RESUMO
Bacteria belonging to the genus Cronobacter have been recognized as causative agents of life-threatening systemic infections primarily in premature and low-birthweight neonates. Validation of putative bacterial virulence components as well as host factors potentially involved in the response to infection has been hampered in the past by the availability of suitable neonatal animal models. In the current study, the zebrafish embryo model was employed to study the interaction of the zinc metalloproteinase Zpx present in Cronobacter turicensis LMG 23827T , with the eukaryotic MMP-9, a proteinase that functions to cleave extracellular matrix gelatin and collagen. Cleavage and activation of the human recombinant pro-MMP-9 by zpx-expressing C. turicensis cells were demonstrated in vitro, and the presence and increase of the processed, active form of zebrafish pro-MMP-9 were shown in vivo. We provided evidence that Zpx induces the expression of the mmp-9 but also increases the levels of processed MMP-9 during infection. The involvement of the MMP-9 in induction of the expression of the bacterial Zpx was shown in zebrafish mmp-9 morphant experiments. This study identified MMP-9 as a substrate of Zpx and demonstrated yet-undescribed mutual cross-talk between these two proteases in infections mediated by C. turicensis LMG 23827T .
Assuntos
Cronobacter/patogenicidade , Infecções por Enterobacteriaceae/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Animais , Carga Bacteriana , Cronobacter/genética , Cronobacter/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero/microbiologia , Ativação Enzimática , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Metaloproteinase 9 da Matriz/genética , Metaloendopeptidases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Peixe-Zebra/embriologiaRESUMO
Several bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals to control biofilm formation and virulence. Previous work showed that in Burkholderia cenocepacia the RpfFBc/RpfR system is involved in sensing and responding to DSF signals and that this signal/sensor gene pair is highly conserved in several bacterial species including Cronobacter spp. Here we show that C. turicensis LMG 23827(T) possesses a functional RpfF/R system that is involved in the regulation of various phenotypes, including colony morphology, biofilm formation and swarming motility. In vivo experiments using the zebrafish embryo model revealed a role of this regulatory system in virulence of this opportunistic pathogen. We provide evidence that the RpfF/R system modulates the intracellular c-di-GMP level of the organism, an effect that may underpin the alteration in phenotype and thus the regulated phenotypes may be a consequence thereof. This first report on an RpfF/R-type QS system of an organism outside the genus Burkholderia revealed that both the underlying molecular mechanisms as well as the regulated functions show a high degree of conservation.
Assuntos
Cronobacter/fisiologia , Percepção de Quorum , Animais , Proteínas de Bactérias/biossíntese , Biofilmes , Técnicas Biossensoriais , Cronobacter/patogenicidade , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Peptídeo Hidrolases/biossíntese , Fenótipo , Peixe-ZebraRESUMO
Members of the genus Cronobacter are responsible for cases of meningitis and bacteremia with high fatality rates in neonates. Macrophage uptake of invading microbes is an innate process, and it has been proposed that macrophage infectivity potentiator (Mip) like proteins enhance the ability of pathogens to survive within macrophages. Cronobacter harbor the mip-like gene fkpA, but its role in intracellular survival of these bacteria in human macrophages has not yet been studied. Application of gentamicin exclusion assays and human THP-1 macrophage cells revealed significant differences in the capablility of Cronobacter species to survive and replicate within macrophages. Analysis to the amino acid level revealed both length and sequence variations in FkpA proteins among species. In this study, we addressed the possible influence of FkpA variants in intracellular survival of Cronobacter spp. in human macrophages, by knocking out the fkpA genes in two different Cronobacter strains and subsequent complementation with variants of the fkpA genes. Our results provide strong evidence that, in Cronobacter spp., FkpA must be considered a virulence factor, but its influence on macrophage survival and replication varies among strains and/or species due to the presence of amino acid variations.
