RESUMEN
The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.
Asunto(s)
Caenorhabditis elegans/microbiología , Shigella flexneri/patogenicidad , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/genética , Modelos Animales de Enfermedad , Disentería Bacilar/genética , Disentería Bacilar/microbiología , Disentería Bacilar/patología , Técnicas de Silenciamiento del Gen , Genes Bacterianos , Genes de Helminto , Especificidad del Huésped , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/fisiología , Humanos , Inmunidad Innata , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Hierro/metabolismo , Microscopía Electrónica de Transmisión , Interferencia de ARN , Shigella flexneri/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Virulencia/genéticaRESUMEN
S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB) to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.
Asunto(s)
Asparaginasa/fisiología , Proteínas Bacterianas/fisiología , Disentería Bacilar/microbiología , Células Epiteliales/microbiología , Proteínas Periplasmáticas/fisiología , Shigella flexneri/enzimología , Animales , Asparaginasa/química , Asparagina/química , Adhesión Bacteriana , Proteínas Bacterianas/química , Caenorhabditis elegans , Línea Celular , Cricetinae , Femenino , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Hidrólisis , Ratones Endogámicos BALB C , Proteínas Periplasmáticas/química , Shigella flexneri/crecimiento & desarrolloRESUMEN
SfII is a serotype-converting temperate bacteriophage of the highly prevalent Shigella flexneri serotype 2a. We isolated the SfII phage from a wild-type strain of S. flexneri serotype 2a. Here, we present the complete genome sequence of this phage.
RESUMEN
The serotype-specific glucosyltransferase, GtrV, is responsible for glucosylation of the O-antigen repeating unit of Shigella flexneri serotype 5a strains. GtrV is an integral inner membrane protein with two essential periplasmic loops: the large Loop 2 and the C-terminal Loop 10. In this study, the full length of the Loop 2 was shown to be necessary for GtrV function. Site-directed mutagenesis within this loop revealed that conserved aromatic and charged amino acids have a critical role in the formation of the active site. Sequential deletions of the C-terminal end indicated that this region may be essential for assembly of the protein in the cytoplasmic membrane. The highly conserved FWAED motif is thought to form the substrate-binding site and was found to be critical in GtrV and GtrX, a serotype-specific glucosyltransferase with homology to GtrV. The data presented constitutes a targeted analysis of the formation of the GtrV active site and highlights the essential role of the large periplasmic Loop 2 in its function.