Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle. Colonization requires the expression of a type 3 secretion (T3S) system that injects a mixture of effector proteins into host cells to promote bacterial attachment and disease progression. The T3S system is tightly regulated by a complex network of transcriptional and post-transcriptional regulators. Using transposon mutagenesis, here we identified the ybeZYX-Int operon as being required for normal T3S levels. Deletion analyses localized the regulation to the endoribonuclease YbeY, previously linked to 16S rRNA maturation and small RNA (sRNA) function. Loss of ybeY in EHEC had pleiotropic effects on EHEC cells, including reduced motility and growth and cold sensitivity. Using UV cross-linking and RNA-Seq (CRAC) analysis, we identified YbeY-binding sites throughout the transcriptome and discovered specific binding of YbeY to the “neck” and “beak” regions of 16S rRNA but identified no significant association of YbeY with sRNA, suggesting that YbeY modulates T3S by depleting mature ribosomes. In E. coli, translation is strongly linked to mRNA stabilization, and subinhibitory concentrations of the translation-initiation inhibitor kasugamycin provoked rapid degradation of a polycistronic mRNA encoding needle filament and needle tip proteins of the T3S system. We conclude that T3S is particularly sensitive to depletion of initiating ribosomes, explaining the inhibition of T3S in the ΔybeY strain. Accessory virulence transcripts may be preferentially degraded in cells with reduced translational capacity, potentially reflecting prioritization in protein production.

YBEY is an essential biogenesis factor for mitochondrial ribosomes

“…Similarly, RT-qPCR did not reveal an accumulation of unprocessed tRNA precursors in YBEY KO mitochondria (Supplementary Figure 5B), and cRT-PCR analysis of the MT-CO2 mRNA termini did not uncover any significant defect in its processing or polyadenylation ( Supplementary Figure 5C-E). The downregulation of select mRNAs observed in YBEY KO cells ( Figure 3C,D) may in fact stem from a lack of translation, as previously demonstrated in E. coli (17). Interestingly, 16S rRNA, although similarly well processed and polyadenylated in both YBEY + and YBEY KO cells, showed frequent irregular truncations, in particular in the domain I, in the latter background ( Supplementary Figure 5F-H).…”

“…The deeply conserved YbeY protein has recently come into limelight as a critical factor required for normal physiology in Bacteria. Its loss has been associated with a wide array of debilitating phenotypes, such as sensitivity to abiotic stresses, inability to establish host-pathogen/symbiont relationships (6,7,(13)(14)(15), metabolic deregulations and a severe growth impairment up to lethality (3,11,17). Widespread among Eukarya, YbeY has so far only been studied in A. thaliana, where it was found indispensable for chloroplast development and photosynthesis (16).…”

“…Based on studies in various bacteria, YbeY has been implicated in ribosome maturation and quality control, with a particularly important role in small subunit (SSU) biogenesis (3-8), and post-transcriptional gene expression regulation (9-14). The deletion of ybeY is often lethal or associated with severe alterations of cellular metabolism and growth, indicating its indispensability for a wide variety of bacterial-type ribosomes (4,6,7,(13)(14)(15)(16)(17).…”

“…Based on studies in various bacteria, YbeY has been implicated in ribosome maturation and quality control, with a particularly important role in small subunit (SSU) biogenesis (3-8), and post-transcriptional gene expression regulation (9-14). The deletion of ybeY is often lethal or associated with severe alterations of cellular metabolism and growth, indicating its indispensability for a wide variety of bacterial-type ribosomes (4,6,7,(13)(14)(15)(16)(17).Mechanistically, YbeY has been described as a metal-dependent endoribonuclease (5,12), and in some bacteria, ybeY mutants accumulate 16S rRNA with an unprocessed 3' end (3,5,7,8). Therefore, YbeY was proposed to be the "missing" 3' endoribonuclease required for 16S rRNA maturation to obtain the correct anti-Shine-Dalgarno sequence, which is needed for translation initiation on most bacterial mRNAs.…”

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus

“…In Brucella abortus, YbeY is also linked to a variety of important phenotypes including virulence, proper cellular morphology, stress response and mRNA transcript levels (Budnick et al, 2018). In enterohemorrhagic Escherichia coli, YbeY was found to stabilize a type 3 secretion system required for virulence in humans (McAteer et al, 2018). A recent study also found that YbeY was crucial for T-DNA transfer and tumour formation in the plant pathogen Agrobacterium tumefaciens (Möller et al, 2019).…”