RESUMO
Yersinia enterocolitica type III secretion machines transport YopQ and other Yop effectors into host immune cells. YopD and its chaperone LcrH are essential components of the Yersinia type III pathway, enabling effector translocation into host cells. YopD, LcrH, and YscM1 also regulate yop expression post-transcriptionally in response to environmental signals; however, the molecular mechanisms for this regulation and Yop secretion are unknown. We show here that YopD associates with 30 S ribosomal particles in a manner requiring LcrH. When added to ribosomes, YopD, LcrH, and YscM1 block the translation of yopQ mRNA. We propose a model whereby LcrH-dependent association of YopD with 30 S ribosomal particles enables YscM1 to block yopQ translation unless type III machines are induced to secrete the effector.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Epistasia Genética , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Biossíntese de Proteínas , Transporte Proteico , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismoRESUMO
LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR(+)] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV(*327)). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR(-) phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV(*327) mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR(+), LCR(-), or dominant negative LCR(-) phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor.