Identification and characterization of autotransporter proteins of Yersinia pestis KIM

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.     

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).     

Genomic comparison of Yersinia pestis and Yersinia pseudotuberculosis by combination of suppression subtractive hybridization and DNA microarray

In order to further figure out the genetic differences between Yersinia pestis and Yersinia pseudotuberculosis, and to provide novel insights into the evolution of Y. pestis, we compared the genomes of Y. pseudotuberculosis serogroup I strain ATCC29833 and Y. pestis Antiqua strain 49006 using a combination of suppression subtractive hybridization (SSH) and comparative genomic hybridization with DNAs from a diverse panel of Y. pestis and Y. pseudotuberculosis strains. SSH followed by BLAST analysis revealed 112 SSH fragments specific to strain ATCC29833, compared to the genomic sequence data of Y. pestis strains CO92, KIM and 91001. We identified 17 SSH fragments that appeared to be newly determined genetic contents of Y. pseudotuberculosis. The combination of SSH and microarray analysis showed that the parallel loss of genes contributed greatly not only to the significant genomic divergence between Y. pestis and Y. pseudotuberculosis but also to the intra-species microevolution of both of species. The results confirmed our earlier hypothesis that Y. pestis Antiqua isolates from the natural plague focus B in China represented the most ancestral strains in China, hence phylogenetically the closest isolates to Y. pseudotuberculosis.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Xiaoyi Wang and Dongsheng Zhou contributed equally to this work.     

The hmu locus of Yersinia pestis is essential for utilization of free haemin and haem-protein complexes as iron sources

Yersinia pestis strains utilize haem and several haem-protein complexes as sole sources of iron. In this study, the haemin uptake locus (hmu) of Y. pestis KIM6+ was selected from a genomic library by trans-duction into an Escherichia coli siderophore synthesis (entC) mutant. Recombinant plasmids containing a common 16 kb BamHI insert were isolated that allowed E. coli entC to use haemin as an iron source. An 8.6 kb region of this insert was found to be essential for haemin utilization and encoded at least five proteins with molecular masses of 79/77, 44, 37, 35, and 30/27.5 kDa. A 10.9 kb Clal fragment containing the hmu locus showed varying degrees of homology to genomic DNA from Yersinia pseudotuberculosis, Yersinia enter-ocolitica, and other genera of Enterobacteriaceae. An E. coli hemA aroB strain harbouring cloned hmu genes used haemin as both an iron and porphyrin source but only on iron-poor medium, suggesting that haemin uptake is tightly iron regulated. Additionally, haemoglobin and myoglobin were used as iron sources by an E. coli entC (pHMU2.2) strain. Deletion of the hmu locus from Y. pestis KIM6+ chromosome generated a mutant that grew poorly on iron-depleted medium containing free haemin as well as mammalian haem-protein complexes including haemoglobin, haemoglobin-haptoglobin, myoglobin, haem-haemopexin, and haem-albumin unless it was complemented with cloned hmu genes.     

Sequence analysis of the yadA, inv, and ail genes and their expression in the main and nonmain Yersinia pestis subspecies and Yersinia pseudotuberculosis

The nucleotide sequences of the inv, yadA, and ail adhesin-invasin genes were analyzed in 24 strains of the main and nonmain Yersinia pestis subspecies, which were isolated from natural plague foci in Russia and neighbor countries, and ten Y. pseudotuberculosis strains. All of the five plague agent subspecies (main, caucasica, altaica, ulegeica, and hissarica) had the inv and yadA genes altered by insertion of the IS element and a single nucleotide deletion, respectively, as was earlier observed for the Y. pestis strains KIM and CO92. Consequently, the strains lacked functional activity of the Inv and YadA proteins. The ail gene of the main and ulegeica subspecies had a missense mutation, which replaced Val138 with Phe in the Ail protein. The strains of the caucasica subspecies had an AGT insertion in the ail gene, resulting in Ser148 insertion in the polypeptide chain. The changes in the ail sequence probably exerted no effect on ail expression, since the strains of all subspecies were resistant to blood serum complement.     

Analysis of insect toxin complex gene variability of <Emphasis Type="Italic">Yersinia pestis</Emphasis> and <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> 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.     

Ail provides multiple mechanisms of serum resistance to Yersinia pestis

Ail, a multifunctional outer membrane protein of Yersinia pestis, confers cell binding, Yop delivery and serum resistance activities. Resistance to complement proteins in serum is critical for the survival of Y. pestis during the septicemic stage of plague infections. Bacteria employ a variety of tactics to evade the complement system, including recruitment of complement regulatory factors, such as factor H, C4b‐binding protein (C4BP) and vitronectin (Vn). Y. pestis Ail interacts with the regulatory factors Vn and C4BP, and Ail homologs from Y. enterocolitica and Y. pseudotuberculosis recruit factor H. Using co‐sedimentation assays, we demonstrate that two surface‐exposed amino acids, F80 and F130, are required for the interaction of Y. pestis Ail with Vn, factor H and C4BP. However, although Ail‐F80A/F130A fails to interact with these complement regulatory proteins, it still confers 10,000‐fold more serum resistance than a Δail strain and prevents C9 polymerization, potentially by directly interfering with MAC assembly. Using site‐directed mutagenesis, we further defined this additional mechanism of complement evasion conferred by Ail. Finally, we find that at Y. pestis concentrations reflective of early‐stage septicemic plague, Ail weakly recruits Vn and fails to recruit factor H, suggesting that this alternative mechanism of serum resistance may be essential during plague infection.     

Generation and Characterization of Murine Monoclonal Antibodies to Recombinant YopM, YopB and LcrV Proteins of Yersinia pestis

Summary YopM, an effector, YopB, a translator, and LcrV, a regulator, are proteins forming important componants of type III secretion system of Yersinia pestis. Recombinant truncated YopM of 32 kDa, YopB of 28 kDa and LcrV of 31 kDa sizes were utilized for priming BALB/c mice for the generation of monoclonal antibodies following standard poly-ethylene glycol (PEG) fusion protocol. Nine, 10 and 6 stabilized hybridoma cell lines could be generated against YopM, YopB and LcrV proteins, respectively. All these monoclonal antibodies were found reactive to Y. pestis strain A1122 and did not show any cross-reactivity to Y. enterocolitica, Y. pseudotuberculosis, Y. kristensenii, Y. frederiksenii, Y. intermedia, Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Salmonella abortus-equi and Staphylococcus aureus tested by ELISA and Western blotting. Monoclonal antibodies also exhibited reactivity to their corressponding native protein antigens in Y. pestis i.e. 42 kDa for YopM, 41 kDa for YopB and 37 kDa for LcrV in immunoblotting. Reactivity of monoclonal antibodies was further assessed on 26 Y. pestis isolates including 18 from 1994 plague outbreak regions (11 from pneumonic patients, 7 from rodents) and 8 from rodents of Deccan plateau of Southern India by Western blotting as well as by sandwich ELISA. The monoclonal antibodies could specifically locate the expression of yopM, yopB and lcrV genes among these Indian Y. pestis strains as well. Results obtained with sandwich ELISA and Western blot were identical to those observed by PCR. Monoclonal antibodies to Yops, therefore, can be employed for an early and reliable identification of virulent Y. pestis strains.     

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.     

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.     

Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis

Autotransporters, the largest family of secreted proteins in Gram‐negative bacteria, perform a variety of functions, including adherence, cytotoxicity and immune evasion. In Yersinia pestis the autotransporter YapE has adhesive properties and contributes to disease in the mouse model of bubonic plague. Here, we demonstrate that omptin cleavage of Y. pestis YapE is required to mediate bacterial aggregation and adherence to eukaryotic cells. We demonstrate that omptin cleavage is specific for the Y. pestis and Y. pseudotuberculosis YapE orthologues but is not conserved in the Yersinia enterocolitica protein. We also show that cleavage of YapE occurs in Y. pestis but not in the enteric Yersinia species, and requires the omptin Pla (plasminogen activator protease), which is encoded on the Y. pestis‐specific plasmid pPCP1. Together, these data show that post‐translation modification of YapE appears to be specific to Y. pestis, was acquired along with the acquisition of pPCP1 during the divergence of Y. pestis from Y. pseudotuberculosis, and are the first evidence of a novel mechanism to regulate bacterial adherence.     

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.     

An additional step in the transmission of Yersinia pestis?

Plague, caused by the bacterium Yersinia pestis, is a mammalian vector-borne disease, transmitted by fleas that serve as the vector between rodent hosts. For many pathogens, including Y. pestis, there are strong evolutionary pressures that lead to a reduction in ‘useless genes'', with only those retained that reflect function in the specific environment inhabited by the pathogen. Genetic traits critical for survival and transmission between two environments, the rodent and the flea, are conserved in epizootic/epidemic plague strains. However, there are genes that remain conserved for which no function in the flea–rodent cycle has yet been observed, indicating an additional environment may exist in the transmission cycle of plague. Here, we present evidence for highly conserved genes that suggests a role in the persistence of Y. pestis after death of its host. Furthermore, maintenance of these genes points to Y. pestis traversing a post-mortem path between, and possibly within, epizootic periods and offering insight into mechanisms that may allow Y. pestis an alternative route of transmission in the natural environment.     

MLVA distribution characteristics of Yersinia pestis in China and the correlation analysis

Background  

Yersinia pestis, the aetiological agent of plague, has been well defined genotypically on local and worldwide scales. In November 2005, five cases of severe pneumonia of unknown causes, resulting in two deaths, were reported in Yulong, Yunnan province. In this study, we compared Y. pestis isolated from the Yulong focus to strains from other areas.     

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.     

Genome Sequence of the Deep-Rooted Yersinia pestis Strain Angola Reveals New Insights into the Evolution and Pangenome of the Plague Bacterium

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.Yersinia pestis, the causative agent of plague, is a nonmotile Gram-negative bacterial pathogen. The genus Yersinia comprises two other pathogens that cause worldwide infections in humans and animals: Y. pseudotuberculosis and Y. enterocolitica (11, 12, 22, 61, 71). Despite their genetic relationship, these species differ radically in their pathogenicity and transmission. Plague is primarily a disease of wild rodents that is transmitted to other mammals through flea bites. In humans it produces the bubonic form of plague. Y. pestis also can be transmitted from human to human by aerosol, especially during pandemics, causing primarily pneumonic plague. Evolutionarily, it is estimated that Y. pestis diverged from the enteric pathogen Y. pseudotuberculosis within the last 20,000 years, while Y. pseudotuberculosis and Y. enterocolitica lineages separated 0.4 to 1.9 million years ago (2). Y. pestis inhabits a distinct ecological niche, and its transmission is anchored in its unique plasmid inventory: the murine toxin (pMT) and plasminogen activator (pPCP) plasmids. In addition, Y. pestis harbors the low-calcium-response plasmid pCD, which it inherited from its closest relative, Y. pseudotuberculosis (pYV) (12), and it also is found in the more distantly related Y. enterocolitica (71). So-called cryptic plasmids have been described in the literature as part of the Y. pestis mobilome (71), but no sequence data are available to decipher the nature and impact of such plasmids in the epidemiology and pathogenicity of Y. pestis (14). Y. pestis isolates have been historically grouped into the biovars Antiqua (ANT), Medievalis (MED), and Orientalis (ORI), based on metabolic properties such as nitrate reduction and fermentation patterns (72). However, we will use the population-based nomenclature for Y. pestis introduced by Achtman et al. (1), as we believe it better reflects the true evolutionary relationship. Due to its young evolutionary age, only a few genetic polymorphisms have been identified within the Y. pestis genomes sequenced to date (1). Here, we report the comparative analysis of the virulent Y. pestis strain Angola, a representative of one of the most ancient Y. pestis lineages thus far sequenced. We studied adaptive microevolutionary traits Y. pestis has acquired and predicted the global Yersinia pangenome. By comparing the genomes of the three human pathogenic Yersinia species (12, 22), we investigated the global- and species-specific gene reservoir, the genome dynamics, and the degree of genetic diversity that is found within these species. Our genotypic and phenotypic analyses, as well as the refined single-nucleotide polymorphism (SNP)-based phylogeny of Y. pestis, indicate that Angola is a deep-rooted isolate with unique genome characteristics intermediate between modern Y. pestis isolates and Y. pseudotuberculosis.     

Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O∶8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10⁷-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD₅₀ of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.