RESUMEN
BACKGROUND: Explanations for the genesis and propagation of cholera pandemics since 1817 have remained elusive. Evolutionary pathogen change is presumed to have been a dominant factor behind the 7th "El Tor" pandemic, but little is known to support this hypothesis for preceding pandemics. The role of anomalous climate in facilitating strain replacements has never been assessed. The question is of relevance to guide the understanding of infectious disease emergence today and in the context of climate change. METHODOLOGY/PRINCIPAL FINDINGS: We investigate the roles of climate and putative strain variation for the 6th cholera pandemic (1899-1923) using newly assembled historical records for climate variables and cholera deaths in provinces of former British India. We compare this historical pandemic with the 7th (El Tor) one and with the temporary emergence of the O139 strain in Bangladesh and globally. With statistical methods for nonlinear time series analysis, we examine the regional synchrony of outbreaks and associations of the disease with regional temperature and rainfall, and with the El Niño Southern Oscillation (ENSO). To establish future expectations and evaluate climate anomalies accompanying historical strain replacements, climate projections are generated with multi-model climate simulations for different 50-year periods. The 6th cholera pandemic featured the striking synchronisation of cholera outbreaks over Bengal during the El Niño event of 1904-07, following the invasion of the Bombay Presidency with a delay of a few years. Accompanying anomalous weather conditions are similar to those related to ENSO during strain replacements and pandemic expansions into Africa and South America in the late 20th century. Rainfall anomalies of 1904-05 at the beginning of the large cholera anomaly fall in the 99th percentile of simulated changes for the regional climate. CONCLUSIONS/SIGNIFICANCE: Evolutionary pathogen change can act synergistically with climatic conditions in the emergence and propagation of cholera strains. Increased climate variability and extremes under global warming provide windows of opportunity for emerging pathogens.
Asunto(s)
Cólera , Pandemias , Cólera/epidemiología , Humanos , Historia del Siglo XIX , Bangladesh/epidemiología , Cambio Climático , India/epidemiología , Historia del Siglo XX , Clima , Vibrio cholerae/genéticaRESUMEN
Cholera is a life-threatening gastrointestinal infection caused by a toxigenic bacterium, Vibrio cholerae. After a lull of almost 30 years, a first case of cholera was detected in Lebanon in October 2022. The outbreak lasted three months, with 8007 suspected cases (671 laboratory-confirmed) and 23 deaths. In this study, we use phenotypic methods and microbial genomics to study 34 clinical and environmental Vibrio cholerae isolates collected throughout this outbreak. All isolates are identified as V. cholerae O1, serotype Ogawa strains from wave 3 of the seventh pandemic El Tor (7PET) lineage. Phylogenomic analysis unexpectedly reveals the presence of two different strains of the seventh pandemic El Tor (7PET) lineage. The dominant strain has a narrow antibiotic resistance profile and is phylogenetically related to South Asian V. cholerae isolates and derived African isolates from the AFR15 sublineage. The second strain is geographically restricted and extensively drug-resistant. It belongs to the AFR13 sublineage and clusters with V. cholerae isolates collected in Yemen. In conclusion, the 2022-2023 Lebanese cholera outbreak is caused by the simultaneous introduction of two different 7PET strains. Genomic surveillance with cross-border collaboration is therefore crucial for the identification of new introductions and routes of circulation of cholera, improving our understanding of cholera epidemiology.
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Cólera , Brotes de Enfermedades , Filogenia , Líbano/epidemiología , Humanos , Cólera/epidemiología , Cólera/microbiología , Genoma Bacteriano/genética , Genómica/métodos , Vibrio cholerae/genética , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/clasificación , Masculino , Antibacterianos/farmacología , Femenino , Vibrio cholerae O1/genética , Vibrio cholerae O1/aislamiento & purificación , Vibrio cholerae O1/clasificación , Adolescente , Adulto , Adulto Joven , Persona de Mediana Edad , Niño , Epidemiología MolecularRESUMEN
Introduction: as cholera, due to toxigenic bacteria Vibrio cholera (serogroups O1 and O139), is a major public health threat in Africa, the aim of this work was to investigate potentially pathogenic Vibrionaceae bacteria firstly from human stool samples, and secondly from various environmental water points of Saint-Louis city in Senegal. Methods: a hospital-based study was conducted between 2013 and 2015. Stool samples were taken and cultured from daily incoming patients or hospitalized for acute diarrhea at Saint-Louis´ regional hospital. For environment, a monthly longitudinal sampling from January to October 2016 was carried out at 10 sites in the city. We used total DNA extracted from APW (alkaline peptone water) broth solutions and on suspect bacterial colonies to run PCR Multiplex targeting specific DNA fragments to detect Vibrio genus and specific species. In case of positivity, a simplex PCR was performed to test for cholera toxins Ctx, and V. parahaemolyticus TRH and TDH. Results: for 43 patients screened, bacterial culture was positive in 6% of cases but no strain of V. cholerae or other Vibrio sp. was isolated. PCR on 90 APW solutions were positive for Vibrio sp.(n = 43), V. cholera(n = 27), V. mimicus(n = 16), V. parahaemolyticus(8), V. alginolyticus(n = 4), and V. vulnificus(n = 2). Unlike for those on suspected colonies which were positive for a majority of V. parahaemolyticus (n = 40) and V. cholerae non-O1 / O139 (n = 35). Six strains of V. parahaemolyticus carried TRH gene, 3 of which expressed simultaneously virulence TRH and TDH genes. For physicochemical parameters, all temperatures varied similarly according to a unimodal seasonality, as well as salinity. Conclusion: despite the presence of natural populations of Vibrionaceae, even toxigenic ones, was noted in water environment, along with favorable habitat conditions that could play a role in transmission of Vibriosis in the Saint Louis population, we did not isolate any of them from patients screened at the hospital.
Asunto(s)
Cólera , Heces , Reacción en Cadena de la Polimerasa , Humanos , Senegal , Cólera/microbiología , Cólera/epidemiología , Heces/microbiología , Diarrea/microbiología , Diarrea/epidemiología , Microbiología del Agua , Vibrionaceae/aislamiento & purificación , Vibrionaceae/genética , Vibrio/aislamiento & purificación , Vibrio/genética , ADN Bacteriano/análisis , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/genética , Adulto , Femenino , MasculinoRESUMEN
Non-O1 and non-O139 Vibrio cholerae (NOVC) are serogroups that do not produce cholera toxin and are not responsible for epidemics. Even though rarely encountered in clinical practice, they can cause a spectrum of different conditions ranging from mild gastrointestinal syndrome to extraintestinal diseases, of which bacteremia and wound infections are the most severe. Risk factors for severe disease are cirrhosis, neoplasms, and diabetes mellitus. The mortality rate of NOVC bacteremia in hospitalized patients ranges from 24 to 61.5%. Incidence of NOVC infections is still rare, and consensus recommendations on treatment are not available. We report a case of NOVC bacteremia associated with severe cellulitis in an immunocompetent 75-year-old man who had eaten raw seafood in a location by the northern Adriatic Sea (Italy). Twenty-four hours after intake, he developed a high fever and vomiting. Afterwards, he started noticing the appearance of cellulitis in his right leg, which worsened in a matter of hours. The patient had a history of compensated type 2 diabetes mellitus. NOVC was isolated from both blood cultures and the leg ulcer. The non-O1, non-O139 serogroup was confirmed, and the detection of the cholera toxin gene was negative. Both tests were performed by the Reference National Laboratory of Istituto Superiore di Sanità (ISS). Multiple antimicrobial regimens were administered, with complete recovery. In conclusion, considering the severity of NOVC-associated manifestations, it is of pivotal importance to reach etiological diagnosis for a target antimicrobial therapy and to consider V. cholerae infection in the differential diagnosis in the presence of risk factors and potential exposure.
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Celulitis (Flemón) , Vibrio cholerae no O1 , Humanos , Masculino , Celulitis (Flemón)/microbiología , Celulitis (Flemón)/tratamiento farmacológico , Anciano , Vibrio cholerae no O1/aislamiento & purificación , Vibrio cholerae no O1/genética , Bacteriemia/microbiología , Bacteriemia/tratamiento farmacológico , Vibriosis/microbiología , Cólera/microbiología , Sepsis/microbiología , Sepsis/tratamiento farmacológico , Antibacterianos/uso terapéutico , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/genéticaRESUMEN
We report the discovery of a persistent presence of Vibrio cholerae at very low abundance in the inlet of a single wastewater treatment plant in Copenhagen, Denmark at least since 2015. Remarkably, no environmental or locally transmitted clinical case of V. cholerae has been reported in Denmark for more than 100 years. We, however, have recovered a near-complete genome out of 115 metagenomic sewage samples taken over the past 8 years, despite the extremely low relative abundance of one V. cholerae read out of 500,000 sequenced reads. Due to the very low relative abundance, routine screening of the individual samples did not reveal V. cholerae. The recovered genome lacks the gene responsible for cholerae toxin production, but although this strain may not pose an immediate public health risk, our finding illustrates the importance, challenges, and effectiveness of wastewater-based pathogen surveillance.
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Aguas del Alcantarillado , Vibrio cholerae , Dinamarca , Aguas del Alcantarillado/microbiología , Vibrio cholerae/genética , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/clasificación , Genoma Bacteriano , Aguas Residuales/microbiología , Cólera/microbiología , Cólera/epidemiologíaRESUMEN
Defense systems that recognize viruses provide important insights into both prokaryotic and eukaryotic innate immunity mechanisms. Such systems that restrict foreign DNA or trigger cell death have recently been recognized, but the molecular signals that activate many of these remain largely unknown. Here, we characterize one such system in pandemic Vibrio cholerae responsible for triggering cell density-dependent death (CDD) of cells in response to the presence of certain genetic elements. We show that the key component is the Lamassu DdmABC anti-phage/plasmid defense system. We demonstrate that signals that trigger CDD were palindromic DNA sequences in phages and plasmids that are predicted to form stem-loop hairpins from single-stranded DNA. Our results suggest that agents that damage DNA also trigger DdmABC activation and inhibit cell growth. Thus, any infectious process that results in damaged DNA, particularly during DNA replication, can in theory trigger DNA restriction and death through the DdmABC abortive infection system.
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ADN Viral , Vibrio cholerae , Vibrio cholerae/genética , ADN Viral/genética , Secuencias Invertidas Repetidas/genética , Plásmidos/genética , Plásmidos/metabolismo , Bacteriófagos/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
Malawi experienced its deadliest Vibrio cholerae (Vc) outbreak following devastating cyclones, with >58,000 cases and >1700 deaths reported between March 2022 and May 2023. Here, we use population genomics to investigate the attributes and origin of the Malawi 2022-2023 Vc outbreak isolates. Our results demonstrate the predominance of ST69 clone, also known as the seventh cholera pandemic El Tor (7PET) lineage, expressing O1 Ogawa (~ 80%) serotype followed by Inaba (~ 16%) and sporadic non-O1/non-7PET serogroups (~ 4%). Phylogenetic reconstruction revealed that the Malawi outbreak strains correspond to a recent importation from Asia into Africa (sublineage AFR15). These isolates harboured known antimicrobial resistance and virulence elements, notably the ICEGEN/ICEVchHai1/ICEVchind5 SXT/R391-like integrative conjugative elements and a CTXφ prophage with the ctxB7 genotype compared to historical Malawian Vc isolates. These data suggest that the devastating cyclones coupled with the recent importation of 7PET serogroup O1 strains, may explain the magnitude of the 2022-2023 cholera outbreak in Malawi.
Asunto(s)
Cólera , Brotes de Enfermedades , Filogenia , Vibrio cholerae , Malaui/epidemiología , Cólera/epidemiología , Cólera/microbiología , Humanos , Vibrio cholerae/genética , Vibrio cholerae/clasificación , Genómica , Genoma Bacteriano/genética , Profagos/genética , Genotipo , SerogrupoRESUMEN
Vibrio cholerae (V. cholerae), the etiological agent of cholera, employs various virulence factors to adapt and thrive within both aquatic and human host environments. Among these factors, the type VI secretion system (T6SS) stands out as one of the crucial determinants of its pathogenicity. Valine glycine repeat protein G1 (VgrG1) and hemolysin coregulated protein (HCP) are considered major effector molecules of T6SS. Previous studies have highlighted that VgrG1 interacts with HCP proteins. Additionally, it has been shown that VgrG1 possesses an actin cross-linking domain (ACD) with actin-binding activity. Interestingly, it was reported that purified HCP protein treatment increased the stress fibers within cells. Therefore, we hypothesize that HCP may interact with host cell actin, potentially playing a role in the cytoskeletal rearrangement during V. cholerae infection. To test this hypothesis, we characterized HCP from the V. cholerae O139 serotype and demonstrated its interaction with actin monomers. In silico analysis and experimental validation revealed the presence of an actin-binding site within HCP. Furthermore, overexpression of HCP resulted in its colocalization with actin stress fibers in host cells. Our findings establish HCP as an effector molecule for potent host cell actin cytoskeleton remodeling during V. cholerae infection, providing new insights into bacterial pathogenicity mechanisms. Understanding the interplay between bacterial effectors and host cell components is crucial for developing targeted therapeutic interventions against cholera and related infectious diseases.
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Citoesqueleto de Actina , Proteínas Bacterianas , Vibrio cholerae , Vibrio cholerae/patogenicidad , Vibrio cholerae/metabolismo , Vibrio cholerae/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Humanos , Citoesqueleto de Actina/metabolismo , Interacciones Huésped-Patógeno , Unión Proteica , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Actinas/metabolismo , Cólera/microbiología , Proteínas Hemolisinas/metabolismoRESUMEN
Vibrio cholerae is a Gram-negative gastrointestinal pathogen responsible for the diarrheal disease cholera. Expression of key virulence factors, cholera toxin and toxin-coregulated pilus, is regulated directly by ToxT and indirectly by two transmembrane transcription regulators (TTRs), ToxR and TcpP, that promote the expression of toxT. TcpP abundance and activity are controlled by TcpH, a single-pass transmembrane protein, which protects TcpP from a two-step proteolytic process known as regulated intramembrane proteolysis (RIP). The mechanism of TcpH-mediated protection of TcpP represents a major gap in our understanding of V. cholerae pathogenesis. The absence of tcpH leads to unimpeded degradation of TcpP in vitro and a colonization defect in a neonate mouse model of V. cholerae colonization. Here, we show that TcpH protects TcpP from RIP via direct interaction. We also demonstrate that α-linolenic acid, a dietary fatty acid, promotes TcpH-dependent inhibition of RIP via co-association of TcpP and TcpH molecules within detergent-resistant membranes (DRMs) in a mechanism requiring the TcpH transmembrane domain. Taken together, our data support a model where V. cholerae cells use exogenous α-linolenic acid to remodel the phospholipid bilayer in vivo, leading to co-association of TcpP and TcpH within DRMs where RIP of TcpP is inhibited by TcpH, thereby promoting V. cholerae pathogenicity. IMPORTANCE: Vibrio cholerae continues to pose a significant global burden on health and an alternative therapeutic approach is needed, due to evolving multidrug resistance strains. Transcription of toxT, stimulated by TcpP and ToxR, is essential for V. cholerae pathogenesis. Our results show that TcpP, one of the major regulators of toxT gene expression, is protected from proteolysis by TcpH, via direct interaction. Furthermore, we identified a gut metabolite, α-linolenic acid, that stimulates the co-association of TcpP and TcpH within detergent-resistant membranes (also known as lipid-ordered membrane domains), thereby supporting TcpH-dependent antagonism of TcpP proteolysis. Data presented here extend our knowledge of RIP, virulence gene regulation in V. cholerae, and, to the best of our knowledge, provides the first evidence that lipid-ordered membranes exist within V. cholerae. The model presented here also suggests that TTRs, common among bacteria and archaea, and co-component signal transduction systems present in Enterobacteria, could also be influenced similarly.
Asunto(s)
Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Proteolisis , Factores de Transcripción , Vibrio cholerae , Factores de Virulencia , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidad , Vibrio cholerae/efectos de los fármacos , Animales , Ratones , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Virulencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Cólera/microbiologíaRESUMEN
The Type VI secretion system (T6SS) is a multicomponent apparatus, present in many Gram-negative bacteria, which can inhibit bacterial prey in various ecological niches. Pseudomonas aeruginosa assembles one of its three T6SS (H1-T6SS) to respond to attacks from adjacent competing bacteria. Surprisingly, repeated assemblies of the H1-T6SS, termed dueling, were described in a monoculture in the absence of an attacker strain; however, the underlying mechanism was unknown. Here, we explored the role of H2-T6SS of P. aeruginosa in triggering H1-T6SS assembly. We show that H2-T6SS inactivation in P. aeruginosa causes a significant reduction in H1-T6SS dueling and that H2-T6SS activity directly triggers retaliation by the H1-T6SS. Intraspecific competition experiments revealed that elimination of H2-T6SS in non-immune prey cells conferred protection from H1-T6SS. Moreover, we show that the H1-T6SS response is triggered independently of the characterized lipase effectors of the H2-T6SS, as well as those of Acinetobacter baylyi and Vibrio cholerae. Our results suggest that H1-T6SS response to H2-T6SS in P. aeruginosa can impact intraspecific competition, particularly when the H1-T6SS effector-immunity pairs differ between strains, and could determine the outcome of multistrain colonization.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa harbors three different Type VI secretion systems (H1, H2, and H3-T6SS), which can translocate toxins that can inhibit bacterial competitors or inflict damage to eukaryotic host cells. Unlike the unregulated T6SS assembly in other Gram-negative bacteria, the H1-T6SS in P. aeruginosa is precisely assembled as a response to various cell damaging attacks from neighboring bacterial cells. Surprisingly, it was observed that neighboring P. aeruginosa cells repeatedly assemble their H1-T6SS toward each other. Mechanisms triggering this "dueling" behavior between sister cells were unknown. In this report, we used a combination of microscopy, genetic and intraspecific competition experiments to show that H2-T6SS initiates H1-T6SS dueling. Our study highlights the interplay between different T6SS clusters in P. aeruginosa, which may influence the outcomes of multistrain competition in various ecological settings such as biofilm formation and colonization of cystic fibrosis lungs.
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Pseudomonas aeruginosa , Sistemas de Secreción Tipo VI , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiología , Sistemas de Secreción Tipo VI/metabolismo , Sistemas de Secreción Tipo VI/genética , Acinetobacter/genética , Acinetobacter/metabolismo , Acinetobacter/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Vibrio cholerae/genética , Vibrio cholerae/fisiología , Vibrio cholerae/metabolismo , Interacciones MicrobianasRESUMEN
Seventh-pandemic Vibrio cholerae strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. In this study, we used cryogenic electron microscopy to determine the mechanistic basis for plasmid defense by DdmDE. The helicase-nuclease DdmD adopts an autoinhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. The structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the nontarget DNA strand. In vitro studies indicate that DdmD translocates in the 5'-to-3' direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination.
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Proteínas Argonautas , Proteínas Bacterianas , Islas Genómicas , Plásmidos , Vibrio cholerae , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , ADN Helicasas/metabolismo , ADN Helicasas/genética , ADN Bacteriano/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Multimerización de Proteína , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMEN
Vibrio cholerae, the causative agent of the diarrheal disease cholera, poses an ongoing health threat due to its wide repertoire of horizontally acquired elements (HAEs) and virulence factors. New clinical isolates of the bacterium with improved fitness abilities, often associated with HAEs, frequently emerge. The appropriate control and expression of such genetic elements is critical for the bacteria to thrive in the different environmental niches they occupy. H-NS, the histone-like nucleoid structuring protein, is the best-studied xenogeneic silencer of HAEs in gamma-proteobacteria. Although H-NS and other highly abundant nucleoid-associated proteins (NAPs) have been shown to play important roles in regulating HAEs and virulence in model bacteria, we still lack a comprehensive understanding of how different NAPs modulate transcription in V. cholerae. By obtaining genome-wide measurements of protein occupancy and active transcription in a clinical isolate of V. cholerae, harboring recently discovered HAEs encoding for phage defense systems, we show that a lack of H-NS causes a robust increase in the expression of genes found in many HAEs. We further found that TsrA, a protein with partial homology to H-NS, regulates virulence genes primarily through modulation of H-NS activity. We also identified few sites that are affected by TsrA independently of H-NS, suggesting TsrA may act with diverse regulatory mechanisms. Our results demonstrate how the combinatorial activity of NAPs is employed by a clinical isolate of an important pathogen to regulate recently discovered HAEs. IMPORTANCE: New strains of the bacterial pathogen Vibrio cholerae, bearing novel horizontally acquired elements (HAEs), frequently emerge. HAEs provide beneficial traits to the bacterium, such as antibiotic resistance and defense against invading bacteriophages. Xenogeneic silencers are proteins that help bacteria harness new HAEs and silence those HAEs until they are needed. H-NS is the best-studied xenogeneic silencer; it is one of the nucleoid-associated proteins (NAPs) in gamma-proteobacteria and is responsible for the proper regulation of HAEs within the bacterial transcriptional network. We studied the effects of H-NS and other NAPs on the HAEs of a clinical isolate of V. cholerae. Importantly, we found that H-NS partners with a small and poorly characterized protein, TsrA, to help domesticate new HAEs involved in bacterial survival and in causing disease. A proper understanding of the regulatory state in emerging isolates of V. cholerae will provide improved therapies against new isolates of the pathogen.
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Proteínas Bacterianas , Cólera , Regulación Bacteriana de la Expresión Génica , Vibrio cholerae , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Vibrio cholerae/metabolismo , Cólera/microbiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Transcripción Genética , Virulencia , Factores de Virulencia/genética , Transferencia de Gen HorizontalRESUMEN
Strain-specific pili enable Vibrio cholerae bacteria to adhere to each other and form aggregates in liquid culture. A new study focuses on strains with less specific, promiscuous pili and suggests a role for contact-dependent bacterial killing in shaping the composition of these aggregates.
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Fimbrias Bacterianas , Vibrio cholerae , Vibrio cholerae/fisiología , Vibrio cholerae/genética , Fimbrias Bacterianas/fisiología , Fimbrias Bacterianas/metabolismo , Adhesión Bacteriana/fisiologíaRESUMEN
CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.
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Vibrio vulnificus , Vibrio , Vibrio/genética , Vibrio vulnificus/genética , Vibrio parahaemolyticus/genética , Regulación Bacteriana de la Expresión Génica , Sistemas CRISPR-Cas , Vibrio cholerae/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Silenciamiento del Gen , Aliivibrio fischeri/genéticaRESUMEN
Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.
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Proteínas Argonautas , Proteínas Bacterianas , Plásmidos , Vibrio cholerae , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Proteínas Argonautas/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Desoxirribonucleasas/ultraestructura , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Modelos Moleculares , Plásmidos/genética , Plásmidos/inmunología , Plásmidos/metabolismo , Dominios Proteicos , Multimerización de Proteína , Vibrio cholerae/genética , Vibrio cholerae/inmunología , Vibrio cholerae/patogenicidadRESUMEN
Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma membrane remained elusive in several bacteria. VCA0625-27, made of periplasmic ligand binding protein (PLBP) VCA0625, an atypical monomeric permease VCA0626, and a cytosolic ATPase VCA0627, recently emerged as hexose-6-phosphate uptake system of Vibrio cholerae. Here we report high resolution crystal structure of VCA0625 in G6P bound state that largely resembles AfuA of Actinobacillus pleuropneumoniae. MD simulations on VCA0625 in apo and G6P bound states unraveled an 'open to close' and swinging bi-lobal motions, which are diminished upon G6P binding. Mutagenesis followed by biochemical assays on VCA0625 underscored that R34 works as gateway to bind G6P. Although VCA0627 binds ATP, it is ATPase deficient in the absence of VCA0625 and VCA0626, which is a signature phenomenon of type-I ABC importer. Further, modeling, docking and systematic sequence analysis allowed us to envisage the existence of similar atypical type-I G6P importer with fused monomeric permease in 27 other gram-negative bacteria.
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Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas , Glucosa-6-Fosfato , Vibrio cholerae , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Glucosa-6-Fosfato/metabolismo , Glucosa-6-Fosfato/química , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Vibrio cholerae/metabolismo , Vibrio cholerae/genéticaRESUMEN
Bacterial genomes are littered with exogenous: competing DNA elements. Here, Sprenger et al. demonstrate that the Vibrio cholerae prophage VP882 modulates host functions via production of regulatory sRNAs to promote phage development. Alternatively, host sRNAs inhibit the VP882 lytic phase by specifically regulating phage genes.
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Profagos , Vibrio cholerae , Vibrio cholerae/genética , Profagos/genética , Profagos/fisiología , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Genoma Bacteriano , Bacteriófagos/genética , Bacteriófagos/fisiología , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , ARN Bacteriano/metabolismoRESUMEN
This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived food samples in Guangdong province from 2015 to 2019. A total of 21 V. cholerae strains were obtained, which exhibited high resistance rates for nalidixic acid (57.14 %, 12/21), ampicillin (33.33 %, 7/21), and ciprofloxacin (19.05 %, 4/21). The quinolone resistance-related gene, qnrVC, was prevalent in 80.95 % (17/21) of the isolates. Additionally, chromosomally mediated quinolone-resistance mutations, including mutations in GyrA at position 83 (S83I) and ParC at position 85 (S85L), were detected in 47.62 % of the isolates. The combination of target mutation and qnrVC genes was shown to mediate resistance or intermediate resistance to ciprofloxacin in V. cholerae. Furthermore, an IncC-type conjugative plasmid carrying thirteen antibiotic resistance genes, including genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. While none of our food isolates harbored the toxigenic CTX- and TCP-encoding genes, they did possess genes encoding toxins such as HlyA and Autoinducer-2. Notably, some V. cholerae strains from this study exhibited a close genetic relationship with clinical strains, suggesting their potential to cause human infections. Taken together, this study provides a comprehensive view of the epidemiological features and genetic basis of antimicrobial resistance and virulence potential of V. cholerae strains isolated from food in southern China, thereby advancing our understanding of this important pathogen.
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Antibacterianos , Farmacorresistencia Bacteriana Múltiple , Microbiología de Alimentos , China/epidemiología , Farmacorresistencia Bacteriana Múltiple/genética , Antibacterianos/farmacología , Animales , Humanos , Pruebas de Sensibilidad Microbiana , Cólera/microbiología , Cólera/epidemiología , Vibrio cholerae/genética , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae no O1/genética , Vibrio cholerae no O1/efectos de los fármacos , Vibrio cholerae no O1/aislamiento & purificación , Plásmidos/genéticaRESUMEN
Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulators in Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in dacA1. A subsequent suppressor screen to restore viability of ΔdacA1 in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed.