RESUMEN
Porcine reproductive and respiratory syndrome (PRRS) outbreaks in pigs are associated with increased susceptibility of pigs to secondary bacterial infections, including Streptococcus suis - an important zoonotic pathogen causing bacterial meningitis in humans. This case-control study examined the association between human S. suis infection and PRRS outbreaks in pigs in northern Vietnam. We included 90 S. suis case-patients and 183 non-S. suis sepsis controls from a referral hospital in Hanoi in 2010, a period of major PRRS epizootics in Vietnam. PRRS exposure was determined using data from the National Centre of Veterinary Diagnosis. By univariate analysis, significantly more S. suis patients were reported residing in or adjacent to a PRRS district compared to controls [odds ratio (OR) 2·82, 95% confidence interval (CI) 1·35-5·89 and OR 3·15, 95% CI 1·62-6·15, respectively]. Only residency in adjacent districts remained significantly associated with risk of S. suis infection after adjusting for sex, occupation, and eating practices. SaTScan analysis showed a possible cluster of S. suis infection in humans around PRRS confirmed locations during the March-August period. The findings indicate an epidemiological association between PRRS in pigs and S. suis infections in humans. Effective strategies to strengthen control of PRRS in pigs may help reduce transmission of S. suis infection to humans.
Asunto(s)
Brotes de Enfermedades/veterinaria , Síndrome Respiratorio y de la Reproducción Porcina/epidemiología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Infecciones Estreptocócicas/epidemiología , Streptococcus suis/fisiología , Animales , Humanos , Síndrome Respiratorio y de la Reproducción Porcina/virología , Factores de Riesgo , Infecciones Estreptocócicas/microbiología , Porcinos , Vietnam/epidemiologíaRESUMEN
In Mekong Delta farms (Vietnam), antimicrobials are extensively used, but limited data are available on levels of antimicrobial resistance (AMR) among Escherichia coli isolates. We performed a structured survey of AMR in E. coli isolates (n = 434) from 90 pig, chicken, and duck farms. The results were compared with AMR among E. coli isolates (n = 234) from 66 small wild animals (rats and shrews) trapped on farms and in forests and rice fields. The isolates were susceptibility tested against eight antimicrobials. E. coli isolates from farmed animals were resistant to a median of 4 (interquartile range [IQR], 3 to 6) antimicrobials versus 1 (IQR, 1 to 2) among wild mammal isolates (P < 0.001). The prevalences of AMR among farmed species isolates (versus wild animals) were as follows: tetracycline, 84.7% (versus 25.6%); ampicillin, 78.9% (versus 85.9%); trimethoprim-sulfamethoxazole, 52.1% (versus 18.8%); chloramphenicol, 39.9% (versus 22.5%); amoxicillin-clavulanic acid, 36.6% (versus 34.5%); and ciprofloxacin, 24.9% (versus 7.3%). The prevalence of multidrug resistance (MDR) (resistance against three or more antimicrobial classes) among pig isolates was 86.7% compared to 66.9 to 72.7% among poultry isolates. After adjusting for host species, MDR was â¼8 times greater among isolates from wild mammals trapped on farms than among those trapped in forests/rice fields (P < 0.001). Isolates were assigned to unique profiles representing their combinations of susceptibility results. Multivariable analysis of variance indicated that AMR profiles from wild mammals trapped on farms and those from domestic animals were more alike (R(2) range, 0.14 to 0.30) than E. coli isolates from domestic animals and mammals trapped in the wild (R(2) range, 0.25 to 0.45). The results strongly suggest that AMR on farms is a key driver of environmental AMR in the Mekong Delta.
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Animales Domésticos/microbiología , Animales Salvajes/microbiología , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Infecciones por Escherichia coli/veterinaria , Escherichia coli/efectos de los fármacos , Animales , Pollos , Patos , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Pruebas de Sensibilidad Microbiana , Ratas , Musarañas/microbiología , Porcinos , VietnamRESUMEN
We investigated the prevalence, diversity, and antimicrobial resistance (AMR) profiles of non-typhoidal Salmonella (NTS) and associated risk factors on 341 pig, chicken, and duck farms in Dong Thap province (Mekong Delta, Vietnam). Sampling was stratified by species, district (four categories), and farm size (three categories). Pooled faeces, collected using boot swabs, were tested using ISO 6575: 2002 (Annex D). Isolates were serogrouped; group B isolates were tested by polymerase chain reaction to detect S. Typhimurium and (monophasic) serovar 4,[5],12:i:- variants. The farm-level adjusted NTS prevalence was 64·7%, 94·3% and 91·3% for chicken, duck and pig farms, respectively. Factors independently associated with NTS were duck farms [odds ratio (OR) 21·2], farm with >50 pigs (OR 11·9), pig farm with 5-50 pigs (OR 4·88) (vs. chickens), and frequent rodent sightings (OR 2·3). Both S. Typhimurium and monophasic S. Typhimurium were more common in duck farms. Isolates had a high prevalence of resistance (77·6%) against tetracycline, moderate resistance (20-30%) against chloramphenicol, sulfamethoxazole-trimethoprim, ampicillin and nalidixic acid, and low resistance (<5%) against ciprofloxacin and third-generation cephalosporins. Multidrug resistance (resistance against ⩾3 classes of antimicrobial) was independently associated with monophasic S. Typhimurium and other group B isolates (excluding S. Typhimurium) and pig farms. The unusually high prevalence of NTS on Mekong Delta farms poses formidable challenges for control.
Asunto(s)
Crianza de Animales Domésticos , Farmacorresistencia Bacteriana , Contaminación Ambiental , Aves de Corral , Salmonelosis Animal/epidemiología , Salmonella/aislamiento & purificación , Porcinos , Animales , Heces/microbiología , Prevalencia , Salmonella/efectos de los fármacos , Salmonelosis Animal/microbiología , Serotipificación , VietnamRESUMEN
Campylobacter are zoonotic pathogens commonly associated with gastroenteritis. To assess the relevance of Campylobacter in Vietnam, an economically transitioning country in SE Asia, we conducted a survey of 343 pig and poultry farms in the Mekong delta, a region characterized by mixed species farming with limited biosecurity. The animal-level prevalence of Campylobacter was 31·9%, 23·9% and 53·7% for chickens, ducks and pigs, respectively. C. jejuni was predominant in all three host species, with the highest prevalence in pigs in high-density production areas. Campylobacter isolates demonstrated high levels of antimicrobial resistance (21% and 100% resistance against ciprofloxacin and erythromycin, respectively). Multilocus sequence type genotyping showed a high level of genetic diversity within C. jejuni, and predicted C. coli inter-species transmission. We suggest that on-going intensification of animal production systems, limited biosecurity, and increased urbanization in Vietnam is likely to result in Campylobacter becoming an increasingly significant cause of human diarrhoeal infections in coming years.
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Infecciones por Campylobacter/veterinaria , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de los Porcinos/epidemiología , Animales , Antibacterianos/farmacología , Campylobacter/clasificación , Campylobacter/efectos de los fármacos , Campylobacter/genética , Campylobacter/aislamiento & purificación , Infecciones por Campylobacter/epidemiología , Infecciones por Campylobacter/microbiología , Estudios Transversales , Farmacorresistencia Bacteriana , Heces/microbiología , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Aves de Corral , Enfermedades de las Aves de Corral/microbiología , Prevalencia , Factores de Riesgo , Porcinos , Enfermedades de los Porcinos/microbiología , Vietnam/epidemiologíaRESUMEN
To examine the prevalence of human pathogens carried by rats in urban areas in Hanoi and Hai Phong, Vietnam, we live-trapped 100 rats in January 2011 and screened them for a panel of bacteria and viruses. Antibodies against Leptospira interrogans (22·0%), Seoul virus (14·0%) and rat hepatitis E virus (23·0%) were detected in rats, but antibodies against Yersinia pestis were not detected. Antibodies against L. interrogans and Seoul virus were found only in adult rats. In contrast, antibodies to rat hepatitis E virus were also found in juvenile and sub-adult rats, indicating that the transmission mode of rat hepatitis E virus is different from that of L. interrogans and Seoul virus. Moreover, phylogenetic analyses of the S and M segments of Seoul viruses found in Rattus norvegicus showed that Seoul viruses from Hai Phong and Hanoi formed different clades. Human exposure to these pathogens has become a significant public health concern.
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Anticuerpos Antibacterianos/sangre , Anticuerpos Antivirales/sangre , Enfermedades de los Roedores/epidemiología , Enfermedades de los Roedores/etiología , Zoonosis/epidemiología , Zoonosis/etiología , Animales , Análisis por Conglomerados , Femenino , Variación Genética , Masculino , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Ratas , Virus Seoul/clasificación , Virus Seoul/genética , Virus Seoul/aislamiento & purificación , Análisis de Secuencia de ADN , Estudios Seroepidemiológicos , Vietnam/epidemiologíaRESUMEN
OBJECTIVES: Central nervous system (CNS) infections are common causes of morbidity and mortality worldwide. We aimed to discover protein biomarkers that could rapidly and accurately identify the likely cause of the infections, essential for clinical management and improving outcome. METHODS: We applied liquid chromatography tandem mass spectrometry on 45 cerebrospinal fluid (CSF) samples from a cohort of adults with and without CNS infections to discover potential diagnostic biomarkers. We then validated the diagnostic performance of a selected biomarker candidate in an independent cohort of 364 consecutively treated adults with CNS infections admitted to a referral hospital in Vietnam. RESULTS: In the discovery cohort, we identified lipocalin 2 (LCN2) as a potential biomarker of bacterial meningitis (BM) other than tuberculous meningitis. The analysis of the validation cohort showed that LCN2 could discriminate BM from other CNS infections (including tuberculous meningitis, cryptococcal meningitis and virus/antibody-mediated encephalitis), with sensitivity of 0.88 (95% confident interval (CI), 0.77-0.94), specificity of 0.91 (95% CI, 0.88-0.94) and diagnostic odds ratio of 73.8 (95% CI, 31.8-171.4). LCN2 outperformed other CSF markers (leukocytes, glucose, protein and lactate) commonly used in routine care worldwide. The combination of LCN2, CSF leukocytes, glucose, protein and lactate resulted in the highest diagnostic performance for BM (area under the receiver operating characteristics curve, 0.96; 95% CI, 0.93-0.99). Data are available via ProteomeXchange with identifier PXD020510. CONCLUSIONS: LCN2 is a sensitive and specific biomarker for discriminating BM from a broad spectrum of other CNS infections. A prospective study is needed to assess the diagnostic utility of LCN2 in the diagnosis and management of CNS infections.
RESUMEN
Salmonellosis is a public health concern in both the developed and developing countries. Although the majority of human non-typhoidal Salmonella enterica (NTS) cases are the result of foodborne infections or person-to-person transmission, NTS infections may also be acquired by environmental and occupational exposure to animals. While a considerable number of studies have investigated the presence of NTS in farm animals and meat/carcasses, very few studies have investigated the risk of NTS colonization in humans as a result of direct animal exposure. We investigated asymptomatic NTS colonization in 204 backyard chicken farms, 204 farmers and 306 matched individuals not exposed to chicken farming, in southern Vietnam. Pooled chicken faeces, collected using boot or handheld swabs on backyard chicken farms, and rectal swabs from human participants were tested. NTS colonization prevalence was 45.6%, 4.4% and 2.6% for chicken farms, farmers and unexposed individuals, respectively. Our study observed a higher prevalence of NTS colonization among chicken farmers (4.4%) compared with age-, sex- and location- matched rural and urban individuals not exposed to chickens (2.9% and 2.0%). A total of 164 chicken NTS strains and 17 human NTS strains were isolated, and 28 serovars were identified. Salmonella Weltevreden was the predominant serovar in both chickens and humans. NTS isolates showed resistance (20-40%) against tetracycline, chloramphenicol, sulfamethoxazole-trimethoprim and ampicillin. Our study reflects the epidemiology of NTS colonization in chickens and humans in the Mekong delta of Vietnam and emphasizes the need of larger, preferably longitudinal studies to study the transmission dynamics of NTS between and within animal and human host populations.
Asunto(s)
Portador Sano , Pollos , Enfermedades de las Aves de Corral/microbiología , Salmonelosis Animal/microbiología , Infecciones por Salmonella/microbiología , Salmonella/clasificación , Animales , Antibacterianos/farmacología , Pollos/microbiología , Farmacorresistencia Bacteriana , Agricultores , Granjas , Humanos , Enfermedades de las Aves de Corral/epidemiología , Prevalencia , Infecciones por Salmonella/epidemiología , Salmonelosis Animal/epidemiología , Vietnam/epidemiología , ZoonosisRESUMEN
We investigated episodes of suspected highly pathogenic avian influenza (HPAI)-like illness among 12 meat duck flocks in two districts in Tien Giang province (Mekong Delta, Vietnam) in November 2013. In total, duck samples from 8 of 12 farms tested positive for HPAI virus subtype A/haemagglutinin 5 and neuraminidase 1 (H5N1) by real-time RT-PCR. Sequencing results confirmed clade of 2.3.2.1.c as the cause of the outbreaks. Most (7/8) laboratory-confirmed positive flocks had been vaccinated with inactivated HPAI H5N1 clade 2.3.4 vaccines <6 days prior to onset of clinical signs. A review of vaccination data in relation to estimated production in the area suggested that vaccination efforts were biased towards larger flocks and that vaccination coverage was low [21.2% ducks vaccinated with two shots (range by district 7.4-34.9%)]. The low-coverage data, the experimental evidence of lack of cross-protection conferred by the currently used vaccines based on clade 2.3.4 together with the short lifespan of meat duck flocks (60-70 days), suggest that vaccination is not likely to be effective as a tool for control of H5N1 infection in meat duck flocks in the area.
Asunto(s)
Brotes de Enfermedades/veterinaria , Patos , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/epidemiología , Animales , Brotes de Enfermedades/prevención & control , Subtipo H5N1 del Virus de la Influenza A/genética , Gripe Aviar/prevención & control , Gripe Aviar/virología , Carne , Vacunación/veterinaria , Vietnam/epidemiologíaRESUMEN
Introduction of double stranded RNA into invertebrate cells often results in posttranscriptional silencing of target genes through a mechanism termed RNA interference (RNAi). Double-stranded RNA is cleaved by an RNAse III-like enzyme, termed dicer, to small interfering RNAs (siRNAs). In Drosophila, these siRNAs are incorporated in the RNA induced silencing complex (RISC) and mediate degradation of target mRNA. The RISC complex contains members of Argonaute (Ago) family of proteins. We show here that RNAi in a hemocyte cell line of Anopheles gambiae, the principal malaria vector in Africa, requires expression of dicer-2, Ago2 and Ago3 proteins. Furthermore, we demonstrate that RNAi in the mosquito does not spread outside of the target region, suggesting that RNA dependent RNA polymerase mediated transitive amplification is absent in the mosquito.
Asunto(s)
Anopheles/genética , Interferencia de ARN/fisiología , Animales , Línea Celular , Silenciador del Gen/fisiología , Genes de Insecto/genética , Luciferasas/genética , Filogenia , Plásmidos/genética , Biosíntesis de Proteínas/genética , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , TransfecciónRESUMEN
In a prospective study conducted between mid 1993 and 1994, 437 adults and children were admitted with community-acquired septicaemia to an infectious diseases hospital in southern Viet Nam. Gram-negative aerobes accounted for 90% of isolates and were predominantly Salmonella typhi (67%), Sal. para-typhi A (3%), Escherichia coli (10%), and Klebsiella spp. (5%). Other Salmonella spp. (1%), Pseudomonas aeruginosa (1%), Neisseria meningitidis (0.5%) and Haemophilus influenzae (0.2%) were uncommon. Staphylococcus aureus (5.5%) and Streptococcus pneumoniae (2%) were the most common Gram-positive isolates. Patients with enteric fever were younger (median age 16 years, range 1-63) than the other patients (median age 43 years, range 1-88) (P < 0.001) and had a lower mortality rate (0.3% vs. 23%; relative risk 69.5, 95% confidence interval 9.5-507.8; P < 0.0001). Over 70% of the Sal. typhi isolated were multi-drug-resistant, and 4% were resistant to nalidixic acid. Multidrug-resistant Sal. typhi is a major cause of community-acquired septicaemia in Viet Nam.
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Salmonella typhi , Sepsis/microbiología , Fiebre Tifoidea/microbiología , Adolescente , Adulto , Técnicas Bacteriológicas , Niño , Preescolar , Resistencia a Múltiples Medicamentos , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Sepsis/epidemiología , Salud Urbana/estadística & datos numéricos , Vietnam/epidemiologíaRESUMEN
Seventy-nine comatose cerebral malaria patients given standard supportive treatment were randomized to receive specific antimalarial chemotherapy of intravenous quinine, intravenous artesunate, or artemisinin suppositories. Artesunate and artemisinin reduced peripheral asexual parasitaemia significantly more rapidly than quinine (90% clearance time 16 h, 18.9 h and 34.5 h respectively), but did not significantly reduce the duration of coma or mortality. The rapid lowering of peripheral parasitaemia may not ameliorate complications already present. These results demonstrate that artemisinin suppositories are as effective as artesunate and quinine given intravenously, and have economic and practical advantages for the treatment of severe malaria in areas remote from major medical centres. However, large numbers of patients will need to be studied if differences in mortality between the 3 treatment groups are to be demonstrated.
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Antimaláricos/administración & dosificación , Artemisininas , Malaria Cerebral/tratamiento farmacológico , Quinina/administración & dosificación , Sesquiterpenos/administración & dosificación , Adolescente , Adulto , Animales , Artesunato , Femenino , Humanos , Inyecciones Intravenosas , Malaria Cerebral/parasitología , Masculino , Persona de Mediana Edad , Plasmodium falciparum , SupositoriosRESUMEN
A method is described for the assessment of the dissolution behavior of solid dosage forms with a high content of a very water insoluble drug. In this method the dissolved drug is continuously extracted from the aqueous dissolution medium into an organic phase. The reported data on the dissolution of artemisinin show that this method guarantees sink conditions in the aqueous dissolution medium during the total duration of the experiment.
Asunto(s)
Antimaláricos/química , Artemisininas , Sesquiterpenos/química , Cloroformo , Sesquiterpenos/análisis , Solubilidad , Comprimidos/análisis , AguaRESUMEN
A series of 65 intracranial tumours operated on, and followed by, the same surgical team during 7 years is presented as a retrospective analysis (1987-1994). Among these patients, only 38 had a diagnosis based upon computed tomography (CT) scan because it was not until 1991 that the first CT scanner was set up in Ho Chi Minh City. Many patients were drowsy or comatose prior to operation due to lack of specialists' intervention or tardy diagnosis and evacuation from remote provincial hospitals. All operations were satisfactory despite the lack of many essential instruments. The histological diagnosis followed the classification proposed by Zulch KJ.(1) Intratumoural calcification has in no case been seen on X-rays or on histological specimens. Operative mortality rate was nil. Although not all the patients with malignant tumours could be followed up, the data available concerning their survival time and the quality of life seems similar to that observed in other reports.
RESUMEN
We report 15 new avian influenza virus A/H5N1 haemagglutinin (HA) sequences sampled from visibly sick domestic poultry in southern Vietnam, between 1 January 2010 and 6 March 2010. These HA sequences form a new sub-clade of the clade 1 H5N1 viruses that have been circulating in Vietnam since 2003/2004. The viruses are characterized by a change from isoleucine to valine at position 514 (I514V) and are 1.8% divergent at the nucleotide level from HA sequences sampled in Vietnam in 2007. Five new amino acid changes were observed at previously identified antigenic sites, and three were located within structural elements of the receptor-binding domain. One new mutation removed a potential N-linked glycosylation site, and a methionine insertion was observed in one virus at the polybasic cleavage site. Five of these viruses were sampled from farms where poultry were vaccinated against H5N1, but there was no association between observed amino acid changes and flock vaccination status. Despite the current lack of evidence for antigenic drift or immune escape in Vietnamese H5N1 viruses, continued surveillance remains a high priority.
Asunto(s)
Pollos , Patos , Evolución Molecular , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/inmunología , Gripe Aviar/virología , Agricultura , Animales , Gripe Aviar/epidemiología , Gripe Aviar/prevención & control , Filogenia , Vietnam/epidemiologíaRESUMEN
Helminth egg contamination of vegetables purchased at suburban market in Hanoi, Vietnam was examined. A total of 317 vegetables were examined and 82 (26%) were revealed to be positive for parasite eggs. Of the 15 varieties, 13 were positive except for horseradish and cucumber. Contamination was highest in leafy vegetables (31%), followed by root vegetables (17%) and fruit vegetables (3%). Throughout the survey, five species of parasite eggs were found: Ascaris sp., Trichuris sp., Toxocara sp., Taenia sp. and Ascaridia galli. In the interview with the villagers, 121 (81%) of 149 adult villagers stated that they usually use not only animal feces but also human feces as a fertilizer. Throughout the survey, a total of 453 eggs were recovered. Number of eggs recovered from vegetables was higher in the dry season (355 eggs) than in the rainy season (98 eggs). The study revealed that vegetables purchased at a market in suburban Hanoi (Vietnam) were highly contaminated with parasite eggs excreted by humans and animals. Considering the eating habits of the Vietnamese and the 17% embryonation rate of detected parasites, vegetables seem to play an important role in soil-transmitted helminth infection in this country.
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Recuento de Huevos de Parásitos , Verduras/parasitología , Animales , Humanos , Factores de Riesgo , Encuestas y Cuestionarios , VietnamRESUMEN
Malaria infection results in increased expression of immune responsive genes, including those encoding antimicrobial peptides such as Gambicin (Gam1) and Cecropin A (Cec1). Understanding how these genes are regulated will provide insights how the mosquito immune system is activated by Plasmodium. We previously have shown that Cec1 was primarily regulated by the Imd-Relish (REL2) pathway in the Anopheles gambiae Sua1B cell line. We show here that expression of Defensin A (Def1) and Gam1 was reduced after RNA interference against components of the Imd-REL2 pathway in An. gambiae cell lines. Interestingly, promoter reporters of these antimicrobial peptides were expressed at very low level in the cell line MSQ43 from Anopheles stephensi. Surprisingly, over-expression of either NF-kappaB transcription factor REL1 or REL2 alone is sufficient to induce the expression of Cec1, Gam1 and Def1. These results suggest that expression of these antimicrobial peptides (AMP) in vivo may be regulated by both the Toll and Imd pathways. We also show here for the first time that Tep4, a gene encoding a thioester containing protein, is regulated by REL2. Taken together, these results suggest that there are significant overlaps of genes regulated by the Toll-Rel1 and Imd-Rel2 pathways. Further, the different expression patterns in two different Anopheline cell lines provide a platform to identify other key positive and negative regulators of the antimicrobial peptide genes.
Asunto(s)
Anopheles/genética , Anopheles/inmunología , Inmunidad/genética , Proteínas de Insectos/genética , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Línea Celular , Escherichia coli , Genes Reporteros , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Transducción de SeñalRESUMEN
The BofC protein acts negatively on intercompartmental signalling of pro-sigma(K) processing in the sigma(K)-checkpoint of Bacillus subtilis. Signalling is brought about by the SpoIVB protein, which is synthesized in the forespore and initiates proteolytic processing of pro-sigmaK to its mature and active form in the opposed mother cell chamber of the developing cell. We have shown here that BofC, like SpoIVB, is secreted across the inner forespore membrane and, from the analysis of a bofC deletion and insertion mutant, is likely to interact with SpoIVB. In the absence of BofC, the amount of SpoIVB found in sporulating cells is substantially reduced, although SpoIVB is still able to activate proteolysis of pro-sigma(K). Conversely, in the absence of SpoIVB, the levels of BofC accumulate suggesting that the fate of each molecule is dependent upon their mutual interaction. Our results suggest that BofC could maintain SpoIVB in a stable but inactive form. Supporting this, we have shown that overproduction of BofC inhibits SpoIVB autoproteolysis and leads to a delay in proteolytic cleavage of pro-sigma(K). Based on our work here, we have proposed a model for BofC's functional role in intercompartmental signalling.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana , Serina Endopeptidasas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Eliminación de Gen , Mutagénesis Insercional , Fenotipo , Fosfolípidos/metabolismo , Serina Endopeptidasas/genéticaRESUMEN
During spore formation in Bacillus subtilis, the SpoIVB protein is a critical component of the sigma(K) regulatory checkpoint. SpoIVB has been shown to be a serine peptidase that is synthesized in the spore chamber and which self-cleaves, releasing active forms. These forms can signal proteolytic processing of the transcription factor sigma(K) in the outer mother cell chamber of the sporulating cell. This forms the basis of the sigma(K) checkpoint and ensures accurate sigma(K)-controlled gene expression. SpoIVB has also been shown to activate a second distinct process, termed the second function, which is essential for the formation of heat-resistant spores. In addition to the serine peptidase domain, SpoIVB contains a PDZ domain. We have altered a number of conserved residues in the PDZ domain by site-directed mutagenesis and assayed the sporulation phenotype and signaling properties of mutant SpoIVB proteins. Our work has revealed that the SpoIVB PDZ domain could be used for up to four distinct processes, (i) targeting of itself for trans proteolysis, (ii) binding to the protease inhibitor BofC, (iii) signaling of pro-sigma(K) processing, and (iv) signaling of the second function of SpoIVB.
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Bacillus subtilis/enzimología , Proteínas Bacterianas/fisiología , Proteínas de la Membrana , Serina Endopeptidasas/fisiología , Alelos , Secuencia de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Temperatura , Factores de Transcripción/biosíntesisRESUMEN
SpoIVB is essential for intercompartmental signalling in the sigma(K)-checkpoint of Bacillus subtilis. SpoIVB is synthesized in the spore chamber and is the signal which activates proteolytic processing of pro-sigma(K) to its mature and active form sigma(K). We show here that SpoIVB is a serine peptidase of the SA clan. Expression of SpoIVB in Escherichia coli has shown that SpoIVB is able to self-cleave into at least three discrete products, and in vitro studies have shown cleavage in trans. Autoproteolysis of SpoIVB is tightly linked to the initiation of the two developmental functions of this protein, signalling of pro-sigma(K) processing and a yet, uncharacterized, second function which is essential for the formation of heat-resistant spores. In B. subtilis, SpoIVB is synthesized as a zymogen and is subject to two levels of proteolysis. First, autoproteolysis generating intermediate products, at least one of which is proposed to be the active form, followed by processing by one or more enzymes to smaller species. This could provide a mechanism for switching off the active SpoIVB intermediate(s) and suggests a similarity to other proteolytic cascades such as those found in blood coagulation.
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Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Serina Endopeptidasas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Sitios de Unión , Membrana Celular/metabolismo , Fosfolípidos/metabolismo , Serina Endopeptidasas/genética , Esporas BacterianasRESUMEN
The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 microg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg(2+)-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg(2+)-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.