ProP is required for the survival of desiccated Salmonella enterica serovar typhimurium cells on a stainless steel surface

Abstract

Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate- and low-moisture foods.

Fig 1

Comparison of statistically significantly (P < 0.05) differentially expressed genes with >5-fold changes. Yellow circles represent desiccated samples, and red circles represent rehydrated samples. (A) Genes differentially expressed with >5-fold changes; (B) genes upregulated >5-fold; (C) genes downregulated >5-fold.

Fig 2

Validation of microarray data using qRT-PCR. The housekeeping gene gapA was used to normalize the data, and the fold change was calculated using the 2^−ΔΔCT method.

Fig 3

The ProP, ProU, and OsmU osmoprotectant systems and the global regulator RpoE are required for optimal survival of desiccation. All deletion mutants showed significant decreases in viability from that for the wild type (P < 0.05). The ΔproP mutant showed the greatest survival defect.

Fig 4

Comparison of the genes identified as upregulated under desiccation by this study with those found upregulated by Gruzdev et al. (19) and by Li et al. (20).

Fig 5

Proposed model of cellular changes occurring during desiccation of a cell on steel for 4 h (a) and during rehydration of a desiccated cell with water (b). Each symbol represents a range of genes involved in particular physiological or regulatory processes. Blue indicates decreased expression; yellow indicates genes that were expressed at similar levels in the planktonic control bacteria; and red indicates increased expression.