The pathobiology of the septin gene family

Septins are an evolutionarily conserved group of GTP-binding and filament-forming proteins that belong to the large superclass of P-loop GTPases. While originally discovered in yeast as cell division cycle mutants with cytokinesis defects, they are now known to have diverse cellular roles which include polarity determination, cytoskeletal reorganization, membrane dynamics, vesicle trafficking, and exocytosis. Septin proteins form homo- and hetero-oligomeric polymers which can assemble into higher-order filaments. They are also known to interact with components of the cytoskeleton, ie actin and tubulin. The precise role of GTP binding is not clear but a current model suggests that it is associated with conformational changes which alter binding to other proteins. There are at least 12 human septin genes, and although information on expression patterns is limited, most undergo complex alternative splicing with some degree of tissue specificity. Nevertheless, an increasing body of data implicates the septin family in the pathogenesis of diverse disease states including neoplasia, neurodegenerative conditions, and infections. Here the known biochemical properties of mammalian septins are reviewed in the light of the data from yeast and other model organisms. The data implicating septins in human disease are considered and a model linking these data is proposed. It is posited that septins can act as regulatable scaffolds where the stoichiometry of septin associations, modifications, GTP status, and the interactions with other proteins allow the regulation of key cellular processes including polarity determination. Derangements of such septin scaffolds thus explain the role of septins in disease states.     

Significant Roles Played by IL-10 in Chlamydia Infections

Chlamydia species are obligate intracellular parasites which cause usually asymptomatic genital tract infections and also are associated with several complications. Previous studies demonstrated that immune responses to Chlamydia species are different and the diseases will be limited to some cases. Additionally, Chlamydia species are able to modulate immune responses via regulating expression of some immune system molecules including cytokines. IL-10, as the main anti-inflammatory cytokine, plays important roles in the induction of immune-tolerance against self-antigen and also immune-homeostasis after microbe elimination. Furthermore, it has been documented that ectopic expression of IL-10 is associated with several chronic infectious diseases. Therefore, it can be hypothesized that changes in the regulation of this cytokine can be associated with infection with several species of Chlamydia and their associated complications. This review collected the recent information regarding the association and relationship of IL-10 with Chlamydia infections. Another aim of this review article is to address recent data regarding the association of genetic variations (polymorphisms) of IL-10 and Chlamydia infections.     

A Coming of Age Story: Chlamydia in the Post-Genetic Era

Chlamydia spp. are ubiquitous, obligate, intracellular Gram-negative bacterial pathogens that undergo a unique biphasic developmental cycle transitioning between the infectious, extracellular elementary body and the replicative, intracellular reticulate body. The primary Chlamydia species associated with human disease are C. trachomatis, which is the leading cause of both reportable bacterial sexually transmitted infections and preventable blindness, and C. pneumoniae, which infects the respiratory tract and is associated with cardiovascular disease. Collectively, these pathogens are a significant source of morbidity and pose a substantial financial burden on the global economy. Past efforts to elucidate virulence mechanisms of these unique and important pathogens were largely hindered by an absence of genetic methods. Watershed studies in 2011 and 2012 demonstrated that forward and reverse genetic approaches were feasible with Chlamydia and that shuttle vectors could be selected and maintained within the bacterium. While these breakthroughs have led to a steady expansion of the chlamydial genetic tool kit, there are still roads left to be traveled. This minireview provides a synopsis of the currently available genetic methods for Chlamydia along with a comparison to the methods used in other obligate intracellular bacteria. Limitations and advantages of these techniques will be discussed with an eye toward the methods still needed, and how the current state of the art for genetics in obligate intracellular bacteria could direct future technological advances for Chlamydia.     

In Contrast to Chlamydia trachomatis,Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis,Fragmenting the Golgi Apparatus,or Diverting Post-Golgi Sphingomyelin Transport

The Chlamydiales are an order of obligate intracellular bacteria sharing a developmental cycle inside a cytosolic vacuole, with very diverse natural hosts, from amoebae to mammals. The clinically most important species is Chlamydia trachomatis. Many uncertainties remain as to how Chlamydia organizes its intracellular development and replication. The discovery of new Chlamydiales species from other families permits the comparative analysis of cell-biological events and may indicate events that are common to all or peculiar to some species and more or less tightly linked to “chlamydial” development. We used this approach in the infection of human cells with Waddlia chondrophila, a species from the family Waddliaceae whose natural host is uncertain. Compared to C. trachomatis, W. chondrophila had slightly different growth characteristics, including faster cytotoxicity. The embedding in cytoskeletal structures was not as pronounced as for the C. trachomatis inclusion. C. trachomatis infection generates proteolytic activity by the protease Chlamydia protease-like activity factor (CPAF), which degrades host substrates upon extraction; these substrates were not cleaved in the case of W. chondrophila. Unlike Chlamydia, W. chondrophila did not protect against staurosporine-induced apoptosis. C. trachomatis infection causes Golgi apparatus fragmentation and redirects post-Golgi sphingomyelin transport to the inclusion; both were absent from W. chondrophila-infected cells. When host cells were infected with both species, growth of both species was reduced. This study highlights differences between bacterial species that both depend on obligate intracellular replication inside an inclusion. Some features seem principally dispensable for intracellular development of Chlamydiales in vitro but may be linked to host adaptation of Chlamydia and the higher virulence of C. trachomatis.     

Conquering the complex world of human septins: implications for health and disease

Peterson EA and Petty EM. Conquering the complex world of human septins: implications for health and disease. Septins are highly conserved filamentous proteins first characterized in budding yeast and subsequently identified in must eukaryotes. Septins can bind and hydrolyze GTP, which is intrinsically related to their formation of septin hexamers and functional protein interactions. The human septin family is composed of 14 loci, SEPT1‐SEPT14, which encode dozens of different septin proteins. Their central GTPase and polybasic domain regions are highly conserved but they diverge in their N‐terminus and/or C‐terminus. The mechanism by which the different isoforms are generated is not yet well understood, but one can hypothesize that the use of different promoters and/or alternative splicing could give rise to these variants. Septins perform diverse cellular functions according to tissue expression and their interacting partners. Functions identified to date include cell division, chromosome segregation, protein scaffolding, cellular polarity, motility, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, and DNA damage response. Their expression is tightly regulated to maintain proper filament assembly and normal cellular functions. Alterations of these proteins, by mutation or expression changes, have been associated with a variety of cancers and neurological diseases. The association of septins with cancer results from alterations of expression in solid tumors or translocations in leukemias [mixed lineage leukemia (MLL)]. Expression changes in septins have also been associated with neurological conditions such as Alzheimer's and Parkinson's disease, as well as retinopathies, hepatitis C, spermatogenesis and Listeria infection. Pathogenic mutations of SEPT9 were identified in the autosomal dominant neurological disorder hereditary neuralgic amyotrophy (HNA). Human septin research over the past decade has established their importance in cell biology and human disease. Further functional characterization of septins is crucial to our understanding of their possible diagnostic, prognostic, and therapeutic applications.     

Tumor necrosis factor-related apoptosis-inducing ligand translates neonatal respiratory infection into chronic lung disease

Respiratory infections in early life can lead to chronic respiratory disease. Chlamydia infections are common causes of respiratory disease, particularly pneumonia in neonates, and are linked to permanent reductions in pulmonary function and the induction of asthma. However, the immune responses that protect against early-life infection and the mechanisms that lead to chronic lung disease are incompletely understood. Here we identify novel roles for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in promoting Chlamydia respiratory infection-induced pathology in early life, and subsequent chronic lung disease. By infecting TRAIL-deficient neonatal mice and using neutralizing antibodies against this factor and its receptors in wild-type mice, we demonstrate that TRAIL is critical in promoting infection-induced histopathology, inflammation, and mucus hypersecretion, as well as subsequent alveolar enlargement and impaired lung function. This suggests that therapeutic agents that target TRAIL or its receptors may be effective treatments for early-life respiratory infections and associated chronic lung disease.     

The regulation of phagosome maturation in Dictyostelium

Macropinocytosis (fluid uptake) and phagocytosis (particle uptake) are processes that result in the formation of intracellular membrane enclosed vacuoles termed macropinosomes and phagosomes, respectively. Macropinosomes and phagosomes are modified by fission and fusion reactions with the endo-lysosomal pathway that eventually transform these vacuoles into a lysosomal environment. Many human bacterial pathogens, including species of Mycobacteria, Legionella, and Chlamydia, are thought to survive by disrupting the normal membrane trafficking events that usually result in the formation of phago-lysosomes and death of the microorganism. In addition, a number of important pathogens facilitate homotypic phagosome fusion in order to generate an intracellular environment conducive for survival. A greater understanding of the regulation of phagosomal maturation and fusion will be critical in designing new therapies to treat infections caused by intracellular pathogens. The genetically tractable phagocyte, D. discoideum, has proven extremely useful in dissecting the signaling pathways regulating macropinocytosis, phagocytosis, phagosomal maturation and phagosome–phagosome fusion. A body of knowledge has accumulated and demonstrates important roles for Rab GTPases, the cytoskeleton, phosphoinositide metabolism and pH regulation in regulating phagosome maturation. This review will summarize the current state of knowledge.     

Characterization of Group B Streptococcal Invasion of Human Chorion and Amnion Epithelial Cells In Vitro

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol. 87:779–784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics 61:171–177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.     

The immunobiology of Chlamydia

Bacteria of the genus Chlamydia cause a wide variety of disorders in animals and people worldwide. The immune response to chlamydiae is poorly understood and, as Daniel Levitt shows here, there is recent evidence that these organisms induce perturbations in immune function that may assist their own survival in infected hosts and that of co-infecting microbes.     

Capillarosclerosis of the lower urinary tract in analgesic (phenacetin) abuse

Summary The ultrastructural appearances in Capillarosclerosis of the lower urinary tract in analgesic abuse are reported. The capillaries show thickened basement membranes consisting of numerous thin basement membrane lamellae. Between the newly formed basement membrane lamellae, masses of empty vacuoles (fat vacuoles) and a variety of membranous and vesicular structures are present. The pathogenesis of this alteration in the basement membrane is unknown. A common pathogenetic mechanism may be operative in the discoloration of renal papillae and mucous membranes of lower urinary tract on the one hand and capillarosclerosis on the other.     

Tissue-Resident T Cells as the Central Paradigm of Chlamydia Immunity

For almost 2 decades, results from Chlamydia pathogenesis investigations have been conceptualized using a cytokine polarization narrative. Recent viral immunity studies identifying protective tissue-resident memory T cells (Trm) suggest an alternative paradigm based on localized immune networks. As Chlamydia vaccines enter the preclinical pipeline and, in the case of an attenuated trachoma vaccine, are given to human subjects, it may be useful to ask whether cytokine polarization is the appropriate framework for understanding and evaluating vaccine efficacy. In this review, we revisit C. trachomatis pathogenesis data from mice and humans using a Trm narrative and note a comfortable concordance with the Chlamydia pathogenesis literature.     

The septin CDCrel-1 binds syntaxin and inhibits exocytosis.

Septins are GTPases required for the completion of cytokinesis in diverse organisms, yet their roles in cytokinesis or other cellular processes remain unknown. Here we describe studies of a newly identified septin, CDCrel-1, which is predominantly expressed in the nervous system. This protein was associated with membrane fractions, and a significant fraction of the protein copurified and coprecipitated with synaptic vesicles. In detergent extracts, CDCrel-1 and another septin, Nedd5, immunoprecipitated with the SNARE protein syntaxin by directly binding to syntaxin via the SNARE interaction domain. Transfection of HIT-T15 cells with wild-type CDCrel-1 inhibited secretion, whereas GTPase dominant-negative mutants enhanced secretion. These data suggest that septins may regulate vesicle dynamics through interactions with syntaxin.     

An atypical CD8 T‐cell response to Chlamydia muridarum genital tract infections includes T cells that produce interleukin‐13

Chlamydia trachomatis urogenital serovars D–K are intracellular bacterial pathogens that replicate almost exclusively in human reproductive tract epithelium. In the C. muridarum mouse model for human Chlamydia genital tract infections CD4 T helper type 1 cell responses mediate protective immunity while CD8 T‐cell responses have been associated with scarring and infertility. Scarring mediated by CD8 T cells requires production of tumour necrosis factor‐α (TNF‐α); however, TNF‐α is associated with protective immunity mediated by CD4 T cells. The latter result suggests that TNF‐α in‐and‐of itself may not be the sole determining factor in immunopathology. CD8 T cells mediating immunopathology presumably do something in addition to producing TNF‐α that is detrimental during resolution of genital tract infections. To investigate the mechanism underlying CD8 immunopathology we attempted to isolate Chlamydia‐specific CD8 T‐cell clones from mice that self‐cleared genital tract infections. They could not be derived with antigen‐pulsed irradiated naive splenocytes; instead derivation required use of irradiated immune splenocyte antigen‐presenting cells. The Chlamydia‐specific CD8 T‐cell clones had relatively low cell surface CD8 levels and the majority were not restricted by MHC class Ia molecules. They did not express Plac8, and had varying abilities to terminate Chlamydia replication in epithelial cells. Two of the five CD8 clones produced interleukin‐13 (IL‐13) in addition to IL‐2, TNF‐α, IL‐10 and interferon‐γ. IL‐13‐producing Chlamydia‐specific CD8 T cells may contribute to immunopathology during C. muridarum genital tract infections based on known roles of TNF‐α and IL‐13 in scar formation.     

Chlamydial effector proteins localized to the host cell cytoplasmic compartment

Disease-causing microbes utilize various strategies to modify their environment in order to create a favorable location for growth and survival. Gram-negative bacterial pathogens often use specialized secretion systems to translocate effector proteins directly into the cytosol of the eukaryotic cells they infect. These bacterial proteins are responsible for modulating eukaryotic cell functions. Identification of the bacterial effectors has been a critical step toward understanding the molecular basis for the pathogenesis of the bacteria that use them. Chlamydiae are obligate intracellular bacterial pathogens that have a type III secretion system believed to translocate virulence effector proteins into the cytosol of their host cells. Selective permeabilization of the eukaryotic cell membrane was used in conjunction with metabolic labeling of bacterial proteins to identify chlamydial proteins that localize within the cytosol of infected cells. More than 20 Chlamydia trachomatis and C. pneumoniae proteins were detected within the cytoplasmic compartment of infected cells. While a number of cytosolic proteins were shared, others were unique to each species, suggesting that variation among cytosolic chlamydial proteins contributes to the differences in the pathogenesis of the chlamydial species. The spectrum of chlamydial proteins exported differed concomitant with the progress of the developmental cycle. These data confirm that a dynamic relationship exists between Chlamydia and its host and that translocation of bacterial proteins into the cytosol is developmentally dependent.     

Chlamydia muridarum-Specific CD4 T-Cell Clones Recognize Infected Reproductive Tract Epithelial Cells in an Interferon-Dependent Fashion

During natural infections Chlamydia trachomatis urogenital serovars replicate predominantly in the epithelial cells lining the reproductive tract. This tissue tropism poses a unique challenge to host cellar immunity and future vaccine development. In the experimental mouse model, CD4 T cells are necessary and sufficient to clear Chlamydia muridarum genital tract infections. This implies that resolution of genital tract infection depends on CD4 T-cell interactions with infected epithelial cells. However, no laboratory has shown that Chlamydia-specific CD4 T cells can recognize Chlamydia antigens presented by major histocompatibility complex class II (MHC-I) molecules on epithelial cells. In this report we show that MHC-II-restricted Chlamydia-specific CD4 T-cell clones recognize infected upper reproductive tract epithelial cells as early as 12 h postinfection. The timing of recognition and degree of T-cell activation are dependent on the interferon (IFN) milieu. Beta IFN (IFN-β) and IFN-γ have different effects on T-cell activation, with IFN-β blunting IFN-γ-induced upregulation of epithelial cell surface MHC-II and T-cell activation. Individual CD4 T-cell clones differed in their degrees of dependence on IFN-γ-regulated MHC-II for controlling Chlamydia replication in epithelial cells in vitro. We discuss our data as they relate to published studies with IFN knockout mice, proposing a straightforward interpretation of the existing literature based on CD4 T-cell interactions with the infected reproductive tract epithelium.Chlamydia trachomatis is the most common bacterial sexually transmitted infection in the developed world, with 2 to 3 million actively infected individuals in the United States (3) and similar numbers in Europe (17). In women C. trachomatis infections can ascend into the upper reproductive tract, causing pelvic inflammatory disease and scarring with resulting infertility and ectopic pregnancies.Histopathology studies show that C. trachomatis replicates predominantly in the reproductive tract epithelium during natural human infections (16, 36) and experimental murine C. muridarum infections (21). Inclusions are not seen in other cell types even though Chlamydia can undergo limited replication in macrophages and dendritic cells (33). It is unlikely that replication in non-epithelial cell lineages makes a major contribution to genital tract shedding. The mouse model for Chlamydia genital tract infections supports a critical role for CD4 T cells in protective immunity, as mice deficient in major histocompatibility complex class II (MHC-II) cannot control C. muridarum genital tract infections (22), and CD4 T-cell depletion is detrimental to resolution of primary genital tract infections (23). Because C. muridarum replicates in epithelial cells lining the reproductive tract, the most straightforward mechanism for clearing the genital tract would involve Chlamydia-specific CD4 T-cell interactions with infected epithelial cells. However in the absence of any data supporting this specific interaction, other indirect mechanisms based on CD4 T-cell production of gamma interferon (IFN-γ) and provision of help to B cells and CD8 T cells have been proposed as the mechanism for clearance (29).C. muridarum-specific CD4 T-cell lines protective in adoptive-transfer studies were shown to control C. muridarum replication in polarized epithelial monolayers (14). The mechanism of control was dependent on IFN-γ and physical interaction of T cells with the infected epithelial cells via LFA-1. In the presence of IFN-γ, T-cell engagement of epithelial cells via LFA-1 was shown to augment epithelial nitric oxide production above that induced by IFN-γ alone, and nitric oxide was shown to be the effector molecule responsible for controlling Chlamydia replication (13). This anti-Chlamydia effector mechanism did not require that the CD4 T-cell clone recognize the infected epithelial monolayer in an antigen-specific fashion, as the same preactivated CD4 T-cell clone controlled Chlamydia psittaci replication in polarized epithelial monolayers even though it does not recognize a C. psittaci antigen.Epithelial cells are semiprofessional antigen-presenting cells (APCs) and, in their unperturbed state, likely play a role in immunotolerance at mucosal surfaces (19). However in inflammatory environments, such as those resulting from transplant rejection and graft-versus-host disease, epithelial cells change their immunophenotype by upregulation of MHC-II (5, 25). In trachoma, an eye infection caused by Chlamydia trachomatis serovars A to C, conjunctival epithelial cells from human clinical specimens showed upregulated cell surface MHC-II and were presumably competent to present antigens to CD4 T cells (11, 12). In vitro studies have shown that rat and murine uterine epithelial cells process and present exogenous ovalbumin to OVA-specific CD4 T cells (28, 37). However in vitro processing and presentation of concentrated extracellular ovalbumin to CD4 T cells by uterine epithelial cells do not directly address whether Chlamydia antigens sequestered in membrane-bound inclusions get processed and presented to Chlamydia-specific CD4 T cells in vivo. The mechanics of CD4 T-cell contributions to resolution of genital tract infections remain unclear.For this study we derived an epithelial cell line from the upper reproductive tract of a female C57BL/6 mouse and a panel of 10 Chlamydia-specific CD4 T-cell clones from immune C57BL/6 mice that previously self-cleared C. muridarum genital tract infections. These reagents gave us the opportunity to directly investigate (i) whether Chlamydia-specific CD4 T cells can recognize C. muridarum-infected reproductive tract epithelial cells, (ii) when during the time course of infection recognition occurs, and (iii) the role of IFNs in modulating epithelial interactions with CD4 T cells. We present the results of those investigations here.(These data were presented in part at the 2009 Chlamydia Basic Research Conference.)     

Mutational Analysis of the Chlamydia muridarum Plasticity Zone

Pathogenically diverse Chlamydia spp. can have surprisingly similar genomes. Chlamydia trachomatis isolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strain Chlamydia muridarum share 99% of their gene content. A region of high genomic diversity between Chlamydia spp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance of C. muridarum compared to C. trachomatis in the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarum PZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarum PZ genes are transcribed—and some may produce functional proteins—but are dispensable for infection of the murine genital tract.     

Life inside and out: making and breaking protein disulfide bonds in Chlamydia

Disulphide bonds are widely used among all domains of life to provide structural stability to proteins and to regulate enzyme activity. Chlamydia spp. are obligate intracellular bacteria that are especially dependent on the formation and degradation of protein disulphide bonds. Members of the genus Chlamydia have a unique biphasic developmental cycle alternating between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body. The proteins in the envelope of the EB are heavily cross-linked with disulphides and this is known to be critical for this infectious phase. In this review, we provide a comprehensive summary of what is known about the redox state of chlamydial envelope proteins throughout the developmental cycle. We focus especially on the factors responsible for degradation and formation of disulphide bonds in Chlamydia and how this system compares with redox regulation in other organisms. Focussing on the unique biology of Chlamydia enables us to provide important insights into how specialized suites of disulphide bond (Dsb) proteins cater for specific bacterial environments and lifecycles.     

Intracellular appearance of Penicillium chrysogenum virus

An electron microscopic study of intracellular Penicillium chrysogenum virus showed virus particles with a dense core distributed as individuals or aggregates in cytoplasm and/or in vacuoles of mycelial cells. In cytoplasm the virus particles were often near, but not in, the nucleus. Electron-dense regions of granular appearance enclosed more complete virus particles in some cells. Lipid bodies, vacuoles, and membranous structures were often prominent in infected cells. Sometimes cylindrical arrays of virus particles occurred in vacuoles and also in degenerated cells. Aberrations consisting of various degrees of degeneration or disappearance of cytoplasm and cellular organelles were often observed in aged cells with large quantities of virus. The virus particles sometimes released fibrous material forming networks from their core region.     

Identification of the Helicobacter pylori VacA Toxin Domain Active in the Cell Cytosol

Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity.