Comparative roles of clpA and clpB in the survival of S. Typhimurium under stress and virulence in poultry

Abstract

By assisting in the proteolysis, disaggregation and refolding of the aggregated proteins, Caseinolytic proteases (Clps) enhance the cellular survival under stress conditions. In the current study, comparative roles of two such Clps, ClpA (involved in proteolysis) and ClpB (involved in protein disaggregation and refolding) in the survival of Salmonella Typhimurium (S. Typhimurium) under different stresses and in virulence have been investigated. clpA and clpB gene deletion mutant strains (∆clpA and ∆clpB) of S. Typhimurium have been hypersensitive to 42 °C, HOCl and paraquat. However, the ∆clpB strain was comparatively much more susceptible (p < 0.001) to the above stresses than ∆clpA strain. ∆clpB strain also showed reduced survival (p < 0.001) in poultry macrophages. The hypersusceptibilities of ∆clpB strain to oxidants and macrophages were restored in plasmid based complemented (∆clpB + pclpB) strain. Further, the ∆clpB strain was defective for colonization in the poultry caecum and showed decreased dissemination to the spleen and liver. Our findings suggest that the role of ClpB is more important than the role of ClpA for the survival of S. Typhimurium under stress and colonization in chickens.

Figure 1

In vitro growth analysis of WT, ∆clpA and ∆clpB strains of S. Typhimurium. Isolated colonies of different strains were inoculated and grown in LB broth for overnight at 37 °C, 180 rpm. Overnight cultures were then diluted (at 1:100) in fresh medium. The optical densities were measured at 600 nm at an interval of one h. Experiment was performed two times. Data are presented as mean ± S.D. of three  replicates.

Figure 2

Growth of WT, ∆clpA and ∆clpB strains of S. Typhimurium at different temperatures. The overnight cultures of different strains were diluted in fresh media (1:100) and grown on shaker incubator either at 37 °C (A) or 42 °C (B). Aliquots were withdrawn at indicated time intervals, ten fold serially diluted and plated on HE agar plates. CFUs/ml were calculated following overnight incubation of plates. Data are presented as mean ± S.D. of three replicates (***denotes p < 0.001).

Figure 3

Sensitivities of WT, ∆clpA and ∆clpB strains of S. Typhimurium to paraquat. Mid-log grown cultures of WT, ∆clpA, ∆clpB mutants and ∆clpA + pclpA (complemented) strains of S. Typhimurium were exposed to 0 or 1% paraquat. Cultures were then ten fold serially diluted and plated on HE agar plates. CFUs/ml were calculated after overnight incubation of plates. Data are presented as mean ± S.D. of three replicates (***denotes p < 0.001).

Figure 4

Sensitivities of ∆clpA and ∆clpB strains of S. Typhimurium to HOCl. Mid-log phase grown cultures of WT, ∆clpA, ∆clpB mutants and complemented (∆clpA + pclpA and ∆clpB + pclpB) strains of S. Typhimurium were exposed to 0, 1.5 and 3 mM HOCl for 2 h. Cultures were then serially diluted and plated on HE agar plates. CFUs/ml were enumerated after overnight incubation of plates. Data are presented as mean ± S.D. of three replicates (***denotes p < 0.001).

Figure 5

Sensitivities of WT, ∆clpA, ∆clpB, ∆clpA + pclpA and ∆clpB + pclpB strains to poultry macrophages stimulated by LPS. The mid log grown cultures were suspended in RPMI-1640. Macrophages were stimulated with LPS as described in materials and methods. The poultry macrophages were infected at MOI of 1:50 (macrophage: bacteria) for 2 h at 37 °C, 5% CO₂. Macrophages were lysed by 0.1% TritonX-100. Bacteria were serially diluted and plated on HE agar plates and the number of bacteria recovered (log₁₀ CFUs/ml as mean S.D.) were counted the next day. Data is representative of two individual experiments (n = 3) (***denotes p < 0.001).

Figure 6

SDS-PAGE analysis of aggregated proteins in WT, ∆clpA and ∆clpB strains of S. Typhimurium. Mid-log grown cultures of various strains were exposed to different concentrations of HOCl for 30 min. Aggregated proteins from such cells were prepared and analysed on 10% SDS-gel. Relative quantification was done using ImageJ software(NIH) bundled with 32 bit Java 1.8.0 and expressed as fold difference from protein aggregates isolated from WT strain incubated with 0 mM HOCl. Quantification is indicated below each lane. Experiment was performed two times.

Figure 7

Bacterial burdens in the spleen (A) and liver (B) of S. Typhimurium, ∆clpA and ∆clpB infected birds. Half of the spleen and 100 mg of liver tissues were homogenized in PBS. One hundred microlitres of the homogenates were plated on HE agar plates. Colonies were counted following incubation of the plates. CFUs/spleen and CFUs/gm of liver were calculated. Data are presented as mean ± S.D. (n = 4) (**denotes p < 0.01, *denotes p < 0.05).