AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways

Abstract

SummaryThe regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long-range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C-terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long-range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.

Bacterial c-di-GMP Is an Immunostimulatory Molecule

“…The cyclic dinucleotide c-di-GMP is now recognized as a widespread intracellular signaling molecule in prokaryotes (2,3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). We have explored the extracellular biological effects of c-di-GMP and its potential use as a therapeutic agent.…”

“…The level of intracellular c-di-GMP in some species is believed to be regulated by the opposing effects of diguanylate cyclases for c-di-GMP synthesis from GTP and c-di-GMP phosphodiesterase for breakdown to 5Ј-GMP and each controlling the level of c-di-GMP in the cell. c-di-GMP has been shown to influence bacterial cell survival, cell differentiation, colonization and biofilm formation, as well as bacteria-host interactions (2,3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Although the role of intracellular c-di-GMP in bacteria is now recognized and is becoming an intense field of research, we have been studying the effect of this microbial molecule in the context of other biological systems and its potential clinical and therapeutic use for the prevention and inhibition of infection and disease (19 -22).…”

“…Briefly, after DNase I (Invitrogen Life Technologies) treatment, 1 g of total RNA from each sample was used as template for the reverse transcription reaction. Fifty microliters of cDNA was synthesized using oligo(dT) 15 , random hexamers, and multiscribe reverse transcriptase (Applied Biosystems). All samples were reverse transcribed under the same conditions (25°C for 10 min, 48°C for 30 min) and from the same reverse transcription master mix to minimize differences in reverse transcription efficiency.…”

Comparative Genetics of the rdar Morphotype inSalmonella

“…In Salmonella, Tafi are produced together with cellulose (70), capsular and extracellular polysaccharides (17,26,67), and BapA (38), all of which come together to form a recalcitrant extracellular matrix that links individual cells together. Extracellular matrix production is associated with multicellular properties of Salmonella, both in the formation of the rdar morphotype (16,52) and in the formation of pellicles at the air-liquid interface in standing culture (51,56). These phenotypes enhance the resistance of Salmonella to antimicrobial stresses (2,55) and contribute to long-term survival (68).…”

“…1). AgfD has an C-terminal DNA binding domain with homology to the LuxR family of transcriptional regulators (27) and an N-terminal putative receiver domain (51). However, no activating signal has yet been identified.…”