Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla

Background  

Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells.     

Structural basis for activation of an integral membrane protease by lipopolysaccharide

Omptins constitute a unique family of outer membrane proteases that are widespread in Enterobacteriaceae. The plasminogen activator (Pla) of Yersinia pestis is an omptin family member that is very important for development of both bubonic and pneumonic plague. The physiological function of Pla is to cleave (activate) human plasminogen to form the plasma protease plasmin. Uniquely, lipopolysaccharide (LPS) is essential for the catalytic activity of all omptins, including Pla. Why omptins require LPS for enzymatic activity is unknown. Here, we report the co-crystal structure of LPS-free Pla in complex with the activation loop peptide of human plasminogen, its natural substrate. The structure shows that in the absence of LPS, the peptide substrate binds deep within the active site groove and displaces the nucleophilic water molecule, providing an explanation for the dependence of omptins on LPS for enzymatic activity.     

Production of Outer Membrane Vesicles by the Plague Pathogen Yersinia pestis

Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.     

The Single Substitution I259T,Conserved in the Plasminogen Activator Pla of Pandemic Yersinia pestis Branches,Enhances Fibrinolytic Activity

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla β-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the β-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in α₂-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution ²⁵⁹IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution ²⁵⁹TIDKN/IIDKT in Pla altered proteolysis in favor of α₂-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.Since the year 540, plague has killed some 200 million humans in three pandemics, i.e., the Justinian plague, the Black Death, and the modern plague (36). Genomic studies have estimated that the etiological agent, Yersinia pestis, evolved from the oral-fecal pathogen Yersinia pseudotuberculosis serotype O1b only shortly before the first pandemic, i.e., 5,000 to 20,000 years ago (1, 2, 46), which has made the bacterium a paradigm of the rapid evolution of a severe bacterial pathogen (57). At least four biovars of Y. pestis have been identified through metabolic and genomic studies; of these biovars, Antiqua, Medievalis, and Orientalis may be associated with the three plague pandemics, whereas the fourth biovar, Microtus, is associated with human-attenuated Y. pestis strains from two geographically distant infection foci in China (36, 59-61). A recent molecular analysis indicated that the biovars are not monophyletic and proposed the subdivision of Y. pestis into eight molecular groupings, which represent different evolutionary branches and histories and are only partially compatible with the biovars (1). Y. pestis evolved from Y. pseudotuberculosis along branch 0, which consists of “atypical” Y. pestis strains designated Angola, Microtus, and Pestoides; these are phylogenetically ancestral to the Antiqua, Medievalis, and Orientalis branches (1).As a disease, plague exhibits various pathologies. Bubonic plague is the zoonotic form of the disease, which is usually acquired by humans from the bite of a flea that has been infected through a blood meal on a diseased rodent (36). The bacteria invade at the intradermal flea bite site and migrate to lymphatic vessels and then to regional draining lymph nodes, where they multiply and cause the development of buboes (44). Without early treatment, bubonic plague progresses to life-threatening septicemic plague, and hematogenous spread of the bacterium to lungs leads to pneumonic plague, a rapidly fatal and highly contagious airborne disease. Occasional injection of Y. pestis cells by the flea directly into the circulatory system leads to primary septicemic plague (43).The plasminogen activator Pla is a cell surface protease encoded by the Y. pestis-specific plasmid pPCP1 (10, 48). Pla is essential in the pathogenesis of bubonic (43, 49) and pneumonic plague (28), whereas it has less of a role in primary septicemic plague (43, 49). The pla gene is highly transcribed in buboes of Y. pestis-infected mice (45), and Pla specifically potentiates migration of the bacteria to lymphatic tissue (43). Pla seems to have a different role in pneumonic plague, where it allows Y. pestis to replicate rapidly in the lungs, causing lethal fulminant pneumonia (28). Virulent Y. pestis strains lacking the Pla-encoding plasmid pPCP1 have been isolated in Asia (3), and they can be associated with primary septicemic plague (43).Pla is an aspartic protease (22, 55) that activates human plasminogen (Plg) to the serine protease plasmin (47) and inactivates the plasmin inhibitor α₂-antiplasmin (α₂AP), thus affecting the main control system for plasmin activity (22). Plg is an abundant circulating zymogen, and its activation is central in the pathogenesis of plague (13, 28, 43), and plasmin is a powerful serine protease associated with cell migration and degradation of fibrin clots (29, 32, 37). In accordance with this, Pla-mediated bacterial adherence directs uncontrolled plasmin proteolysis onto basement membranes to enhance bacterial metastasis through tissue barriers (25, 27), and fibrinolysis by Pla-generated plasmin activity plays a role in the pathogenesis of bubonic plague (8).Compared to those of other Y. pestis biovars, Microtus isolates have several unique genomic features that may be involved in their inherent inability to attack the human host, and specific losses of genes or gene functions are thought to be responsible for the human attenuation (59). Interestingly, the attenuation does not apply to the murine host. The predicted amino acid sequence of the Pla polypeptide is remarkably conserved: in the branches Antiqua, Medievalis, and Orientalis, the Pla sequences are completely identical, whereas a single amino acid substitution, T259I, has been detected in atypical Angola and Microtus strains (6, 38, 50). A genetic analysis of 260 isolates of Y. pestis showed that the T259I substitution in Pla is shared by all isolates of biovar Microtus but absent in those of other biovars (59). Many of the Pestoides strains lack the pPCP1 plasmid and hence also the pla gene (12), and pla sequences from Pestoides are not available.Pla is a member of the omptin family of conserved outer membrane proteases/adhesins detected in several gram-negative bacterial pathogens (15, 17, 21). The omptins have the same molecular size, a β-barrel fold of 10 transmembrane β strands, and five surface-exposed loops, L1 to L5 (Fig. ​(Fig.1).1). The catalytic residues and the residues interacting with lipid A in the outer membrane are completely conserved (17, 21-23, 41, 55). The omptins cleave peptide substrates at basic residues (17) but show dramatic heterogeneity in the recognition of biologically important polypeptides, such as Plg, the antiprotease α₂AP, gelatin, and progelatinases. Analyses of hybrid proteins created between Pla and the omptins PgtE of Salmonella enterica and OmpT of Escherichia coli have indicated that the differing polypeptide substrate selectivity of omptins is dictated by sequence variation in the mobile loop structures of the β-barrel (22, 40). Residue T259 in Pla is located at surface loop 5 and oriented inward in the active-site groove of the Pla barrel, close to residue K262, where Pla is autoprocessed (22, 23) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Model of Pla structure (23) and location of residue Thr259. Side (top drawing) and top (bottom drawing) views of the transmembrane β-barrel are shown. L1 to L5 are the surface loops. Catalytic residues Asp84, Asp86, Asp206, and His208 are indicated in green, Thr259 is in red, and the autoprocessing site Lys262 is in yellow. OM is the outer membrane. (C) Amino acid sequence of residues 254 to 273 at L5 and the termini of β-strands 9 and 10 in Pla, Microtus Pla, and PgtE are shown.The omptin β-barrel has spread by horizontal gene transfer in gram-negative bacteria and adapted to the life-styles of host bacteria (15, 17, 21, 22, 40). Overall, the omptins give an example of an evolvable, robust enzyme fold (34) that easily acquires novel or improved functions. The fact that the single substitution T259I associates with ancestral Y. pestis Microtus and Angola populations suggests that Microtus Pla represents a form of the protein that preceded the common Pla protein. The central role of Plg activation in the pathogenesis of plague led us to analyze whether the single substitution T259I affects the fibrinolytic activities of the Pla molecule.     

The Omptins of Yersinia pestis and Salmonella enterica Cleave the Reactive Center Loop of Plasminogen Activator Inhibitor 1

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.Plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the mammalian fibrinolytic/plasminogen system (29, 37). The fibrinolytic system comprises the serine protease zymogen plasminogen, urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), PAI-1, and plasmin inhibitor α₂-antiplasmin (α₂AP) (for a review, see reference 52). Plasminogen is converted to plasmin, which is a broad-spectrum serine protease that dissolves fibrin in blood clots, degrades laminin of basement membranes, and activates matrix metalloproteinases that degrade collagens and gelatins in tissue barriers. Herewith, plasmin controls physiological processes such as fibrinolysis/coagulation, cell migration and invasion, and tumor metastasis (29, 37). PAI-1 maintains normal hemostasis by inhibiting the function of the plasminogen activators tPA and uPA, which are serine proteases and highly specific for cleavage of the plasminogen molecule. tPA binds to fibrin and is associated with plasmin-mediated breakdown of fibrin clots, whereas uPA has low affinity for fibrin and associates with cell surface proteolysis, cellular migration, and damage of tissue barriers (52).The mammalian fibrinolytic and coagulation systems are targeted by invasive bacterial pathogens during infection (reviewed in references 6, 11, 34, and 61). In bacterial sepsis, increased production of fibrin clots at a damaged endothelium results from enhanced thrombin-catalyzed fibrin generation and from an increased serum level of PAI-1. Coagulation can protect the host by activating immune systems or by physically restraining the bacteria (6, 15, 25, 41). On the other hand, several invasive bacterial pathogens enhance fibrinolysis either through direct plasminogen activation or by immobilizing plasminogen/plasmin on the surface (6, 34, 61). Activation of the plasminogen system by bacteria enhances bacterial dissemination and invasiveness through release of bacteria from fibrin deposits and through degradation of tissue barriers. Bacterial plasminogen activators and receptors have been under extensive structural and functional studies, but much less is known about interactions of bacteria with the regulatory proteins of fibrinolysis.PAI-1 is present in a large variety of tissues and is secreted by several human cells (37). In healthy individuals, the level of PAI-1 antigen in human plasma is low (6 to 85 ng/ml), but synthesis and secretion of PAI-1 are strongly elevated in disease states and induced by, e.g., inflammatory cytokines and endotoxin of Gram-negative bacteria (37). PAI-1 is a serine protease inhibitor (serpin), which exists in two forms. In its active form, PAI-1 rapidly inactivates both tPA and uPA by forming a covalent bond between the hydroxyl group of a catalytic serine residue of tPA/uPA and the carboxyl group of the residue R346 at the reactive center loop (RCL) of PAI-1 (52). The RCL of PAI-1 is a 19-amino-acid-long flexible loop which inserts into the catalytic center of tPA/uPA and contains the “bait” residues R346 and M347, which mimic the normal target of tPA/uPA. PAI-1 induces distortion of the active site of tPA/uPA, which prevents completion of the catalytic cycle (70). The active form of PAI-1 is unstable, with a half-life of 2 to 3 h at 37°C, and it changes spontaneously and irreversibly into a latent form, where the RCL is incorporated into a central β-sheet of the PAI-1 molecule and therefore cannot react with tPA or uPA. This conformational change takes place also after proteolytic cleavage of PAI-1 at the R346-M347 bond. The active form of PAI-1 binds with high affinity to vitronectin (Vn), and PAI-1/Vn complex formation increases the half-life of PAI-1 2- to 4-fold (10, 46, 69). Most circulating PAI-1 is thought to be in a complex with Vn, and the complex serves as the reservoir of physiologically active PAI-1 (44).Plague disease caused by Yersinia pestis is associated with imbalance of the fibrinolytic system, and decreased fibrin(ogen) deposition has been observed in both bubonic and pneumonic plague (11, 36). The plasminogen activator Pla, which is encoded by a Y. pestis-specific 9.5-kb virulence plasmid, pPCP1 (59), does not degrade fibrin directly but mimics the action of tPA and uPA in converting plasminogen to plasmin by cleavage at R561-V562. Pla also degrades the serpin α₂AP and thus creates uncontrolled plasmin activity (32, 60). Pla belongs to the omptin superfamily of bacterial β-barrel outer membrane proteases (for reviews of omptins, see references 21 and 23). The omptins share molecular size and transmembrane fold but differ markedly in their substrate selectivities. In their catalytic centers, omptins combine structural features of aspartic and serine proteases (66).Increased fibrinolysis observed in plague led us to investigate whether Y. pestis increases plasminogen activation also indirectly by controlling the activity of PAI-1. We compared Y. pestis to Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis, and the study also included omptins of other enterobacterial species.     

Yersinia pestis and plague: an updated view on evolution,virulence determinants,immune subversion,vaccination and diagnostics

Plague is a vector-borne disease caused by Yersinia pestis. Transmitted by fleas from rodent reservoirs, Y. pestis emerged less than 6000 years ago from an enteric bacterial ancestor through events of gene gain and genome reduction. It is a highly remarkable model for the understanding of pathogenic bacteria evolution, and a major concern for public health as highlighted by recent human outbreaks. A complex set of virulence determinants, including the Yersinia outer membrane proteins (Yops), the broad range protease Pla, pathogen-associated molecular patterns (PAMPs) and iron capture systems play critical roles in the molecular strategies that Y. pestis employs to subvert the human immune system, allowing unrestricted bacterial replication in lymph nodes (bubonic plague) and in lungs (pneumonic plague). Some of these immunogenic proteins as well as the capsular antigen F1 are exploited for diagnostic purposes, which are critical in the context of the rapid onset of death in the absence of antibiotic treatment (less than a week for bubonic plague and less than 48 h for pneumonic plague). In here, we review recent research advances on Y. pestis evolution, virulence factors function, bacterial strategies to subvert mammalian innate immune responses, vaccination and problems associated to pneumonic plague diagnosis.     

Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis

Autotransporters, the largest family of secreted proteins in Gram‐negative bacteria, perform a variety of functions, including adherence, cytotoxicity and immune evasion. In Yersinia pestis the autotransporter YapE has adhesive properties and contributes to disease in the mouse model of bubonic plague. Here, we demonstrate that omptin cleavage of Y. pestis YapE is required to mediate bacterial aggregation and adherence to eukaryotic cells. We demonstrate that omptin cleavage is specific for the Y. pestis and Y. pseudotuberculosis YapE orthologues but is not conserved in the Yersinia enterocolitica protein. We also show that cleavage of YapE occurs in Y. pestis but not in the enteric Yersinia species, and requires the omptin Pla (plasminogen activator protease), which is encoded on the Y. pestis‐specific plasmid pPCP1. Together, these data show that post‐translation modification of YapE appears to be specific to Y. pestis, was acquired along with the acquisition of pPCP1 during the divergence of Y. pestis from Y. pseudotuberculosis, and are the first evidence of a novel mechanism to regulate bacterial adherence.     

Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host.     

Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica

The O-antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express beta-barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O-antigen repeats on wild-type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O-antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6-Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla-mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O-antigen prevented PgtE-mediated bacterial adhesion to basement membrane. Substitution of Arg-138 and Arg-171 of the motif for protein binding to lipid A 4'-phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O-antigen sterically prevents recognition of large-molecular-weight substrates. Loss of O-antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.     

Human single‐chain urokinase is activated by the omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues

Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single‐chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158‐Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in omptin proteolysis.     

A Surface-Focused Biotinylation Procedure Identifies the Yersinia pestis Catalase KatY as a Membrane-Associated but Non-Surface-Located Protein

This study identified major surface proteins of the plague bacterium Yersinia pestis. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium Y. pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis cells grown at 37°C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10⁹ cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the Y. pestis pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.     

Complete Protection against Pneumonic and Bubonic Plague after a Single Oral Vaccination

Background

No efficient vaccine against plague is currently available. We previously showed that a genetically attenuated Yersinia pseudotuberculosis producing the Yersinia pestis F1 antigen was an efficient live oral vaccine against pneumonic plague. This candidate vaccine however failed to confer full protection against bubonic plague and did not produce F1 stably.

Methodology/Principal Findings

The caf operon encoding F1 was inserted into the chromosome of a genetically attenuated Y. pseudotuberculosis, yielding the VTnF1 strain, which stably produced the F1 capsule. Given orally to mice, VTnF1 persisted two weeks in the mouse gut and induced a high humoral response targeting both F1 and other Y. pestis antigens. The strong cellular response elicited was directed mostly against targets other than F1, but also against F1. It involved cells with a Th1—Th17 effector profile, producing IFNγ, IL-17, and IL-10. A single oral dose (10⁸ CFU) of VTnF1 conferred 100% protection against pneumonic plague using a high-dose challenge (3,300 LD₅₀) caused by the fully virulent Y. pestis CO92. Moreover, vaccination protected 100% of mice from bubonic plague caused by a challenge with 100 LD₅₀ Y. pestis and 93% against a high-dose infection (10,000 LD₅₀). Protection involved fast-acting mechanisms controlling Y. pestis spread out of the injection site, and the protection provided was long-lasting, with 93% and 50% of mice surviving bubonic and pneumonic plague respectively, six months after vaccination. Vaccinated mice also survived bubonic and pneumonic plague caused by a high-dose of non-encapsulated (F1^-) Y. pestis.

Significance

VTnF1 is an easy-to-produce, genetically stable plague vaccine candidate, providing a highly efficient and long-lasting protection against both bubonic and pneumonic plague caused by wild type or un-encapsulated (F1-negative) Y. pestis. To our knowledge, VTnF1 is the only plague vaccine ever reported that could provide high and durable protection against the two forms of plague after a single oral administration.     

Dynamics of CRISPR Loci in Microevolutionary Process of Yersinia pestis Strains

The potential use of CRISPR loci genotyping to elucidate population dynamics and microevolution of 146 Yersinia pestis strains from different biovars and locations was investigated in this work. The majority of strains from the Orientalis biovar presented specific spacer arrays, allowing for the establishment of a CRISPR signature for their respective isolates. Twenty-one new spacers were found in the Y. pestis strains from plague foci in Brazil. Ninety-three (64%) strains were grouped in the G1 genotype, whereas the others were distributed in 35 genotypes. This study allowed observing a microevolutionary process in a group of Y. pestis isolated from Brazil. We also identified specific genotypes of Y. pestis that were important for the establishment of the bacteria in plague foci in Brazil. The data have provided supporting evidence for the diversity and dynamics of CRISPR loci present in the genome of Y. pestis strains from plague foci in Brazil.     

Molecular recognition of chymotrypsin by the serine protease inhibitor ecotin from Yersinia pestis

Resistance to antibiotics is a problem not only in terms of healthcare but also biodefense. Engineering of resistance into a human pathogen could create an untreatable biothreat pathogen. One such pathogen is Yersinia pestis, the causative agent of plague. Previously, we have used a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. The serine protease inhibitor ecotin was identified as one such target. We have carried out mutational analyses in the closely related Yersinia pseudotuberculosis, validating that the ecotin gene is a virulence-associated gene in this bacterium. Y. pestis ecotin inhibits chymotrypsin. Here, we present the structure of ecotin in complex with chymotrypsin to 2.74 Å resolution. The structure features a biologically relevant tetramer whereby an ecotin dimer binds to two chymotrypsin molecules, similar to what was observed in related serine protease inhibitor structures. However, the vast majority of the interactions in the present structure are distinctive, indicating that the broad specificity of the inhibitor for these proteases is based largely on its capacity to recognize features unique to each of them. These findings will have implications for the development of small ecotin inhibitors for therapeutic use.     

Duration of Plague (Yersinia pestis) Outbreaks in Black-Tailed Prairie Dog (Cynomys ludovicianus) Colonies of Northern Colorado

Plague, caused by the bacterium Yersinia pestis, triggers die-offs in colonies of black-tailed prairie dogs (Cynomys ludovicianus), but the time-frame of plague activity is not well understood. We document plague activity in fleas from prairie dogs and their burrows on three prairie dog colonies that suffered die-offs. We demonstrate that Y. pestis transmission occurs over periods from several months to over a year in prairie dog populations before observed die-offs.     

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.     

Dynamics of Yersinia pestis and Its Antibody Response in Great Gerbils (Rhombomys opimus) by Subcutaneous Infection

Background

Rhombomys opimus (great gerbil) is a reservoir of Yersinia pestis in the natural plague foci of Central Asia. Great gerbils are highly resistant to Y. pestis infection. The coevolution of great gerbils and Y. pestis is believed to play an important role in the plague epidemics in Central Asia plague foci. However, the dynamics of Y. pestis infection and the corresponding antibody response in great gerbils have not been evaluated. In this report, animal experiments were employed to investigate the bacterial load in both the liver and spleen of infected great gerbils. The dynamics of the antibody response to the F1 capsule antigen of Y. pestis was also determined.

Methodology

Captured great gerbils that tested negative for both anti-F1 antibodies and bacterial isolation were infected subcutaneously with different doses (10⁵ to 10¹¹ CFU) of a Y. pestis strain isolated from a live great gerbil during routine plague surveillance in the Junggar Basin, Xinjiang, China. The clinical manifestations, changes in body weight, anal temperature, and gross anatomy of the infected animals were observed. The blood cell count, bacterial load, and anti-F1 antibody titers were determined at different time points after infection using a blood analyzer, plate counts, and an indirect hemagglutination assay, respectively.

Conclusions/Significance

The dynamics of bacterial load and the anti-F1 antibody concentration in great gerbils are highly variable among individuals. The Y. pestis infection in great gerbils could persist as long as 15 days. They act as an appropriate reservoir for plague in the Junggar Basin, which is part of the natural plague foci in Central Asia. The dynamics of the Y. pestis susceptibility of great gerbil will improve the understanding of its variable resistance, which would facilitate the development of more effective countermeasures for controlling plague epidemics in this focus.     

Plague Meningitis—A Retrospective Analysis of Cases Reported in the United States, 1970-1979

Meningitis caused by Yersinia pestis developed in 6 (6%) of a total of 105 patients with plague reported to the Centers for Disease Control from 1970 to 1979. Five of the six cases occurred in children aged 10 to 15 years. All six patients received antibiotic therapy before meningitis developed, which appeared between the 9th and 14th days after the onset of acute illness in five of the six patients. There were no neurologic sequelae. The antigenic and biochemical profiles of the Y pestis strains isolated from cerebrospinal fluid in the meningitis cases did not differ from those of the Y pestis strains obtained from blood and bubo aspirates in the other 99 patients, and neither did in vitro studies suggest antibiotic resistance. While plague meningitis is an uncommon complication of acute plague infection, physicians in the western United States should be aware that it may develop as much as 14 days after antibiotic therapy for the acute plague infection has been initiated.