Two Distinct Yersinia pestis Populations Causing Plague among Humans in the West Nile Region of Uganda

Background

Plague is a life-threatening disease caused by the bacterium, Yersinia pestis. Since the 1990s, Africa has accounted for the majority of reported human cases. In Uganda, plague cases occur in the West Nile region, near the border with Democratic Republic of Congo. Despite the ongoing risk of contracting plague in this region, little is known about Y. pestis genotypes causing human disease.

Methodology/Principal Findings

During January 2004–December 2012, 1,092 suspect human plague cases were recorded in the West Nile region of Uganda. Sixty-one cases were culture-confirmed. Recovered Y. pestis isolates were analyzed using three typing methods, single nucleotide polymorphisms (SNPs), pulsed field gel electrophoresis (PFGE), and multiple variable number of tandem repeat analysis (MLVA) and subpopulations analyzed in the context of associated geographic, temporal, and clinical data for source patients. All three methods separated the 61 isolates into two distinct 1.ANT lineages, which persisted throughout the 9 year period and were associated with differences in elevation and geographic distribution.

Conclusions/Significance

We demonstrate that human cases of plague in the West Nile region of Uganda are caused by two distinct 1.ANT genetic subpopulations. Notably, all three typing methods used, SNPs, PFGE, and MLVA, identified the two genetic subpopulations, despite recognizing different mutation types in the Y. pestis genome. The geographic and elevation differences between the two subpopulations is suggestive of their maintenance in highly localized enzootic cycles, potentially with differing vector-host community composition. This improved understanding of Y. pestis subpopulations in the West Nile region will be useful for identifying ecologic and environmental factors associated with elevated plague risk.     

Adhesion pili in Yersinia pestis

Y. pestis cells cultivated at 37 degrees C are capable of agglutinating red blood cells of some animals, which is due to the appearance of pili. The adhesion pili consist of protein subunits with a molecular weight of the order of 12000 daltons, their isoionic point being at pH 4.7. The reaction of hemagglutination was inhibited by the mixture of ganglyosides, while the preliminary treatment of red blood cells with neuraminidases increased its effectiveness. The pili are supposed to take part in the expression of virulence.     

Yersinia pestis lineages in Mongolia

Background

Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia.

Methodology/Principal Findings

Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches.

Conclusions/Significance

We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years.     

Cyclic nucleotides in the dynamic development of shock caused by the murine toxin of Yersinia pestis

The authors have studied the effect of Y. pestis "mouse" toxin (LD50), injected intravenously to rats, on cAMP and cGMP content in the tissues of different organs (the lungs, liver, heart, spleen, kidneys, small intestine) and in the blood in the course of the development of toxinfection shock. The effect of Y. pestis "mouse" toxin on cyclic nucleotide content in the organs of experimental animals is determined by the sum of oppositely directed effects produced by the thermostable and thermolabile fractions of the toxin. Its thermostable fraction, when introduced in the dose used in the experiments, did not kill the animals. The most pronounced changes in the cyclic nucleotide content have been detected in the lungs which appear to be the main target organ for Y. pestis "mouse" toxin.     

Genome sequence of Yersinia pestis KIM

We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.     

Immunobiological properties of Yersinia pestis antigens

The present review contains information concerning immunobiological properties of plague microbe antigens. All of the identified antigens are evaluated in relation to pathogenicity of Yersinia pestis namely a resistance to phagocytosis, toxicity, adhesiveness etc. as well as persistence ability and adaptation to variable environment. In addition, the role of antigens in immunogenicity of living plague microbe for experimental animals is considered. The data concerning mechanisms of antigenic contribution to the development of adaptive immunity are presented.     

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

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

Proteomic characterization of Yersinia pestis virulence

The Yersinia pestis proteome was studied as a function of temperature and calcium by two-dimensional differential gel electrophoresis. Over 4,100 individual protein spots were detected, of which hundreds were differentially expressed. A total of 43 differentially expressed protein spots, representing 24 unique proteins, were identified by mass spectrometry. Differences in expression were observed for several virulence-associated factors, including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as several putative virulence factors and membrane-bound and metabolic proteins. Differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants.     

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.     

Magnetic biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.     

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.     

Yersinia genome diversity disclosed by Yersinia pestis genome-wide DNA microarray

The genus Yersinia includes 11 species, 3 of which (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) are pathogenic for humans. The remaining 8 species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) are merely opportunistic pathogens found mostly in the environment. In this work, the genomic differences among Yersinia were determined using a Y. pestis-specific DNA microarray. The results revealed 292 chromosomal genes that were shared by all Yersinia species tested, constituting the conserved gene pool of the genus Yersinia. Hierarchical clustering analysis of the microarray data revealed the genetic relationships among all 11 species in this genus. The microarray analysis in conjunction with PCR screening greatly reduced the number of chromosomal genes (32) specific for Y. pestis to 16 genes and uncovered a high level of genomic plasticity within Y. pseudotuberculosis, indicating that its different serotypes have undergone an extensively parallel loss or acquisition of genetic content, which is likely to be important for its adaptation to changes in environmental niches.     

Magnetic immunoenzyme analysis of Yersinia pestis antigens

The detection of Y. pestis cells in magnetic enzyme immunoassay is carried out with the use of magnetic polyacrylamide microgranules. In the assay system for the determination of the antigen commercial Y. pestis antigens, peroxidase-labeled antibodies, the substrate mixture consisting of sodium salt of 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and H2O2 in citrate-phosphate buffer solution, pH 4.5, are used. The sensitivity of the method is 5 X 10(4) microbial bodies per ml.     

Survival of Yersinia pestis on environmental surfaces

The survival of two strains of Yersinia pestis (avirulent A1122 and virulent Harbin) on the surfaces of four materials was investigated. Viability was evaluated with epifluorescence microscopy by using the metabolic stain cyanoditolyl tetrazolium chloride and plate counts. Small numbers of cells suspended in phosphate buffer survived 2 to 4 h after visible drying on stainless steel, polyethylene, or glass and beyond 48 h on paper. Cells suspended in brain heart infusion broth (BHI) persisted more than 72 h on stainless steel, polyethylene, and glass. Small numbers of cells suspended in BHI were still viable at 120 h on paper. These data suggest that Y. pestis maintains viability for extended periods (last measured at 5 days) under controlled conditions.     

鼠疫菌毒力调控研究

鼠疫菌通过一系列转录调控子(如CRP、PhoP、RovA和Fur)控制着一些关键毒力因子(如Pla、强毒力岛、III型分泌系统等)的基因表达。鼠疫菌可感应宿主体内信号刺激,紧密调控毒力因子的表达。在这个紧密调控过程中,调控子、毒力相关基因构成了一个动态网络。鼠疫菌在从假结核菌祖先演化的进程中,基因表达调控网络的重塑在鼠疫菌毒力进化过程中发挥着不可取代的作用。     

Survival of Yersinia pestis on Environmental Surfaces

The survival of two strains of Yersinia pestis (avirulent A1122 and virulent Harbin) on the surfaces of four materials was investigated. Viability was evaluated with epifluorescence microscopy by using the metabolic stain cyanoditolyl tetrazolium chloride and plate counts. Small numbers of cells suspended in phosphate buffer survived 2 to 4 h after visible drying on stainless steel, polyethylene, or glass and beyond 48 h on paper. Cells suspended in brain heart infusion broth (BHI) persisted more than 72 h on stainless steel, polyethylene, and glass. Small numbers of cells suspended in BHI were still viable at 120 h on paper. These data suggest that Y. pestis maintains viability for extended periods (last measured at 5 days) under controlled conditions.