A Novel sRNA in Shigella flexneri That Regulates Tolerance and Virulence Under Hyperosmotic Pressure

Abstract

Regulation of the environmental stress response and virulence of Shigella flexneri may involve multiple signaling pathways; however, these mechanisms are not well-defined. In bacteria, small regulatory RNAs (sRNAs) regulate bacterial growth, metabolism, virulence, and environmental stress response. Therefore, identifying novel functional sRNAs in S. flexneri could help elucidate pathogenic adaptations to host micro-environmental stresses and associated virulence. The aim of this study was to confirm the presence of an sRNA, Ssr54, in S. flexneri and to determine its functions and possible mechanism of action. Ssr54 was found to regulate tolerance and virulence under hyperosmotic pressure. Its expression was verified by qRT-PCR and Northern blotting, and its genomic position was confirmed by 5'-rapid amplification of cDNA ends. Ssr54 expression was significantly decreased (~ 80%) under hyperosmotic conditions (680 mM NaCl), and the survival rate of the Ssr54 deletion strain increased by 20% under these conditions. This suggested that Ssr54 has been selected to promote host survival under hyperosmotic conditions. Additionally, virulence assessment, including guinea pig Sereny test and competitive invasion assays in mouse lungs, revealed that Ssr54 deletion significantly decreased S. flexneri virulence. Two-dimensional gel analyses suggest that Ssr54 may modulate the expression of tolC, ompA, and treF genes, which may affect the virulence and survival of S. flexneri under osmotic pressures. Furthermore, treF expression has been shown to improve the survival of S. flexneri under osmotic pressures. These results suggest that Ssr54 has a broad range of action in S. flexneri response to hyperosmotic environmental stresses and in controlling its virulence to adapt to environmental stresses encountered during host infection.

Keywords: Shigella flexneri; Ssr54; environmental stress; sRNA; virulence.

Figure 1

Experimental verification and expression of S. flexneri small RNAs (sRNAs). (A) Northern blot of the novel sRNA (Ssr54) and 5S rRNA in S. flexneri wild-type, Ssr54 mutant, and Ssr54 complement strains. Ssr54 expression in the S. flexneri strains grown to logarithmic growth phase was verified by Northern blotting; (B) Ssr54 genomic position in S. flexneri was assessed via 5′ rapid amplification of cDNA ends.

Figure 2

Levels of the small RNA Ssr54 in S. flexneri strain 301 when exposed to high osmotic conditions for 30 min. (A) Northern blot of Ssr54 and 5S rRNA in the S. flexneri wild-type strain under hyperosmotic conditions. Control cultured in 160 mM NaCl; (B) RNA levels of Ssr54, qRT-PCR analyses of the relative Ssr54 levels in S. flexneri under high osmotic conditions compared with that in 160 mM NaCl. Relative abundance was calculated using the 2^−ΔΔCt method. Bars indicate fold changes calculated as the mean of triplicate experiments, representing the ratios of Ssr54 expression levels. Black bars represent the control transcript values; Red bars represent results under stress conditions. Error bars indicate standard deviations. Statistical analyses were performed using Student's t-test.

Figure 3

Osmotic stress of the Ssr54 mutant. Growth characteristics of the wild-type, Ssr54 mutant, and complement strains, as well as the mutant strain carrying the pACYC184 empty vector, in LB under different osmotic conditions: (A) 0 mM NaCl; (B) 680 mM NaCl. The error bars indicate standard deviations based on triplicate experiments. Statistical analyses were performed using Student's t-test.

Figure 4

Survival of the sRNA Ssr54 mutant strain (ΔSsr54) relative to that of the wild-type S. flexneri 301 strain when exposed to high osmotic stress. Wild-type S. flexneri 301, ΔSsr54, the complement (ΔSsr54 + pSsr54) strain, and mutant strain carrying the pACYC184 empty vector were grown in Luria-Bertani medium (160 mM NaCl) up to the exponential phase and then subjected to high osmotic stress (680 mM NaCl). Viability was determined by counting bacteria plated in serial dilutions. Bars represent the mean percent survival compared with untreated controls. Statistical analyses were performed using Student's t-test. Data represent the average of biological triplicates, and errors bars indicate one standard deviation. *Denotes a significant difference with P < 0.01.

Figure 5

Phenotypes of wild-type S. flexneri and sRNA mutant ΔSsr54 in guinea pigs. More inflammatory cells were present in the corneas of guinea pigs inoculated with the wild-type and complement strains than those inoculated with the mutant ΔSsr54 strain, whereas the NaCl (control) group showed no inflammatory cells.

Figure 6

Mit4ce were infected with wild-type, mutant ΔSsr54, and complement strains. A competitive index (CI) of 1 represents recovery of equivalent amounts of wild-type and mutant bacteria. The CI of the Ssr54 mutant and wild-type was lower than 1 (P < 0.05); The CI of the Ssr54 complement strain and wild-type was ~ 1 (P > 0.05).

Figure 7

HeLa cell competitive invasion assays of wild-type, mutant ΔSsr54, and complement strains. The results show the competitive index (CI) of the Ssr54 mutant. A CI of 1 represents recovery of equivalent amounts of wild-type and mutant bacteria. The CI of the Ssr54 mutant and wild-type was significantly lower than 1 (P < 0.05); The CI of the Ssr54 complement strain and wild-type was ~1 (P > 0.05).

Figure 8

qRT-PCR for three mRNA targets of Ssr54. Level of three mRNA targets in the ΔSsr54 mutant strain under hyperosmotic conditions expressed relative to the level in wild-type. Statistical analyses were performed using Student's t-test. Data are representative of three independent experiments. Error bars indicate one standard deviation. *Denotes a significant difference with P < 0.01.