Proteolysis of V antigen from Yersinia pestis

Brubaker, Robert R.; Sample, Allen K.; Yu, Dong; Zahorchak, Robert J.; Hu, Ping; Fowler, Janet M.

doi:10.1016/0882-4010(87)90114-8

Cited by 32 publications

(25 citation statements)

References 27 publications

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“…Expression and purification of recombinant protein was performed essentially as described by us previously (Motin et al , 1996). We analyzed purified proteins by using SDS-PAGE followed by silver stain or immunoblot with the monoclonal antibody to LcrV (Brubaker et al , 1987; Motin et al , 1994). Protein sample buffer contained a reducing agent dithiothreitol (DTT) at a concentration of 100 mM.…”

Section: Methodsmentioning

confidence: 99%

Amino acid and structural variability of Yersinia pestis LcrV protein

Anisimov

Dentovskaya

Panfertsev

et al. 2010

Infection, Genetics and Evolution

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The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium and is generally conserved between the epidemic strains of Yersinia pestis. We investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from 19 Y. pestis strains belonging to different phylogenetic groups (“subspecies”) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324–326. These major variations, together with other minor substitutions in amino acid sequences, allowed us to classify the LcrV alleles into five sequence types (A-E). We observed that the strains of different Y. pestis “subspecies” can have the same type of LcrV, including that conserved in epidemic strains, and different types of LcrV can exist within the same natural plague focus. Therefore, the phenomenon of “selective virulence” characteristic of the strains of the microtus biovar is unlikely to be the result of polymorphism of the V antigen. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in the oligomerization of LcrV.

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Section: Methodsmentioning

confidence: 99%

Amino acid and structural variability of Yersinia pestis LcrV protein

Anisimov

Dentovskaya

Panfertsev

et al. 2010

Infection, Genetics and Evolution

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“…Its role is uncertain because no one has yet constructed a clean V-mutant and because of the inability to completely purify this labile protein (9). It is possible that V antigen has regulatory functions both within Y. pestis and upon mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

lcrR, a low-Ca2(+)-response locus with dual Ca2(+)-dependent functions in Yersinia pestis

Barve

Straley

1990

J Bacteriol

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The low-Ca2e response (Lcr) of Yersinia includes a regulatory cascade and a set of virulence-related proteins, one of which is the V antigen. The regulatory genes modulate both bacterial growth and expression of the virulence-related proteins in response to temperature and the presence of Ca2+ and nucleotides. In this study we defined a new Lcr locus, kcrR, in Yersinia pestis KIM. An kcrR mutant, obtained by insertion mutagenesis, failed to grow at 37C whether Ca2+ was present or not. However, it grew normally in the presence of ATP, showing that the Ca2+-and nucleotide-responsive mechanisms are separate in Y. pestis. The IcrR mutant was avirulent in mice, probably due to its compromised growth at 37°C. ,B-Galactosidase measurements and Northern (RNA blot) analysis revealed that kcrR transcription was regulated primarily by temperature. The DNA sequence of the IcrR locus contained a single open reading frame of 441 bases that could encode a protein with a molecular weight of 16,470 and a pI of 10.73. Expression of an IcrR-containing clone in Escherichia coli yielded a 16,000-molecular-weight protein. At 37°C, the IcrR mutant strongly expressed V antigen and initiated IcrGVH transcription whether Ca2+ was present or not, indicating that this mutant had lost the transcriptional downregulation of IcrGVH shown by the parent in the presence of Ca2+. In the absence of Ca2+, the mutant failed to express LcrG, even though kcrGVH mRNA initiated upstream of IcrG at the normal sites. These data suggest that the IcrR locus is necessary for the regulation of LcrG expression in the absence of Ca2+. Therefore, this locus has a dual regulatory role in the low-Ca2+ response.Yersinia pestis, the causative agent of bubonic plague, exhibits a virulence property called the low-calcium response (Lcr) that is unique to the Yersinia species pathogenic for humans. The Lcr is encoded by highly homologous Lcr plasmids in these yersiniae (39), exemplified by the 75-kilobase (kb) plasmid pCD1 in Yersinia pestis KIM (2,15,19), and is manifested in vitro as two coordinately expressed properties: the requirement for millimolar quantities of Ca2+ for maximal growth at 37°C and the temperature-and Ca2+-regulated expression of virulence-associated proteins, including the V and W antigens and Yops (yersinial plasmidencoded outer membrane proteins) (4,8,10,38,39,47). As a consequence of the Lcr, Y. pestis, when shifted from 26 to 37°C, ceases growth within two generations (56), a phenomenon known as growth restriction. Reinitiation of growth occurs if the cultures are returned to 26°C; at 37°C, growth restriction is prevented if a variety of nucleotides, including ATP (57), or one of the divalent cations Ca2e, Sr+, and Zn2+ (56) is present in the growth medium. Zahorchak and Brubaker (57) showed that nucleotides are neither hydrolyzed exogenously nor transported into the bacterium and suggested that they might function by chelating Mg2+; however, their mechanism of action has not been clarified further. Mg2e potentiates the Lcr, with the most ra...

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“…pestis strain KIM8 is a nonpigmented variant with a 100-kb chromosomal deletion of the high-pathogenicity island that also lacks pPCP1, the 7.5-kb virulence plasmid encoding the surface protease Pla (13,14,25). Strain KIM8 harbors the pCD1 virulence plasmid, however, and promotes type III secretion in a manner that is indistinguishable from that of Y. pestis wild-type strain KIM (71).…”

Section: Yope-dhfr and Yope-lacz Blockmentioning

confidence: 99%