Abstract
The transition from a planktonic lifestyle to a host-attached state is often critical for bacterial virulence. Upon attachment to host cells, enteropathogenic Escherichia coli (EPEC) employs a type III secretion system (T3SS) to inject into the host cells ∼20 effector proteins, including Tir. CesT, which is encoded from the same operon downstream of tir, is a Tir-bound chaperone that facilitates Tir translocation. Upon Tir translocation, the liberated CesT remains in the bacterial cytoplasm and antagonizes the posttranscriptional regulator CsrA, thus eliciting global regulation in the infecting pathogen. Importantly, tight control of the Tir/CesT ratio is vital, since an uncontrolled surge in free CesT levels may repress CsrA in an untimely manner, thus abrogating colonization. We investigated how fluctuations in Tir translation affect the regulation of this ratio. By creating mutations that cause the premature termination of Tir translation, we revealed that the untranslated tir mRNA becomes highly unstable, resulting in a rapid drop in cesT mRNA levels and, thus, CesT levels. This mechanism couples Tir and CesT levels to ensure a stable Tir/CesT ratio. Our results expose an additional level of regulation that enhances the efficacy of the initial interaction of EPEC with the host cell, providing a better understanding of the bacterial switch from the planktonic to the cell-adherent lifestyle.IMPORTANCE Host colonization by extracellular pathogens often entails the transition from a planktonic lifestyle to a host-attached state. Enteropathogenic E. coli (EPEC), a Gram-negative pathogen, attaches to the intestinal epithelium of the host and employs a type III secretion system (T3SS) to inject effector proteins into the cytoplasm of infected cells. The most abundant effector protein injected is Tir, whose translocation is dependent on the Tir-bound chaperon CesT. Upon Tir injection, the liberated CesT binds to and inhibits the posttranscriptional regulator CsrA, resulting in reprogramming of gene expression in the host-attached bacteria. Thus, adaptation to the host-attached state involves dynamic remodeling of EPEC gene expression, which is mediated by the relative levels of Tir and CesT. Fluctuating from the optimal Tir/CesT ratio results in a decrease in EPEC virulence. Here we elucidate a posttranscriptional circuit that prevents sharp variations from this ratio, thus improving host colonization.
Highlights
The transition from a planktonic lifestyle to a host-attached state is often critical for bacterial virulence
These findings indicate that abrogating Tir translation does not result in the CesT-dependent repression of CsrA
These results indicate that specific enteropathogenic Escherichia coli (EPEC) factors are not required for the degradation of the LEE5 mRNA, further confirming that tir translation is required for maintaining high levels of LEE5 mRNA, and indicate that the cloned fragment contains all the sequences required for this regulation
Summary
The transition from a planktonic lifestyle to a host-attached state is often critical for bacterial virulence. Upon attachment to host cells, enteropathogenic Escherichia coli (EPEC) employs a type III secretion system (T3SS) to inject into the host cells ϳ20 effector proteins, including Tir. CesT, which is encoded from the same operon downstream of tir, is a Tir-bound chaperone that facilitates Tir translocation. Most Escherichia coli strains are commensal nonpathogenic strains, yet some strains, including enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) strains, evolved into pathogens that cause conditions ranging from asymptomatic colonization to acute life-threatening disease [1,2,3] Both commensal and pathogenic E. coli strains colonize the intestinal epithelium; while commensal E. coli strains remain planktonic, residing within the mucous layer that lines the epithelium, the pathogens establish a cell-adherent lifestyle. Intimin, encoded by eae, is an outer membrane protein that binds to the host surface-exposed loop of Tir, leading to tight bacterial attachment and clustering of Tir beneath the attached bacteria [14], triggering localized actin polymerization and formation of an actin structure termed pedestal [5, 15]
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