Klebsiella pneumoniae DedA family proteins have redundant roles in divalent cation homeostasis and resistance to phagocytosis.

The DedA superfamily is a highly conserved family of membrane proteins. Deletion of Escherichia coli yqjA and yghB, encoding related DedA family proteins, results in sensitivity to elevated temperature, antibiotics, and alkaline pH. The human pathogen Klebsiella pneumoniae possesses genes encoding DedA family proteins with >90% amino acid identity to E. coli YqjA and YghB. We hypothesized that the deletion of K. pneumoniae yqjA and yghB will impact its physiology and may reduce its virulence. The K. pneumoniae ΔyqjA ΔyghB mutant (strain VT101) displayed a growth defect at 42°C and alkaline pH sensitivity, not unlike its E. coli counterpart. However, VT101 retained mostly wild-type resistance to antibiotics. We found VT101 was sensitive to the chelating agent EDTA, the anionic detergent SDS, and agents capable of alkalizing the bacterial cytoplasm such as bicarbonate or chloroquine. We could restore growth at alkaline pH and at elevated temperature by addition of 0.5-2 mM Ca2+ or Mg2+ to the culture media. VT101 displayed a slower uptake of calcium, which was dependent upon calcium channel activity. VT201, with similar deletions as VT101 but derived from a virulent K. pneumoniae strain, was highly susceptible to phagocytosis by alveolar macrophages and displayed a defect in the production of capsule. These findings suggest divalent cation homeostasis and virulence are interlinked by common functions of the DedA family.IMPORTANCEKlebsiella pneumoniae is a dangerous human pathogen. The DedA protein family is found in all bacteria and is a membrane transporter often required for virulence and antibiotic resistance. K. pneumoniae possesses homologs of E. coli YqjA and YghB, with 60% amino acid identity and redundant functions, which we have previously shown to be required for tolerance to biocides and alkaline pH. A K. pneumoniae strain lacking yqjA and yghB was found to be sensitive to alkaline pH, elevated temperature, and EDTA/SDS and displayed a defect in calcium uptake. Sensitivity to these conditions was reversed by addition of calcium or magnesium to the growth medium. Introduction of ΔyqjA and ΔyghB mutations into virulent K. pneumoniae resulted in the loss of capsule, increased phagocytosis by macrophages, and a partial loss of virulence. These results show that targeting the Klebsiella DedA family results in impaired divalent cation transport and, in turn, loss of virulence.

Cardiolipin coordinates inflammatory metabolic reprogramming through regulation of Complex II disassembly and degradation.

Macrophage metabolic plasticity enables repurposing of electron transport from energy generation to inflammation and host defense. Altered respiratory complex II function has been implicated in cancer, diabetes, and inflammation, but regulatory mechanisms are incompletely understood. Here, we show that macrophage inflammatory activation triggers Complex II disassembly and succinate dehydrogenase subunit B loss through sequestration and selective mitophagy. Mitochondrial fission supported lipopolysaccharide-stimulated succinate dehydrogenase subunit B degradation but not sequestration. We hypothesized that this Complex II regulatory mechanism might be coordinated by the mitochondrial phospholipid cardiolipin. Cardiolipin synthase knockdown prevented lipopolysaccharide-induced metabolic remodeling and Complex II disassembly, sequestration, and degradation. Cardiolipin-depleted macrophages were defective in lipopolysaccharide-induced pro-inflammatory cytokine production, a phenotype partially rescued by Complex II inhibition. Thus, cardiolipin acts as a critical organizer of inflammatory metabolic remodeling.

The IRE1α-XBP1 Signaling Axis Promotes Glycolytic Reprogramming in Response to Inflammatory Stimuli.

Immune cells must be able to adjust their metabolic programs to effectively carry out their effector functions. Here, we show that the endoplasmic reticulum (ER) stress sensor Inositol-requiring enzyme 1 alpha (IRE1α) and its downstream transcription factor X box binding protein 1 (XBP1) enhance the upregulation of glycolysis in classically activated macrophages (CAMs). The IRE1α-XBP1 signaling axis supports this glycolytic switch in macrophages when activated by lipopolysaccharide (LPS) stimulation or infection with the intracellular bacterial pathogen Brucella abortus. Importantly, these different inflammatory stimuli have distinct mechanisms of IRE1α activation; while Toll-like receptor 4 (TLR4) supports glycolysis under both conditions, TLR4 is required for activation of IRE1α in response to LPS treatment but not B. abortus infection. Though IRE1α and XBP1 are necessary for maximal induction of glycolysis in CAMs, activation of this pathway is not sufficient to increase the glycolytic rate of macrophages, indicating that the cellular context in which this pathway is activated ultimately dictates the cell's metabolic response and that IRE1α activation may be a way to fine-tune metabolic reprogramming. IMPORTANCE The immune system must be able to tailor its response to different types of pathogens in order to eliminate them and protect the host. When confronted with bacterial pathogens, macrophages, frontline defenders in the immune system, switch to a glycolysis-driven metabolism to carry out their antibacterial functions. Here, we show that IRE1α, a sensor of ER stress, and its downstream transcription factor XBP1 support glycolysis in macrophages during infection with Brucella abortus or challenge with Salmonella LPS. Interestingly, these stimuli activate IRE1α by independent mechanisms. While the IRE1α-XBP1 signaling axis promotes the glycolytic switch, activation of this pathway is not sufficient to increase glycolysis in macrophages. This study furthers our understanding of the pathways that drive macrophage immunometabolism and highlights a new role for IRE1α and XBP1 in innate immunity.

Human neutrophil IL1ß directs intestinal epithelial cell extrusion during Salmonella infection.

Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and seeded with primary human polymorphonuclear leukocytes (PMN-HIOs). PMNs did not significantly alter luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed TUNEL+ epithelial cells that was driven by PMN Caspase-1 activity. Inhibition of Caspases-1, -3 or -4 abrogated epithelial cell death and extrusion in the infected PMN-HIOs but only Caspase-1 inhibition significantly increased bacterial burden in the PMN-HIO epithelium. Thus, PMNs promote cell death in human intestinal epithelial cells through multiple caspases as a protective response to infection. IL-1ß was necessary and sufficient to induce cell shedding in the infected HIOs. These data support a critical innate immune function for human neutrophils in amplifying cell death and extrusion of human epithelial cells from the Salmonella-infected intestinal monolayer.

Amino acids suppress macropinocytosis and promote release of CSF1 receptor in macrophages.

The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to growth factors and other stimuli. To test the hypothesis that nutrient environments modulate solute uptake by macropinocytosis, this study analyzed the effects of extracellular amino acids on the accumulation of fluorescent fluid-phase probes in murine macrophages. Nine amino acids, added individually or together, were capable of suppressing macropinocytosis in murine bone marrow-derived macrophages stimulated with the growth factors colony stimulating factor 1 (CSF1) or interleukin 34, both ligands of the CSF1 receptor (CSF1R). The suppressive amino acids did not inhibit macropinocytosis in response to lipopolysaccharide, the chemokine CXCL12, or the tumor promoter phorbol myristate acetate. Suppressive amino acids promoted release of CSF1R from cells and resulted in the formation of smaller macropinosomes in response to CSF1. This suppression of growth factor-stimulated macropinocytosis indicates that different nutrient environments modulate CSF1R levels and bulk ingestion by macropinocytosis, with likely consequences for macrophage growth and function.

Comparative transcriptional profiling of the early host response to infection by typhoidal and non-typhoidal Salmonella serovars in human intestinal organoids.

Salmonella enterica represents over 2500 serovars associated with a wide-ranging spectrum of disease; from self-limiting gastroenteritis to invasive infections caused by non-typhoidal serovars (NTS) and typhoidal serovars, respectively. Host factors strongly influence infection outcome as malnourished or immunocompromised individuals can develop invasive infections from NTS, however, comparative analyses of serovar-specific host responses have been constrained by reliance on limited model systems. Here we used human intestinal organoids (HIOs), a three-dimensional "gut-like" in vitro system derived from human embryonic stem cells, to elucidate similarities and differences in host responses to NTS and typhoidal serovars. HIOs discriminated between the two most prevalent NTS, Salmonella enterica serovar Typhimurium (STM) and Salmonella enterica serovar Enteritidis (SE), and typhoidal serovar Salmonella enterica serovar Typhi (ST) in epithelial cell invasion, replication and transcriptional responses. Pro-inflammatory signaling and cytokine output was reduced in ST-infected HIOs compared to NTS infections, consistent with early stages of NTS and typhoidal diseases. While we predicted that ST would induce a distinct transcriptional profile from the NTS strains, more nuanced expression profiles emerged. Notably, pathways involved in cell cycle, metabolism and mitochondrial functions were downregulated in STM-infected HIOs and upregulated in SE-infected HIOs. These results correlated with suppression of cellular proliferation and induction of host cell death in STM-infected HIOs and in contrast, elevated levels of reactive oxygen species production in SE-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to infection by individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection.

The IRE1α Stress Signaling Axis Is a Key Regulator of Neutrophil Antimicrobial Effector Function.

Activation of the endoplasmic reticulum stress sensor, IRE1α, is required for effective immune responses against bacterial infection and is associated with human inflammatory diseases in which neutrophils are a key immune component. However, the specific role of IRE1α in regulating neutrophil effector function has not been studied. In this study, we show that infection-induced IRE1α activation licenses neutrophil antimicrobial capacity, including IL-1ß production, formation of neutrophil extracellular traps (NETs), and methicillin-resistant Staphylococcus aureus (MRSA) killing. Inhibition of IRE1α diminished production of mitochondrial reactive oxygen species and decreased CASPASE-2 activation, which both contributed to neutrophil antimicrobial activity. Mice deficient in CASPASE-2 or neutrophil IRE1α were highly susceptible to MRSA infection and failed to effectively form NETs in the s.c. abscess. IRE1α activation enhanced calcium influx and citrullination of histone H3 independently of mitochondrial reactive oxygen species production, suggesting that IRE1α coordinates multiple pathways required for NET formation. Our data demonstrate that the IRE1α-CASPASE-2 axis is a major driver of neutrophil activity against MRSA infection and highlight the importance of IRE1α in neutrophil antibacterial function.

Salmonella enterica Serovar Typhimurium SPI-1 and SPI-2 Shape the Global Transcriptional Landscape in a Human Intestinal Organoid Model System.

The intestinal epithelium is a primary interface for engagement of the host response by foodborne pathogens, like Salmonella enterica Typhimurium. While the interaction of S Typhimurium with the mammalian host has been well studied in transformed epithelial cell lines or in the complex intestinal environment in vivo, few tractable models recapitulate key features of the intestine. Human intestinal organoids (HIOs) contain a polarized epithelium with functionally differentiated cell subtypes, including enterocytes and goblet cells and a supporting mesenchymal cell layer. HIOs contain luminal space that supports bacterial replication, are more amenable to experimental manipulation than animals and are more reflective of physiological host responses. Here, we use the HIO model to define host transcriptional responses to S Typhimurium infection, also determining host pathways dependent on Salmonella pathogenicity island-1 (SPI-1)- and -2 (SPI-2)-encoded type 3 secretion systems (T3SS). Consistent with prior findings, we find that S Typhimurium strongly stimulates proinflammatory gene expression. Infection-induced cytokine gene expression was rapid, transient, and largely independent of SPI-1 T3SS-mediated invasion, likely due to continued luminal stimulation. Notably, S Typhimurium infection led to significant downregulation of host genes associated with cell cycle and DNA repair, leading to a reduction in cellular proliferation, dependent on SPI-1 and SPI-2 T3SS. The transcriptional profile of cell cycle-associated target genes implicates multiple miRNAs as mediators of S Typhimurium-dependent cell cycle suppression. These findings from Salmonella-infected HIOs delineate common and distinct contributions of SPI-1 and SPI-2 T3SSs in inducing early host responses during enteric infection and reinforce host cell proliferation as a process targeted by SalmonellaIMPORTANCESalmonella enterica serovar Typhimurium (S Typhimurium) causes a significant health burden worldwide, yet host responses to initial stages of intestinal infection remain poorly understood. Due to differences in infection outcome between mice and humans, physiological human host responses driven by major virulence determinants of Salmonella have been more challenging to evaluate. Here, we use the three-dimensional human intestinal organoid model to define early responses to infection with wild-type S Typhimurium and mutants defective in the SPI-1 or SPI-2 type-3 secretion systems. While both secretion system mutants show defects in mouse models of oral Salmonella infection, the specific contributions of each secretion system are less well understood. We show that S Typhimurium upregulates proinflammatory pathways independently of either secretion system, while the downregulation of the host cell cycle pathways relies on both SPI-1 and SPI-2. These findings lay the groundwork for future studies investigating how SPI-1- and SPI-2-driven host responses affect infection outcome and show the potential of this model to study host-pathogen interactions with other serovars to understand how initial interactions with the intestinal epithelium may affect pathogenesis.

Endoplasmic reticulum stress sensor IRE1α propels neutrophil hyperactivity in lupus.

Neutrophils amplify inflammation in lupus through the release of neutrophil extracellular traps (NETs). The endoplasmic reticulum stress sensor inositol-requiring enzyme 1 α (IRE1α) has been implicated as a perpetuator of inflammation in various chronic diseases; however, IRE1α has been little studied in relation to neutrophil function or lupus pathogenesis. Here, we found that neutrophils activated by lupus-derived immune complexes demonstrated markedly increased IRE1α ribonuclease activity. Importantly, in neutrophils isolated from patients with lupus, we also detected heightened IRE1α activity that was correlated with global disease activity. Immune complex-stimulated neutrophils produced both mitochondrial ROS (mitoROS) and the activated form of caspase-2 in an IRE1α-dependent fashion, whereas inhibition of IRE1α mitigated immune complex-mediated NETosis (in both human neutrophils and a mouse model of lupus). Administration of an IRE1α inhibitor to lupus-prone MRL/lpr mice over 8 weeks reduced mitoROS levels in peripheral blood neutrophils, while also restraining plasma cell expansion and autoantibody formation. In summary, these data identify a role for IRE1α in the hyperactivity of lupus neutrophils and show that this pathway is upstream of mitochondrial dysfunction, mitoROS formation, and NETosis. We believe that inhibition of the IRE1α pathway is a novel strategy for neutralizing NETosis in lupus, and potentially other inflammatory conditions.

The Mitochondrial Fission Regulator DRP1 Controls Post-Transcriptional Regulation of TNF-α.

The mitochondrial network plays a critical role in the regulation of innate immune signaling and subsequent production of proinflammatory cytokines such as IFN-ß and IL-1ß. Dynamin-related protein 1 (DRP1) promotes mitochondrial fission and quality control to maintain cellular homeostasis during infection. However, mechanisms by which DRP1 and mitochondrial dynamics control innate immune signaling and the proinflammatory response are incompletely understood. Here we show that macrophage DRP1 is a positive regulator of TNF-α production during sterile inflammation or bacterial infection. Silencing macrophage DRP1 decreased mitochondrial fragmentation and TNF-α production upon stimulation with lipopolysaccharide (LPS) or methicillin-resistant Staphylococcus aureus (MRSA) infection. The defect in TNF-α induction could not be attributed to changes in gene expression. Instead, DRP1 was required for post-transcriptional control of TNF-α. In contrast, silencing DRP1 enhanced IL-6 and IL-1ß production, indicating a distinct mechanism for DRP1-dependent TNF-α regulation. Our results highlight DRP1 as a key player in the macrophage pro-inflammatory response and point to its involvement in post-transcriptional control of TNF-α production.

Mitochondria-Derived Vesicles Deliver Antimicrobial Reactive Oxygen Species to Control Phagosome-Localized Staphylococcus aureus.

Pathogenic bacteria taken up into the macrophage phagosome are the target of many anti-microbial mechanisms. Although mitochondria-derived antimicrobial effectors like reactive oxygen species (mROS) aid in bacterial killing, it is unclear how these effectors reach bacteria within the phagosomal lumen. We show here that endoplasmic reticulum stress triggered upon methicillin-resistant Staphylococcus aureus (MRSA) infection induces mROS that are delivered to bacteria-containing phagosomes via mitochondria-derived vesicles (MDVs). The endoplasmic reticulum stress sensor IRE1α induces mROS, specifically hydrogen peroxide (mH2O2), upon MRSA infection. MRSA infection also stimulates the generation of MDVs, which require the mitochondrial stress response factor Parkin, and contributes to mH2O2 accumulation in bacteria-containing phagosomes. Accumulation of phagosomal H2O2 requires Toll-like receptor signaling and the mitochondrial enzyme superoxide dismutase-2 (Sod2), which is delivered to phagosomes by MDVs. Sod2 depletion compromises mH2O2 production and bacterial killing. Thus, mitochondrial redox capacity enhances macrophage antimicrobial function by delivering mitochondria-derived effector molecules into bacteria-containing phagosomes.

Endoplasmic Reticulum Stress Activates the Inflammasome via NLRP3- and Caspase-2-Driven Mitochondrial Damage.

Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1α ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1α activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity.

The Endoplasmic Reticulum Stress Sensor Inositol-Requiring Enzyme 1α Augments Bacterial Killing through Sustained Oxidant Production.

UNLABELLED: Bacterial infection can trigger cellular stress programs, such as the unfolded protein response (UPR), which occurs when misfolded proteins accumulate within the endoplasmic reticulum (ER). Here, we used the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) as an infection model to probe how ER stress promotes antimicrobial function. MRSA infection activated the most highly conserved unfolded protein response sensor, inositol-requiring enzyme 1α (IRE1α), which was necessary for robust bacterial killing in vitro and in vivo. The macrophage IRE1-dependent bactericidal activity required reactive oxygen species (ROS). Viable MRSA cells excluded ROS from the nascent phagosome and strongly triggered IRE1 activation, leading to sustained generation of ROS that were largely Nox2 independent. In contrast, dead MRSA showed early colocalization with ROS but was a poor activator of IRE1 and did not trigger sustained ROS generation. The global ROS stimulated by IRE1 signaling was necessary, but not sufficient, for MRSA killing, which also required the ER resident SNARE Sec22B for accumulation of ROS in the phagosomal compartment. Taken together, these results suggest that IRE1-mediated persistent ROS generation might act as a fail-safe mechanism to kill bacterial pathogens that evade the initial macrophage oxidative burst. IMPORTANCE: Cellular stress programs have been implicated as important components of the innate immune response to infection. The role of the IRE1 pathway of the ER stress response in immune secretory functions, such as antibody production, is well established, but its contribution to innate immunity is less well defined. Here, we show that infection of macrophages with viable MRSA induces IRE1 activation, leading to bacterial killing. IRE1-dependent bactericidal activity required generation of reactive oxygen species in a sustained manner over hours of infection. The SNARE protein Sec22B, which was previously demonstrated to control ER-phagosome trafficking, was dispensable for IRE1-driven global ROS production but necessary for late ROS accumulation in bacteria-containing phagosomes. Our study highlights a key role for IRE1 in promoting macrophage bactericidal capacity and reveals a fail-safe mechanism that leads to the concentration of antimicrobial effector molecules in the macrophage phagosome.

Listeria exploits damage and death to spread bad news.

Pathogenic Listeria monocytogenes replicates within the host cytosol; little is known about how it transits from cell to cell, spreading infection. A recent study implicates infection-induced membrane damage as a trigger for efferocytosis, the recognition and uptake of dead cells, thereby tricking neighboring cells into taking up the invader.

Zebrafish as a natural host model for Vibrio cholerae colonization and transmission.

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival.

Termination of Vibrio cholerae virulence gene expression is mediated by proteolysis of the major virulence activator, ToxT.

Vibrio cholerae is the causative agent of cholera, a severe diarrhoeal illness. V. cholerae produces two major virulence factors: the cholera toxin, which directly causes diarrhoea, and the toxin-coregulated pilus, which is required for intestinal colonization. Production of these virulence factors is dependent on the major virulence regulator, ToxT. Under virulence-inducing growth conditions, transcription factors ToxR and TcpP initially activate transcription of toxT. However, once ToxT has been expressed, it produces more of itself independent of ToxR and TcpP by activating transcription of the long tcpA operon, within which toxT is located. It is known that V. cholerae terminates virulence gene expression prior to escape from the host, but it is unknown how this ToxT-positive feedback loop is broken, an essential step in terminating virulence gene expression. To better understand how ToxT protein activity is regulated, we monitored ToxT accumulation and activity under virulence-inducing and -repressing growth conditions. Our results suggest that ToxT protein undergoes proteolytic degradation to terminate virulence gene expression. This directed degradation of ToxT supports a model for terminating V. cholerae virulence gene expression late in infection, with both ToxT and TcpP undergoing proteolysis prior to escape from the host.

Genetic screening for bacterial mutants in liquid growth media by fluorescence-activated cell sorting.

Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and makes screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction.

Bicarbonate Induces Vibrio cholerae virulence gene expression by enhancing ToxT activity.

Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO(2) into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.