Mutations in Salmonella pathogenicity island 2 (SPI2) genes affecting transcription of SPI1 genes and resistance to antimicrobial agents - PubMed (original) (raw)

Mutations in Salmonella pathogenicity island 2 (SPI2) genes affecting transcription of SPI1 genes and resistance to antimicrobial agents

J Deiwick et al. J Bacteriol. 1998 Sep.

Abstract

The Salmonella typhimurium genome contains two pathogenicity islands (SPI) with genes encoding type III secretion systems for virulence proteins. SPI1 is required for the penetration of the epithelial layer of the intestine. SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts. Although most mutations in SPI2 lead to a strong reduction of virulence, they have different effects in vitro, with some mutants having significantly increased sensitivity to gentamicin and the antibacterial peptide polymyxin B. Previously we showed that certain mutations in SPI2 affect the ability of S. typhimurium to secrete SPI1 effector proteins and to invade cultured eukaryotic cells. In this study, we show that these SPI2 mutations affect the expression of the SPI1 invasion genes. Analysis of reporter fusions to various SPI1 genes reveals highly reduced expression of sipC, prgK, and hilA, the transcriptional activator of SPI1 genes. These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.

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Figures

FIG. 1

FIG. 1

SPI2. The positions and orientations of insertions of mTn_5_ insertions (open arrows) and the aphT gene cassette (solid arrow) in mutant strains used in this study are shown. The transcriptional orientation of SPI2 genes encoding the type III secretion system apparatus (ssa) and the two-component regulatory system (ssr) is indicated by arrows. cs, centisomes.

FIG. 2

FIG. 2

Protein secreted by S. typhimurium and detection of SipC. (A) Secreted protein was precipitated from 2 ml of culture supernatant, separated by SDS-PAGE, and stained with Coomassie brilliant blue R-250. Positions of the SPI1 effector proteins SipABC (right) and the molecular weight marker (in thousands on the left) are indicated. (B) As for panel A except that protein was transferred onto nitrocellulose membranes after electrophoretic separation. The presence of SipC was detected with a polyclonal antiserum raised against rSipC. (C) Detection of SipC in total cell fractions of S. typhimurium. Equal amounts of protein (about 20 μg) of cells from overnight cultures were separated on SDS–12% polyacrylamide gels and subsequently transferred onto nitrocellulose membranes. SipC was detected after incubation with the rabbit antiserum against rSipC as described above. Lanes: M, marker; wt, wild-type S. typhimurium 1 to 7, SPI2 mutant strains P2D6, P11C3, P9B6, P11D10, P9B7, P8G12, and NP_ssaV_, respectively; 8 to 10, SPI1 mutant strains P4H2, P6E11, and P7B12, respectively.

FIG. 3

FIG. 3

Effect of SPI2 mutations on expression of SPI1 genes. The expression of lacZ fusions to prgK (hatched bars) and sipC (open bars) was analyzed in various SPI1 and SPI2 backgrounds. Bacterial cultures were grown overnight under inducing conditions, and enzyme activity was determined as described in Materials and Methods. wt, wild type.

FIG. 4

FIG. 4

Effect of SPI2 mutations on expression of hilA. The expression of a lacZ fusion to hilA was analyzed in various SPI1 and SPI2 backgrounds as indicated for Fig. 3. wt, wild type.

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