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.     

Acute oral toxicity of Yersinia pseudotuberculosis to fleas: implications for the evolution of vector-borne transmission of plague

Yersinia pestis diverged from Yersinia pseudotuberculosis相似文献   

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.     

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.     

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.     

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.     

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.     

Yersinia pestis pFra shows biovar-specific differences and recent common ancestry with a Salmonella enterica serovar Typhi plasmid

Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y. pestis-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different Y. pestis strains which distinguish Y. pestis bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between Y. pestis and the exclusively human pathogen Salmonella enterica serovar Typhi.     

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.     

Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation

Yersinia pestis is an important human pathogen that is maintained in flea-rodent enzootic cycles in many parts of the world. During its life cycle, Y. pestis senses host-specific environmental cues such as temperature and regulates gene expression appropriately to adapt to the insect or mammalian host. For example, Y. pestis synthesizes different forms of lipid A when grown at temperatures corresponding to the in vivo environments of the mammalian host and the flea vector. At 37 degrees C, tetra-acylated lipid A is the major form; but at 26 degrees C or below, hexa-acylated lipid A predominates. In this study, we show that the Y. pestis msbB (lpxM) and lpxP homologs encode the acyltransferases that add C12 and C(16:1) groups, respectively, to lipid IV(A) to generate the hexa-acylated form, and that their expression is upregulated at 21 degrees C in vitro and in the flea midgut. A Y. pestis deltamsbB deltalpxP double mutant that did not produce hexa-acylated lipid A was more sensitive to cecropin A, but not to polymyxin B. This mutant was able to infect and block fleas as well as the parental wild-type strain, indicating that the low-temperature-dependent change to hexa-acylated lipid A synthesis is not required for survival in the flea gut.     

A Yersinia pestis-specific DNA fragment encodes temperature-dependent coagulase and fibrinolysin associated phenotypes

The effect of temperature on coagulase and fibrinolysin expression (Pla) by Yersinia pestis has been implicated in the transmission of plague by fleas. In an attempt to improve our understanding of this process, we have cloned, sequenced and characterized the gene encoding the Pla phenotypes in Y. pestis, and examined its temperature-dependent regulation. The coding region for this gene overlaps a 900bp Y. pestis-specific DNA fragment that we have previously shown to be capable of detecting plague bacilli in fleas. The pla gene contains a single open reading frame encoding 312 amino acids with a predicted molecular weight of 34.7 kD and a putative signal sequence of 20 amino acids. This coding region appears to be sufficient for both coagulase and fibrinolytic activities. In Y. pestis, modulation between coagulase and fibrinolytic activities is temperature-dependent: coagulase activity is most evident at temperatures below 30 degrees C but fibrinolytic activity increases with higher temperatures (greater than 30 degrees C), regardless of the temperature at which the bacteria are grown. Our results lead us to believe that this regulation occurs post-translationally. It is possible that the alternative forms of the Pla protein are essential to 'flea blockage' and subsequent transmission of the plague bacillus to animals.     

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.     

Yersinia pestis kills Caenorhabditis elegans by a biofilm-independent process that involves novel virulence factors

It is known that Yersinia pestis kills Caenorhabditis elegans by a biofilm-dependent mechanism that is similar to the mechanism used by the pathogen to block food intake in the flea vector. Using Y. pestis KIM 5, which lacks the genes that are required for biofilm formation, we show that Y. pestis can kill C. elegans by a biofilm-independent mechanism that correlates with the accumulation of the pathogen in the intestine. We used this novel Y. pestis-C. elegans pathogenesis system to show that previously known and unknown virulence-related genes are required for full virulence in C. elegans. Six Y. pestis mutants with insertions in genes that are not related to virulence before were isolated using C. elegans. One of the six mutants carried an insertion in a novel virulence gene and showed significantly reduced virulence in a mouse model of Y. pestis pathogenesis. Our results indicate that the Y. pestis-C. elegans pathogenesis system that is described here can be used to identify and study previously uncharacterized Y. pestis gene products required for virulence in mammalian systems.     

Factors providing the blocking activity of Yersinia pestis

Discusses published data on the specific mechanism of Y. pestis transfer by "blocked" fleas. Special attention is paid to individual phenotypical signs and genetic determinants of Y. pestis whose expression correlates with the blocking activity of bacteria. Prospects for further research are outlined.     

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 potential role of swift foxes (Vulpes velox) and their fleas in plague outbreaks in prairie dogs

Swift foxes (Vulpes velox) have been proposed as potential carriers of fleas infected with the bacterium Yersinia pestis between areas of epizootics in black-tailed prairie dogs (Cynomys ludovicianus). We examined antibody prevalence rates of a population of swift foxes in Colorado, USA, and used polymerase chain reaction (PCR) assays to examine their flea biota for evidence of Y. pestis. Fifteen of 61 (24%) captured foxes were seropositive, and antibody prevalence was spatially correlated with epizootic plague activity in prairie dog colonies in the year of, and previous to, the study. Foxes commonly harbored the flea Pulex simulans, though none of the fleas was positive for Y. pestis.     

Physiological and regulatory characterization of KatA and KatY in Yersinia pestis

The catalase or catalase-peroxidase activity commonly exists in many pathogens and plays an important role in resisting the oxidative burst of phagocytes helping the pathogen persistently colonize in the host. Yersinia pestis is a facultative pathogen and the causative agent of plague. KatY has been identified as a thermosensing antigen with modest catalase activity in this pathogen. Here Y. pestis KatA and KatY were experimentally confirmed as a monofunctional catalase and bifunctional catalase-peroxidase, respectively. Their expression induced by H2O2 was proven to be mediated by the oxidative regulator, OxyR. Expression of KatA changed with growth phases and was crucial to its traditional physiological role in protecting Y. pestis cells against toxicity of exogenous H2O2. KatY was regulated by temperature and H2O2, two major elements of phagolysosomal microenvironments. Consistent with the above results, gene expression of katY increased significantly during intracellular growth of Y. pestis compared with that in vitro growth. However, a DeltakatY mutant was fully virulent to mice, suggesting that KatY is not required for Y. pestis virulence.     

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.     

Increased invasiveness as one of the manifestations of phenotype variability of the plague microbe in fleas

Experimental studies conducted on genetically connected virulent subcultures of Y. pestis showed that the death of albino mice infected by flea bite occurred earlier than in the animals infected by a syringe subcutaneously. A high invasiveness of Y. pestis subcultures isolated from fleas (in comparison with the initial strains and subcultures from the animals) persisted for 2--3 passages in their cultivation on artificial nutrient media.