Impaired synthesis and secretion of SopA in Salmonella Typhimurium dam mutants”.

GIACOMODONATO MN; SARNACKI, SH; NOTO LLANA M; GARCIA CATTANEO; UZZAU; RUBINO; CERQUETTI MC

FEMS MICROBIOLOGY LETTERS

WILEY-BLACKWELL PUBLISHING, INC

Año: 2009 p. 71 - 77

0378-1097

DNA adenine methylation regulates virulence gene expression in certain bacteria, including Salmonella Typhimurium. The aim of this study was to investigate the involvement of DNA adenine methylase (Dam) methylation in the expression and secretion of the SPI-1 effector protein SopA. For this purpose, SopA–FLAG-tagged wild-type and dam strains of Salmonella Typhimurium were constructed. The expression and secretion of SopA were determined in bacterial culture and in intracellular bacteria recovered from infected HEp-2 epithelial cells. Bacterial culture supernatants and pellets were used to investigate secreted proteins and cell-associated proteins, respectively. Western blot and quantitative reverse transcriptase PCR analysis showed that the dam mutant expresses lower levels of SopA than the wild-type strain. Interestingly, the strain lacking Dam synthesizes SopA under nonpermissive conditions (28 1C). In addition, SopA secretion was drastically impaired in the dam mutant. In vivo experiments showed that the intracellularSalmonella Typhimurium. The aim of this study was to investigate the involvement of DNA adenine methylase (Dam) methylation in the expression and secretion of the SPI-1 effector protein SopA. For this purpose, SopA–FLAG-tagged wild-type and dam strains of Salmonella Typhimurium were constructed. The expression and secretion of SopA were determined in bacterial culture and in intracellular bacteria recovered from infected HEp-2 epithelial cells. Bacterial culture supernatants and pellets were used to investigate secreted proteins and cell-associated proteins, respectively. Western blot and quantitative reverse transcriptase PCR analysis showed that the dam mutant expresses lower levels of SopA than the wild-type strain. Interestingly, the strain lacking Dam synthesizes SopA under nonpermissive conditions (28 1C). In addition, SopA secretion was drastically impaired in the dam mutant. In vivo experiments showed that the intracellulardam strains of Salmonella Typhimurium were constructed. The expression and secretion of SopA were determined in bacterial culture and in intracellular bacteria recovered from infected HEp-2 epithelial cells. Bacterial culture supernatants and pellets were used to investigate secreted proteins and cell-associated proteins, respectively. Western blot and quantitative reverse transcriptase PCR analysis showed that the dam mutant expresses lower levels of SopA than the wild-type strain. Interestingly, the strain lacking Dam synthesizes SopA under nonpermissive conditions (28 1C). In addition, SopA secretion was drastically impaired in the dam mutant. In vivo experiments showed that the intracellulardam mutant expresses lower levels of SopA than the wild-type strain. Interestingly, the strain lacking Dam synthesizes SopA under nonpermissive conditions (28 1C). In addition, SopA secretion was drastically impaired in the dam mutant. In vivo experiments showed that the intracellular1C). In addition, SopA secretion was drastically impaired in the dam mutant. In vivo experiments showed that the intracellulardam mutant. In vivo experiments showed that the intracellular Salmonella dam mutant synthesizes SopA although in lower amounts than the wild-type strain. Taken together, our results suggest that Dam methylation modulates the expression and secretion of SopA in Salmonella Typhimurium.mutant synthesizes SopA although in lower amounts than the wild-type strain. Taken together, our results suggest that Dam methylation modulates the expression and secretion of SopA in Salmonella Typhimurium.Salmonella Typhimurium.