RESUMEN
Vibrio cholerae causes fatal diarrheal disease cholera in humans due to consumption of contaminated water and food. To instigate the disease, the bacterium must evade the host intestinal innate immune system; penetrate the mucus layer of the small intestine, adhere and multiply on the surface of microvilli and produce toxin(s) through the action of virulence associated genes. V. cholerae O1 that has caused a major cholera outbreak in Haiti contained several unique genetic signatures. These novel traits are used to differentiate them from the canonical El Tor strains. Several studies reported the spread of these Haitian variant strains in different parts of the world including Asia and Africa, but there is a paucity of information on the clinical consequence of these genetic changes. To understand the impact of these changes, we undertook a study involving mice and rabbit models to evaluate the pathogenesis. The colonization ability of Haitian variant strain in comparison to canonical El Tor strain was found to be significantly more in both suckling mice and rabbit model. Adult mice also displayed the same results. Besides that, infection patterns of Haitian variant strains showed a completely different picture. Increased mucosal damaging, colonization, and inflammatory changes were observed through hematoxylin-eosin staining and transmission electron microscopy. Fluid accumulation ability was also significantly higher in rabbit model. Our study indicated that these virulence features of the Haitian variant strain may have some association with the severe clinical outcome of the cholera patients in different parts of the world.
RESUMEN
Mycobacterium fortuitum is a natural fish pathogen. It induces apoptosis in headkidney macrophages (HKM) of catfish, Clarias sp though the mechanism remains largely unknown. We observed M. fortuitum triggers calcium (Ca2+) insult in the sub-cellular compartments which elicits pro-apototic ER-stress factor CHOP. Alleviating ER-stress inhibited CHOP and attenuated HKM apoptosis implicating ER-stress in the pathogenesis of M. fortuitum. ER-stress promoted calpain activation and silencing the protease inhibited caspase-12 activation. The study documents the primal role of calpain/caspase-12 axis on caspase-9 activation in M. fortuitum-pathogenesis. Mobilization of Ca2+ from ER to mitochondria led to increased mitochondrial Ca2+ (Ca2+)m load,, mitochondrial permeability transition (MPT) pore opening, altered mitochondrial membrane potential (ΔΨm) and cytochrome c release eventually activating the caspase-9/-3 cascade. Ultra-structural studies revealed close apposition of ER and mitochondria and pre-treatment with (Ca2+)m-uniporter (MUP) blocker ruthenium red, reduced Ca2+ overload suggesting (Ca2+)m fluxes are MUP-driven and the ER-mitochondria tethering orchestrates the process. This is the first report implicating role of sub-cellular Ca2+ in the pathogenesis of M. fortuitum. We summarize, the dynamics of Ca2+ in sub-cellular compartments incites ER-stress and mitochondrial dysfunction, leading to activation of pro-apoptotic calpain/caspase-12/caspase-9 axis in M. fortuitum-infected HKM.
RESUMEN
Objectives: Because of the rise in antimicrobial resistance, an inexpensive, diet-based treatment against Helicobacter pylori infection would be of great interest. The present study was performed to assess the in vitro effects of ellagic acid against clinical H. pylori strains that were resistant to antibiotics used for therapy and also to observe the morphological structure following treatment with ellagic acid. The effectiveness of ellagic acid in eradicating H. pylori infection in a murine (C57BL/6) infection model, one of the standard inbred mouse lines often used for experimental infection, was also assessed. Methods: A total of 55 strains were screened. The agar dilution method was used to determine the susceptibility of isolates to test compounds. Transmission electron microscopy was used to observe the morphology following treatment with ellagic acid. The antibacterial activity of ellagic acid in an H. pylori SS1-infected mouse model and its effect on gastric mucosal injury were determined by histology and PCR. Results: Ellagic acid inhibited the growth of all 55 of the H. pylori strains tested. The MIC of ellagic acid ranged from 5 to 30 mg/L, showing its bactericidal properties in vitro. Ellagic acid also demonstrated anti-H. pylori efficacy in eradication of this organism in an in vivo model, as well as restitution and repair of H. pylori-induced gastric mucosal damage. Conclusions: The present study paves the way for the preventive and therapeutic use of ellagic acid against H. pylori infection and, thus, ellagic acid can be considered a promising antibacterial agent against H. pylori-associated gastroduodenal diseases in humans.
Asunto(s)
Ácido Elágico/farmacología , Mucosa Gástrica/patología , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Animales , Antibacterianos/farmacología , Modelos Animales de Enfermedad , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/ultraestructura , Humanos , India , Masculino , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Organismos Libres de Patógenos EspecíficosRESUMEN
Colicinogenic plasmids encode toxic proteins which have antagonistic activity against closely related bacteria. This study describes the molecular characterization of three colicinogenic plasmids designated as pSSE3, pSSE and pSSE2, each with a molecular size of â¼6 kb, identified in clinical isolates of Shigella sonnei. Sequence analysis revealed that pSSE and pSSE2 shared extensive sequence homology with each other and with Escherichia coli E-type colicinogenic plasmids. The plasmid pSSE3 lacked an additional gene imparting immunity to colicin E8, a unique feature not observed in any of the previously reported sequences of colicin E3 plasmids. Incomplete digestion of colicinogenic plasmids by restriction endonucleases, metachromatic staining with acridine orange and presence of single stranded initiation (ssi) region confirmed the coexistence of ssDNA along with dsDNA. Plasmid copy number as determined by real-time PCR was found to be about 20. Transmission electron microscopy revealed DNA impairment in test bacteria after colicin exposure. We hypothesize that S. sonnei has acquired E-group colicin plasmids from its close relative E. coli, with their sequences undergoing subtle changes depending on the cohabitation in the same milieu.
Asunto(s)
Colicinas/genética , Plásmidos , Shigella sonnei/genética , Colicinas/farmacología , Enzimas de Restricción del ADN/metabolismo , ADN de Cadena Simple , Disentería Bacilar/microbiología , Escherichia coli/genética , Microscopía Electrónica de Transmisión , Reacción en Cadena en Tiempo Real de la Polimerasa , Shigella sonnei/efectos de los fármacos , Shigella sonnei/ultraestructuraRESUMEN
Inflammatory bowel disease (IBD) is a group of inflammatory disorders of the intestine caused by dysregulated T-cell mediated immune response against commensal microflora. Probiotics are reported as therapeutically effective against IBD. However, variable efficacy of the live probiotic strains, difference in survival and persistence in the gut between the strains and the lack of insight into the mechanisms of probiotic action limit optimal therapeutic efficacy. Our aims were to evaluate the lactobacillus strains isolated from the North Indian population for the generation of regulatory cells and cytokines in the intestine, to study their effects on pro-inflammatory mediators in the mouse model of inflammatory bowel disease and to explore the underlying mechanisms of their actions. Among the selected lactobacillus strains, Lactobacillus casei Lbs2 (MTCC5953) significantly suppressed lipopolysaccharide-induced pro-inflammatory cytokine (TNF-alpha, IL-6) secretion. Both live and heat-killed Lbs2 polarized Th0 cells to T-regulatory (Treg) cells in vitro, increased the frequency of FoxP3(+) Treg cells in the mesenteric lymph nodes (MLNs) and alleviated macroscopic and histopathological features of colitis in probiotic-fed mice. Moreover, the levels of IL-12, TNF-alpha and IL-17A were suppressed, while IL-10 and TGF-beta levels were augmented in the colonic tissues of Lbs2-treated mice. The induced Treg (iTreg) cells secreted IL-10 and TGF-beta and exerted suppressive effects on the proliferation of effector T-cells. Adoptive transfer of iTreg cells ameliorated the disease manifestations of murine colitis and suppressed the levels of TNF-alpha and IL-17A. Finally, Lbs2 effects were mediated by Toll-like receptor 2 (TLR2) activation on the dendritic cells. This study identified live and heat-killed Lbs2 as putative therapeutic candidates against IBD and highlighted their Toll-like receptor 2-dependent immunomodulatory and regulatory function.
Asunto(s)
Colitis/terapia , Células Dendríticas/efectos de los fármacos , Inmunoterapia/métodos , Mucosa Intestinal/inmunología , Lacticaseibacillus casei/inmunología , Probióticos/uso terapéutico , Linfocitos T Reguladores/efectos de los fármacos , Receptor Toll-Like 2/metabolismo , Animales , Células Cultivadas , Colitis/inducido químicamente , Citocinas/metabolismo , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Calor , Humanos , Terapia de Inmunosupresión , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Ácido TrinitrobencenosulfónicoRESUMEN
BACKGROUND: Almost all Helicobacter pylori infected person develop gastritis and severe gastritis is supposed to be the denominator of peptic ulcer diseases, which may lead to gastric cancer. However, it is still an enigma why few strains are associated with ulcer formation, while others are not related with any disease outcome. Although a number of putative virulence factors have been reported for H. pylori, there are contradictory results regarding their connotation with diseases. Recently, there has been a significant attention in strain-specific genes outside the cag pathogenicity island, especially genes within plasticity regions. Studies demonstrated that certain genes in this region may play important roles in the pathogenesis of H. pylori-associated diseases. The aim of this study was to assess the role of selected genes (jhp0940, jhp0945, jhp0947 and jhp0949) in the plasticity region in relation to risk of H. pylori-related diseases in Indian population. METHODS: A total of 113 H. pylori strains isolated from duodenal ulcer (DU) (n = 61) and non-ulcer dyspepsia (NUD) subjects (n = 52) were screened by PCR and Dot-Blot to determine the presence of these genes. The comparative study of IL-8 production and apoptosis were also done by co-culturing the AGS cells with H. pylori strains of different genotype. RESULTS: PCR and Dot-Blot results indicated that the prevalence rates of jhp0940, jhp0945, jhp0947 and jhp0949 in the H. pylori strains were 9.8, 47.5, 50.8, 40.9 % and 17.3, 28.8, 26.9, 19.2 % isolated from DU and NUD, respectively. IL-8 production and apoptotic cell death were significantly higher in H. pylori strains containing jhp0945, jhp0947 and jhp0949 than the strains lacking those genes. Results indicated that the prevalence of jhp0945, jhp0947 and jhp0949 are associated with increased risk of severe diseases in India. CONCLUSION: Our study showed that presence of jhp0945, jhp0947 and jhp0949 were significantly associated with symptomatic expressions along with the increased virulence during in vitro study whereas jhp0940 seems to be negatively associated with the disease. These results suggest that jhp0945, jhp0947 and jhp0949 could be useful prognostic markers for the development of duodenal ulcer in India.
RESUMEN
Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses.
Asunto(s)
Exosomas/enzimología , Inflamación/patología , Péptido Hidrolasas/metabolismo , Vibrio cholerae/enzimología , Vibrio cholerae/patogenicidad , Factores de Virulencia/metabolismo , Animales , Muerte Celular , Línea Celular , Células Epiteliales/patología , Humanos , Íleon/microbiología , Íleon/patología , Ratones Endogámicos BALB CRESUMEN
Mycobacterium fortuitum causes 'mycobacteriosis' in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.
Asunto(s)
Apoptosis/fisiología , Calcio/metabolismo , Macrófagos/metabolismo , Mycobacterium fortuitum/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animales , Bagres/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
In aquatic environments, Vibrio cholerae colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of chiA2 is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of V. cholerae chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for V. cholerae survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that V. cholerae could utilize mucin as a nutrient source. In comparison to the wild type strain, ΔchiA2 mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also â¼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the ΔchiA2 mutant caused â¼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the ΔchiA2 mutant and wild type V. cholerae was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by V. cholerae in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by V. cholerae for growth and survival in the host intestine.
Asunto(s)
Quitinasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Intestinos/microbiología , Vibrio cholerae/metabolismo , Animales , Quitinasas/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Mucinas/metabolismo , Conejos , Vibrio cholerae/genéticaRESUMEN
A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the ß-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.
Asunto(s)
Pandemias , Vibriosis/epidemiología , Vibriosis/microbiología , Vibrio parahaemolyticus/aislamiento & purificación , Antibacterianos/farmacología , ADN Bacteriano/análisis , ADN Bacteriano/genética , Diarrea , Farmacorresistencia Bacteriana , Heces/microbiología , Humanos , India/epidemiología , Modelos Lineales , Pruebas de Sensibilidad Microbiana , Serotipificación , Vibrio parahaemolyticus/clasificación , Vibrio parahaemolyticus/efectos de los fármacos , Vibrio parahaemolyticus/genéticaRESUMEN
In this study, we have established an oral phage cocktail therapy in adult mice model and also performed a comparative analysis between phage cocktail, antibiotic and oral rehydration treatment for orally developed Vibrio cholerae infection. Four groups of mice were orally infected with Vibrio cholerae MAK 757 strain. Phage cocktail and antibiotic treated groups received 1×10(8) plaque forming unit/ml (once a daily) and 40mg/kg (once a daily) as an oral dose respectively for consecutive three days after bacterial infection. In case of oral rehydration group, the solution was supplied after bacterial infection mixed with the drinking water. To evaluate the better and safer approach of treatment, tissue and serum samples were collected. Here, phage cocktail treated mice reduced the log10 numbers of colony per gram by 3log10 (p<0.05); however, ciprofloxacin treated mice reduced the viable numbers up to 5log10 (p<0.05). Whereas, the oral rehydration solution application was not able to reduce the viable bacterial count but the disease progress was much more diminished (p>0.05). Besides, it was evident that antibiotic and phage cocktail treated group had a gradual decrease in both IL-6 and TNF-α level for 3 days (p<0.05) but the scenario was totally opposite in bacterial control and oral hydration treated group. Histological examinations also endorsed the phage cocktail and ciprofloxacin treatment in mice. Although, in this murine model of cholera ciprofloxacin was found to be a better antimicrobial agent, but from the safety and specificity point of view, a better method of application could fill the bridge and advances the phages as a valuable agent in treating Vibrio cholerae infection.
Asunto(s)
Antibacterianos/administración & dosificación , Bacteriófagos , Terapia Biológica/métodos , Cólera/tratamiento farmacológico , Fluidoterapia/métodos , Animales , Carga Bacteriana , Modelos Animales de Enfermedad , Femenino , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/patología , Histocitoquímica , Interleucina-6/metabolismo , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/virologíaRESUMEN
BACKGROUND: This study was conducted to determine the role of enteroaggregative Escherichia coli (EAEC) in inflammatory diarrhea among hospitalized patients in Kolkata. The inflammatory pathogenesis of EAEC was established in mice model and histopathological studies. Presence of fecal leucocytes (FLCs) can be suspected for EAEC infection solely or as a mixed with other enteric pathogens. METHODS: Active surveillance was conducted for 2 years on 2 random days per week with every 5th patient admitted to the Infectious Diseases Hospital (IDH). Diarrheal samples were processed by conventional culture, microscopy, ELISA and molecular methods. Two EAEC isolated as sole pathogens were examined in mice after induced intestinal infection. The intestinal tissue samples were processed to analyze the histological changes. RESULTS: Of the 2519 samples screened, fecal leucocytes, erythrocytes and occult blood were detected in 1629 samples. Most of the patients had acute watery diarrhea (75%) and vomiting (78%). Vibrio cholerae O1 was the main pathogen in patients of 5-10 years age group (33%). Shigellosis was more in children from 2-5 years of age (19%), whereas children <2 years appeared to be susceptible for infection caused by EAEC (16%). When tested for the pathogenicity, the EAEC strains colonized well and caused inflammatory infection in the gut mucosa of BALB/C mice. CONCLUSION: This hospital-based surveillance revealed prevalence of large number of inflammatory diarrhea. EAEC was the suspected pathogen and <2 years children appeared to be the most susceptible age group. BALB/C mice may be a suitable animal model to study the EAEC-mediated pathogenesis.
RESUMEN
The composition of Escherichia coli in the neonatal gut has rarely been studied in developing countries. To gain insight into the composition of E. coli in the neonatal gut and to assess factors that could influence colonization by E. coli, analysis of the phylogenetic groups and virulence determinants of E. coli isolated from the guts of neonates in a tertiary care hospital was carried out. The distribution of the phylogroups of 124 E. coli isolates recovered showed that phylogroups A (23â%) and B1 (49â%) accounted for 72â% of the isolates. Isolates of the phylogenetic group B2 were rare (8â%). Virulence factors were also rare with the exception of aerobactin (iucC), which was detected in 45â% of the isolates and was significantly associated with phylogroup B1. Multinomial logistic regression established that colonization with phylogroup B1 was associated with a stay in the neonatal intensive care unit; phylogroup A was associated with a stay on the ward; and phylogroups B2 and D were associated with neonates delivered vaginally. Evaluation of the effect of different E. coli phylogroups, with and without identified virulence determinants, on the gut of neonatal mice showed histopathological changes in the mucosa. The severity of the changes could be correlated with the presence of virulence determinants, irrespective of the phylogroup.
Asunto(s)
Escherichia coli/clasificación , Escherichia coli/genética , Tracto Gastrointestinal/microbiología , Filogenia , Factores de Virulencia/genética , Animales , Análisis por Conglomerados , Modelos Animales de Enfermedad , Escherichia coli/aislamiento & purificación , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/patología , Genotipo , Humanos , Recién Nacido , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Estudios Prospectivos , Centros de Atención TerciariaRESUMEN
The aim of the present study was to explore a new approach based on the hemagglutination (HA) assay to understand the colonization ability of Shigella spp. To study colonization ability, an animal model of 4-day-old suckling mouse, was exploited. We characterized the HA activity of 48 Shigella strains, with erythrocytes collected from rabbit, guinea pig, chicken, and sheep. Only rabbit and guinea pig erythrocytes showed positive HA reactions in most of the cases. On the basis of HA pattern, 4 strains from each serogroup were selected for in vivo colonization studies. Our results showed a positive correlation between HA activity and colonization ability of the strains belonging to different serogroups (groups A, B, C, and D) of Shigella. In all 4 serogroups, high HA titer was associated with greater intestinal colonization.
Asunto(s)
Eritrocitos/metabolismo , Eritrocitos/microbiología , Hemaglutinación/fisiología , Intestinos/microbiología , Shigella/fisiología , Animales , Animales Lactantes , Pollos , Cobayas , Pruebas de Hemaglutinación , Ratones , Conejos , Ovinos , Shigella/metabolismoRESUMEN
Like most other Gram-negative bacteria, Shigella releases outer membrane vesicles (OMVs) into the surrounding environment during growth. In this study, we have exploited OMVs of Shigella as a protective immunogen in a mice model against Shigellosis. Distinctive vesicle secretion was noticed from different Shigella strains. Among them, Shigella boydii type 4 (BCH612) was secreting relatively higher amounts. We immunized female adult mice orally with 32 µg of purified Shigella boydii type 4 (BCH612) OMVs four times at 1-week intervals. Antibodies against these vesicles were detected in immunized sera until 120 days, indicating a persistent immune response. To observe whether the passive immunity had been transferred to the neonates, the immunized female mice were mated and the offspring were challenged orally, with wild-type homologous and heterologous Shigella strains. All offspring of immunized mothers survived the challenge with homologous strain BCH612 and up to 81% protective efficacy was noted against heterologous strains Shigella dysenteriae 1, Shigella flexneri 2a, Shigella flexneri 3a, Shigella flexneri 6 and Shigella sonnei. Our results exhibited for the first time that oral immunization of adult female mice with purified OMVs of Shigella, without any adjuvant, conferred passive protection to their offspring against shigellosis. These findings will contribute to the future development of a potential non-living vaccine candidate against shigellosis.
Asunto(s)
Disentería Bacilar/prevención & control , Inmunización Pasiva/métodos , Vesículas Secretoras/inmunología , Shigella boydii/inmunología , Administración Oral , Animales , Animales Recién Nacidos , Anticuerpos Antibacterianos/sangre , Modelos Animales de Enfermedad , Disentería Bacilar/microbiología , Femenino , Inmunidad Materno-Adquirida , Masculino , Ratones , Shigella dysenteriae/inmunología , Shigella dysenteriae/patogenicidad , Shigella flexneri/inmunología , Shigella flexneri/patogenicidad , Shigella sonnei/inmunología , Shigella sonnei/patogenicidad , Análisis de SupervivenciaRESUMEN
Shigellosis is a major form of bacillary dysentery caused by Shigella spp. To date, there is no suitable animal model to evaluate the protective efficacy of vaccine candidates against this pathogen. Here, we describe a successful experimental shigellosis in the guinea-pig model, which has shown the characteristic features of human shigellosis. This model yielded reproducible results without any preparatory treatment besides cecal ligation. In this study, guinea-pigs were discretely infected with virulent Shigella dysenteriae type 1 and Shigella flexneri type 2a into the cecocolic junction after ligation of the distal cecum. All the experimental animals lost â¼10% of their body weight and developed typical dysentery within 24-h postinfection. In the histological analysis, distal colon showed edema, hemorrhage, exudation and inflammatory infiltrations in the lamina propria. Orally immunized animals with heat-killed S. dysenteriae type 1 and S. flexneri type 2a strains showed high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies and conferred significant homologous protective immunity against subsequent challenges with the live strains. The direct administration of shigellae into the cecocolic junction induces acute inflammation, making this animal model useful for assessing shigellosis and evaluating the protective immunity of Shigella vaccine candidates.
Asunto(s)
Modelos Animales de Enfermedad , Disentería Bacilar , Cobayas , Animales , Colon/patología , Histocitoquímica , Reproducibilidad de los Resultados , Shigella dysenteriae/patogenicidad , Shigella flexneri/patogenicidadRESUMEN
Current therapy-regimens against Helicobacter pylori (Hp) infections have considerable failure rates and adverse side effects that urge the quest for an effective alternative therapy. We have shown that curcumin is capable of eradicating Hp-infection in mice. Here we examine the mechanism by which curcumin protects Hp infection in cultured cells and mice. Since, MMP-3 and -9 are inflammatory molecules associated to the pathogenesis of Hp-infection, we investigated the role of curcumin on inflammatory MMPs as well as proinflammatory molecules. Curcumin dose dependently suppressed MMP-3 and -9 expression in Hp infected human gastric epithelial (AGS) cells. Consistently, Hp-eradication by curcumin-therapy involved significant downregulation of MMP-3 and -9 activities and expression in both cytotoxic associated gene (cag)(+ve) and cag(-ve) Hp-infected mouse gastric tissues. Moreover, we demonstrate that the conventional triple therapy (TT) alleviated MMP-3 and -9 activities less efficiently than curcumin and curcumin's action on MMPs was linked to decreased pro-inflammatory molecules and activator protein-1 activation in Hp-infected gastric tissues. Although both curcumin and TT were associated with MMP-3 and -9 downregulation during Hp-eradication, but unlike TT, curcumin enhanced peroxisome proliferator-activated receptor-γ and inhibitor of kappa B-α. These data indicate that curcumin-mediated healing of Hp-infection involves regulation of MMP-3 and -9 activities.
Asunto(s)
Curcumina/farmacología , Inhibidores Enzimáticos/farmacología , Infecciones por Helicobacter/tratamiento farmacológico , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Animales , Células Cultivadas , Curcumina/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Humanos , Inflamación , Mediadores de Inflamación , Ratones , Estómago/patologíaRESUMEN
Aeromonas hydrophila being a ubiquitous bacterium is prone to arsenic exposure. The present study was designed to determine the role of arsenic on growth and virulence of A. hydrophila. Exposure to arsenic (1 mg L(-1) and 2 mg L(-1)) had no effect on growth but significantly inhibited the hemolytic and cytotoxic potential of exposed bacteria. Transmission electron microscopy revealed loss of membrane integrity and presence of condensed cytoplasm suggestive of acute stress in bacteria exposed to arsenic. Arsenic-adapted bacteria were developed by repeated sub-culturing in presence of arsenic. Arsenic-adaptation led to significant recovery in hemolytic and cytotoxic potential. The arsenic-adapted bacteria exhibited normal membrane integrity, decreased cytoplasmic condensation and possessed scattered polysome like structures in the cytoplasm. A positive correlation was observed between arsenic tolerance and resistance to several antimicrobials. Arsenic-adaptation failed to confer cross-protection to mercury and cadmium stress. SDS-PAGE analysis revealed the expression of two new proteins of approximately 85 kDa and 79 kDa respectively in arsenic-adapted A. hydrophila. Plasmid-curing and transformation studies clearly indicate plasmid has no role on arsenic resistance trait of the bacteria. Our study, for the first time, reports a structure and function relationship of xenobiotics on bacteria.
Asunto(s)
Aeromonas hydrophila/crecimiento & desarrollo , Aeromonas hydrophila/patogenicidad , Arsénico/toxicidad , Bagres/microbiología , Enfermedades de los Peces/microbiología , Aeromonas hydrophila/efectos de los fármacos , Aeromonas hydrophila/ultraestructura , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Virulencia/efectos de los fármacosRESUMEN
BACKGROUND: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). METHODOLOGY/PRINCIPAL FINDINGS: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. CONCLUSION: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.
Asunto(s)
Cólera/patología , Íleon/patología , Metaloendopeptidasas/deficiencia , Péptido Hidrolasas/deficiencia , Serina Proteasas/metabolismo , Vibrio cholerae O1/enzimología , Secuencia de Aminoácidos , Animales , Cólera/microbiología , Modelos Animales de Enfermedad , Eliminación de Gen , Hemorragia , Humanos , Íleon/microbiología , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Péptido Hidrolasas/genética , Conejos , Serina Proteasas/química , Serina Proteasas/genética , Vibrio cholerae O1/química , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad , VirulenciaRESUMEN
We show here that oral immunization with purified outer membrane vesicles (OMVs) of Vibrio cholerae induces a prolonged high rise in the protective antibody titre. Rabbit immune sera were vibriocidal against the homologous and against several heterologous V. cholerae strains in vitro. In addition, OMV immunization conferred significant protective immunity against subsequent bacterial challenges. Thirty OMV-immunized rabbits were challenged with different V. cholerae strains; each challenged group contained five immunized and three unimmunized animals. All the immunized rabbits survived bacterial challenges and were healthy after 24 h, except the two from each group that received the SG24 and SG06 strains, respectively, which developed watery diarrhoea. In contrast, all the unimmunized animals developed cholera-like symptoms, with a death toll of eight within 24 h of challenge. This is the first report of the induction of protective immunity by V. cholerae OMVs in a rabbit model (removable intestinal tie-adult rabbit diarrhoea) that mimics the human disease. Finally, OMVs were found to be significantly less reactogenic than the live and the heat-killed bacteria. Our studies show that oral immunization with OMVs of V. cholerae may induce long-term immunity and may be useful as a 'nonliving' vaccine candidate for the future.