Salmonella enterica highly expressed genes are disease specific

Abstract

During in vitro broth culture, bacterial gene expression is typically dominated by highly expressed factors involved in protein biosynthesis, maturation, and folding, but it is unclear if this also applies to conditions in natural environments. Here, we used a promoter trap strategy with an unstable green fluorescent protein reporter that can be detected in infected mouse tissues to identify 21 Salmonella enterica promoters with high levels of activity in a mouse enteritis model. We then measured the activities of these and 31 previously identified Salmonella promoters in both the enteritis and a murine typhoid fever model. Surprisingly, the data reveal that instead of protein biosynthesis genes, disease-specific genes such as Salmonella pathogenicity island 1 (SPI-1)-associated genes and genes involved in anaerobic respiration (enteritis) or SPI-2-associated genes and genes of the PhoP regulon (typhoid fever), respectively, dominate Salmonella in vivo gene expression. The overall functional profile of highly expressed genes suggests a marked shift in major transcriptional activities to nutrient utilization during enteritis or to fighting against the host during typhoid fever. The large proportion of known and novel essential virulence factors among the identified genes suggests that high expression levels during infection may correlate with functional relevance.

FIG. 1.

GFP_OVA content in Salmonella cells carrying a transcriptional P_rpsA-gfp_ova fusion during enteritis and typhoid fever, as determined by flow cytometry. (A) Mice were infected with initially nonfluorescing Salmonella cells from a stationary in vitro culture. Salmonella were recovered 8 h after oral infection from infected ceca (Β) or 4 days after systemic infection from spleen (C). The shaded areas represent GFP_OVA levels below the detection threshold in the respective tissue homogenates. Similar data were obtained in three independent experiments. To assess GFP maturation and degradation, Salmonella cells expressing stable GFP (open symbols) or nonstable GFP_OVA (solid symbols) were freshly prepared from infected ceca (D), in vitro cultures with limited aeration (E), or infected spleen (F) and incubated in fully aerated LB medium containing chloramphenicol to block de novo synthesis. At different time intervals, GFP fluorescence was determined by flow cytometry.

FIG. 2.

(A) Salmonella promoter activities in murine colitis (cecum) and typhoid fever (spleen) models. Promoter fusions were originally obtained from flow cytometric sorts of infected cecal lumen (open triangles) or spleen (gray triangles). For comparison, activities of five ribosomal promoters are also shown (black squares). b.t., below detection threshold; com, commonly active promoters; ent.i., enteritis induced; t.f.i., typhoid fever induced. Error bars indicate the range of repeated measurements. (B) Coefficient of variation of results obtained from repeated measurements of individual promoter-gfp_ova fusions in typhoid fever (spleen) and enteritis (cecum) models. The plots shows ranges (whiskers) and quartiles (the boxes extend from the 25th percentile to the 75th percentile, and the lines within boxes depict the medians).

FIG. 3.

Homology of highly in vivo-expressed Salmonella genes to E. coli K-12. A majority of Salmonella genes with disease-specific expression (enteritis, open circles; typhoid fever, gray circles; for classifications, see the legend to Fig. 2A) lack E. coli homologues, whereas most commonly expressed genes (black circles) are conserved in E. coli. Promoters that were only partially detected in one of the models were assumed to have median expression values close to the respective detection thresholds (6,000 molecules for typhoid fever and 4,000 molecules for enteritis) for calculating expression ratios. Promoters with undetectable expression in one of the disease models were assumed to have median expression values of about a quarter of the respective detection thresholds, based on the typical bandwidths of GFP measurements. n.h., homology below 5%.

FIG. 4.

Functional profile of highly expressed Salmonella genes during enteritis (A) or typhoid fever (B). Expression levels determined as the number of GFP_OVA copies per cell of promoters associated with physiology, virulence, or unknown function were separately summed. The cumulative activity of promoters associated with nutrient utilization and energy conversion (UTIL), SPI-1, SPI-2, or the PhoPQ regulon (PhoP) is also shown.