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.     

Loss of a biofilm-inhibiting glycosyl hydrolase during the emergence of Yersinia pestis

Yersinia pestis, the bacterial agent of plague, forms a biofilm in the foregut of its flea vector to produce a transmissible infection. The closely related Yersinia pseudotuberculosis, from which Y. pestis recently evolved, can colonize the flea midgut but does not form a biofilm in the foregut. Y. pestis biofilm in the flea and in vitro is dependent on an extracellular matrix synthesized by products of the hms genes; identical genes are present in Y. pseudotuberculosis. The Yersinia Hms proteins contain functional domains present in Escherichia coli and Staphylococcus proteins known to synthesize a poly-beta-1,6-N-acetyl-D-glucosamine biofilm matrix. In this study, we show that the extracellular matrices (ECM) of Y. pestis and staphylococcal biofilms are antigenically related, indicating a similar biochemical structure. We also characterized a glycosyl hydrolase (NghA) of Y. pseudotuberculosis that cleaved beta-linked N-acetylglucosamine residues and reduced biofilm formation by staphylococci and Y. pestis in vitro. The Y. pestis nghA ortholog is a pseudogene, and overexpression of functional nghA reduced ECM surface accumulation and inhibited the ability of Y. pestis to produce biofilm in the flea foregut. Mutational loss of this glycosidase activity in Y. pestis may have contributed to the recent evolution of flea-borne transmission.     

鼠疫耶尔森氏菌环二鸟苷酸代谢及生物被膜形成调控研究进展

鼠疫耶尔森氏菌(Yersinia pestis,以下简称"鼠疫菌")是烈性传染病鼠疫的病原菌,以鼠蚤作为传播媒介。鼠疫菌在其传播媒介鼠蚤的前胃中形成生物被膜从而促进其在宿主间传播。鼠疫菌生物被膜的形成受第二信使分子环二鸟苷酸(c-di-GMP)的正向调控。鼠疫菌中c-di-GMP由二鸟苷酸环化酶(DGC)HmsT和HmsD合成,由磷酸二酯酶(PDE)HmsP降解。文中主要介绍影响鼠疫菌环二鸟苷酸代谢及生物被膜形成的调控因子,并对其作用机制进行讨论和总结。     

Yersinia--flea interactions and the evolution of the arthropod-borne transmission route of plague

Yersinia pestis, the causative agent of plague, is unique among the enteric group of Gram-negative bacteria in relying on a blood-feeding insect for transmission. The Yersinia-flea interactions that enable plague transmission cycles have had profound historical consequences as manifested by human plague pandemics. The arthropod-borne transmission route was a radical ecologic change from the food-borne and water-borne transmission route of Yersinia pseudotuberculosis, from which Y. pestis diverged only within the last 20000 years. Thus, the interactions of Y. pestis with its flea vector that lead to colonization and successful transmission are the result of a recent evolutionary adaptation that required relatively few genetic changes. These changes from the Y. pseudotuberculosis progenitor included loss of insecticidal activity, increased resistance to antibacterial factors in the flea midgut, and extending Yersinia biofilm-forming ability to the flea host environment.     

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.     

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.     

Modeling plague persistence in host-vector communities in California

Plague is an enzootic disease in the western United States, even though long-term persistent infections do not seem to occur. Enzootic persistence may occur as a function of dynamic interactions between flea vectors and transiently infected hosts, but the specific levels of vector competence, host competence, and transmission and recovery rates that would promote persistence and emergence among wild hosts and vectors are not known. We developed a mathematical model of enzootic plague in the western United States and implemented the model with the following objectives: 1) to use matrix manipulation within a classic susceptible-->infective-->resistant-->susceptible (SIRS) model framework to describe transmission of the plague bacterium Yersinia pestis among rodents and fleas in California, 2) to perform sensitivity analysis with model parameters and variables to indicate which values tended to dominate model output, and 3) to determine whether enzootic maintenance would be predicted with realistic parameter values obtained from the literature for Y. pestis in California rodents and fleas. The model PlagueSIRS was implemented in discrete time as a computer simulation incorporating environmental stochasticity and seasonality, by using matrix functions in the computer language R, allowing any number of rodent and flea species to interact through parasitism and disease transmission. Sensitivity analysis indicated that the model was sensitive to flea attack rate, host recovery rate, and rodent host carrying capacity but relatively insensitive to changes in the duration of latent infection in the flea, host and vector competence, flea recovery from infection, and host mortality attributable to plague. Realistic parameters and variable values did allow for the model to predict enzootic plague in some combinations, specifically when rodent species that were susceptible to infection but resistant to morbidity were parasitized by multiple poorly competent flea species, including some that were present year-round. This model could be extended to similar vectorborne disease systems and could be used iteratively with data collection in sylvatic plague studies to better understand plague persistence and emergence in nature.     

Flea diversity as an element for persistence of plague bacteria in an East African plague focus

Plague is a flea-borne rodent-associated zoonotic disease that is caused by Yersinia pestis and characterized by long quiescent periods punctuated by rapidly spreading epidemics and epizootics. How plague bacteria persist during inter-epizootic periods is poorly understood, yet is important for predicting when and where epizootics are likely to occur and for designing interventions aimed at local elimination of the pathogen. Existing hypotheses of how Y. pestis is maintained within plague foci typically center on host abundance or diversity, but little attention has been paid to the importance of flea diversity in enzootic maintenance. Our study compares host and flea abundance and diversity along an elevation gradient that spans from low elevation sites outside of a plague focus in the West Nile region of Uganda (~725-1160 m) to higher elevation sites within the focus (~1380-1630 m). Based on a year of sampling, we showed that host abundance and diversity, as well as total flea abundance on hosts was similar between sites inside compared with outside the plague focus. By contrast, flea diversity was significantly higher inside the focus than outside. Our study highlights the importance of considering flea diversity in models of Y. pestis persistence.     

Transmission shifts underlie variability in population responses to Yersinia pestis infection

Host populations for the plague bacterium, Yersinia pestis, are highly variable in their response to plague ranging from near deterministic extinction (i.e., epizootic dynamics) to a low probability of extinction despite persistent infection (i.e., enzootic dynamics). Much of the work to understand this variability has focused on specific host characteristics, such as population size and resistance, and their role in determining plague dynamics. Here, however, we advance the idea that the relative importance of alternative transmission routes may vary causing shifts from epizootic to enzootic dynamics. We present a model that incorporates host and flea ecology with multiple transmission hypotheses to study how transmission shifts determine population responses to plague. Our results suggest enzootic persistence relies on infection of an off-host flea reservoir and epizootics rely on transiently maintained flea infection loads through repeated infectious feeds by fleas. In either case, early-phase transmission by fleas (i.e., transmission immediately following an infected blood meal) has been observed in laboratory studies, and we show that it is capable of driving plague dynamics at the population level. Sensitivity analysis of model parameters revealed that host characteristics (e.g., population size and resistance) vary in importance depending on transmission dynamics, suggesting that host ecology may scale differently through different transmission routes enabling prediction of population responses in a more robust way than using either host characteristics or transmission shifts alone.     

Epizootological role of fleas in the Gorno-Altai natural plague focus (a review)

Epizootological role of fleas in the Gorno-Altai natural plague focus (Sailugemsk focus) and numerous data on the flea viability are analyzed and generalized. Information concerning the flea natural infectivity with Yersinia pestis altaica is represented. Ecological peculiarities of some flea species parasitizing the main host, Mongolian pika Ochotona pallasi, and nature of their interrelations with Y. pestis are investigated. It is shown that the flea taxocenosis provides the permanent all year-round circulation of Y. pestis in the Gorno-Altai natural focus. Certain combinations of structural elements of the flea taxocenosis have a dominant significance in determination the circulation process at different phases of the annual epizootic cycle.     

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.     

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.     

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.     

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.     

Nutrient depletion may trigger the Yersinia pestis OmpR‐EnvZ regulatory system to promote flea‐borne plague transmission

The flea’s lumen gut is a poorly documented environment where the agent of flea‐borne plague, Yersinia pestis, must replicate to produce a transmissible infection. Here, we report that both the acidic pH and osmolarity of the lumen’s contents display simple harmonic oscillations with different periods. Since an acidic pH and osmolarity are two of three known stimuli of the OmpR‐EnvZ two‐component system in bacteria, we investigated the role and function of this Y. pestis system in fleas. By monitoring the in vivo expression pattern of three OmpR‐EnvZ‐regulated genes, we concluded that the flea gut environment triggers OmpR‐EnvZ. This activation was not, however, correlated with changes in pH and osmolarity but matched the pattern of nutrient depletion (the third known stimulus for OmpR‐EnvZ). Lastly, we found that the OmpR‐EnvZ and the OmpF porin are needed to produce the biofilm that ultimately obstructs the flea’s gut and thus hastens the flea‐borne transmission of plague. Taken as a whole, our data suggest that the flea gut is a complex, fluctuating environment in which Y. pestis senses nutrient depletion via OmpR‐EnvZ. Once activated, the latter triggers a molecular program (including at least OmpF) that produces the biofilm required for efficient plague transmission.     

Ecological characteristics of flea species relate to their suitability as plague vectors

The ability of vector-borne diseases to persist and spread is closely linked to the ecological characteristics of the vector species they use. Yet there have been no investigations of how species used as vectors by pathogens such as the plague bacterium differ from closely related species that are not used as vectors. The plague bacterium uses mammals as reservoir hosts and fleas as vectors. The ability of different fleas to serve as vectors is assumed to depend on how likely they are to experience gut blockage following bacterial multiplication; the blockage causes fleas to regurgitate blood into a wound and thus inject bacteria into new hosts. Beyond these physiological differences, it is unclear whether there exist fundamental ecological differences between fleas that are effective vectors and those that are not. Here, using a comparative analysis, we identify clear associations between the ability of flea species to transmit plague and their ecological characteristics. First, there is a positive relationship between the abundance of flea species on their hosts and their potential as vectors. Second, although the number of host species exploited by a flea is not associated with its potential as a vector, there is a negative relationship between the ability of fleas to transmit plague and the taxonomic diversity of their host spectrum. This suggests a correlation between some ecological characteristics of fleas and their ability to develop the plague blockage. The plague pathogen thus uses mainly abundant fleas specialized on a narrow taxonomic range of mammals, features that should maximize the persistence of the disease in the face of high flea mortality, and its transmission to suitable hosts only. This previously unrecognized pattern of vector use is of importance for the persistence and transmission of the disease.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

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).