Prevalence of Yersinia pestis in rodents and fleas associated with black-tailed prairie dogs (Cynomys ludovicianus) at Thunder Basin National Grassland, Wyoming

Rodents (and their fleas) that are associated with prairie dogs are considered important for the maintenance and transmission of the bacterium (Yersinia pestis) that causes plague. Our goal was to identify rodent and flea species that were potentially involved in a plague epizootic in black-tailed prairie dogs at Thunder Basin National Grassland. We collected blood samples and ectoparasites from rodents trapped at off- and on-colony grids at Thunder Basin National Grassland between 2002 and 2004. Blood samples were tested for antibodies to Y. pestis F-1 antigen by a passive hemagglutination assay, and fleas were tested by a multiplex polymerase chain reaction, for the presence of the plague bacterium. Only one of 1,421 fleas, an Oropsylla hirsuta collected in 2002 from a deer mouse, Peromyscus maniculatus, tested positive for Y. pestis. Blood samples collected in summer 2004 from two northern grasshopper mice, Onychomys leucogaster, tested positive for Y. pestis antibodies. All three positive samples were collected from on-colony grids shortly after a plague epizootic occurred. This study confirms that plague is difficult to detect in rodents and fleas associated with prairie dog colonies, unless samples are collected immediately after a prairie dog die-off.     

Yersinia pestis YrbH is a multifunctional protein required for both 3-deoxy-D-manno-oct-2-ulosonic acid biosynthesis and biofilm formation

Bubonic plague is transmitted by fleas whose feeding is blocked by a Yersinia pestis biofilm in the digestive tract. Y. pestis also block feeding of Caenorhabditis elegans by forming a biofilm on the nematode head, making the nematode an experimentally tractable surrogate for fleas to study plague transmission. Arabinose 5-phosphate isomerase (API), encoded by Y. pestis yrbH, catalyses the conversion of ribulose 5-phosphate into arabinose 5-phosphate (A5P), the first committed step in the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) biosynthesis pathway. Here we show that Y. pestis YrbH is a multifunctional protein required for both Kdo biosynthesis and biofilm formation on C. elegans. The YrbH protein contains four functional components: biofilm-related region 1 (B1), a sugar isomerase domain (SIS), biofilm-related region 2 (B2) and a cystathionine beta-synthase domain pair (CBS). B1, SIS and B2 are all required for API function, but any of the three is sufficient for a biofilm-related function. The CBS domain appears to negatively regulate the biofilm-related function.     

Protein regions important for plasminogen activation and inactivation of alpha2-antiplasmin in the surface protease Pla of Yersinia pestis

The plasminogen activator, surface protease Pla, of the plague bacterium Yersinia pestis is an important virulence factor that enables the spread of Y. pestis from subcutaneous sites into circulation. Pla-expressing Y. pestis and recombinant Escherichia coli formed active plasmin in the presence of the major human plasmin inhibitor, alpha2-antiplasmin, and the bacteria were found to inactivate alpha2-antiplasmin. In contrast, only poor plasminogen activation and no cleavage of alpha2-antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A beta-barrel topology model for Pla and OmpT predicted 10 transmembrane beta-strands and five surface-exposed loops L1-L5. Hybrid Pla-OmpT proteins were created by substituting each of the loops between Pla and OmpT. Analysis of the hybrid molecules suggested a critical role of L3 and L4 in the substrate specificity of Pla towards plasminogen and alpha2-antiplasmin. Substitution analysis at 25 surface-located residues showed the importance of the conserved residues H101, H208, D84, D86, D206 and S99 for the proteolytic activity of Pla-expressing recombinant E. coli. The mature alpha-Pla of 292 amino acids was processed into beta-Pla by an autoprocessing cleavage at residue K262, and residues important for the self-recognition of Pla were identified. Prevention of autoprocessing of Pla, however, had no detectable effect on plasminogen activation or cleavage of alpha2-antiplasmin. Cleavage of alpha2-antiplasmin and plasminogen activation were influenced by residue R211 in L4 as well as by unidentified residues in L3. OmpT, which is not associated with invasive bacterial disease, was converted into a Pla-like protease by deleting residues D214 and P215, by substituting residue K217 for R217 in L4 of OmpT and also by substituting the entire L3 with that from Pla. This simple modification of the surface loops and the substrate specificity of OmpT exemplifies the evolution of a housekeeping protein into a virulence factor by subtle mutations at critical protein regions. We propose that inactivation of alpha2-antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.     

Possible vector dissemination by swift foxes following a plague epizootic in black-tailed prairie dogs in northwestern Texas

To determine whether swift foxes (Vulpes velox) could facilitate transmission of Yersinia pestis to uninfected black-tailed prairie dog (Cynomys ludovicianus) colonies by acquiring infected fleas, ectoparasite and serologic samples were collected from swift foxes living adjacent to prairie dog towns during a 2004 plague epizootic in northwestern Texas, USA. A previous study (1999-2001) indicated that these swift foxes were infested almost exclusively with the flea Pulex irritans. Black-tailed prairie dogs examined from the study area harbored only Pulex simulans and Oropsylla hirsuta. Although P. irritans was most common, P. simulans and O. hirsuta were collected from six swift foxes and a single coyote (Canis latrans) following the plague epizootic. Thus, both of these canids could act as transport hosts (at least temporarily) of prairie dog fleas following the loss of their normal hosts during a plague die-off. All six adult swift foxes tested positive for antibodies to Y. pestis. All 107 fleas from swift foxes tested negative for Y. pestis by mouse inoculation. Although swift foxes could potentially carry Y. pestis to un-infected prairie dog colonies, we believe they play only a minor role in plague epidemiology, considering that they harbored just a few uninfected prairie dog fleas (P. simulans and O. hirsuta).     

The evolution of flea-borne transmission in Yersinia pestis

Transmission by fleabite is a recent evolutionary adaptation that distinguishes Yersinia pestis, the agent of plague, from Yersinia pseudotuberculosis and all other enteric bacteria. The very close genetic relationship between Y. pestis and Y. pseudotuberculosis indicates that just a few discrete genetic changes were sufficient to give rise to flea-borne transmission. Y. pestis exhibits a distinct infection phenotype in its flea vector, and a transmissible infection depends on genes that are specifically required in the flea, but not the mammal. Transmission factors identified to date suggest that the rapid evolutionary transition of Y. pestis to flea-borne transmission within the last 1,500 to 20,000 years involved at least three steps: acquisition of the two Y. pestis-specific plasmids by horizontal gene transfer; and recruitment of endogenous chromosomal genes for new functions. Perhaps reflective of the recent adaptation, transmission of Y. pestis by fleas is inefficient, and this likely imposed selective pressure favoring the evolution of increased virulence in this pathogen.     

Role of the Yersinia pestis yersiniabactin iron acquisition system in the incidence of flea-borne plague

Plague is a flea-borne zoonosis caused by the bacterium Yersinia pestis. Y. pestis mutants lacking the yersiniabactin (Ybt) siderophore-based iron transport system are avirulent when inoculated intradermally but fully virulent when inoculated intravenously in mice. Presumably, Ybt is required to provide sufficient iron at the peripheral injection site, suggesting that Ybt would be an essential virulence factor for flea-borne plague. Here, using a flea-to-mouse transmission model, we show that a Y. pestis strain lacking the Ybt system causes fatal plague at low incidence when transmitted by fleas. Bacteriology and histology analyses revealed that a Ybt-negative strain caused only primary septicemic plague and atypical bubonic plague instead of the typical bubonic form of disease. The results provide new evidence that primary septicemic plague is a distinct clinical entity and suggest that unusual forms of plague may be caused by atypical Y. pestis strains.     

No evidence of persistent Yersina pestis infection at prairie dog colonies in north-central Montana

Sylvatic plague is a flea-borne zoonotic disease caused by the bacterium Yersinia pestis, which can cause extensive mortality among prairie dogs (Cynomys) in western North America. It is unclear whether the plague organism persists locally among resistant host species or elsewhere following epizootics. From June to August 2002 and 2003 we collected blood and flea samples from small mammals at prairie dog colonies with a history of plague, at prairie dog colonies with no history of plague, and from off-colony sites where plague history was unknown. Blood was screened for antibody to Y. pestis by means of enzyme-linked immunosorbent assay or passive hemagglutination assay and fleas were screened for Y. pestis DNA by polymerase chain reaction. All material was negative for Y. pestis including 156 blood samples and 553 fleas from colonies with a known history of plague. This and other studies provide evidence that Y. pestis may not persist at prairie dog colonies following an epizootic.     

Uniquely insidious: Yersinia pestis biofilms

Bubonic plague, one of history's deadliest infections, is transmitted by fleas infected with Yersinia pestis. The bacteria can starve fleas by blocking their digestive tracts, which stimulates the insects to bite repeatedly and thereby infect new hosts. Direct examination of infected fleas, aided by in vitro studies and experiments with the nematode Caenorhabditis elegans, have established that Y. pestis forms a biofilm in the insect. The extracellular matrix of the biofilm seems to contain a homopolymer of N-acetyl-d-glucosamine, which is a constituent of many bacterial biofilms. A regulatory mechanism involved in Y. pestis biofilm formation, cyclic-di-GMP signaling, is also widespread in bacteria; yet only Y. pestis forms biofilms in fleas. Here, the historical background of bubonic plague is briefly described and recent studies investigating the mechanisms by which these unique and deadly biofilms are formed are discussed.     

Molecular and physiological insights into plague transmission, virulence and etiology

Plague is caused by Yersinia pestis, which evolved from the enteric pathogen Y. pseudotuberculosis, which normally causes a chronic and relatively mild disease. Y. pestis is not only able to parasitize the flea but also highly virulent to rodents and humans, causing epidemics of a systemic and often fatal disease. Y. pestis could be used as a bio-weapon and for bio-terrorism. It uses a number of strategies that allow the pathogen to change its lifestyle rapidly to survive in fleas and to grow in the mammalian hosts. Extensive studies reviewed here give an overall picture of the determinants responsible for plague pathogenesis in mammalians and the transmission by fleas. The availability of multiple genomic sequences and more extensive use of genomics and proteomics technologies should allow a comprehensive dissection of the complex of host-adaptation and virulence in Y. pestis.     

Pesticins. 3. Expression of coagulase and mechanism of fibrinolysis

Mutational loss of pesticin I, a bacteriocin-like substance produced by Pasteurella pestis, is known to result in concomitant loss of a coagulase and fibrinolytic factor. No relationship was detected between pesticinogeny and other tested properties either associated with virulence or peculiar to P. pestis. Pesticin I was distinguished from the coagulase and fibrinolytic activities on the basis of anatomical distribution, behavior during gel filtration, and sensitivity to heat. Coagulase and the fibrinolytic factor were not differentiated by these criteria. Spontaneous suppressor mutations causing reversion to pesticinogeny were not detected, nor were such mutants obtained by treatment with ultraviolet light or 2-aminopurine. Attempts to demonstrate a common activator of pesticin I, coagulase, or the fibrinolytic factor in extracts of pesticinogenic cells were not successful. These results are in accord with the hypothesis that at least two structural genes for the three activities reside on a replicon distinct from the chromosome proper. Fibrinolytic activity was significantly reduced in the presence of 0.003 m epsilon-aminocaproic acid and was nonexistent on fibrin films freed from endogenous plasminogen by treatment with heat. Fibrinolytic activity on heated films could be restored by addition of plasma or serum from six mammalian species. Accordingly, the plague fibrinolytic factor, like staphylokinase or urokinase, promotes the conversion of plasminogen to plasmin.     

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.     

Serotype differences and lack of biofilm formation characterize Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.     

Use of protein microarray to identify gene expression changes of Yersinia pestis at different temperatures

Yersinia pestis is a bacterium that is transmitted between fleas, which have a body temperature of 26 °C, and mammalian hosts, which have a body temperature of 37 °C. To adapt to the temperature shift, phenotype variations, including virulence, occur. In this study, an antigen microarray including 218 proteins of Y. pestis was used to evaluate antibody responses in a pooled plague serum that was unadsorbed, adsorbed by Y. pestis cultivated at 26 °C, or adsorbed by Y. pestis cultivated at 26 and 37 °C to identify protein expression changes during the temperature shift. We identified 12 proteins as being expressed at 37 °C but not at 26 °C, or expressed at significantly higher levels at 37 °C than at 26 °C. The antibodies against 7 proteins in the serum adsorbed by Y. pestis cultivated at 26 and 37 °C remained positive, suggesting that they were not expressed on the surface of Y. pestis in LB broth in vitro or specifically expressed in vivo. This study proved that protein microarray and antibody profiling comprise a promising technique for monitoring gene expression at the protein level and for better understanding pathogenicity, to find new vaccine targets against plague.     

Prevalence and abundance of fleas in black-tailed prairie dog burrows: implications for the transmission of plague (Yersinia pestis)

Plague, the disease caused by the bacterium Yersinia pestis, can have devastating impacts on North American wildlife. Epizootics, or die-offs, in prairie dogs (Cynomys ludovicianus) occur sporadically and fleas (Siphonaptera) are probably important in the disease's transmission and possibly as maintenance hosts of Y. pestis between epizootics. We monitored changes in flea abundance in prairie dog burrows in response to precipitation, temperature, and plague activity in shortgrass steppe in northern Colorado. Oropsylla hirsuta was the most commonly found flea, and it increased in abundance with temperature. In contrast, Oropsylla tuberculata cynomuris declined with rising temperature. During plague epizootics, flea abundance in burrows increased and then subsequently declined after the extirpation of their prairie dog hosts.     

Insights into Yersinia pestis biofilm development: topology and co-interaction of Hms inner membrane proteins involved in exopolysaccharide production

Primarily, three operons, hmsHFRS, hmsT and hmsP, are responsible for the development of a Yersinia pestis biofilm, which is essential for blockage-dependent transmission of plague from fleas to mammals. Here, using specific antibodies, a polymeric beta-1,6-N-acetyl-d-glucosamine-like polysaccharide was detected in the extracellular matrix of hmsHFRS-dependent Y. pestis biofilm. The production of this exopolysaccharide (EPS) was controlled by diguanylate cyclase HmsT and EAL domain phosphodiesterase HmsP, acting as positive and negative regulators respectively. Cellular compartmentalization of soluble segments of Hms inner membrane proteins, including the putative glycosyltransferase domain of HmsR, the diguanylate cyclase/GGDEF domain of HmsT and the phosphodiesterase/EAL domain of HmsP, was determined by a combination of topology prediction algorithms and construction of C-terminal translational fusions with beta-galactosidase and alkaline phosphatase. Multiple interactions of Hms inner membrane proteins were detected using bacterial cAMP based two-hybrid system. Biochemical analyses confirmed some of these protein-protein interactions. Our results indicate that synthesis and regulation of the Y. pestis biofilm EPS occurs in the cytoplasm by a proposed Hms enzymatic complex.     

High-frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut

The acquisition of foreign DNA by horizontal transfer from unrelated organisms is a major source of variation leading to new strains of bacterial pathogens. The extent to which this occurs varies widely, due in part to lifestyle factors that determine exposure to potential donors. Yersinia pestis, the plague bacillus, infects normally sterile sites in its mammalian host, but forms dense aggregates in the non-sterile digestive tract of its flea vector to produce a transmissible infection. Here we show that unrelated co-infecting bacteria in the flea midgut are readily incorporated into these aggregates, and that this close physical contact leads to high-frequency conjugative genetic exchange. Transfer of an antibiotic resistance plasmid from an Escherichia coli donor to Y. pestis occurred in the flea midgut at a frequency of 10-3 after only 3 days of co-infection, and after 4 weeks 95% of co-infected fleas contained an average of 103 antibiotic-resistant Y. pestis transconjugants. Thus, transit in its arthropod vector exposes Y. pestis to favourable conditions for efficient genetic exchange with microbial flora of the flea gut. Horizontal gene transfer in the flea may be the source of antibiotic-resistant Y. pestis strains recently isolated from plague patients in Madagascar.