Mutations in yscC, yscD, and yscG prevent high-level expression and secretion of V antigen and Yops in Yersinia pestis.

The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca(2+)-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degrees C in the absence of Ca2+. In this study, we constructed and characterized mutants with in-frame deletions in yscC, yscD, and yscG of the ysc operon that contains yscA through yscM. All three mutants lost the Ca2+ requirement for growth at 37 degrees c, expressed only basal levels of V antigen and YopM in the presence or absence of Ca2+, and failed to secrete these proteins to the culture supernatant. Overproduction of YopM in these mutants failed to restore YopM export, showing that the mutations had a direct effect on secretion. The protein products of yscC, yscD, and yscG were identified and localized by immunoblot analysis. YscC was localized to the outer membrane of Y. pestis, while YscD was found in the inner membrane. YscG was distributed equally between the soluble and total membrane fractions. Double mutants were characterized to assess where YscC and YscD act in low-Ca2+ response (LCR) regulation. lcrH::cat-yscC and lcrH::cat-yscD double mutants were constitutively induced for expression of V antigen and YopM; however, these proteins were not exported. This finding showed that the ysc mutations did not directly decrease induction of LCR stimulon genes. In contrast, lcrE-yscC, lcrG-yscC, lcrE-yscD, and lcrG-yscD double mutants as well as an lcrE-lcrD double mutant expressed only basal levels of V antigen and YopM and also failed to secrete these proteins to the culture supernatant. These results indicated that a functional LCR secretion system was necessary for high-level expression of LCR stimulon proteins in the lcrE and lcrG mutants but not in an lcrH::cat mutant. Possible models of regulation which incorporate these results are discussed.     

LcrG, a secreted protein involved in negative regulation of the low-calcium response in Yersinia pestis.

The purpose of this study was to define the function of LcrG, the product of the first gene in the lcrGVHyopBD operon of the low-Ca(2+)-response (LCR) virulence plasmid of Yersinia pestis. We created a Y. pestis strain having an in-frame deletion in lcrG. This nonpolar mutant had an abnormal LCR growth phenotype: it was unable to grow at 37 degrees C in the presence of 2.5 mM Ca2+ ("Ca2+ blind") but was able to grow at 37 degrees C when 18 mM ATP was present. At 37 degrees C it failed to downregulate the expression and secretion of its truncated product (LcrG), V antigen, and YopM. All of these mutant properties were complemented by plasmids carrying normal lcrG. However, a nonpolar lcrE mutation and an lcrH mutation (both also causing a Ca(2+)-blind phenotype) were not complemented in this way. The Y. pestis parent strain expressed LcrG at 37 degrees C in the presence and absence of Ca2+ and transported it to the medium when Ca2+ was absent. We identified two LCR-regulated loci, lcrD and yscDEF, required for this transport. Complementation analysis of the Y. pestis lcrR strain previously shown to lack the expression of LcrG showed that the loss of LcrG but not of LcrR caused the Ca(2+)-blind phenotype of that mutant. Taken together, the results show that LcrG is a negative regulator of the LCR, perhaps functioning in Ca2+ sensing along with LcrE.     

Cloning of the locus encoding synthesis of an outer membrane protein (Omp2) of the calcium dependence plasmid of Yersinia pestis (Lehmann, Neumann)

The genetic locus of Yersinia pestis encoding synthesis of a 46-kDa heat-inducible outer membrane protein (Omp2) was cloned into pBR322 plasmid. The Omp2 was shown to be analogous to previously described YopH and Yop2b proteins. The fifth HindIII fragment of 48-MDa calcium dependence plasmid pCad358 mediates production of 31- and 28-kDa proteins, irrespective of orientation of the insertion. A 31-kDa polypeptide seems to correspond to the YopJ described elsewhere. The maps of BamHI and HindIII of pCad358 region studied differed from those described for pCD1 plasmid of Y. pestis KIM. The products encoded by genes from the fragment cloned in the Pgm+ background give rise to considerable growth of Y. pestis within mouse peritoneal macrophages but were not sufficient to cause lethal infectious process.     

The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha.

In Yersinia pestis KIM, there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only Yops M and N are found in easily detectable amounts in the culture medium. In this study, we located and characterized the yopM gene to obtain clues about its role in the virulence of Y. pestis. Rabbit antibody was raised against Yops M and H, copurified from the supernatant of Y. pseudotuberculosis 43(pGW600, pCD1 yopE::Mu dI1[Apr lac]). This antiserum was adsorbed with an Escherichia coli clone that strongly expressed YopH. The resulting YopM-specific antibody was used to screen a HindIII library of pCD1. HindIII-F and several subclones from it expressed YopM in E. coli minicells. A DNA fragment of 1.39 kilobases from HindIII-F was sequenced and found to contain a 367-amino-acid open reading frame capable of encoding a protein with molecular mass (41,566 daltons) and isoelectric point (4.06) similar to those of YopM. The +1 site of the yopM gene was determined by primer extension. The DNA sequence contained repeating structures: 11 pairs of exact direct repeats, two exact inverted repeats, and three palindromes, ranging from 10 to 42 bases in size. One consensus 14-amino-acid sequence was repeated six times in the predicted protein sequence. The YopM sequence shares some significant homology with the von Willebrand factor- and thrombin-binding domain of the alpha chain of human platelet membrane glycoprotein Ib. These findings suggested a testable hypothesis for the function of YopM.     

The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes.

The low-Ca2+ response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37 degrees C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response. An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of lcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2+ response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2+ response virulence operon, and virulence in mice. The mutant had lost its Ca2+ and nucleotide requirement for growth, showed diminished expression of Ca2(+)-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2+ response virulence genes in addition to its putative role as a secreted virulence protein.     

Identification and characterization of autotransporter proteins of Yersinia pestis KIM

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.     

Genome-wide transcriptional response of Yersinia pestis to stressful conditions simulating phagolysosomal environments

Yersinia pestis is a Gram-negative coccobacillus causing the dangerous disease, plague. Survival of Y. pestis within host macrophages is important in the initial stages of infection. In our present work, DNA microarray was used to determine the expression profiles of Y. pestis strain 201 in response to in vitro simulating conditions of Mg(2+) limitation, polymyxin treatment and oxidative stress that could be found in phagolysosomal environment. It was demonstrated that Y. pestis made appropriate adaptive/protective responses to survive the stressful environments. There are the induced expression of antiphagocytic factors and Mg(2+) transporters under Mg(2+) limitation condition, the stimulation of drug/analogue sensitivity and glycerol assimilation after polymyxin treatment, and the differential expression in genes encoding stress-responsive proteins, components of cell envelope, iron assimilation and regulatory functions in response to both Mg(2+) limitation and polymyxin treatment. Under oxidative stress, Y. pestis uses several mechanisms, especially including the induced expression of detoxification enzymes and DNA repair proteins, to protect from or repair the oxidative cell damages. This microarray analysis would provide the candidates for identifying genes or pathways required for growth and proliferation of Y. pestis in macrophages.     

Identification and cloning of a fur regulatory gene in Yersinia pestis.

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.     

Evidence for two evolutionary lineages of highly pathogenic Yersinia species.

Sensitivity to Yersinia pestis bacteriocin pesticin correlates with the existence of two groups of human pathogenic yersiniae, mouse lethal and mouse nonlethal. The presence of the outer membrane pesticin receptor (FyuA) in mouse-lethal yersiniae is a prerequisite for pesticin sensitivity. Genes that code for FyuA (fyuA) were identified and sequenced from pesticin-sensitive bacteria, including Y. enterocolitica biotype 1B (serotypes O8; O13, O20, and O21), Y. pseudotuberculosis serotype O1, Y. pestis, two known pesticin-sensitive Escherichia coli isolates (E. coli Phi and E. coli CA42), and two newly discovered pesticin-sensitive isolates, E. coli K49 and K235. A 2,318-bp fyuA sequence was shown to be highly conserved in all pesticin-sensitive bacteria, including E. coli strains (DNA sequence homology was 98.5 to 99.9%). The same degree of DNA homology (97.8 to 100%) was established for the sequenced 276-bp fragment of the irp2 gene that encodes high-molecular-weight protein 2, which is also thought to be involved in the expression of virulence by Yersinia species. Highly conserved irp2 was also found in all pesticin-sensitive E. coli strains. On the basis of the fyuA and irp2 sequence homologies, two evolutionary groups of highly pathogenic Yersinia species can be established. One group includes Y. enterocolitica biotype 1B strains, while the second includes Y. pestis, Y. pseudotuberculosis serotype O1, and irp2-positive Y. pseudotuberculosis serotype O3 strains. E. coli Phi, CA42, K49, and K235 belong to the second group. The possible proximity of these two iron-regulated genes (fyuA and irp2), as well as their high levels of sequence conservation and similar G+C contents (56.2 and 59.8 mol%), leads to the assumption that these two genes may represent part of an unstable pathogenicity island that has been acquired by pesticin-sensitive bacteria as a result of a horizontal transfer.     

Detection and identification of virulence factors in Yersinia pestis using SELDI ProteinChip system

A rapid method for the detection, purification, and identification of proteins in bacterial extracts was developed using surface enhanced laser desorption/ionization (SELDI) ProteinChip technology. The effectiveness of this technique for monitoring the expression and identification of temperature- and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mammalian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degrees C (the temperature of the flea vector) to 37 degrees C (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degrees C and 37 degrees C in a Ca(2+)-deficient medium. Through an initial protein profiling of the crude bacterial extract on strong anion exchange and copper affinity, ProteinChip arrays detected five proteins that were up-regulated and three proteins that were down-regulated at 37 degrees C. Two of the proteins predominately expressed at 37 degrees C were semi-purified in less than two days. The two proteins were identified as catalase-peroxidase and Antigen 4. Aside from its speed, a salient feature of the SELDI technique is the microgram amounts of crude sample required for analysis.     

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.