Optimal type three secretion system activity of the pathogenic Yersiniae require polynucleotide phosphorylase: Mediated by its S1 RNA binding domain

Both low temperatures and host cell encounters are two stresses that have been well studied in many species of bacteria. Polynucleotide phosphorylase (PNPase), a ubiquitous exoribonuclease found in bacteria, chloroplasts, yeast, and mammalian cells, is required for both cold growth and optimum functioning of the antiphagocytic type three secretion system (TTSS) of some Gram-negative bacteria. In this work, the pathogenic yersiniae required PNPase during the host cell induced stress response (HCISR) during which TTSS transcripts and the effector proteins they encode become upregulated. In efforts of identifying PNPase-sensitive transcripts and their encoded proteins required for optimal TTSS activity during the HCISR, global transcriptome and proteome studies of the YP Deltapnp strain were undertaken. Additionally, PNPase was concretely established as a virulence-associated gene of the Yersinia pseudotuberculosis Deltapnp strain's by virtue of its diminished virulence in a murine model of infection. Surprisingly, the catalytic activity of PNPase was not required for optimal TTSS function in cell culture infections; moreover, over-expression of S1 RNA binding domains from various proteins, including PNPase, was sufficient for functional complementation of the yersiniae Deltapnp strains' TTSS deficiencies. Furthermore, a 15 residue region of the full-length 90-aa residue S1 domain of RNase E was found to be absolutely requisite for optimal TTSS function. Within this 15 residue region required for optimal TTSS function lies a phenylalanine residue conserved in the S1 domain of PNPase (F638). Interestingly, the PNPaseF638G variant, mutated for the conserved phenylalanine residue, was unable to functionally rescue the suboptimally functioning TTSS of the Yersinia pestis (YP) Delta pnp strain. Moreover, PNPaseF638G was also unable to confer cold growth ability to the YP Deltapnp strain unlike the unaltered PNPase which does enable cold growth. Our findings suggest that a highly conserved phenylalanine residue, shown to facilitate RNA contact in a RNase E-RNA co-crystal, serves as the link between PNPase's and the S1 domain's affect on TTSS function as well as linking two bacterial stresses, namely cold growth and cytotoxicity. Hypothetical models of what PNPase might be accomplishing mechanistically during the HCISR are discussed.