Papers by Stefan Pukatzki
Vibrio cholerae Alkalizes Its Environment via Citrate Metabolism to Inhibit Enteric Growth In Vitro
Microbiology Spectrum
Vibrio cholerae must compete with other bacteria in order to cause disease. Here, we show that V.... more Vibrio cholerae must compete with other bacteria in order to cause disease. Here, we show that V. cholerae creates an alkaline environment, which is able to inhibit the growth of other enteric bacteria. We demonstrate that V. cholerae environmental alkalization is linked to the capacity of the bacteria to metabolize citrate. This behavior could potentially contribute to V. cholerae’s ability to colonize the human intestine.
ABSTRACTBackgroundVibrio cholerae,the causative agent of cholera, is a human pathogen that thrive... more ABSTRACTBackgroundVibrio cholerae,the causative agent of cholera, is a human pathogen that thrives in estuarine environments.V. choleraecompetes with neighboring microbes by the contact-dependent translocation of toxic effectors with the type VI secretion system (T6SS). Effector types are highly variable acrossV. choleraestrains, but all pandemic isolates encode the same set of distinct effectors. It is possible that acquisition of these effectors via horizontal gene transfer played a role in the development of pandemicV. cholerae.ResultsWe assessed the distribution ofV. choleraeT6SS loci across multipleVibriospecies. We showed that the fish-pathogenV. anguillarumencodes all threeV. choleraecore loci as well as two of the four additional auxiliary clusters. We further demonstrated thatV. anguillarumshares T6SS effectors withV. cholerae,including every pandemic-associatedV. choleraeeffector. We identified a novel T6SS cluster (Accessory Aux1) that is widespread inV. anguillarumand en...
Microbiology Resource Announcements, 2021
Vibrio cholerae is the etiologic agent of cholera, an acute and often fatal diarrheal disease tha... more Vibrio cholerae is the etiologic agent of cholera, an acute and often fatal diarrheal disease that affects millions globally. We report the draft genome sequences of 13 non-O1/O139 V. cholerae strains isolated from the Rio Grande Delta, TX, USA. These genomes will aid future analyses of environmental serovars.
The human pathogen Pseudo utilizes conserved virulence ( the social amoeba Dictyostel
under which the human pathogen Pseudomonas aeruginosa in- ni, fects Dictyostelium discoideum, a g... more under which the human pathogen Pseudomonas aeruginosa in- ni, fects Dictyostelium discoideum, a genetically tractable eukaryotic vii organism. When D. discoideum is plated on nutrient agar plates ta] with different P. aeruginosa strains, the bacteria form lawns on ca these plates with amoebae embedded in them. Virulent P. aerugi- pa nosa strains kill these amoebae and leave an intact bacterial lawn. th A number of P. aeruginosa mutants have been identified that are th avirulent in this assay. Amoebae feed on these bacteria and form sti plaques in their bacterial lawns. One avirulent mutant strain carries of an insertional mutation in the lasR gene. LasR is a transcription ha factor that controls a number of virulence genes in a density- ml dependent fashion. Another class of avirulent P. aeruginosa mu- sr tants is defective in type III secretion. One mutant lacks the PscJ no protein, a structural component of the secretion apparatus, sug- m gesting that cytotoxins are injected into ...
Nature Communications, 2021
The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease chol... more The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.
Microbial Cell, 2021
Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a microbe capable of in... more Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a microbe capable of inhabiting two different ecosystems: chitinous surfaces in brackish, estuarine waters and the epithelial lining of the human gastrointestinal tract. V. cholerae defends against competitive microorganisms with a contact-dependent, contractile killing machine called the type VI secretion system (T6SS) in each of these niches. The T6SS resembles an inverted T4 bacteriophage tail and is used to deliver toxic effector proteins into neighboring cells. Pandemic strains of V. cholerae encode a unique set of T6SS effector proteins, which may play a role in pathogenesis or pandemic spread. In our recent study (Santoriello et al. (2020), Nat Commun, doi: 10.1038/s41467-020-20012-7), using genomic and molecular biology tools, we demonstrated that the T6SS island Auxiliary Cluster 3 (Aux3) is unique to pandemic strains of V. cholerae. We went on to show that Aux3 is related to a phage-like element circ...
Cell Reports, 2020
Highlights d The T6SS promotes epithelial damage in the Drosophila model of Vibrio cholerae infec... more Highlights d The T6SS promotes epithelial damage in the Drosophila model of Vibrio cholerae infection d Infection with Vibrio cholerae with a T6SS impairs intestinal growth d The T6SS inhibits intestinal repair via interactions with the natural fly microbiome Authors
Proceedings of the National Academy of Sciences, 2018
Significance Enteric pathogens including the causative agent of cholera, Vibrio cholerae , use th... more Significance Enteric pathogens including the causative agent of cholera, Vibrio cholerae , use the type-six secretion system (T6SS) to kill commensal microbes in the host intestine. Eradicating competing microbes allows pathogens to improve colonization. However, it is not known whether commensal destruction has additional consequences on host viability. We used the Drosophila model of cholera to determine the impacts of T6SS on fly health and longevity. We found that T6SS-dependent competition with the symbiotic Acetobacter pasteurianus intensified disease symptoms, and accelerated host death. Gnotobiotic flies without A. pasteurianus abolished T6SS-dependent death, and reintroduction of A. pasteurianus alone was sufficient to restore accelerated death. These observations implicate T6SS-dependent interactions with commensal bacteria as a factor for the progression of cholera.
Trends in microbiology, Apr 24, 2016
Microbial species often exist in complex communities where they must avoid predation and compete ... more Microbial species often exist in complex communities where they must avoid predation and compete for favorable niches. The type VI secretion system (T6SS) is a contact-dependent bacterial weapon that allows for direct killing of competitors through the translocation of proteinaceous toxins. Vibrio cholerae is a Gram-negative pathogen that can use its T6SS during antagonistic interactions with neighboring prokaryotic and eukaryotic competitors. The T6SS not only promotes V. cholerae's survival during its aquatic and host life cycles, but also influences its evolution by facilitating horizontal gene transfer. This review details the recent insights regarding the structure and function of the T6SS as well as the diverse signals and regulatory pathways that control its activation in V. cholerae.
PLOS Neglected Tropical Diseases, 2015
The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thriv... more The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen's arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7 th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection.
Proceedings of the National Academy of Sciences of the United States of America, Jan 13, 2015
Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-... more Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells...
Proceedings of the National Academy of Sciences of the United States of America, Jan 9, 2010
The acute diarrheal disease cholera is caused by the marine bacterium Vibrio cholerae. A type VI ... more The acute diarrheal disease cholera is caused by the marine bacterium Vibrio cholerae. A type VI secretion system (T6SS), which is structurally similar to the bacteriophage cell-puncturing device, has been recently identified in V. cholerae and is used by this organism to confer virulence toward phagocytic eukaryotes, such as J774 murine macrophages and Dictyostelium discoideum. We tested the interbacterial virulence of V. cholerae strain V52, an O37 serogroup with a constitutively active T6SS. V52 was found to be highly virulent toward multiple Gram-negative bacteria, including Escherichia coli and Salmonella Typhimurium, and caused up to a 100,000-fold reduction in E. coli survival. Because the T6SS-deficient mutants V52ΔvasK and V52ΔvasH showed toxicity defects that could be complemented, virulence displayed by V. cholerae depends on a functional T6SS. V. cholerae V52 and strains of the O1 serogroup were resistant to V52, suggesting that V. cholerae has acquired immunity independ...
Frontiers in Microbiology, 2010
Vibrio cholerae, the marine bacterium responsible for the diarrheal disease cholera, utilizes a m... more Vibrio cholerae, the marine bacterium responsible for the diarrheal disease cholera, utilizes a multitude of virulence factors to cause disease. The importance of two of these factors, the toxin co-regulated pilus (TCP) and cholera toxin (CT), has been well documented for pandemic O1 and epidemic O139 serogroups. In contrast, endemic non-O1 and non-O139 serogroups can cause localized outbreaks of cholera-like illness, often in the absence of TCP and CT. One virulence mechanism used by these strains is the type VI secretion system (T6SS) to export toxins across the cell envelope and confer toxicity toward eukaryotic and prokaryotic organisms. The V. cholerae strain V52 (an O37 serogroup strain) possesses a constitutively active T6SS and was responsible for an outbreak of gastroenteritis in Sudan in 1968. To evaluate a potential role of the T6SS in the disease cholera, we compared the T6SS clusters of V. cholerae strains with sequenced genomes. We found that the majority of V. cholerae strains, including one pandemic strain, contain intact T6SS gene clusters; thus, we propose that the T6SS is a conserved mechanism that allows pandemic and endemic V. cholerae to persist both in the host and in the environment.
Nature Communications, 2014
Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with d... more Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with differing pathogenic potential. Only strains that express the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) are capable of pandemic spread of cholera diarrhoea. Regardless, all V. cholerae strains sequenced to date harbour genes for the type VI secretion system (T6SS) that translocates effectors into neighbouring eukaryotic and prokaryotic cells. Here we report that the effectors encoded within these conserved gene clusters differ widely among V. cholerae strains, and that immunity proteins encoded immediately downstream from the effector genes protect their host from neighbouring bacteria producing corresponding effectors. As a consequence, strains with matching effector-immunity gene sets can coexist, while strains with different sets compete against each other. Thus, the V. cholerae T6SS contributes to the competitive behaviour of this species.
Frontiers in Microbiology, 2013
Diarrheal diseases are the second-most common cause of death among children under the age of five... more Diarrheal diseases are the second-most common cause of death among children under the age of five worldwide. Cholera alone, caused by the marine bacterium Vibrio cholerae, is responsible for several million cases and over 120,000 deaths annually. When contaminated water is ingested, V. cholerae passes through the gastric acid barrier, penetrates the mucin layer of the small intestine, and adheres to the underlying epithelial lining. V. cholerae multiplies rapidly, secretes cholera toxin, and exits the human host in vast numbers during diarrheal purges. How V. cholerae rapidly reaches such high numbers during each purge is not clearly understood. We propose that V. cholerae employs its bactericidal type VI secretion system to engage in intraspecies and intraguild predation for nutrient acquisition to support rapid growth and multiplication.
Journal of Visualized Experiments, 2013
Electroporation has become a widely used method for rapidly and efficiently introducing foreign D... more Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.
Proceedings of the National Academy of Sciences, 2002
Genetically accessible host models are useful for studying microbial pathogenesis because they of... more Genetically accessible host models are useful for studying microbial pathogenesis because they offer the means to identify novel strategies that pathogens use to evade immune mechanisms, cause cellular injury, and induce disease. We have developed conditions under which the human pathogen Pseudomonas aeruginosa infects Dictyostelium discoideum , a genetically tractable eukaryotic organism. When D. discoideum is plated on nutrient agar plates with different P. aeruginosa strains, the bacteria form lawns on these plates with amoebae embedded in them. Virulent P. aeruginosa strains kill these amoebae and leave an intact bacterial lawn. A number of P. aeruginosa mutants have been identified that are avirulent in this assay. Amoebae feed on these bacteria and form plaques in their bacterial lawns. One avirulent mutant strain carries an insertional mutation in the lasR gene. LasR is a transcription factor that controls a number of virulence genes in a density-dependent fashion. Another cl...
Proceedings of the National Academy of Sciences, 2006
The bacterium Vibrio cholerae , like other human pathogens that reside in environmental reservoir... more The bacterium Vibrio cholerae , like other human pathogens that reside in environmental reservoirs, survives predation by unicellular eukaryotes. Strains of the O1 and O139 serogroups cause cholera, whereas non-O1/non-O139 strains cause human infections through poorly defined mechanisms. Using Dictyostelium discoideum as a model host, we have identified a virulence mechanism in a non-O1/non-O139 V. cholerae strain that involves extracellular translocation of proteins that lack N-terminal hydrophobic leader sequences. Accordingly, we have named these genes “VAS” genes for virulence-associated secretion, and we propose that these genes encode a prototypic “type VI” secretion system. We show that vas genes are required for cytotoxicity of V. cholerae cells toward Dictyostelium amoebae and mammalian J774 macrophages by a contact-dependent mechanism. A large number of Gram-negative bacterial pathogens carry genes homologous to vas genes and potential effector proteins secreted by this pa...
Proceedings of the National Academy of Sciences, 2007
Genes encoding type VI secretion systems (T6SS) are widely distributed in pathogenic Gram-negativ... more Genes encoding type VI secretion systems (T6SS) are widely distributed in pathogenic Gram-negative bacterial species. In Vibrio cholerae , T6SS have been found to secrete three related proteins extracellularly, VgrG-1, VgrG-2, and VgrG-3. VgrG-1 can covalently cross-link actin in vitro , and this activity was used to demonstrate that V. cholerae can translocate VgrG-1 into macrophages by a T6SS-dependent mechanism. Protein structure search algorithms predict that VgrG-related proteins likely assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate “tail spike” of Escherichia coli bacteriophage T4. VgrG-1 was shown to interact with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form. Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, we propose that the VgrG components of the T6SS appar...
PLoS Pathogens, 2013
The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects... more The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipidbinding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.
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Papers by Stefan Pukatzki