An inducible model of chronic hyperglycemia.

Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation tools have been reported in zebrafish, which have significantly impacted regeneration studies. However, NTR1.0-based tools are not appropriate for modeling chronic cell loss as prolonged application of the required MTZ dose (10 mM) is deleterious to zebrafish health. We established that this dose corresponds to the median lethal dose (LD50) of MTZ in larval and adult zebrafish and that it induced intestinal pathology. NTR2.0 is a more active nitroreductase engineered from Vibrio vulnificus NfsB that requires substantially less MTZ to induce cell ablation. Here, we report on the generation of two new NTR2.0-based zebrafish lines in which acute ß-cell ablation can be achieved without MTZ-associated intestinal pathology. For the first time, we were able to sustain ß-cell loss and maintain elevated glucose levels (chronic hyperglycemia) in larvae and adults. Adult fish showed significant weight loss, consistent with the induction of a diabetic state, indicating that this paradigm will allow the modeling of diabetes and associated pathologies.

Cultivating Healthy Connections: Exploring and Engineering the Microbial Flow That Shapes Microbiomes.

Our view of the microbial world has undergone a radical transformation over the past decade. For most of the 20th century, medical microbiological research was focused on understanding the virulent nature of disease-causing pathogens. More recently, advances in DNA sequencing methodologies have exposed a wider diversity of microscopic wildlife that associate with our bodies and the environments around us, and the unexpected roles they play in supporting our health. Our expanding view of the microbial world is now motivating therapeutic interventions that are based not just on the elimination of nefarious pathogens but the nurturing of beneficial microbiomes. In this Commentary, I consider how our historically pathogen-based view of host-microbe interactions may be limiting the scope of new and alternative strategies for engineering microbiomes. I suggest that recognizing the therapeutic potential of the ongoing microbial transmission that connects microbiomes could illuminate unexplored opportunities for cultivating healthy host-microbe relationships.

Swimming motility of a gut bacterial symbiont promotes resistance to intestinal expulsion and enhances inflammation.

Some of the densest microbial ecosystems in nature thrive within the intestines of humans and other animals. To protect mucosal tissues and maintain immune tolerance, animal hosts actively sequester bacteria within the intestinal lumen. In response, numerous bacterial pathogens and pathobionts have evolved strategies to subvert spatial restrictions, thereby undermining immune homeostasis. However, in many cases, it is unclear how escaping host spatial control benefits gut bacteria and how changes in intestinal biogeography are connected to inflammation. A better understanding of these processes could uncover new targets for treating microbiome-mediated inflammatory diseases. To this end, we investigated the spatial organization and dynamics of bacterial populations within the intestine using larval zebrafish and live imaging. We discovered that a proinflammatory Vibrio symbiont native to zebrafish governs its own spatial organization using swimming motility and chemotaxis. Surprisingly, we found that Vibrio's motile behavior does not enhance its growth rate but rather promotes its persistence by enabling it to counter intestinal flow. In contrast, Vibrio mutants lacking motility traits surrender to host spatial control, becoming aggregated and entrapped within the lumen. Consequently, nonmotile and nonchemotactic mutants are susceptible to intestinal expulsion and experience large fluctuations in absolute abundance. Further, we found that motile Vibrio cells induce expression of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) in gut-associated macrophages and the liver. Using inducible genetic switches, we demonstrate that swimming motility can be manipulated in situ to modulate the spatial organization, persistence, and inflammatory activity of gut bacterial populations. Together, our findings suggest that host spatial control over resident microbiota plays a broader role in regulating the abundance and persistence of gut bacteria than simply protecting mucosal tissues. Moreover, we show that intestinal flow and bacterial motility are potential targets for therapeutically managing bacterial spatial organization and inflammatory activity within the gut.

Patterns of partnership: surveillance and mimicry in host-microbiota mutualisms.

The repertoire of microbial cues monitored by animal and plant tissues encompasses not just molecules but also microbial activities. These include typical pathogen strategies of injuring membranes, degrading cellular material, and scavenging resources. These activities, however, are not exclusive to pathogens. Instead, they characterize the competitive strategies of microbes living in multispecies communities, like those typically found colonizing host tissues. Similar activities are also deployed by host tissues to keep microbes in check. We propose that host surveillance and mimicry of Microbial-Associated Competitive Activities (MACAs), derived from an evolutionary history of living in mixed microbial communities, has shaped contemporary animal and plant tissue programs of defense, repair, metabolism, and development.

Sublethal antibiotics collapse gut bacterial populations by enhancing aggregation and expulsion.

Antibiotics induce large and highly variable changes in the intestinal microbiome even at sublethal concentrations, through mechanisms that remain elusive. Using gnotobiotic zebrafish, which allow high-resolution examination of microbial dynamics, we found that sublethal doses of the common antibiotic ciprofloxacin cause severe drops in bacterial abundance. Contrary to conventional views of antimicrobial tolerance, disruption was more pronounced for slow-growing, aggregated bacteria than for fast-growing, planktonic species. Live imaging revealed that antibiotic treatment promoted bacterial aggregation and increased susceptibility to intestinal expulsion. Intestinal mechanics therefore amplify the effects of antibiotics on resident bacteria. Microbial dynamics are captured by a biophysical model that connects antibiotic-induced collapses to gelation phase transitions in soft materials, providing a framework for predicting the impact of antibiotics on the intestinal microbiome.

The Other Side of the Coin: What Beneficial Microbes Can Teach Us about Pathogenic Potential.

Koch's postulates and molecular Koch's postulates have made an indelible mark on how we study and classify microbes, particularly pathogens. However, rigid adherence to these historic postulates constrains our view of not only microbial pathogenesis but also host-microbe relationships in general. Collectively, the postulates imply that a "microbial pathogen" is a clearly identifiable organism with the exclusive capacity to elicit disease through an arsenal of pathogen-specific "virulence factors." This narrow definition has been repeatedly contradicted. Advances in DNA sequencing technologies and new experimental systems have revealed that the outcomes of host-microbe interactions are highly contextual and dynamic, especially those involving resident microbiota and variable aspects of host biology. Clarifying what differentiates pathogenic from non-pathogenic microbes, including their paradoxical ability to masquerade as one another, is critical to developing targeted diagnostics and treatments for infectious disease. Such endeavors will also inform the design of therapeutic strategies based on microbiome engineering by providing insights into how manipulating entire host-microbe systems may directly or indirectly alter the pathogenic potential of microbial communities. With these goals in mind, we discuss the need to develop experimental models that better capture the contexts that determine the nature of host-microbe relationships. To demonstrate the potential of one such model-the zebrafish and its resident microbiota-we describe recent work that has revealed the thin line between pathogenic and mutualistic relationships, how the intestine physically shapes bacterial populations and inflammation, and the ability of microbial transmission to override the host's innate immune system.

Bacterial Cohesion Predicts Spatial Distribution in the Larval Zebrafish Intestine.

Are there general biophysical relationships governing the spatial organization of the gut microbiome? Despite growing realization that spatial structure is important for population stability, interbacterial competition, and host functions, it is unclear in any animal gut whether such structure is subject to predictive, unifying rules or if it results from contextual, species-specific behaviors. To explore this, we used light sheet fluorescence microscopy to conduct a high-resolution comparative study of bacterial distribution patterns throughout the entire intestinal volume of live, larval zebrafish. Fluorescently tagged strains of seven bacterial symbionts, representing six different species native to zebrafish, were each separately monoassociated with animals that had been raised initially germ-free. The strains showed large differences in both cohesion-the degree to which they auto-aggregate-and spatial distribution. We uncovered a striking correlation between each strain's mean position and its cohesion, whether quantified as the fraction of cells existing as planktonic individuals, the average aggregate size, or the total number of aggregates. Moreover, these correlations held within species as well; aggregates of different sizes localized as predicted from the pan-species observations. Together, our findings indicate that bacteria within the zebrafish intestine are subject to generic processes that organize populations by their cohesive properties. The likely drivers of this relationship-peristaltic fluid flow, tubular anatomy, and bacterial growth and aggregation kinetics-are common throughout animals. We therefore suggest that the framework introduced here of biophysical links between bacterial cohesion and spatial organization should be useful for directing explorations in other host-microbe systems, formulating detailed models that can quantitatively map onto experimental data, and developing new tools that manipulate cohesion to engineer microbiome function.

Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages.

Correlating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis and in vitro assays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCE A great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.

The enteric nervous system promotes intestinal health by constraining microbiota composition.

Sustaining a balanced intestinal microbial community is critical for maintaining intestinal health and preventing chronic inflammation. The gut is a highly dynamic environment, subject to periodic waves of peristaltic activity. We hypothesized that this dynamic environment is a prerequisite for a balanced microbial community and that the enteric nervous system (ENS), a chief regulator of physiological processes within the gut, profoundly influences gut microbiota composition. We found that zebrafish lacking an ENS due to a mutation in the Hirschsprung disease gene, sox10, develop microbiota-dependent inflammation that is transmissible between hosts. Profiling microbial communities across a spectrum of inflammatory phenotypes revealed that increased levels of inflammation were linked to an overabundance of pro-inflammatory bacterial lineages and a lack of anti-inflammatory bacterial lineages. Moreover, either administering a representative anti-inflammatory strain or restoring ENS function corrected the pathology. Thus, we demonstrate that the ENS modulates gut microbiota community membership to maintain intestinal health.

Host Gut Motility Promotes Competitive Exclusion within a Model Intestinal Microbiota.

The gut microbiota is a complex consortium of microorganisms with the ability to influence important aspects of host health and development. Harnessing this "microbial organ" for biomedical applications requires clarifying the degree to which host and bacterial factors act alone or in combination to govern the stability of specific lineages. To address this issue, we combined bacteriological manipulation and light sheet fluorescence microscopy to monitor the dynamics of a defined two-species microbiota within a vertebrate gut. We observed that the interplay between each population and the gut environment produces distinct spatiotemporal patterns. As a consequence, one species dominates while the other experiences sudden drops in abundance that are well fit by a stochastic mathematical model. Modeling revealed that direct bacterial competition could only partially explain the observed phenomena, suggesting that a host factor is also important in shaping the community. We hypothesized the host determinant to be gut motility, and tested this mechanism by measuring colonization in hosts with enteric nervous system dysfunction due to a mutation in the ret locus, which in humans is associated with the intestinal motility disorder known as Hirschsprung disease. In mutant hosts we found reduced gut motility and, confirming our hypothesis, robust coexistence of both bacterial species. This study provides evidence that host-mediated spatial structuring and stochastic perturbation of communities can drive bacterial population dynamics within the gut, and it reveals a new facet of the intestinal host-microbe interface by demonstrating the capacity of the enteric nervous system to influence the microbiota. Ultimately, these findings suggest that therapeutic strategies targeting the intestinal ecosystem should consider the dynamic physical nature of the gut environment.

Strengths and Limitations of Model Systems for the Study of Urinary Tract Infections and Related Pathologies.

Urinary tract infections (UTIs) are some of the most common bacterial infections worldwide and are a source of substantial morbidity among otherwise healthy women. UTIs can be caused by a variety of microbes, but the predominant etiologic agent of these infections is uropathogenic Escherichia coli (UPEC). An especially troubling feature of UPEC-associated UTIs is their high rate of recurrence. This problem is compounded by the drastic increase in the global incidence of antibiotic-resistant UPEC strains over the past 15 years. The need for more-effective treatments for UTIs is driving research aimed at bettering our understanding of the virulence mechanisms and host-pathogen interactions that occur during the course of these infections. Surrogate models of human infection, including cell culture systems and the use of murine, porcine, avian, teleost (zebrafish), and nematode hosts, are being employed to define host and bacterial factors that modulate the pathogenesis of UTIs. These model systems are revealing how UPEC strains can avoid or overcome host defenses and acquire scarce nutrients while also providing insight into the virulence mechanisms used by UPEC within compromised individuals, such as catheterized patients. Here, we summarize our current understanding of UTI pathogenesis while also giving an overview of the model systems used to study the initiation, persistence, and recurrence of UTIs and life-threatening sequelae like urosepsis. Although we focus on UPEC, the experimental systems described here can also provide valuable insight into the disease processes associated with other bacterial pathogens both within the urinary tract and elsewhere within the host.

Identification of Population Bottlenecks and Colonization Factors during Assembly of Bacterial Communities within the Zebrafish Intestine.

UNLABELLED: The zebrafish, Danio rerio, is a powerful model for studying bacterial colonization of the vertebrate intestine, but the genes required by commensal bacteria to colonize the zebrafish gut have not yet been interrogated on a genome-wide level. Here we apply a high-throughput transposon mutagenesis screen to Aeromonas veronii Hm21 and Vibrio sp. strain ZWU0020 during their colonization of the zebrafish intestine alone and in competition with each other, as well as in different colonization orders. We use these transposon-tagged libraries to track bacterial population sizes in different colonization regimes and to identify gene functions required during these processes. We show that intraspecific, but not interspecific, competition with a previously established bacterial population greatly reduces the ability of these two bacterial species to colonize. Further, using a simple binomial sampling model, we show that under conditions of interspecific competition, genes required for colonization cannot be identified because of the population bottleneck experienced by the second colonizer. When bacteria colonize the intestine alone or at the same time as the other species, we find shared suites of functional requirements for colonization by the two species, including a prominent role for chemotaxis and motility, regardless of the presence of another species. IMPORTANCE: Zebrafish larvae, which are amenable to large-scale gnotobiotic studies, comprehensive sampling of their intestinal microbiota, and live imaging, are an excellent model for investigations of vertebrate intestinal colonization dynamics. We sought to develop a mutagenesis and tagging system in order to understand bacterial population dynamics and functional requirements during colonization of the larval zebrafish intestine. We explored changes in bacterial colonization dynamics and functional requirements when bacteria colonize a bacterium-free intestine, one previously colonized by their own species, or one colonized previously or simultaneously with a different species. This work provides a framework for rapid identification of colonization factors important under different colonization conditions. Furthermore, we demonstrate that when colonizing bacterial populations are very small, this approach is not accurate because random sampling of the input pool is sufficient to explain the distribution of inserts recovered from bacteria that colonized the intestines.

Combining quantitative genetic footprinting and trait enrichment analysis to identify fitness determinants of a bacterial pathogen.

Strains of Extraintestinal Pathogenic Escherichia c oli (ExPEC) exhibit an array of virulence strategies and are a major cause of urinary tract infections, sepsis and meningitis. Efforts to understand ExPEC pathogenesis are challenged by the high degree of genetic and phenotypic variation that exists among isolates. Determining which virulence traits are widespread and which are strain-specific will greatly benefit the design of more effective therapies. Towards this goal, we utilized a quantitative genetic footprinting technique known as transposon insertion sequencing (Tn-seq) in conjunction with comparative pathogenomics to functionally dissect the genetic repertoire of a reference ExPEC isolate. Using Tn-seq and high-throughput zebrafish infection models, we tracked changes in the abundance of ExPEC variants within saturated transposon mutant libraries following selection within distinct host niches. Nine hundred and seventy bacterial genes (18% of the genome) were found to promote pathogen fitness in either a niche-dependent or independent manner. To identify genes with the highest therapeutic and diagnostic potential, a novel Trait Enrichment Analysis (TEA) algorithm was developed to ascertain the phylogenetic distribution of candidate genes. TEA revealed that a significant portion of the 970 genes identified by Tn-seq have homologues more often contained within the genomes of ExPEC and other known pathogens, which, as suggested by the first axiom of molecular Koch's postulates, is considered to be a key feature of true virulence determinants. Three of these Tn-seq-derived pathogen-associated genes--a transcriptional repressor, a putative metalloendopeptidase toxin and a hypothetical DNA binding protein--were deleted and shown to independently affect ExPEC fitness in zebrafish and mouse models of infection. Together, the approaches and observations reported herein provide a resource for future pathogenomics-based research and highlight the diversity of factors required by a single ExPEC isolate to survive within varying host environments.

A phyletically rare gene promotes the niche-specific fitness of an E. coli pathogen during bacteremia.

In bacteria, laterally acquired genes are often concentrated within chromosomal regions known as genomic islands. Using a recently developed zebrafish infection model, we set out to identify unique factors encoded within genomic islands that contribute to the fitness and virulence of a reference urosepsis isolate-extraintestinal pathogenic Escherichia coli strain CFT073. By screening a series of deletion mutants, we discovered a previously uncharacterized gene, neaT, that is conditionally required by the pathogen during systemic infections. In vitro assays indicate that neaT can limit bacterial interactions with host phagocytes and alter the aggregative properties of CFT073. The neaT gene is localized within an integrated P2-like bacteriophage in CFT073, but was rarely found within other proteobacterial genomes. Sequence-based analyses revealed that neaT homologues are present, but discordantly conserved, within a phyletically diverse set of bacterial species. In CFT073, neaT appears to be unameliorated, having an exceptionally A+T-rich composition along with a notably altered codon bias. These data suggest that neaT was recently brought into the proteobacterial pan-genome from an extra-phyletic source. Interestingly, even in G+C-poor genomes, as found within the Firmicutes lineage, neaT-like genes are often unameliorated. Sequence-level features of neaT homologues challenge the common supposition that the A+T-rich nature of many recently acquired genes reflects the nucleotide composition of their genomes of origin. In total, these findings highlight the complexity of the evolutionary forces that can affect the acquisition, utilization, and assimilation of rare genes that promote the niche-dependent fitness and virulence of a bacterial pathogen.

The RTX pore-forming toxin α-hemolysin of uropathogenic Escherichia coli: progress and perspectives.

Members of the RTX family of protein toxins are functionally conserved among an assortment of bacterial pathogens. By disrupting host cell integrity through their pore-forming and cytolytic activities, this class of toxins allows pathogens to effectively tamper with normal host cell processes, promoting pathogenesis. Here, we focus on the biology of RTX toxins by describing salient properties of a prototype member, α-hemolysin, which is often encoded by strains of uropathogenic Escherichia coli. It has long been appreciated that RTX toxins can have distinct effects on host cells aside from outright lysis. Recently, advances in modeling and analysis of host-pathogen interactions have led to novel findings concerning the consequences of pore formation during host-pathogen interactions. We discuss current progress on longstanding questions concerning cell specificity and pore formation, new areas of investigation that involve toxin-mediated perturbations of host cell signaling cascades and perspectives on the future of RTX toxin investigation.

The repeat-in-toxin family member TosA mediates adherence of uropathogenic Escherichia coli and survival during bacteremia.

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC. tosA, found in strains within the B2 phylogenetic subgroup of E. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence of tosA in an E. coli isolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function of tosA revealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.

Use of zebrafish to probe the divergent virulence potentials and toxin requirements of extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, alpha-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.

Origins and virulence mechanisms of uropathogenic Escherichia coli.

Strains of uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections, including both cystitis and pyelonephritis. These bacteria have evolved a multitude of virulence factors and strategies that facilitate bacterial growth and persistence within the adverse settings of the host urinary tract. Expression of adhesive organelles like type 1 and P pili allow UPEC to bind and invade host cells and tissues within the urinary tract while expression of iron-chelating factors (siderophores) enable UPEC to pilfer host iron stores. Deployment of an array of toxins, including hemolysin and cytotoxic necrotizing factor 1, provide UPEC with the means to inflict extensive tissue damage, facilitating bacterial dissemination as well as releasing host nutrients and disabling immune effector cells. These toxins also have the capacity to modulate, in more subtle ways, host signaling pathways affecting myriad processes, including inflammatory responses, host cell survival, and cytoskeletal dynamics. Here, we discuss the mechanisms by which these and other virulence factors promote UPEC survival and growth within the urinary tract. Comparisons are also made between UPEC and other strains of extraintestinal pathogenic E. coli that, although closely related to UPEC, are distinct in their abilities to colonize the host and cause disease.

Inactivation of host Akt/protein kinase B signaling by bacterial pore-forming toxins.

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs), and they have the capacity to induce the death and exfoliation of target uroepithelial cells. This process can be facilitated by the pore-forming toxin alpha-hemolysin (HlyA), which is expressed and secreted by many UPEC isolates. Here, we demonstrate that HlyA can potently inhibit activation of Akt (protein kinase B), a key regulator of host cell survival, inflammatory responses, proliferation, and metabolism. HlyA ablates Akt activation via an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Inhibitor studies indicate that Akt inactivation by HlyA involves aberrant stimulation of host protein phosphatases. We found that two other bacterial pore-forming toxins (aerolysin from Aeromonas species and alpha-toxin from Staphylococcus aureus) can also markedly attenuate Akt activation in a dose-dependent manner. These data suggest a novel mechanism by which sublytic concentrations of HlyA and other pore-forming toxins can modulate host cell survival and inflammatory pathways during the course of a bacterial infection.