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.
Highlights
Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle
We previously reported that the LEE4 and LEE5 polycistronic transcripts are bound by the small RNA chaperone Hfq and RNase E under type 3 secretion (T3S)-inducing conditions in EHEC [37, 38]
Using UV cross-linking and stringent, denaturing purification of YbeY-RNA complexes in EHEC, we found that YbeY binds 16S rRNA in vivo
Summary
Regulators of type 3 secretion in enterohemorrhagic E. coli identified by transposon mutagenesis. The repressive effect seen in the ybeY clean deletion may be partially dependent on YbeZ This conclusion is supported by the observation that inserting tetRA after the ybeY coding sequence (to introduce affinity tags into YbeY for UV cross-linking and analysis of cDNAs (CRAC)) reduced T3S, and WT expression could be restored by removing the cassette (Fig. 2B, lanes 10 and 11). These results demonstrate that YbeY is required for T3S and indicate that an interplay between YbeY-YbeZ and downstream genes may exacerbate the T3S phenotype in the ybeY deletion. Our recent analysis of Hfq- and RNase E– binding sites in EHEC [37, 38] (Fig. 6E) indicates that espD binds RNase E, and we propose that the polycistronic LEE4 transcript is preferentially degraded by RNase E when mature 70S ribosomes are depleted as in a ⌬ybeY background or when translation initiation is inhibited by antibiotics
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