that the starvation-dependent increase in the S25 and L17 mRNAs is retained within the nucleus and not is available for translation. Amino acid refeeding of the cells caused the translocation of the stored nuclear mRNAs to the polysomal fraction.

Amino acid-regulated gene expression in eukaryotic cells.

Given the central role of protein synthesis in cellular function, it is likely that intricate mechanisms exist to detect and respond to amino acid deprivation. However, the current understanding of amino acid-dependent control of gene expression in mammalian cells is limited. A few examples of enzyme, transporters, and unidentified mRNA species subject to amino acid availability have been reported and some examples are summarized here. Each example chosen-asparagine synthetase, system A transport activity, and ribosomal protein L17--are associated with different aspects of amino acid metabolism, and therefore reflect the spectrum of metabolic pathways influenced by substrate control. Most of the data accumulated thus far suggest that a general control response exists such that these various activities are induced when any one of several amino acids becomes limiting. Consistent with observations in yeast, it appears that the degree of tRNA acylation and its resultant effect on protein synthesis may play an important role in initiating the starvation signal. De novo protein synthesis is required for starvation-dependent increases in several mRNA species, which suggests that the amino acid signaling pathway is composed of a series of intermediate steps before activation of specific structural genes.

Identification of an amino acid-regulated mRNA from rat liver as the mammalian equivalent of bacterial ribosomal protein L22.

Amino acid deprivation of rat hepatoma cells induced the levels of a 612-base pair mRNA termed ASI (Shay, N. F., Nick, H. S., and Kilberg, M. S. (1990) J. Biol. Chem. 265, 17844-17848). The ASI mRNA was present at levels equal to or greater than actin in every rat tissue tested. The corresponding full-length cDNA was cloned, and the present report demonstrates that the deduced 184-residue amino acid sequence shares greater than 30% identity to a number of bacterial and chloroplast L22 ribosomal proteins, including those from Escherichia coli and Halobacterium halobium. A monospecific anti-peptide antibody was produced that upon immunochemical analysis of subcellular fractions of rat liver recognized a band in the microsomal fraction and, more specifically, reacted with a single polypeptide in the ribosomal large subunit fraction. The antibody did not react with any proteins of the mitochondrial large subunit, but did recognize a protein in human liver homogenate at the same relative mobility (23 kDa) as that observed for rat liver.

Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): implications for actin-based Listeria motility.

Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization. We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM. This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEBS Lett. 333, 123-126], exhibits little or no evidence of saturable GP5GP5GP5 binding. When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.

Enhanced mRNA content in response to amino acid starvation for a 73 kDa protein of the inner mitochondrial membrane.

Biosynthesis of several rat liver proteins is enhanced by amino acid deprivation of cultured hepatocytes or hepatoma cells. One of these proteins, MP-73, was synthesized at a rate 2- to 3-fold greater when cells were incubated for 3-9 h under conditions of amino acid deprivation versus amino acid supplementation. Immunoblotting with polyclonal antibodies prepared against MP-73 localized it to the inner mitochondrial membrane. MP-73 appears to be a hydrophobic, integral membrane protein. MP-73 antibody was used to identify a partial cDNA (NS3.2) of approximately 2 kb. A probe prepared from pNS3.2 identified a transcript in rat Fao hepatoma cells of approximately 4.4 kb that was increased in abundance by more than 20-fold following amino acid starvation of the cells.

Identification of the protein responsible for hepatic system N amino acid transport activity.

In the liver, glutamine utilization may be limited by the rate of transport across the plasma membrane by the System N carrier. System N-mediated transport activity has been solubilized from rat liver plasma membrane, partially purified, and then reconstituted into proteoliposomes. To identify the System N carrier protein, monoclonal antibodies were generated against the protein fraction enriched for System N activity. Two antibodies , 3E1-2 and 1E7-3, inhibited System N activity in hepatocytes. These antibodies also immunoprecipitated System N activity from a mixture of solubilized proteins and were specific for antigen recognition in that neither immunoprecipitated System A activity. The antibody recognized a single protein of molecular size 100 kDa by immunoblot analysis. Recognition of this protein by the antibody increased in parallel with the enrichment of System N activity in solubilized membrane fractions. These data suggest that a 100-kDa plasma membrane protein mediates System N transport activity in rat hepatocytes.

Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.

The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.

Plasma membrane clustering of system y+ (CAT-1) amino acid transporter as detected by immunohistochemistry.

Transport of cationic amino acids in fully differentiated mammalian cells is mediated primarily by system y1+ [cationic amino acid transporter (CAT)-1 gene product]. Antibodies, prepared against synthetic peptide sequences predicted to be extracellular loops of the CAT-1 transporter protein, detected the transporter on the surface of cultured cells. In human fibroblasts, porcine pulmonary artery endothelial cells, and cultured rat hepatoma cells, the CAT-1 transporter protein was clustered in an apparent random pattern throughout the plasma membrane. In contrast, labeling of the fibroblasts with antibodies against the epidermal growth factor receptor or the GLUT-1 glucose transporter demonstrated a uniform staining pattern covering the entire cell surface. The CAT-1 antibody labeling was specific, as demonstrated by peptide inhibition and the lack of staining by preimmune serum. Furthermore, hepatocytes did not exhibit specific antibody binding consistent with the lack of system y1+ activity. Disruption of the microtubule assembly resulted in a reversible loss of the CAT-1 transporter clusters and a more generalized labeling of the cell body. The data demonstrate the existence of microdomains within the plasma membrane that contain the CAT-1 transporter protein.