RESUMO
In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.
Assuntos
Fímbrias Bacterianas , Osmorregulação , Trealose , Bexiga Urinária , Infecções Urinárias , Animais , Trealose/metabolismo , Camundongos , Bexiga Urinária/microbiologia , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Infecções Urinárias/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Modelos Animais de Doenças , Feminino , Pressão Osmótica , Escherichia coli Extraintestinal Patogênica/metabolismo , Escherichia coli Extraintestinal Patogênica/genética , Ureia/metabolismo , Trealase/metabolismo , Trealase/genética , Deleção de Genes , Glucose/metabolismoRESUMO
Klebsiella pneumoniae is a Gram-negative pathogen that has become a worldwide concern due to the emergence of multidrug-resistant isolates responsible for various invasive infectious diseases. Biofilm formation constitutes a major virulence factor for K. pneumoniae and relies on the expression of fimbrial adhesins and aggregation of bacterial cells on biotic or abiotic surfaces in a coordinated manner. During biofilm aggregation, bacterial cells communicate with each other through inter- or intra-species interactions mediated by signallng molecules, called autoinducers, in a mechanism known as quorum sensing (QS). In most Gram-negative bacteria, intra-species communication typically involves the LuxI/LuxR system: LuxI synthase produces N-acyl homoserine lactones (AHLs) as autoinducers and the LuxR transcription factor is their cognate receptor. However, K. pneumoniae does not produce AHL but encodes SdiA, an orphan LuxR-type receptor that responds to exogenous AHL molecules produced by other bacterial species. While SdiA regulates several cellular processes and the expression of virulence factors in many pathogens, the role of this regulator in K. pneumoniae remains unknown. In this study, we describe the characterization of sdiA mutant strain of K. pneumoniae. The sdiA mutant strain has increased biofilm formation, which correlates with the increased expression of type 1 fimbriae, thus revealing a repressive role of SdiA in fimbriae expression and bacterial cell adherence and aggregation. On the other hand, SdiA acts as a transcriptional activator of cell division machinery assembly in the septum, since cells lacking SdiA regulator exhibited a filamentary shape rather than the typical rod shape. We also show that K. pneumoniae cells lacking SdiA regulator present constant production of QS autoinducers at maximum levels, suggesting a putative role for SdiA in the regulation of AI-2 production. Taken together, our results demonstrate that SdiA regulates cell division and the expression of virulence factors such as fimbriae expression, biofilm formation, and production of QS autoinducers in K. pneumoniae.
RESUMO
Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for various infections outside the gastrointestinal tract in humans and other animals. ExPEC strain MT78 is invasive to various nonphagocytic cells and highly virulent in vivo To identify genes required for invasion of nonphagocytic cells by this strain, we applied signature-tagged mutagenesis to generate a library of mutants and tested them for invasion of avian fibroblasts. Mutants showing reduced cellular invasion included those with insertions in the fim operon, encoding type 1 fimbriae. Another attenuated mutant showed a disruption in the treA gene, which encodes a periplasmic trehalase. The substrate of TreA, trehalose, can be metabolized and used as a carbon source or can serve as an osmoprotectant under conditions of osmotic stress in E. coli K-12. We generated and characterized mutant MT78ΔtreA In contrast to the wild type, MT78ΔtreA was able to grow under osmotic stress caused by 0.6 M urea but not in minimal M9 medium with trehalose as the only carbon source. It presented decreased association and invasion of avian fibroblasts, decreased yeast agglutination titer, and impaired type 1 fimbria production. In a murine model of urinary tract infection, MT78ΔtreA was less able to colonize the bladder. All phenotypes were rescued in the complemented mutant. Our results show that the treA gene is needed for optimal production of type 1 fimbriae in ExPEC strain MT78 and that loss of treA significantly reduces its cell invasion capacity and colonization of the bladder in a murine model of urinary tract infection.
Assuntos
Infecções por Escherichia coli/patologia , Escherichia coli Extraintestinal Patogênica/enzimologia , Escherichia coli Extraintestinal Patogênica/patogenicidade , Fímbrias Bacterianas/metabolismo , Proteínas Periplásmicas/metabolismo , Trealase/metabolismo , Fatores de Virulência/metabolismo , Animais , Aves , Células Cultivadas , Meios de Cultura/química , Modelos Animais de Doenças , Endocitose , Infecções por Escherichia coli/microbiologia , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/crescimento & desenvolvimento , Fibroblastos/microbiologia , Fímbrias Bacterianas/genética , Deleção de Genes , Teste de Complementação Genética , Camundongos Endogâmicos CBA , Mutagênese , Proteínas Periplásmicas/genética , Trealase/genética , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Infecções Urinárias/patologia , Virulência , Fatores de Virulência/genéticaRESUMO
Type-1 fimbriae are associated with most Salmonella enterica serovars and are an essential factor for host colonization. Mannanoligosaccharides (MOS), a prebiotic that is agglutinated by type-1 fimbriae, are proposed for the control of enterobacteria colonization and may be an alternative to Salmonella control in pigs. The aim of this study was to evaluate the capability of porcine Salmonella strains to adhere to MOS in vitro. A total of 108 strains of Salmonella sp. isolated from carrier pigs were evaluated for the amplification of fimA and fimH genes, agglutination of MOS and hemagglutination. In all tested strains, amplicons of expected size were detected for both fimA and fimH gene. In the hemagglutination assays, 31 (28.7 percent) strains presented mannose-sensitive agglutination of erythrocytes, indicating that the strains were expressing type-1 fimbriae. Considering only strains expressing the type-1 fimbriae, 23 (74.2 percent) presented a strong agglutination of MOS, 3 (9.6 percent) a weak reaction and 5 (16.2 percent) none. The results indicate that Salmonella enterica strains expressing type-1 fimbriae can agglutinate effectively in vitro to MOS.
Fímbrias tipo 1 estão presentes na maioria dos sorovares de Salmonella enterica e são fatores essenciais para a colonização do hospedeiro. Mananoligossacarídeo (MOS), um prebiótico que aglutina com fímbria tipo 1, tem sido proposto para o controle da colonização de enterobactérias e pode ser uma alternativa para o controle da infecção por Salmonella sp. em suínos. O objetivo desse estudo foi avaliar a capacidade in vitro de aglutinação ao MOS em cepas de Salmonella sp. isoladas de suínos. Um total de 108 cepas de Salmonella sp. foram avaliadas quanto à presença dos genes fimA e fimH, aglutinação ao MOS e hemaglutinação. Em todas as cepas testadas, fragmentos de tamanho esperado foram amplificados para ambos os genes. Nos testes de hemaglutinação, 31 (28,7 por cento) cepas apresentaram aglutinação de hemácias inibida pela manose, indicando que havia expressão de fímbria tipo 1. Considerando apenas as cepas com a expressão de fímbria tipo 1, 23 (74,2 por cento) apresentaram uma aglutinação forte ao MOS, 3 (9,6 por cento) uma reação fraca e 5 (16,2 por cento) não aglutinaram. Os resultados indicam que MOS pode aglutinar in vitro de forma efetiva com cepas de Salmonella enterica que estejam expressando fímbria tipo 1.
RESUMO
Type-1 fimbriae are associated with most Salmonella enterica serovars and are an essential factor for host colonization. Mannanoligosaccharides (MOS), a prebiotic that is agglutinated by type-1 fimbriae, are proposed for the control of enterobacteria colonization and may be an alternative to Salmonella control in pigs. The aim of this study was to evaluate the capability of porcine Salmonella strains to adhere to MOS in vitro. A total of 108 strains of Salmonella sp. isolated from carrier pigs were evaluated for the amplification of fimA and fimH genes, agglutination of MOS and hemagglutination. In all tested strains, amplicons of expected size were detected for both fimA and fimH gene. In the hemagglutination assays, 31 (28.7%) strains presented mannose-sensitive agglutination of erythrocytes, indicating that the strains were expressing type-1 fimbriae. Considering only strains expressing the type-1 fimbriae, 23 (74.2%) presented a strong agglutination of MOS, 3 (9.6%) a weak reaction and 5 (16.2%) none. The results indicate that Salmonella enterica strains expressing type-1 fimbriae can agglutinate effectively in vitro to MOS.
RESUMO
Type-1 fimbriae are associated with most Salmonella enterica serovars and are an essential factor for host colonization. Mannanoligosaccharides (MOS), a prebiotic that is agglutinated by type-1 fimbriae, are proposed for the control of enterobacteria colonization and may be an alternative to Salmonella control in pigs. The aim of this study was to evaluate the capability of porcine Salmonella strains to adhere to MOS in vitro. A total of 108 strains of Salmonella sp. isolated from carrier pigs were evaluated for the amplification of fimA and fimH genes, agglutination of MOS and hemagglutination. In all tested strains, amplicons of expected size were detected for both fimA and fimH gene. In the hemagglutination assays, 31 (28.7%) strains presented mannose-sensitive agglutination of erythrocytes, indicating that the strains were expressing type-1 fimbriae. Considering only strains expressing the type-1 fimbriae, 23 (74.2%) presented a strong agglutination of MOS, 3 (9.6%) a weak reaction and 5 (16.2%) none. The results indicate that Salmonella enterica strains expressing type-1 fimbriae can agglutinate effectively in vitro to MOS.
Fímbrias tipo 1 estão presentes na maioria dos sorovares de Salmonella enterica e são fatores essenciais para a colonização do hospedeiro. Mananoligossacarídeo (MOS), um prebiótico que aglutina com fímbria tipo 1, tem sido proposto para o controle da colonização de enterobactérias e pode ser uma alternativa para o controle da infecção por Salmonella sp. em suínos. O objetivo desse estudo foi avaliar a capacidade in vitro de aglutinação ao MOS em cepas de Salmonella sp. isoladas de suínos. Um total de 108 cepas de Salmonella sp. foram avaliadas quanto à presença dos genes fimA e fimH, aglutinação ao MOS e hemaglutinação. Em todas as cepas testadas, fragmentos de tamanho esperado foram amplificados para ambos os genes. Nos testes de hemaglutinação, 31 (28,7%) cepas apresentaram aglutinação de hemácias inibida pela manose, indicando que havia expressão de fímbria tipo 1. Considerando apenas as cepas com a expressão de fímbria tipo 1, 23 (74,2%) apresentaram uma aglutinação forte ao MOS, 3 (9,6%) uma reação fraca e 5 (16,2%) não aglutinaram. Os resultados indicam que MOS pode aglutinar in vitro de forma efetiva com cepas de Salmonella enterica que estejam expressando fímbria tipo 1.