RNase-mediated reprogramming of Yersinia virulence

RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we report that of 11 tested potential RNases of the intestinal pathogen Yersinia pseudotuberculosis, two, the endoribonuclease RNase III and the exoribonuclease PNPase, repress the synthesis of the master virulence regulator LcrF. LcrF activates the expression of virulence plasmid genes encoding the type III secretion system (Ysc-T3SS) and its substrates (Yop proteins), that are employed to inhibit immune cell functions during infection. Loss of both RNases led to an increase in lcrF mRNA levels and stability. Our work indicates that PNPase exerts its influence through YopD, known to accelerate lcrF mRNA degradation. Loss of RNase III results in the downregulation of the CsrB and CsrC RNAs, leading to increased availability of active CsrA, which has previously been shown to enhance lcrF mRNA translation and stability. Other factors that influence the translation process and were found to be differentially expressed in the RNase III-deficient mutant could support this process. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal towards virulence plasmid-encoded genes. A similar extensive transcriptional reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. This illustrated that RNase III enables immediate coordination of virulence traits, such as Ysc-T3SS/Yops, with other functions required for host-pathogen interactions and survival in the host. RNA sequencing was used to determine the global transcriptome of the wild-type (YPIII) and YP356 (RNase III-deficient mutant) grown under different conditions. For this purpose, cells were grown O/D for 2 h at 25°C with a subsequent shift to 37°C. Samples were taken in triplicates before and at different time points after the shift to 37°C and total RNA was isolated using hot-phenol RNA extraction. A first set of samples was taken after 2 h at 25°C (Time 0) before the shift to 37°C. The second and third sample were taken 1 h (Time 1) and 4 h (Time 2) after the shift to 37°C, either with (“-Ca2+”) or without (“37°C”) induction of secretion. Five independent cultures (10 ml each) were prepared from independent O/N cultures in LB medium for each sample and pooled for RNA extraction. Harvesting of the cultures took place at 4,000 rpm for 3 min with the centrifuge set to the respective cultivation temperature. Triplicates for all time points were prepared for the wild-type (YPIII) and YP356 (RNase III-deficient mutant)