FimA, FimF, and FimH are necessary for assembly of type 1 fimbriae on Salmonella enterica serovar Typhimurium

Abstract

Salmonella enterica serovar Typhimurium is a Gram-negative member of the family Enterobacteriaceae and is a common cause of bacterial food poisoning in humans. The fimbrial appendages are found on the surface of many enteric bacteria and enable the bacteria to bind to eukaryotic cells. S. Typhimurium type 1 fimbriae are characterized by mannose-sensitive hemagglutination and are assembled via the chaperone/usher pathway. S. Typhimurium type 1 fimbrial proteins are encoded by the fim gene cluster (fimAICDHFZYW), with fimAICDHF expressed as a single transcriptional unit. The structural components of the fimbriae are FimA (major subunit), FimI, FimH (adhesin), and FimF (adaptor). In order to determine which components are required for fimbrial formation in S. Typhimurium, mutations in fimA, fimI, fimH, and fimF were constructed and examined for their ability to produce surface-assembled fimbriae. S. Typhimurium SL1344ΔfimA, -ΔfimH, and -ΔfimF mutants were unable to assemble fimbriae, indicating that these genes are necessary for fimbrial production in S. Typhimurium. However, SL1344ΔfimI was able to assemble fimbriae. In Escherichia coli type 1 and Pap fimbriae, at least two adaptors are expressed in addition to the adhesins. However, E. coli type 1 and Pap fimbriae have been reported to be able to assemble fimbriae in the absence of these proteins. These results suggest differences between the S. Typhimurium type 1 fimbrial system and the E. coli type 1 and Pap fimbrial systems.

Fig 1

Genetic organization of the S. Typhimurium fim gene cluster and plasmids used in this study. The arrows indicate promoter regions and directions of transcription. Solid lines indicate DNA carrying the fim genes retained on recombinant plasmids used in this study.

Fig 2

(A) Transcriptional analysis of the intergenic regions of the fim gene cluster. The intergenic regions within fimAICDHF are linked on one transcript. RT-PCRs were carried out on RNA isolated from wild-type SL1344. The intergenic regions amplified are indicated by the underlined genes. Primers amplifying rpoD were included as a control. RT +/− indicates the presence or absence, respectively, of reverse transcriptase in reaction mixtures. (B) Transcription of fim genes in S. Typhimurium SL1344 mutants. RT-PCRs were carried out on RNA isolated from wild-type S. Typhimurium SL1344 and Fim mutants. The numbers indicate the strains from which RNA was prepared, and RT-PCRs were performed with (+) or without (−) reverse transcriptase: 1, S. Typhimurium SL1344; 2, SL1344ΔfimA; 3, SL1344fimA::TT; 4, SL1344ΔfimH; 5, SL1344ΔfimF; 6, SL1344ΔfimA+ (pfimA). RT-PCRs were performed with fimA-specific primers (a), fimH primers (b), or fimF primers (c).

Fig 3

Nonfimbriate and fimbriate strains of S. Typhimurium. (A) S. Typhimurium SL1344ΔfimH; (B) S. Typhimurium SL1344.

Fig 4

Production of FimA by S. Typhimurium SL1344 mutants. Immunoblot assays were carried out on lysates from wild-type S. Typhimurium SL1344 and various mutants with antibody raised against SL1344 FimA or bacterial GroEL. Strains are indicated by numbers above the blots: 1, S. Typhimurium SL1344; 2, SL1344ΔfimA; 3, SL1344ΔfimH; 4, SL1344ΔfimF; 5, SL1344ΔfimA + pfimA; 6, SL1344fimA::TT::fimA; 7, E. coli + pfimA.