RTX Toxin Research Articles

Identification and Characterization of an RTX Toxin–Like Gene and Its Operon from Avibacterium paragallinarum

Identification and Characterization of an RTX Toxin–Like Gene and Its Operon from Avibacterium paragallinarum

Multilocus Sequence Typing and rtxA Toxin Gene Sequencing Analysis of Kingella kingae Isolates Demonstrates Genetic Diversity and International Clones

Background Kingella kingae, a normal component of the upper respiratory flora, is being increasingly recognized as an important invasive pathogen in young children. Genetic diversity of this species has not been studied.MethodsWe analyzed 103 strains from different countries and clinical origins by a new multilocus sequence-typing (MLST) schema. Putative virulence gene rtxA, encoding an RTX toxin, was also sequenced, and experimental virulence of representative strains was assessed in a juvenile-rat model.ResultsThirty-six sequence-types (ST) and nine ST-complexes (STc) were detected. The main STc 6, 14 and 23 comprised 23, 17 and 20 strains respectively, and were internationally distributed. rtxA sequencing results were mostly congruent with MLST, and showed horizontal transfer events. Of interest, all members of the distantly related ST-6 (n = 22) and ST-5 (n = 4) harboured a 33 bp duplication or triplication in their rtxA sequence, suggesting that this genetic trait arose through selective advantage. The animal model revealed significant differences in virulence among strains of the species.ConclusionMLST analysis reveals international spread of ST-complexes and will help to decipher acquisition and evolution of virulence traits and diversity of pathogenicity among K. kingae strains, for which an experimental animal model is now available.

Additive Function of Vibrio vulnificus MARTXVv and VvhA Cytolysins Promotes Rapid Growth and Epithelial Tissue Necrosis During Intestinal Infection

Vibrio vulnificus is a pathogen that causes both severe necrotizing wound infections and life-threatening food-borne infections. Food-borne infection is particularly lethal as the infection can progress rapidly to primary septicemia resulting in death from septic shock and multiorgan failure. In this study, we use both bioluminescence whole animal imaging and V. vulnificus bacterial colonization of orally infected mice to demonstrate that the secreted multifunctional-autoprocessing RTX toxin (MARTXVv) and the cytolysin/hemolysin VvhA of clinical isolate CMCP6 have an important function in the gut to promote early in vivo growth and dissemination of this pathogen from the small intestine to other organs. Using histopathology, we find that both cytotoxins can cause villi disruption, epithelial necrosis, and inflammation in the mouse small intestine. A double mutant deleted of genes for both cytotoxins was essentially avirulent, did not cause intestinal epithelial tissue damage, and was cleared from infected mice by 36 hours by an effective immune response. Therefore, MARTXVv and VvhA seem to play an additive role for pathogenesis of CMCP6 causing intestinal tissue damage and inflammation that then promotes dissemination of the infecting bacteria to the bloodstream and other organs. In the absence of these two secreted factors, we propose that this bacterium is unable to cause intestinal infection in humans.

Protective mechanism of curcumin againstVibrio vulnificusinfection

Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., has many beneficial biological activities. However, there are relatively few reports of the effects of curcumin on pathogen infections. This study examined the effect of curcumin on a Vibrio vulnificus infection. The cytotoxicity of V. vulnificus to HeLa cells was significantly inhibited by curcumin (at 10 or 30μM). To further examine the inhibitory mechanism of curcumin against V. vulnificus-mediated cytotoxicity, the level of bacterial growth, bacterial motility, cell adhesion, RTX toxin expression and host cell reactions were evaluated. Curcumin inhibited V. vulnificus growth in HI broth. Curcumin inhibited both bacterial adhesion and RTX toxin binding to the host cells, which can be considered the major protective mechanisms for the decrease in V. vulnificus cytotoxicity. Curcumin also inhibited host cell rounding and actin aggregation, which are the early features of cell death caused by V. vulnificus. In addition, curcumin decreased the V. vulnificus-induced NF-κB translocation in HeLa cells. Finally, curcumin protected mice from V. vulnificus-induced septicemia. In conclusion, curcumin may be an alternative antimicrobial agent against fatal bacterial infections.

Pathogenic Potential of Campylobacter ureolyticus

The recent detection and isolation of the aflagellate Campylobacter ureolyticus (previously known as Bacteroides ureolyticus) from intestinal biopsy specimens and fecal samples of children with newly diagnosed Crohn's disease led us to investigate the pathogenic potential of this bacterium. Adherence and gentamicin protection assays were employed to quantify the levels of adherence to and invasion into host cells. C. ureolyticus UNSWCD was able to adhere to the Caco-2 intestinal epithelial cell line with a value of 5.341% ± 0.74% but was not able to invade the Caco-2 cells. The addition of two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), to the cell line did not affect attachment or invasion, with attachment levels being 4.156% ± 0.61% (P = 0.270) for TNF-α and 6.472% ± 0.61% (P = 0.235) for IFN-γ. Scanning electron microscopy visually confirmed attachment and revealed that C. ureolyticus UNSWCD colonizes and adheres to intestinal cells, inducing cellular damage and microvillus degradation. Purification and identification of the C. ureolyticus UNSWCD secretome detected a total of 111 proteins, from which 29 were bioinformatically predicted to be secretory proteins. Functional classification revealed three putative virulence and colonization factors: the surface antigen CjaA, an outer membrane fibronectin binding protein, and an S-layer RTX toxin. These results suggest that C. ureolyticus has the potential to be a pathogen of the gastrointestinal tract.

Genomic basis of endosymbiont-conferred protection against an insect parasitoid

Bacterial endosymbionts exert a variety of beneficial effects on insect hosts. In pea aphids (Acyrthosiphon pisum), several inherited endosymbiont species protect their hosts against parasitoid wasps, which are major natural enemies. However, strains of these symbiont species vary in their ability to confer protection against parasitoids, with some conferring almost complete protection and others conferring almost none. In this study, two strains of the endosymbiont Regiella insecticola (R. insecticola 5.15 and R. insecticola LSR1) were found to differ in ability to protect pea aphids attacked by the parasitoid Aphidius ervi. Parasitism trials reveal that R. insecticola 5.15, but not R. insecticola LSR1, significantly reduced parasitoid success and increased aphid survivorship. To address the potential genetic basis of protection conferred by R. insecticola 5.15 we sequenced the genome of this symbiont strain, and then compared its gene repertoire with that of the already sequenced nonprotective strain R. insecticola LSR1. We identified striking differences in gene sets related to eukaryote pathogenicity. The protective strain R. insecticola 5.15 encoded five categories of pathogenicity factors that were missing or inactivated in R. insecticola LSR1. These included genes encoding the O-antigen biosynthetic pathway, an intact Type 1 Secretion System and its secreted RTX toxins, an intact SPI-1 Type 3 Secretion System and its effectors, hemin transport, and the two-component system PhoPQ. These five pathogenicity factors and translocation systems are hypothesized to collectively play key roles in the endosymbiont's virulence against parasitoids, resulting in aphid protection. Mechanisms through which these factors may target parasitoids are discussed.

Comparative analysis of proteins in the culture supernatants of human intestinal epithelial cells infected with the wild-type and rtxE mutant of Vibrio vulnificus

Bacterial virulence factors and secreted extracellular proteins from damaged host cells following infection have been recognized as key mediators in the pathophysiological alterations observed in septic shock, and have also been shown to have a synergistic influence on bacterial pathogenicity. We hypothesized that during infections, virulence factors as well as host-shed proteins may synergistically influence aspects of the pathogenicity of V. vulnificus, such as primary septic shock and overproduction of proinflammatory cytokines. However, virulence factors and host-derived proteins have yet to be clearly evaluated during V. vulnificus infection. In this study, we analyzed and compared the proteins in conditioned supernatants generated from co-cultures of host cells and either wild-type or rtxE mutant V. vulnificus using LC-QTOF-MS/MS analysis. In a previous study, we determined that the culture supernatants of the rtxE mutant V. vulnificus-infected INT-407 cells induced significantly lower levels of IL-8 production from human intestinal epithelial cells than did the culture supernatants of wild-type V. vulnificus-infected INT-407 cells. LC-QTOF-MS/MS analysis results demonstrated that levels of proteins such as HSP90 α/β, 14-3-3 γ, PRX II, hnRNP K, β-actin, α-tubulin and V. vulnificus flagellin were significantly lower in the culture supernatants of rtxE mutant V. vulnificus-infected INT-407 cells than in the culture supernatants of wild-type V. vulnificus-infected INT-407 cells. These results demonstrate that V. vulnificus RTX toxins acting via rtxE, a transporter of virulence factors, play a very important role in the pathogenesis of V. vulnificus, as well as in its initial role in inducing pathogenic mediators from host cells.

RTX Toxin Enhances the Survival of Vibrio vulnificus During Infection by Protecting the Organism From Phagocytosis

Vibrio vulnificus is a marine bacterium causing serious septicemia and wound infection in humans. It produces an RTX toxin that can lyse a variety of cells and is important for virulence in mice. In this study, we explored the role of RTX in pathogenesis by characterizing an RTX-deficient mutant. This mutant showed an ∼2-log reduction in virulence for mice infected by various routes. Survival of the mutant at the infection site and subsequent spread into the bloodstream were impaired. In mice pretreated with cyclophosphamide to deplete the neutrophils, both the virulence and survival at the infection site of this mutant were enhanced. This mutant was further shown to be more readily cleared from the macrophage-rich mouse peritoneal cavity and phagocytosed by murine macrophages. These findings suggest that the RTX of V. vulnificus is required for bacterial survival during infection by protecting the organism from phagocytosis.

Common Ancestry and Novel Genetic Traits of Francisella novicida-Like Isolates from North America and Australia as Revealed by Comparative Genomic Analyses

Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains.

Actin as target for modification by bacterial protein toxins

Various bacterial protein toxins and effectors target the actin cytoskeleton. At least three groups of toxins/effectors can be identified, which directly modify actin molecules. One group of toxins/effectors causes ADP-ribosylation of actin at arginine-177, thereby inhibiting actin polymerization. Members of this group are numerous binary actin-ADP-ribosylating exotoxins (e.g. Clostridium botulinum C2 toxin) as well as several bacterial ADP-ribosyltransferases (e.g. Salmonella enterica SpvB) which are not binary in structure. The second group includes toxins that modify actin to promote actin polymerization and the formation of actin aggregates. To this group belongs a toxin from the Photorhabdus luminescens Tc toxin complex that ADP-ribosylates actin at threonine-148. A third group of bacterial toxins/effectors (e.g. Vibrio cholerae multifunctional, autoprocessing RTX toxin) catalyses a chemical crosslinking reaction of actin thereby forming oligomers, while blocking the polymerization of actin to functional filaments. Novel findings about members of these toxin groups are discussed in detail.

Virulence Evolution of the Human Pathogen Neisseria meningitidis by Recombination in the Core and Accessory Genome

Background Neisseria meningitidis is a naturally transformable, facultative pathogen colonizing the human nasopharynx. Here, we analyze on a genome-wide level the impact of recombination on gene-complement diversity and virulence evolution in N. meningitidis. We combined comparative genome hybridization using microarrays (mCGH) and multilocus sequence typing (MLST) of 29 meningococcal isolates with computational comparison of a subset of seven meningococcal genome sequences.Principal FindingsWe found that lateral gene transfer of minimal mobile elements as well as prophages are major forces shaping meningococcal population structure. Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA) island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages. By computationally screening also the core genome for signs of recombination, we provided evidence that about 40% of the meningococcal core genes are affected by recombination primarily within metabolic genes as well as genes involved in DNA replication and repair. By comparison with the results of previous mCGH studies, our data indicated that genetic structuring as revealed by mCGH is stable over time and highly similar for isolates from different geographic origins.ConclusionsRecombination comprising lateral transfer of entire genes as well as homologous intragenic recombination has a profound impact on meningococcal population structure and genome composition. Our data support the hypothesis that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence.

Kingella kingae: An Emerging Pathogen in Young Children

Kingella kingae is being recognized increasingly as a common etiology of pediatric osteoarticular infections, bacteremia, and endocarditis, which reflects improved culture methods and use of nucleic acid-amplification techniques in clinical microbiology laboratories. K kingae colonizes the posterior pharynx of young children and is transmitted from child to child through close personal contact. Day care attendance increases the risk for colonization and transmission, and clusters of K kingae infections among day care center attendees have been reported. Key virulence factors in K kingae include type IV pili and a potent RTX toxin. In previously healthy children, >95% of K kingae infections are diagnosed between the ages of 6 and 48 months. Among children with underlying medical conditions, K kingae disease may occur at older ages as well. The clinical presentation of K kingae disease is often subtle and may be associated with normal levels of acute-phase reactants, which underscores the importance of a high index of suspicion. K kingae is usually susceptible to ß-lactam antibiotics, and infections typically respond well to medical treatment, with the exception of cases of endocarditis.

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

BackgroundLaribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements.ResultsFor acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases.ConclusionsThe L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.

Molecular and virulence characteristics of an outer membrane-associated RTX exoprotein in Pasteurella pneumotropica

BackgroundPasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system.ResultsThe RTX exoprotein, PnxIIIA, contains only a few copies of the RTX repeat-like sequence and 3 large repeat sequences that are partially similar to the outer membrane protein found in several prokaryotes. Recombinant PnxIIIA protein (rPnxIIIA) was cytotoxic toward J774A.1 mouse macrophage cells, whereas cytotoxicity was attenuated by the addition of anti-CD11a monoclonal antibody. rPnxIIIA could bind to extracellular matrices (ECMs) and cause hemagglutination of sheep erythrocytes. Binding was dependent on the 3 large repeat sequences in PnxIIIA. Protein interaction analyses indicated that PnxIIIA is mainly localized in the outer membrane of P. pneumotropica ATCC 35149 in a self-assembled oligomeric form. PnxIIIA is less cytotoxic to J774A.1 cells than PnxIA and PnxIIA.ConclusionsThe results implicate that PnxIIIA is located on the cell surface and participates in adhesion to ECMs and enhanced hemagglutination in the rodent pathogen P. pneumotropica.

Comparative Genome Biology of a Serogroup B Carriage and Disease Strain Supports a Polygenic Nature of Meningococcal Virulence

Neisseria meningitidis serogroup B strains are responsible for most meningococcal cases in the industrialized countries, and strains belonging to the clonal complex ST-41/44 are among the most prevalent serogroup B strains in carriage and disease. Here, we report the first genome and transcriptome comparison of a serogroup B carriage strain from the clonal complex ST-41/44 to the serogroup B disease strain MC58 from the clonal complex ST-32. Both genomes are highly colinear, with only three major genome rearrangements that are associated with the integration of mobile genetic elements. They further differ in about 10% of their gene content, with the highest variability in gene presence as well as gene sequence found for proteins involved in host cell interactions, including Opc, NadA, TonB-dependent receptors, RTX toxin, and two-partner secretion system proteins. Whereas housekeeping genes coding for metabolic functions were highly conserved, there were considerable differences in their expression pattern upon adhesion to human nasopharyngeal cells between both strains, including differences in energy metabolism and stress response. In line with these genomic and transcriptomic differences, both strains also showed marked differences in their in vitro infectivity and in serum resistance. Taken together, these data support the concept of a polygenic nature of meningococcal virulence comprising differences in the repertoire of adhesins as well as in the regulation of metabolic genes and suggest a prominent role for immune selection and genetic drift in shaping the meningococcal genome.

Dynamics of genome evolution in facultative symbionts of aphids

Aphids are sap-feeding insects that host a range of bacterial endosymbionts including the obligate, nutritional mutualist Buchnera plus several bacteria that are not required for host survival. Among the latter, ‘Candidatus Regiella insecticola’ and ‘Candidatus Hamiltonella defensa’ are found in pea aphids and other hosts and have been shown to protect aphids from natural enemies. We have sequenced almost the entire genome of R. insecticola (2.07 Mbp) and compared it with the recently published genome of H. defensa (2.11 Mbp). Despite being sister species the two genomes are highly rearranged and the genomes only have ∼55% of genes in common. The functions encoded by the shared genes imply that the bacteria have similar metabolic capabilities, including only two essential amino acid biosynthetic pathways and active uptake mechanisms for the remaining eight, and similar capacities for host cell toxicity and invasion (type 3 secretion systems and RTX toxins). These observations, combined with high sequence divergence of orthologues, strongly suggest an ancient divergence after establishment of a symbiotic lifestyle. The divergence in gene sets and in genome architecture implies a history of rampant recombination and gene inactivation and the ongoing integration of mobile DNA (insertion sequence elements, prophage and plasmids).

In-Depth Analysis of Exoproteomes from Marine Bacteria by Shotgun Liquid Chromatography-Tandem Mass Spectrometry: the Ruegeria pomeroyi DSS-3 Case-Study

Microorganisms secrete into their extracellular environment numerous compounds that are required for their survival. Many of these compounds could be of great interest for biotechnology applications and their genes used in synthetic biology design. The secreted proteins and the components of the translocation systems themselves can be scrutinized in-depth by the most recent proteomic tools. While the secretomes of pathogens are well-documented, those of non-pathogens remain largely to be established. Here, we present the analysis of the exoproteome from the marine bacterium Ruegeria pomeroyi DSS-3 grown in standard laboratory conditions. We used a shotgun approach consisting of trypsin digestion of the exoproteome, and identification of the resulting peptides by liquid chromatography coupled to tandem mass spectrometry. Three different proteins that have domains homologous to those observed in RTX toxins were uncovered and were semi-quantified as the most abundantly secreted proteins. One of these proteins clearly stands out from the catalogue, representing over half of the total exoproteome. We also listed many soluble proteins related to ABC and TRAP transporters implied in the uptake of nutrients. The Ruegeria pomeroyi DSS-3 case-study illustrates the power of the shotgun nano-LC-MS/MS strategy to decipher the exoproteome from marine bacteria and to contribute to environmental proteomics.

Inositol Hexakisphosphate-Induced Autoprocessing of Large Bacterial Protein Toxins

Large bacterial protein toxins autotranslocate functional effector domains to the eukaryotic cell cytosol, resulting in alterations to cellular functions that ultimately benefit the infecting pathogen. Among these toxins, the clostridial glucosylating toxins (CGTs) produced by Gram-positive bacteria and the multifunctional-autoprocessing RTX (MARTX) toxins of Gram-negative bacteria have distinct mechanisms for effector translocation, but a shared mechanism of post-translocation autoprocessing that releases these functional domains from the large holotoxins. These toxins carry an embedded cysteine protease domain (CPD) that is activated for autoprocessing by binding inositol hexakisphosphate (InsP6), a molecule found exclusively in eukaryotic cells. Thus, InsP6-induced autoprocessing represents a unique mechanism for toxin effector delivery specifically within the target cell. This review summarizes recent studies of the structural and molecular events for activation of autoprocessing for both CGT and MARTX toxins, demonstrating both similar and potentially distinct aspects of autoprocessing among the toxins that utilize this method of activation and effector delivery.

Population-based analysis of Actinobacillus pleuropneumoniae ApxIVA for use as a DIVA antigen

APXIVA is an RTX toxin of Actinobacillus pleuropneumoniae that is a candidate antigen to differentiate infected from vaccinated animals (DIVA). Insertion of IS Apl1 into the apxIVA gene is known to compromise an APXIVA-based DIVA approach, as is potentially a TGG to TGA mutation in the apxIVA gene. IS Apl1 was found in 63/349 (18.1%) A. pleuropneumoniae isolates from England and Wales including serovars 2, 3, 6–8 and 12. No IS Apl1 insertions into apxIVA were found. Only two serovar 3 isolates contained the TGG to TGA mutation. We conclude that an ApxIVA-based DIVA approach would potentially be viable in England and Wales.

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin

Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of beta-lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the beta-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.