Genetic bases of methionine dependence in <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains of main and non-main subspecies

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and non-main subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and non-main subspecies revealed that the reason for the methionine dependence of strains belonging to the main subspecies is a deletion of a single nucleotide (−G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.     

Determination of genetic bases of auxotrophy in <Emphasis Type="Italic">Yersinia pestis</Emphasis> ssp. <Emphasis Type="Italic">caucasica</Emphasis> strains

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG aroF thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.     

Immunization of mice with YscF provides protection from <Emphasis Type="Italic">Yersinia pestis</Emphasis> infections

Background  

Yersinia pestis, the causative agent of plague, is a pathogen with a tremendous ability to cause harm and panic in populations. Due to the severity of plague and its potential for use as a bioweapon, better preventatives and therapeutics for plague are desirable. Subunit vaccines directed against the F1 capsular antigen and the V antigen (also known as LcrV) of Y. pestis are under development. However, these new vaccine formulations have some possible limitations. The F1 antigen is not required for full virulence of Y. pestis and LcrV has a demonstrated immunosuppressive effect. These limitations could damper the ability of F1/LcrV based vaccines to protect against F1-minus Y. pestis strains and could lead to a high rate of undesired side effects in vaccinated populations. For these reasons, the use of other antigens in a plague vaccine formulation may be advantageous.     

A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in <Emphasis Type="Italic">Yersinia pestis</Emphasis>

Background  

Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis.     

Genotyping of Indian <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains by MLVA and repetitive DNA sequence based PCRs

India experienced two plague outbreaks in Gujarat and Maharastra during 1994 and then in the Shimla district of Himachal Pradesh during 2002. Yersinia pestis strains recovered from rodents and pneumonic patients during the 1994 outbreaks, pneumonic patients from the 2002 Shimla outbreak and rodents trapped on the Deccan Plateau during a surveillance activity carried out in 1998 were characterized by MLVA, ERIC-PCR and ERIC-BOX-PCR. MLVA genotyping of Indian Y. pestis strains revealed strains of 2 Orientalis, 1 Mediaevalis and 1 Antiqua genotypes distributed in three distinct branches corresponding to their biovar. The Orientalis genotype strains recovered from the 1994 outbreaks and 1998 surveillance activity clustered in one branch while the Antiqua biovar strains from the Shimla outbreak and the Mediaevalis strain recovered from a rodent trapped on the Deccan Plateau region during surveillance formed the other branches. The Orientalis Y. pestis strains recovered from rodents and patients from the 1994 plague outbreaks exhibited similar MLVA, ERIC-PCR and ERIC-BOX-PCR profiles and these were closely related to the Orientalis strains recovered from the rodents trapped on the Deccan Plateau. These data provide evidence for the possible linkage between the Y. pestis strains resident in the endemic region and those that were associated with the 1994 plague outbreaks. Mediaevalis and Antiqua biovars also were recovered from the environmental reservoir on the Deccan Plateau and from the pneumonic patients of 2002 plague outbreak. Therefore, as in Central Asian and African regions, Antiqua and Mediaevalis biovars seem to be well established in the Indian subcontinent as well. ERIC-PCR DNA fingerprinting delineated genotypes similar to those defined by MLVA. Thus ERIC-PCR appears to have the potential to be used as a molecular marker in the molecular epidemiological investigations of plague.     

Seasonal dynamics of epizootic process of the plague agent <Emphasis Type="Italic">Yersinia pestis</Emphasis> transmission to the long-tailed suslik <Emphasis Type="Italic">Citellus undulatus</Emphasis> by the flea <Emphasis Type="Italic">Citellophilus tesquorum</Emphasis> in Tuva

Transmission of Yersinia pestis to the long-tailed suslik (Citellus undulatus) by fleas (Citellophilus tesquorum) in the Tuva natural plague focus in different seasons (spring, summer, and autumn) was studied experimentally. Between feeding periods, insects were kept in an artificial nest under temperature and humidity closely corresponding to seasonal ones. The character of the agent transmission was estimated according to the fraction of fleas with the agent in the aggregated state (bacterial lumps, partial blocks of proventriculus), the fraction of blocked individuals, and the fraction of infected susliks and of those with the generalized form of infection. Seasonal dynamics of epizootic process of the Y. pestis transmission corresponded to the results obtained in the epizootic examination of the Tuva natural plague focus and reflected the dynamics of the epizootic process (increase-peak-decline). The activity of the formation of a proventriculus block in C. t. altaicus, the infection ability of the fleas, and the sensitivity of long-tailed Siberian susliks to Y. pestis were the highest in mid-summer (July-first ten days of August), during the period of epizooty activation in the focus. The maximal number of C. t. altaicus with the plague agent at the aggregated state was observed in the cold period, before wintering of insects and after their hibernation.     

A comprehensive study on the role of the <Emphasis Type="Italic">Yersinia pestis</Emphasis> virulence markers in an animal model of pneumonic plague

We determined the role of Yersinia pestis virulence markers in an animal model of pneumonic plague. Eleven strains of Y. pestis were characterized using PCR assays to detect the presence of known virulence genes both encoded by the three plasmids as well as chromosomal markers. The virulence of all Y. pestis strains was compared in a mouse model for pneumonic plague. The presence of all known virulence genes correlated completely with virulence in the Balb/c mouse model. Strains which lacked HmsF initially exhibited visible signs of disease whereas all other strains (except wild-type strains) did not exhibit any disease signs. Forty-eight hours post-infection, mice which had received HmsF^– strains regained body mass and were able to control infection; those infected with strains possessing a full complement of virulence genes suffered from fatal disease. The bacterial loads observed in the lung and other tissues reflected the observed clinical signs as did the cytokine changes measured in these animals. We can conclude that all known virulence genes are required for the establishment of pneumonic plague in mammalian animal models, the role of HmsF being of particular importance in disease progression.     

Analysis of insect toxin complex gene variability of Yersinia pestis and Yersinia pseudotuberculosis strains

The nucleotide sequences of the Tc's insect toxin complex genes have been analyzed in 18 natural strains of the main and non-main subspecies of Yersinia pestis isolated in different natural foci in the Russian Federation, as well as neighboring and more remote countries, as compared to the data on Y. pestis and Y. pseudotuberculosis strains stored in the NCBI GenBank database. The nucleotide sequences of these genes in plague agent strains have been found to be highly conserved, in contrast to those of the pseudotuberculosis agent. The sequences of two genes, tcaC and tccC2, have been found to be almost identical in Y. pestis strains, whereas other three genes (tcaA, tcaB, and tccC1) contain a few mutations, which, however, are not common for all strains of the plague agent. Exceptions are only strains of the Y. pestis biovar orientalis, whose tcaB gene is in a nonfunctional state due to a nucleotide deletion. The results suggest that the formation of the species Y. pestis as an agent of a natural focal infection with a transmissive mechanism has not resulted in degradation of the Tc's complex genes. Instead, these genes are likely to have been altered as the plague agent have been adapting to the new environment.     

Tandem repeats analysis for the high resolution phylogenetic analysis of <Emphasis Type="Italic">Yersinia pestis</Emphasis>

Background

Yersinia pestis, the agent of plague, is a young and highly monomorphic species. Three biovars, each one thought to be associated with the last three Y. pestis pandemics, have been defined based on biochemical assays. More recently, DNA based assays, including DNA sequencing, IS typing, DNA arrays, have significantly improved current knowledge on the origin and phylogenetic evolution of Y. pestis. However, these methods suffer either from a lack of resolution or from the difficulty to compare data. Variable number of tandem repeats (VNTRs) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses in a growing number of pathogens and have given promising results for Y. pestis as well.

Results

In this study we have genotyped 180 Y. pestis isolates by multiple locus VNTR analysis (MLVA) using 25 markers. Sixty-one different genotypes were observed. The three biovars were distributed into three main branches, with some exceptions. In particular, the Medievalis phenotype is clearly heterogeneous, resulting from different mutation events in the napA gene. Antiqua strains from Asia appear to hold a central position compared to Antiqua strains from Africa. A subset of 7 markers is proposed for the quick comparison of a new strain with the collection typed here. This can be easily achieved using a Web-based facility, specifically set-up for running such identifications.

Conclusion

Tandem-repeat typing may prove to be a powerful complement to the existing phylogenetic tools for Y. pestis. Typing can be achieved quickly at a low cost in terms of consumables, technical expertise and equipment. The resulting data can be easily compared between different laboratories. The number and selection of markers will eventually depend upon the type and aim of investigations.

     

Rapid identification and typing of <Emphasis Type="Italic">Yersinia pestis</Emphasis> and other <Emphasis Type="Italic">Yersinia</Emphasis> species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

Background  

Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

Proteomic analysis of iron acquisition,metabolic and regulatory responses of <Emphasis Type="Italic">Yersinia pestis</Emphasis> to iron starvation

Background  

The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).     

The phenomenon of <Emphasis Type="Italic">Yersinia pestis</Emphasis> biofilm formation in the organism of fleas

The paper considers, for the first time, the formation of the extracellular matrix envelope (EME), or the biofilm, by Yersinia pestis as the basis determining the nature of interaction of the plague agent with the flea organism. The significance of the insect proventriculus in the process of biofilm formation is shown. The ultrastructure of the conglomerates of the plague microbe in the flea proventriculus and midgut was studied and the uniform mechanism of their formation was established. The role of Yersinia pestis biofilm in preservation of the plague microbe in the intestine of ectoparasites and in the soil of rodent burrows was discussed. PCR analysis confirmed the presence of the agent in plague infected corpses and flea feces stored at +8−10°C for 7 years and 9 months.     

The many and varied niches occupied by <Emphasis Type="Italic">Yersinia pestis</Emphasis> as an arthropod-vectored zoonotic pathogen

Yersinia pestis, the causative agent of bubonic and pneumonic plague, has a complex lifestyle, cycling between both arthropod and mammalian hosts. This pathogen has previously been shown to survive intracellularly within macrophages and to be capable of biofilm formation within the flea, suggesting the development of a range of strategies to ensure survival throughout its life cycle, including expression of virulence factors and tight regulation of its genes.     

Transmission Efficiency of Two Flea Species (<Emphasis Type="Italic">Oropsylla tuberculata cynomuris</Emphasis> and <Emphasis Type="Italic">Oropsylla hirsuta</Emphasis>) Involved in Plague Epizootics among Prairie Dogs

Plague, caused by Yersinia pestis, is an exotic disease in North America circulating predominantly in wild populations of rodents and their fleas. Black-tailed prairie dogs (Cynomys ludovicianus) are highly susceptible to infection, often experiencing mortality of nearly all individuals in a town as a result of plague. The fleas of black-tailed prairie dogs are Oropsylla tuberculata cynomuris and Oropsylla hirsuta. We tested the efficiency of O. tuberculata cynomuris to transmit Y. pestis daily from 24 to 96 h postinfection and compared it to previously collected data for O. hirsuta. We found that O. tuberculata cynomuris has over threefold greater transmission efficiency (0.18 infected fleas transmit Y. pestis at 24 h postinfection) than O. hirsuta (0.05 fleas transmit). Using a simple model of flea-borne transmission, we combine these laboratory measurements with field data on monthly flea loads to compare the seasonal vectorial capacity of these two flea species. Coinciding with seasonal patterns of flea abundance, we find a peak in potential for flea-borne transmission in March, during high O. tuberculata cynomuris abundance, and in September–October when O. hirsuta is common. Our findings may be useful in determining the timing of insecticidal dusting to slow plague transmission in black-tailed prairie dogs.     

Susceptibility to <Emphasis Type="Italic">Yersinia pestis</Emphasis> Experimental Infection in Wild <Emphasis Type="Italic">Rattus rattus</Emphasis>, Reservoir of Plague in Madagascar

In Madagascar, the black rat, Rattus rattus, is the main reservoir of plague (Yersinia pestis infection), a disease still responsible for hundreds of cases each year in this country. This study used experimental plague challenge to assess susceptibility in wild-caught rats to better understand how R. rattus can act as a plague reservoir. An important difference in plague resistance between rat populations from the plague focus (central highlands) and those from the plague-free zone (low altitude area) was confirmed to be a widespread phenomenon. In rats from the plague focus, we observed that sex influenced plague susceptibility, with males slightly more resistant than females. Other individual factors investigated (weight and habitat of sampling) did not affect plague resistance. When infected at high bacterial dose (more than 10⁵ bacteria injected), rats from the plague focus died mainly within 3–5 days and produced specific antibodies, whereas after low-dose infection (< 5,000 bacteria), delayed mortality was observed and surviving seronegative rats were not uncommon. These results concerning plague resistance level and the course of infection in the black rat would contribute to a better understanding of plague circulation in Madagascar.     

Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

Discrimination among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> H serotypes,serovars and strains based on 16S rRNA, <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">aroE</Emphasis> gene sequence analyses

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.