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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 via 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 illustrates 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.

Author Summary

Bacterial pathogens need to quickly adapt the expression of virulence- and fitness-relevant traits in response to host defenses. Pathogenic Yersinia species rapidly upregulate a type III secretion system (T3SS) to inject antiphagocytic and cell toxic effector proteins, named Yersinia outer proteins (Yops), into attacking immune cells. For this purpose, they display complex and resilient regulatory mechanisms. At the post-transcriptional level, this is mediated by different RNA-binding regulators including YopD and CsrA, while the fate of mRNAs is balanced by ribonucleases. Here, we demonstrate that out of 11 tested putative RNases of Yersinia , two major RNases, the endoribonuclease RNase III, and the exonuclease and degradosome component PNPase play a crucial role in the activation of the Ysc-T3SS/Yop machinery. We show that they promote the decay of the lcrF mRNA encoding the common transcriptional activator LcrF of the Ysc-T3SS/Yop components. PNPase seems to act through the control of the effector YopD, known to promote the decay of the lcrF transcript. In contrast, RNase III triggers processes that reduce lcrF mRNA translation and stability, and involve CsrA.

A transcriptome analysis further revealed that RNase III controls a series of events that include rapid and massive genetic reprogramming from mainly chromosomal-encoded genes to virulence-plasmid-encoded ysc -T3SS/ yop genes. This control process does not only ensure immediate counter-measures during an immune attack, it also helps to overcome accompanying energetic and stress burdens and allows to rapidly readjust the genetic program after a successful defense.