Contribution of the Type III Secretion System (T3SS2) of Vibrio parahaemolyticus in Mitochondrial Stress in Human Intestinal Cells.

Vibrio parahaemolyticus is an important human pathogen that is currently the leading cause of shellfish-borne gastroenteritis in the world. Particularly, the pandemic strain has the capacity to induce cytotoxicity and enterotoxicity through its Type 3 Secretion System (T3SS2) that leads to massive cell death. However, the specific mechanism by which the T3SS2 induces cell death remains unclear and its contribution to mitochondrial stress is not fully understood. In this work, we evaluated the contribution of the T3SS2 of V. parahaemolyticus in generating mitochondrial stress during infection in human intestinal HT-29 cells. To evaluate the contribution of the T3SS2 of V. parahaemolyticus in mitochondrial stress, infection assays were carried out to evaluate mitochondrial transition pore opening, mitochondrial fragmentation, ATP quantification, and cell viability during infection. Our results showed that the Δvscn1 (T3SS2+) mutant strain contributes to generating the sustained opening of the mitochondrial transition pore. Furthermore, it generates perturbations in the ATP production in infected cells, leading to a significant decrease in cell viability and loss of membrane integrity. Our results suggest that the T3SS2 from V. parahaemolyticus plays a role in generating mitochondrial stress that leads to cell death in human intestinal HT-29 cells. It is important to highlight that this study represents the first report indicating the possible role of the V. parahaemolyticus T3SS2 and its effector proteins involvement in generating mitochondrial stress, its impact on the mitochondrial pore, and its effect on ATP production in human cells.

Presence of Zonula Occludens Toxin-Coding Genes among Vibrio parahaemolyticus Isolates of Clinical and Environmental Origin.

In recent studies, emphasis has been placed on the zonula occludens toxin (Zot) from the non-toxigenic Vibrio parahaemolyticus strain PMC53.7 as an agent inducing alterations in the actin cytoskeleton of infected Caco-2 cells and which appears as a relevant virulence factor. Universal zot primers were designed by the alignment of different types of zot gene and identification of conserved sequences to investigate the presence in diverse environmental and clinical V. parahaemolyticus isolates, in co-occurrence with virulence factors, such a hemolysins and secretion systems. The study screened a total of 390 isolates from environmental sources from Chile and Italy and 95 Chilean clinical isolates. The results revealed that around 37.2% of Chilean environmental strains and 25.9% of Italian strains, and 24.2% of clinical isolates carried the zot gene. The Zot-C2 cluster was present in 71.4% of Chilean environmental strains but absent in clinical isolates, while the Zot-C4 cluster was identified in 28.6% of environmental and 100% of clinical isolates. Understanding the role of zot in V. parahaemolyticus virulence is crucial, especially considering the risk associated with consuming diverse isolates from bivalves and the co-occurrence with virulence factors such as TDH, TRH or T3SS2.

Fenofibrate Decreases Ethanol-Induced Neuroinflammation and Oxidative Stress and Reduces Alcohol Relapse in Rats by a PPAR-α-Dependent Mechanism.

High ethanol consumption triggers neuroinflammation, implicated in sustaining chronic alcohol use. This inflammation boosts glutamate, prompting dopamine release in reward centers, driving prolonged drinking and relapse. Fibrate drugs, activating peroxisome proliferator-activated receptor alpha (PPAR-α), counteract neuroinflammation in other contexts, prompting investigation into their impact on ethanol-induced inflammation. Here, we studied, in UChB drinker rats, whether the administration of fenofibrate in the withdrawal stage after chronic ethanol consumption reduces voluntary intake when alcohol is offered again to the animals (relapse-type drinking). Furthermore, we determined if fenofibrate was able to decrease ethanol-induced neuroinflammation and oxidative stress in the brain. Animals treated with fenofibrate decreased alcohol consumption by 80% during post-abstinence relapse. Furthermore, fenofibrate decreased the expression of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukins IL-1ß and IL-6, and of an oxidative stress-induced gene (heme oxygenase-1), in the hippocampus, nucleus accumbens, and prefrontal cortex. Animals treated with fenofibrate showed an increase M2-type microglia (with anti-inflammatory proprieties) and a decrease in phagocytic microglia in the hippocampus. A PPAR-α antagonist (GW6471) abrogated the effects of fenofibrate, indicating that they are dependent on PPAR-α activation. These findings highlight the potential of fenofibrate, an FDA-approved dyslipidemia medication, as a supplementary approach to alleviating relapse severity in individuals with alcohol use disorder (AUD) during withdrawal.

Alcohol use disorder, neuroinflammation, and intake of dietary fibers: a new approach for treatment.

A close communication exists between the microorganisms that live in the intestine and the brain, the so-called microbiota-gut-brain axis. This interaction occurs at different levels, such as the induction by bacteria of an inflammatory state in the intestine that produces (i) stimulation of the vagus nerve that conducts information to the brain, and (ii) the release of proinflammatory cytokines - such as TNF-α- to circulation, which can then be transported to the brain and trigger neuroinflammation. Ethanol-induced neuroinflammation is produced, in part, by the impairment of the epithelial barrier function of the intestine, since acetaldehyde generated in the gut from ethanol oxidation produces a disassembly of the tight junctions (TJ), which allows diffusion of bacterial components into the blood; these events trigger a systemic inflammatory response that crosses the blood-brain barrier and induce neuroinflammation. Some short-chain fatty acids (SCFAs) such as butyrate, propionate, and acetate, are produced from indigestible polysaccharides (fibers) by certain bacteria in the microbiota. These SCFAs have shown a protective function of the TJ against intestinal injuries, preserving the intestinal barrier function. In this perspective article, we discuss the therapeutic possibility to increase the production of SCFAs in the intestine of patients with AUD, to protect the integrity of TJ and thus modulate the neuroinflammatory process that helps to the establishment of chronic alcohol intake. In this way, a new therapeutic alternative is proposed here through a simple dietary intervention in the patient, increasing their fiber consumption.

Use of Short-Chain Fatty Acids for the Recovery of the Intestinal Epithelial Barrier Affected by Bacterial Toxins.

Short-chain fatty acids (SCFAs) are carboxylic acids produced as a result of gut microbial anaerobic fermentation. They activate signaling cascades, acting as ligands of G-protein-coupled receptors, such as GPR41, GPR43, and GPR109A, that can modulate the inflammatory response and increase the intestinal barrier integrity by enhancing the tight junction proteins functions. These junctions, located in the most apical zone of epithelial cells, control the diffusion of ions, macromolecules, and the entry of microorganisms from the intestinal lumen into the tissues. In this sense, several enteric pathogens secrete diverse toxins that interrupt tight junction impermeability, allowing them to invade the intestinal tissue and to favor gastrointestinal colonization. It has been recently demonstrated that SCFAs inhibit the virulence of different enteric pathogens and have protective effects against bacterial colonization. Here, we present an overview of SCFAs production by gut microbiota and their effects on the recovery of intestinal barrier integrity during infections by microorganisms that affect tight junctions. These properties make them excellent candidates in the treatment of infectious diseases that cause damage to the intestinal epithelium.

Fenofibrate (a PPAR-α Agonist) Administered During Ethanol Withdrawal Reverts Ethanol-Induced Astrogliosis and Restores the Levels of Glutamate Transporter in Ethanol-Administered Adolescent Rats.

High-ethanol intake induces a neuroinflammatory response, which has been proposed as responsible for the maintenance of chronic ethanol consumption. Neuroinflammation decreases glutamate transporter (GLT-1) expression, increasing levels of glutamate that trigger dopamine release at the corticolimbic reward areas, driving long-term drinking behavior. The activation of peroxisome proliferator-activated receptor alpha (PPARα) by fibrates inhibits neuroinflammation, in models other than ethanol consumption. However, the effect of fibrates on ethanol-induced neuroinflammation has not yet been studied. We previously reported that the administration of fenofibrate to ethanol-drinking rats decreased ethanol consumption. Here, we studied whether fenofibrate effects are related to a decrease in ethanol-induced neuroinflammation and to the normalization of the levels of GLT-1. Rats were administered ethanol on alternate days for 4 weeks (2 g/kg/day). After ethanol withdrawal, fenofibrate was administered for 14 days (50 mg/kg/day) and the levels of glial fibrillary acidic protein (GFAP), phosphorylated NF-κB-inhibitory protein (pIκBα) and GLT-1, were quantified in the prefrontal cortex, hippocampus, and hypothalamus. Ethanol treatment increased the levels of GFAP in the hippocampus and hypothalamus, indicating a clear astrocytic activation. Similarly, ethanol increased the levels of pIκBα in the three areas. The administration of fenofibrate decreased the expression of GFAP and pIκBα in the three areas. These results indicate that fenofibrate reverts both astrogliosis and NF-κB activation. Finally, ethanol decreased GLT-1 expression in the prefrontal cortex and hippocampus. Fenofibrate normalized the levels of GLT-1 in both areas, suggesting that its effect in reducing ethanol consumption could be due to the normalization of glutamatergic tone.

Analysis of the Zonula occludens Toxin Found in the Genome of the Chilean Non-toxigenic Vibrio parahaemolyticus Strain PMC53.7.

Vibrio parahaemolyticus non-toxigenic strains are responsible for about 10% of acute gastroenteritis associated with this species, suggesting they harbor unique virulence factors. Zonula occludens toxin (Zot), firstly described in Vibrio cholerae, is a secreted toxin that increases intestinal permeability. Recently, we identified Zot-encoding genes in the genomes of highly cytotoxic Chilean V. parahaemolyticus strains, including the non-toxigenic clinical strain PMC53.7. To gain insights into a possible role of Zot in V. parahaemolyticus, we analyzed whether it could be responsible for cytotoxicity. However, we observed a barely positive correlation between Caco-2 cell membrane damage and Zot mRNA expression during PMC53.7 infection and non-cytotoxicity induction in response to purified PMC53.7-Zot. Unusually, we observed a particular actin disturbance on cells infected with PMC53.7. Based on this observation, we decided to compare the sequence of PMC53.7-Zot with Zot of human pathogenic species such as V. cholerae, Campylobacter concisus, Neisseria meningitidis, and other V. parahaemolyticus strains, using computational tools. The PMC53.7-Zot was compared with other toxins and identified as an endotoxin with conserved motifs in the N-terminus and a variable C-terminal region and without FCIGRL peptide. Notably, the C-terminal diversity among Zots meant that not all of them could be identified as toxins. Structurally, PMC53.7-Zot was modeled as a transmembrane protein. Our results suggested that it has partial 3D structure similarity with V. cholerae-Zot. Probably, the PMC53.7-Zot would affect the actin cytoskeletal, but, in the absence of FCIGRL, the mechanisms of actions must be elucidated.

Conservation of Small Regulatory RNAs in Vibrio parahaemolyticus: Possible role of RNA-OUT Encoded by the Pathogenicity Island (VPaI-7) of Pandemic Strains.

Small regulatory RNAs (sRNAs) are molecules that play an important role in the regulation of gene expression. sRNAs in bacteria can affect important processes, such as metabolism and virulence. Previous studies showed a significant role of sRNAs in the Vibrio species, but knowledge about Vibrio parahaemolyticus is limited. Here, we examined the conservation of sRNAs between V. parahaemolyticus and other human Vibrio species, in addition to investigating the conservation between V. parahaemolyticus strains differing in pandemic origin. Our results showed that only 7% of sRNAs were conserved between V. parahaemolyticus and other species, but 88% of sRNAs were highly conserved within species. Nonetheless, two sRNAs coding to RNA-OUT, a component of the Tn10/IS10 system, were exclusively present in pandemic strains. Subsequent analysis showed that both RNA-OUT were located in pathogenicity island-7 and would interact with transposase VPA1379, according to the model of pairing of IS10-encoded antisense RNAs. According to the location of RNA-OUT/VPA1379, we also investigated if they were expressed during infection. We observed that the transcriptional level of VPA1379 was significantly increased, while RNA-OUT was decreased at three hours post-infection. We suggest that IS10 transcription increases in pandemic strains during infection, probably to favor IS10 transposition and improve their fitness when they are facing adverse conditions.

Accessory Toxins of Vibrio Pathogens and Their Role in Epithelial Disruption During Infection.

Gastrointestinal episodes associated with Vibrio species have been rising worldwide in the last few years. Consequently, it is important to comprehend how occurs the production of diarrhea, to establish new preventive and therapeutic measures. Besides the classical CT and TCP toxins, Zot, RTX, and Ace among others have been deeply studied in V. cholerae. However, in other Vibrio species of clinical interest, where some of these toxins have been reported, there is practically no information. Zot activates a cascade of signals inside of the cell that increase the permeability of epithelial barrier, while RTX causes depolymerization of the actin cytoskeleton and Ace increases the permeability of intestinal cell monolayers. The goal of this study is to acquire information about the distribution of these toxins in human pathogenic Vibrios and to review the progress in the study of their role in the intestinal epithelium during infection.

Exploring the Genomic Traits of Non-toxigenic Vibrio parahaemolyticus Strains Isolated in Southern Chile.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence of tdh and trh genes has been used for diagnostic purposes and as a proxy of the virulence of V. parahaemolyticus isolates, the understanding of virulence in V. parahaemolyticus strains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-, trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome of V. parahaemolyticus of strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with the zot sequence found in V. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity of V. parahaemolyticus strains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism.

Bacteriophages in the control of pathogenic vibrios

Vibrios are common inhabitants of marine and estuarine environments. Some of them can be pathogenic to humans and/or marine animals using a broad repertory of virulence factors. Lately, several reports have indicated that the incidence of Vibrio infections in humans is rising and also in animals constitute a continuing threat for aquaculture. Moreover, the continuous use of antibiotics has been accompanied by an emergence of antibiotic resistance in Vibrio species, implying a necessity for efficient treatments. One promising alternative that emerges is the use of lytic bacteriophages; however, there are some drawbacks that should be overcome to make phage therapy a widely accepted method. In this work, we discuss about the major pathogenic Vibrio species and the progress, benefits and disadvantages that have been detected during the experimental use of bacteriophages to their control.

Galleria mellonella: A model of infection to discern novel mechanisms of pathogenesis of non-toxigenic Vibrio parahaemolyticus strains.

Vibrio parahaemolyticus is a leading cause of raw seafood-associated bacterial gastroenteritis in the world. Its pathogenesis is likely to be multifactorial, although the most characteristic virulence-associated factors are the toxins TDH and TRH, in addition to the Type-III Secretion System-2, which codes for diverse effectors involved in cytotoxicity and enterotoxicity. However, diarrhea cases produced by clinical strains lacking all of these main virulence factors (non-toxigenic strains) have been reported in many countries and they can represent up to 9-10% of the clinical isolations. So far, although there have been significant advances in the description of the virulence factors of V. parahaemolyticus, the ability of non-toxigenic strains to cause illness is still not completely understood. To elucidate this question it is necessary to have adequate infection models. The susceptibility of G. mellonella to the infection with non-toxigenic strains seems to be the response to identifying new virulence factors and consequently providing new insights into mechanisms of the virulence of non-toxigenic strains. This new model means an invaluable contribution to public health, since the understanding of virulence in strains lacking the traditional major toxins is essential to detect these strains present in waters and marine products and avoid possible food-borne infection.

Gene expression of Vibrio parahaemolyticus growing in laboratory isolation conditions compared to those common in its natural ocean environment.

BACKGROUND: Vibrio parahaemolyticus is an autochthonous marine bacterial species comprising strains able to grow in broth containing bile salts at 37 °C, a condition seldom found in the ocean. However, this condition is used for isolation in the laboratory because it is considered a necessary property for pathogenesis. In this context, revealing how gene expression enables V. parahaemolyticus to adapt to this particular condition -common to almost all V. parahaemolyticus isolates- will improve our understanding of the biology of this important pathogen. To determine the genes of V. parahaemolyticus differentially expressed when growing in isolation condition (37 °C, 0.9% NaCl, and 0.04% bile salts) referred to those at the temperature and salt concentration prevailing in ocean south of Chile (marine-like condition; 12 °C, 3% NaCl, and absence of bile salts) we used high-throughput sequencing of RNA. RESULTS: Our results showed that in the isolation condition, among the 5034 genes annotated in the V. parahaemolyticus RIMD2210633 genome, 344 were upregulated and 433 downregulated referred to the marine-like condition, managing an adjusted P-value (Padj) < E-5. Between the 50 more highly expressed genes, among the small RNAs (sRNA), the three carbon storage regulators B (CsrB) were up four to six times, while RyhB, related to iron metabolism besides motility control, was down about eight times. Among proteins, BfdA, a hemolysin-co-regulated protein (Hcp1) secreted by T6SS1, one of the most highly expressed genes, was about 140 times downregulated in isolation condition. The highest changes in relative expression were found among neighboring genes coding for proteins related to respiration, which were about 40 times upregulated. CONCLUSIONS: When V. parahaemolyticus is grown in conditions used for laboratory isolation 777 genes are up- or downregulated referred to conditions prevailing in the marine-like condition; the most significantly overrepresented categories among upregulated processes were those related to transport and localization, while secretion and pathogenesis were overrepresented among downregulated genes. Genes with the highest differential expression included the sRNAs CsrB and RhyB and the mRNAs related with secretion, nutritional upshift, respiration and rapid growing.

Role of Non-coding Regulatory RNA in the Virulence of Human Pathogenic Vibrios.

In recent decades, the identification of small non-coding RNAs in bacteria has revealed an important regulatory mechanism of gene expression involved in the response to environmental signals and to the control of virulence. In the family Vibrionaceae, which includes several human and animal pathogens, small non-coding RNAs (sRNAs) are closely related to important processes including metabolism, quorum sensing, virulence, and fitness. Studies conducted in silico and experiments using microarrays and high-throughput RNA sequencing have led to the discovery of an unexpected number of sRNAs in Vibrios. The present review discusses the most relevant reports regarding the mechanisms of action of sRNAs and their implications in the virulence of the main human pathogens in the family Vibrionaceae: Vibrio parahaemolyticus, V. vulnificus and V. cholerae.

Estrategia de verificación de calidad de las cepas de Escherichia coli conservadas en la Colección del Centro de Ingeniería Genética y Biotecnología / Strategy to verify quality of Escherichia coli strains kept in the collection of the Center of Genetic Engineering and Biotechnology

La colección de microorganismos de interés biotecnológico del Centro de Ingeniería Genética y Biotecnología incluye diferentes cepas de bacterias y levaduras utilizables en la manipulación de genes mediante el empleo de la ingeniería genética y para la producción de proteínas recombinantes por métodos biotecnológicos. La colección de hospederos de Escherichia coli centraliza todas las cepas que se utilizan en los proyectos de investigación-desarrollo de la institución. Este microorganismo constituye una de las herramientas biológicas imprescindibles para el trabajo en ingeniería genética y la investigación molecular. La fidelidad de los resultados experimentales depende en parte de la calidad de los bancos de estas cepas, que està determinada por las condiciones de almacenamiento y las estrategias de evaluación de los mismos. La complejidad del manejo de esta colección està influida en gran medida por el hecho de que en los medios de propagación pueden crecer un gran número de contaminantes ambientales de difícil identificación. Mientras màs similar es el contaminante a la cepa de interés, màs difícil es lograr bancos puros con una estabilidad confiable para la conservación. En este trabajo se describe cómo se garantiza el control de calidad de los bancos de Escherichia coli. Los métodos y estrategias de verificación que se describen han sido desarrollados en nuestro laboratorio(AU)

The Collection of Biotechnological-Interest Microorganisms of the CIGB includes several bacteria and yeast strains used in gene manipulation by means of genetic engineering and the production of recombinant proteins. The E coli host collection contains all the strains used in the research projects of the institution. This microorganism is an important tool for genetic engineering and molecular research. Accuracy of results depends on the quality of the microorganism banks, which it is determined by the storage conditions and the evaluation strategies of these microorganisms. The complexity of the management of this collection is influenced by the fact that a great number of contaminants may grow in the propagation media and they are difficult to identify. The more similar is the pollutant to the strain of interest, the harder it is to obtain pure banks with reliable stability and conservation. This work describes how quality control is ensured in the E. coli banks. The verification methods and strategies described here have been developed in our laboratory(AU)

Evaluación del método de siembra en placa "Traza de la dilución" en el control de calidad de bancos de mutantes de escherichia coli K12 / Evaluation of the track dilution plating method in the quality control of mutant banks of escherichia coli K12

El método que más se usa para la enumeración de microorganismos viables requiere de diluciones seriadas de la muestra seguida de la dispersión de 50 a 100 uL de cada dilución en medio sólido. En general para alcanzar una buena exactitud las diluciones se hacen por triplicado. Despúes de un tiempo de incubación, las colonias se cuentan y se estima la concentración bacteriana en la muestra original. Esta técnica es relativamente exacta pero tiene la desventaja de implicar el uso de una gran cantidad de materiales y de tiempo. Recientemente se desarrolló un método alternativo para la cuantificación de microorganismos viables llamado "método de la traza de la dilución" que reduce el tiempo y la cantidad de materiales. Con esta técnica la muestra con un grupo de bacterias desconocidas se siembra en una sola placa con agar nutriente(AU)