SalmonellaProduces an O-Antigen Capsule Regulated by AgfD and Important for Environmental Persistence

Abstract

In this study, we show that Salmonella produces an O-antigen capsule coregulated with the fimbria-and cellulose-associated extracellular matrix. Structural analysis of purified Salmonella extracellular polysaccharides yielded predominantly a repeating oligosaccharide unit similar to that of Salmonella enterica serovar Enteritidis lipopolysaccharide O antigen with some modifications. Putative carbohydrate transport and regulatory operons important for capsule assembly and translocation, designated yihU-yshA and yihVW, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various isogenic ⌬yih mutants, where yihQ and yihO were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that AgfD regulates the yih operons in coordination with extracellular matrix genes coding for thin aggregative fimbriae and cellulose. Although the capsule did not appear to be important for multicellular behavior, we demonstrate that it was important for survival during desiccation stress. Since the yih genes are conserved in salmonellae and the O-antigen capsule was important for environmental persistence, the formation of this surface structure may represent a conserved survival strategy.

Comparative Genetics of the rdar Morphotype inSalmonella

“…Conservation of Tafi production and rdar morphotype formation in these other subgroups suggests an important role in the life cycles of diverse Salmonella isolates. Due to characteristic growth conditions (growth under 30°C, nutrient limitation, low osmolarity) (16,25) and increased resistance to desiccation (26,68) and antimicrobial agents (2,55,56,68), we hypothesize that the rdar morphotype has a fundamental role for Salmonella survival outside host environments. This may contribute to contamination of different food products (4,30) and passage between susceptible hosts.…”

“…With the discovery of BapA (38) and an O-antigen capsule (26), the network of AgfD-regulated extracellular components is growing. Our results demonstrate that loss of the rdar morphotype in Salmonella results primarily from regulatory mutations affecting AgfD expression and not from mutation in genes for Tafi or cellulose biosynthesis.…”

“…agfB and agfD, mlrA, or rpoS promoter-containing fragments were PCR amplified using primers agfD1 and agfD2 (68) (36). Since BapA (38) and capsular polysaccharides (26) were only recently discovered, their precise roles in multicellular aggregation are not well understood. CPS, extracellular polysaccharide.…”

“…Virtually identical operons have also been identified in Escherichia coli and other enterobacterial species (8,49,69). 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).…”

“…AgfD directly activates transcription of the agfBAC operon and adrA, which, in turn, activates cellulose biosynthesis (70). Salmonella capsular polysaccharide (26) and BapA (38) are also regulated by AgfD, but the exact mechanisms have not been determined. AgfD also positively regulates glyA (15) and represses transcription of several genes that inhibit biofilm formation (12).…”