Abstract
Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague.
Highlights
Yersinia pestis, the causative agent of plague, is one of three Yersinia species pathogenic to humans
Calcium does not prevent growth or Yop expression during cell culture, and bacteriostasis can be uncoupled from induction of the Yersinia type III secretion system (TTSS) by manipulating levels of glutamate, sodium, and pH to reflect the presumed in vivo conditions of a necrotic lesion [19,28,29,30]
The pool of mutagenized Y. pestis cells was plated directly onto Blood Base agar containing oxalate, and ampicillin to select for mutants unable to undergo growth restriction at elevated temperatures
Summary
The causative agent of plague, is one of three Yersinia species pathogenic to humans. YopN, TyeA, SycN, YscB and LcrG prevent transport of Yop effectors until contact with a target cell is made [9,10,11,12,13,14,15,16,17,18] These conditions can be simulated in vitro, and upon a shift to 37uC in the absence of calcium, yersiniae undergo growth cessation and massively secrete all of the type III substrates into the medium [19,20,21,22,23]. It has been suggested that yersiniae sense environmental calcium concentrations in the extracellular fluids of their host and in the cytoplasm of host cells via insertion of the type III secretion needle into the plasma membrane of target host cells [16,31,32]; the mechanism for sensing calcium levels remains a mystery
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