RESUMO
A cosmid DNA library had been constructed previously from 40-kb fragments of genomic DNA from a virulent invasive strain of Salmonella enterica serotype Typhimurium (TML) in an avirulent hypo-invasive Typhimurium strain (LT7). Selection of invasive clones from the library was attempted by iterative passage through a rabbit ileal organ culture. After the fourth passage, a clone, designated LT7(pHC20-2), was isolated. Exposure to both gut tissue and Caco-2 cells enhanced the growth, invasiveness for gut and Caco-2 cells, and flagellin expression of LT7(pHC20-2) although its invasiveness was less than that of strain TML. Expression of appendages (surface structures c. 60-70 nm diameter) was shown to play a role in but not to confer invasiveness, and was demonstrated in the absence of direct contact with eukaryotic cells. Exposure to gut tissue also affected the expression of several outer-membrane proteins (OMPs) in all four Salmonella strains--TML, LT7, LT7(pHC79), LT7(pHC20 2)--used in this work. As the genes involved in flagella, invasin and porin expression are distributed around the salmonella chromosome, it is possible that pHC20-2 encodes a pleiotropic regulator of genes involved in gastro-enteritic virulence and adaptation to the in-vivo gut environment. pHC20-2 mapped at c. centisome 25 on the salmonella chromosome close to, but distinct from, SPI-5.
Assuntos
Adesinas Bacterianas , Cromossomos Bacterianos , Íleo/microbiologia , Salmonella typhimurium/genética , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Células CACO-2 , Cosmídeos , Eletroforese em Gel de Poliacrilamida , Flagelina/biossíntese , Flagelina/genética , Humanos , Microscopia Eletrônica , Técnicas de Cultura de Órgãos , Porinas/biossíntese , Porinas/genética , Coelhos , Recombinação Genética , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/ultraestrutura , Análise de Sequência de DNARESUMO
An organ culture system involving explants of distal rabbit ileum was used to study the roles of lipopolysaccharide (LPS) and plasmids in primary invasiveness for enterocytes in situ of strains of Salmonella serotypes Typhimurium and Enteritidis. Long-chain LPS per se does not confer invasiveness on Typhimurium, as known avirulent, hypo-invasive strains express smooth LPS. However, the invasiveness of a naturally occurring rough isogenic derivative of Salmonella serotype Enteritidis PT 4 was about half that of its wild-type parent. Therefore, smooth LPS appears to play a secondary role in maximising invasiveness. No evidence was found to correlate primary invasiveness for gut of 18 strains of Typhimurium with plasmid profiles in general or with the 60-MDa serovar-specific virulence plasmid in particular. Evidence is presented that strongly suggests a seasonal variability in susceptibility of rabbit gut to invasion by Typhimurium. Although no explanation is given for this summer insusceptibility, the data indicate the importance of the physiological status of the host in relation to susceptibility to invasion by Salmonella.
Assuntos
Gastroenterite/microbiologia , Íleo/microbiologia , Infecções por Salmonella/microbiologia , Salmonella enteritidis/fisiologia , Salmonella typhimurium/fisiologia , Animais , Humanos , Lipopolissacarídeos/análise , Plasmídeos/fisiologia , Coelhos , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Estações do Ano , Virulência/genéticaRESUMO
Ten recent clinical isolates of Salmonella serotype Typhimurium from man that were tested for their invasiveness in rabbit ileal explants in vitro, were compared with Typhimurium strain TML, a well-characterised invasive strain isolated from a case of human gastro-enteritis. Nine of the 10 strains showed invasiveness that was comparable to that of strain TML. One isolate (GM3) was apparently substantially less invasive; electron microscopy showed this strain to be histotoxic - the probable reason for its reduced recovery from ileal mucosa and thus apparent 'low' invasiveness. Salmonella serotype Choleraesuis strain A50, isolated from a case of systemic salmonellosis in pigs, and serotype Dublin strain 3246, isolated from a case of systemic salmonellosis in calves, were also examined. Dublin strain 3246, when grown at 37 degrees C and used immediately in the invasion assay, damaged the mucosa in a manner similar to that of Typhimurium strain GM3, whereas Dublin strain 3246 grown at 37 degrees C and stored overnight at 4 degrees C did not. This was reflected in an apparently lower invasiveness of freshly grown organisms compared with that of organisms stored at 4 degrees C. In contrast, the histotoxicity of Typhimurium strain GM3 was not affected by storage at 4 degrees C. When stored at 4 degrees C, the levels of invasiveness of Choleraesuis strain A50 and Dublin strain 3246 were not significantly different from each other or from Typhimurium strain TML.