Immunochemical characterization of Fraction I of Yersinia pestis strains by CAF1M gene

The role of caf1M gene in biogenesis of Yersinia pestis capsule was studied in natural strains of the agent with Fra+/- phenotypes and recombinant variants with ycaA (caf1+;caf1M;caf1A+;caf1R+) locus defect. These bacteria did not form a clearly discernible capsule stained by classical methods but synthesized Cafl, whose content in the cells was many times higher than in lysates, in external cell wall, and in the medium with reference Y. pestis EV NIIEG culture (caf1+;caf1M;caf1A+;caf1R+). However Caf1 was not detected on the surface or culture fluid of natural and mutant Y. pestis cells. Exclusive role of Caf1M in Caf1 delivery to Y. pestis cell surface, but not in F1 monomer folding, was proven. Retention of lipopolysaccharide (LPS), a typical SR-LPS configuration and epitope specificity of its components was demonstrated, ensuring similar reactivity in solid-phase enzyme immunoassay with a panel of monoclonal antibodies to Y. pestis LPS. Study of immunochemical properties of antigenic substances derived from caf1M-defective Y. pestis cells by isolation of F1 showed that these substances represent an envelope protein involved in the caf1+ strains (together with Caf1) in assembly of "mature" F1 molecule as a result of posttranslation modification of various genes products. Variants of identification of Y. pestis with Fra+ phenotype by means of monoclonal antibodies to F1, fibrinolysis/coagulase, or LPS in solid-phase enzyme immunoassay are discussed.     

Calcium-binding crystallins from Yersinia pestis. Characterization of two single betagamma-crystallin domains of a putative exported protein

Betagamma-crystallin is a superfamily with diverse members from vertebrate lens to microbes. However, not many members have been identified and studied. Here, we report the identification of a putative exported protein from Yersinia pestis as a member of the betagamma-crystallin superfamily. Even though calcium has been known to play an important role in the physiology and virulence of the Yersinia genus, calcium-binding proteins have not yet been identified. We have studied the calcium-binding properties of two of the three crystallin domains present in this putative exported protein designated "Yersinia crystallin." These two domains (D1 and D2) have unique AA and BB types of arrangement of their Greek key motifs unlike the domains of other members of the betagamma-crystallin superfamily, which are either AB or BA types. These domains bind two calcium ions with low and high affinity-binding sites. We showed their calcium-binding properties using various probes for calcium and the effect of calcium on their secondary and tertiary structures. Although both domains bind calcium, D1 underwent drastic changes in secondary and tertiary structure and hydrodynamic volume upon calcium binding. Domain D1, which is intrinsically unstructured in the apo form, requires calcium for the typical betagamma-crystallin fold. Calcium exerted an extrinsic stabilization effect on domain D1 but not on D2, which is also largely unstructured. We suggest that this protein might be involved in calcium-dependent processes, such as stress response or physiology in the Yersinia genus, similar to its microbial relatives and mammalian lens crystallins.     

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.     

Identification of a thermo-regulated glutamine-binding protein from Yersinia pestis

Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity ( approximately 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38. The identification and characterization of an unknown protein function using the strategy presented here has applicability to a variety of research areas, including functional genomics and proteomics efforts.     

Chaperone Caf1M Stabilizes Hybrid Proteins Containing Sequences of F1 Antigen Subunit from Yersinia pestis

The Yersinia pestis(causative agent of plague) capsule antigen is a homopolymer of Caf1 protein. Export of the subunits is mediated by the periplasmic chaperone Caf1M. To study the mechanism of Caf1M activity, two hybrid genes including coding sequences for the Caf1 signal peptide, human granulocyte–macrophage colony-stimulating factor (GM-CSF) or interleukin-1 (IL-1) receptor antagonist, and mature Caf1 were constructed and expressed in Escherichia coli.We have shown that in the absence of Caf1M the majority of Caf1 moieties within the hybrid proteins undergo proteolysis in the periplasmic space, presumably by the DegP protease. The coexpression of a gene for chaperone Caf1M significantly increased the amount of full-size hybrid proteins in the periplasm, probably as a result of stabilization of the subunit's spatial structure within the hybrid. This effect was not observed in JCB571 cells, which lack periplasmic disulfide isomerase DsbA, essential for Caf1M activity.     

Chaperone Caf1M stabilizes hybrid proteins containing sequences of F1 antigen subunit from Yersinia pestis

The Yersinia pestis (causative agent of plague) capsule antigen is a homopolymer of Caf1 protein. Export of the subunits is mediated by the periplasmic chaperone Caf1M. To study the mechanism of Caf1M activity, two hybrid genes including coding sequences for the Caf1 signal peptide, human granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-1 (IL-1) receptor antagonist, and mature Caf1 were constructed and expressed in Escherichia coli. We have shown that in the absence of Caf1M the majority of Caf1 moieties within the hybrid proteins undergo proteolysis in the periplasmic space, presumably by the DegP protease. The coexpression of a gene for chaperone Caf1M significantly increased the amount of full-size hybrid proteins in the periplasm, probably as a result of stabilization of the subunits spatial structure within the hybrid. This effect was not observed in JCB571 cells, which lack periplasmic disulfide isomerase DsbA, essential for Caf1M activity.     

Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.     

Crystal structure of the Yersinia pestis GTPase activator YopE

Yersinia pestis, the causative agent of bubonic plague, evades the immune response of the infected organism by using a type III (contact-dependent) secretion system to deliver effector proteins into the cytosol of mammalian cells, where they interfere with signaling pathways that regulate inflammation and cytoskeleton dynamics. The cytotoxic effector YopE functions as a potent GTPase-activating protein (GAP) for Rho family GTP-binding proteins, including RhoA, Rac1, and Cdc42. Down-regulation of these molecular switches results in the loss of cell motility and inhibition of phagocytosis, enabling Y. pestis to thrive on the surface of macrophages. We have determined the crystal structure of the GAP domain of YopE (YopE(GAP); residues 90-219) at 2.2-A resolution. Apart from the fact that it is composed almost entirely of alpha-helices, YopE(GAP) shows no obvious structural similarity with eukaryotic RhoGAP domains. Moreover, unlike the catalytically equivalent arginine fingers of the eukaryotic GAPs, which are invariably contained within flexible loops, the critical arginine in YopE(GAP) (Arg144) is part of an alpha-helix. The structure of YopE(GAP) is strikingly similar to the GAP domains from Pseudomonas aeruginosa (ExoS(GAP)) and Salmonella enterica (SptP(GAP)), despite the fact that the three amino acid sequences are not highly conserved. A comparison of the YopE(GAP) structure with those of the Rac1-ExoS(GAP) and Rac1-SptP complexes indicates that few, if any, significant conformational changes occur in YopE(GAP) when it interacts with its G protein targets. The structure of YopE(GAP) may provide an avenue for the development of novel therapeutic agents to combat plague.     

Various physico-chemical properties of the Yersinia pestis capsular protein

Some properties of the structure of Y. pestis capsular antigen macromolecules have been studied. The aminoacid composition of F1 protein, the aminoacid sequence of the N-terminal fragment of antigen polipeptide chain were determined. Some peculiarities in the dissociation of capsular antigen macromolecules have been studied. The formation of the product resulting from unterminated thermodissociation of F1 protein oligomeric form, consisting of four subunits, has been registered. The aspects of F1 protein association are discussed.     

Nucleotide sequence of the Yersinia pestis gene encoding F1 antigen and the primary structure of the protein. Putative T and B cell epitopes.

The plasmid-located gene caf1 encoding the capsular antigen fraction 1 (F1) of Yersinia pestis was cloned and sequenced. The gene codes for a 170 amino acid peptide with a deduced Mr of 17.6 kDa. The signal peptide sequence was highly homologous to the E. coli consensus signal sequence. The F1 was assumed to have beta-sheet structure for the most part. The region located between amino acids 100 and 150 was suggested to contain putative antigenic determinants and to stimulate T cells.     

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: preserved folding energy drives fiber formation

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.     

Screening of plasmids in museum strains of Yersinia pestis isolated from various natural foci

92 strains of Yersinia pestis isolated from different natural foci and stored for 3-40 years in the museum of live cultures have been studied. The strains having three typical plasmids, their different combinations, plasmidless strains or the strains carrying nontypical plasmids with the molecular masses 9, 15, 55, 80, 90 and 150 Md were found. The old museum strains are proposed to be used as a source of plasmids for the genetical research. The current control of plasmid contents in the museum strains is suggested by the plasmid changes in course of storage.     

Thioesterase portability and peptidyl carrier protein swapping in yersiniabactin synthetase from Yersinia pestis

Multimodular enzymes, including polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and mixed PKS/NRPS systems, contain functional domains with similar functions. Domain swapping and module fusion are potential powerful strategies for creating hybrid enzymes to synthesize modified natural products. To explore these strategies, yersiniabactin (Ybt) synthetase containing two subunits, HMWP2 [two NRPS modules (N-terminus-ArCP-Cy1-A-PCP1 and Cy2-PCP2-C-terminus)] and HMWP1 [one PKS (N-terminus-KS-AT-MT1-KR-ACP) one NRPS module (Cy3-MT2-PCP3-TE-C-terminus)], was used as a model system to study peptidyl carrier protein (PCP) domain swapping, thioesterase (TE) portability, and module-module fusion. The PCP1 domain of the N-terminal NRPS module of HMWP2 was swapped with either PCP2 or PCP3. The fusion proteins were 3-8-fold less active than the wild-type protein. The swapping of PCP2 of HMWP2 abolished the heterocyclization activity of the Cy2 domain while retaining its condensation function. When the two PCPs of HMWP2 were swapped by PCP3TE, it created two active fusion proteins: one or two NRPS modules fused to the TE domain. The internal TE domain of the two fusion proteins catalyzed the hydrolysis of enzyme-bound intermediates HPT-S-PCP3 to form HPT-COOH and HPTT-S-PCP3 to form HPTT-COOH. The TE activity was eliminated by the S2980A point mutation at its active site. Therefore, the three PCPs of the Ybt synthetase were swappable, and its lone TE domain was portable. The reasons for the observed low activities of the fusion proteins and lessons for protein engineering in generating novel modular enzymes were discussed.     

Structural and functional similarity between Yersinia pestis capsular protein Caf1 and human interleukin-1 beta

A comparative study of the structural and functional properties of recombinant Yersinia pestis Caf1 and human IL-1beta was performed. According to Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) data, IL-1beta and Caf1 are typical beta-structural proteins. Neither protein interacts with the hydrophobic probe ANS (8-anilino-1-naphthalenesulfonate) under physiological conditions. Specific binding of Caf1 [K(d) = (5.4 +/- 0.1) x 10(-10) M] to interleukin-1 receptors (IL-1Rs) on the surface of finite mouse fibroblasts (line NIH 3T3) was observed. Caf1 is able to inhibit high-affinity binding of (125)I-labeled IL-1beta to NIH 3T3 cells, and in the presence of Caf1, the binding of [(125)I]IL-1beta is characterized by a K(d) of (2.0 +/- 0.3) x 10(-9) M. Caf1 binding to IL-1R could reflect adhesive properties of the capsular subunits responsible for the contact of bacteria with the host immunocompetent cells. In its turn, this may represent a signal for the initiation of the expression and secretion of the proteins of Y. pestis Yop virulon. Thus, these results help to explain the importance of Caf1 in the interaction of Y. pestis with the host immune system.