Extracellular resistance of Yersinia pestis to phagocytosis

The authors were the first to perform stimulation of parasitizing condition of plague bacilli in mammalian bloodstream by addition of adequate quantity of glucose and calcium ions into the routine bacteriological medium, as well as growing the plague agent in RPMI-1640, isotonic to the serum of man and mammals susceptible to plague. Comparison of proliferative, phenotypic, and biochemical properties of fully virulent and vaccine Y. pestis strains demonstrated the advantages of RPMI-1640 medium, which provided extensive in vitro multiplication of the mentioned microorganisms similar to the bacterioemic stage of plague. Using methods of molecular microbiology and immunology, including a panel of monoclonal antibodies, the researchers demonstrated an abrupt fall of production of main plague surface species-specific antigens such as F1, 'murine' toxin/phospholipase D and fibrinolysin, followed by inhibition of biochemical activities associated with these antigenic substances in Y. pestis, as well as specific components of lipopolysaccharide. Possible molecular mechanisms of virulence and adaptive variability of plague bacteria in the extracellular conditions in mammalian organism are discussed.     

Role of Yops and adhesins in resistance of Yersinia enterocolitica to phagocytosis

Yersinia enterocolitica is a pathogen endowed with two adhesins, Inv and YadA, and with the Ysc type III secretion system, which allows extracellular adherent bacteria to inject Yop effectors into the cytosol of animal target cells. We tested the influence of all of these virulence determinants on opsonic and nonopsonic phagocytosis by PU5-1.8 and J774 mouse macrophages, as well as by human polymorphonuclear leukocytes (PMNs). The adhesins contributed to phagocytosis in the absence of opsonins but not in the presence of opsonins. In agreement with previous results, YadA counteracted opsonization. In every instance, the Ysc-Yop system conferred a significant level of resistance to phagocytosis. Nonopsonized single-mutant bacteria lacking either YopE, -H, -T, or -O were phagocytosed significantly more by J774 cells and by PMNs. Opsonized bacteria were phagocytosed more than nonopsonized bacteria, and mutant bacteria lacking either YopH, -T, or -O were phagocytosed significantly more by J774 cells and by PMNs than were wild-type (WT) bacteria. Opsonized mutants lacking only YopE were phagocytosed significantly more than were WT bacteria by PMNs but not by J774 cells. Thus, YopH, -T, and -O were involved in all of the phagocytic processes studied here but YopE did not play a clear role in guarding against opsonic phagocytosis by J774. Mutants lacking YopP and YopM were, in every instance, as resistant as WT bacteria. Overexpression of YopE, -H, -T, or -O alone did not confer resistance to phagocytosis, although it affected the cytoskeleton. These results show that YopH, YopT, YopO, and, in some instances, YopE act synergistically to increase the resistance of Y. enterocolitica to phagocytosis by macrophages and PMNs.     

Anti-V antigen antibody protects macrophages from Yersinia pestis -induced cell death and promotes phagocytosis

The pathogenic Yersinia spp. harbor a common plasmid (pYV) essential for virulence. The plasmid encodes a type III secretion system that functions to translocate Yersinia outer proteins (Yops) into the host cytosol. Within the host cell, the Yops act to inhibit phagocytosis and induce apoptosis. One of the plasmid-encoded proteins, virulence antigen (V), is a major protective immunogen that is involved in Yop translocation. Yersinia pestis, like the enteric Yersinia spp., was both resistant to phagocytosis by and cytotoxic for J774.A1, a murine macrophage cell line. Both of these activities were dependent on culture of the bacteria at 37 degrees C for 1.5-2 h before infection. However, extending the preculture period at 37 degrees C to 24 h, which induced formation of a capsule, completely blocked cytotoxicity. Treating the bacteria with either rabbit polyclonal anti-V antibodies (R anti-V) or monoclonal antibody (MAb) 7.3, antibodies specific for V and protective against plague in vivo, protected J774.A1 cells from Y. pestis -induced cell death and also reversed the inhibition of phagocytosis. Whereas protection against cell cytotoxicity was afforded by the F(ab')(2) portion of R anti-V, the ability of anti-V to induce uptake of Y. pestis appeared to be dependent on the Fc portion of the Ab. The protective epitope(s) recognized by R anti-V was contained in the central region of Y. pestis V (aa 135-275) and were partially cross reactive with Y. pseudotuberculosis and Y. enterocolitica serotype 08 V antigens.     

Substrains of 129 mice are resistant to Yersinia pestis KIM5: implications for interleukin-10-deficient mice

Interleukin-10 (IL-10)-deficient mice are resistant to several pathogens, including Yersinia pestis. Surprisingly, we observed that heterozygous IL-10(+/-) mice also survive high-dose intravenous infection with Y. pestis KIM5 (Pgm(-)). Analysis of commercial IL-10(-/-) mice revealed that at least 30 cM of genomic DNA from the original 129 strain remains, including a functional Slc11a1 (Nramp1) gene. Interestingly, two substrains of 129 mice were resistant to high-dose Y. pestis KIM5. Resistance does not appear to be recessive, as F(1) mice (C57BL/6J x 129) also survived a high-dose challenge. A QTL-based genetic scan of chromosome 1 with 35 infected F(1) backcrossed mice revealed that resistance to KIM5 maps to a region near IL-10. Two novel IL-10(+/+) mouse strains which each possess most of the original 30-cM stretch of 129 DNA maintained resistance to high-dose infection with Y. pestis KIM5 even in a heterozygous state. Conversely, a novel IL-10(-/-) mouse strain in which most of the 129 DNA has been crossed out exhibited intermediate resistance to KIM5, while the corresponding IL-10(+/-) strain was completely susceptible. Taken together, these results demonstrate that 129-derived genomic DNA near IL-10 confers resistance to Yersinia pestis KIM5 and contributes to the observed resistance of IL-10(-/-) mice.     

Complete DNA Sequence and Detailed Analysis of the Yersinia pestis KIM5 Plasmid Encoding Murine Toxin and Capsular Antigen

Yersinia pestis, the causative agent of plague, harbors at least three plasmids necessary for full virulence of the organism, two of which are species specific. One of the Y. pestis-specific plasmids, pMT1, is thought to promote deep tissue invasion, resulting in more acute onset of symptoms and death. We determined the entire nucleotide sequence of Y. pestis KIM5 pMT1 and identified potential open reading frames (ORFs) encoded by the 100,990-bp molecule. Based on codon usage for known yersinial genes, homology with known proteins in the databases, and potential ribosome binding sites, we determined that 115 of the potential ORFs which we considered could encode polypeptides in Y. pestis. Five of these ORFs were genes previously identified as being necessary for production of the classic virulence factors, murine toxin (MT), and the fraction 1 (F1) capsule antigen. The regions of pMT1 encoding MT and F1 were surrounded by remnants of multiple transposition events and bacteriophage, respectively, suggesting horizontal gene transfer of these virulence factors. We identified seven new potential virulence factors that might interact with the mammalian host or flea vector. Forty-three of the remaining 115 putative ORFs did not display any significant homology with proteins in the current databases. Furthermore, DNA sequence analysis allowed the determination of the putative replication and partitioning regions of pMT1. We identified a single 2,450-bp region within pMT1 that could function as the origin of replication, including a RepA-like protein similar to RepFIB, RepHI1B, and P1 and P7 replicons. Plasmid partitioning function was located ca. 36 kb from the putative origin of replication and was most similar to the parABS bacteriophage P1 and P7 system. Y. pestis pMT1 encoded potential genes with a high degree of similarity to a wide variety of organisms, plasmids, and bacteriophage. Accordingly, our analysis of the pMT1 DNA sequence emphasized the mosaic nature of this large bacterial virulence plasmid and provided implications as to its evolution.

The facultative intracellular parasite Yersinia pestis harbors at least three plasmids, one of which is common to the enteropathogenic species Yersinia pseudotuberculosis and Yersinia enterocolitica (30, 72). The other two plasmids, designated pMT1 and pPCP1, are unique to Y. pestis (10) and are thought to promote the ability of this organism to penetrate deep tissues and to contribute to the acute infection associated with this species. In fact, the Y. pestis genome shares much homology with that of Y. pseudotuberculosis (2, 63), yet the infection caused by the latter organism is usually mild and self-limiting (15). Accordingly, a logical starting point to understanding the difference in the pathogenesis of Y. pestis and Y. pseudotuberculosis is to study the genes encoded on the plasmids unique to plague, pMT1 and pPCP1.The 9.5-kb plasmid pPCP1 encodes a bacteriocin termed pesticin, a pesticin immunity protein, and a plasminogen activator (89). Loss of this plasmid increases the 50% lethal dose of the organism by a factor of 10⁵ when the organism is injected subcutaneously in the mouse model of infection (90). The only characterized virulence determinant encoded by pPCP1, the plasminogen activator, has been implicated in deep tissue invasion by Y. pestis (11) and functions in the flea vector (58). These facts demonstrate that a plasmid, specifically harbored by Y. pestis, encodes a virulence factor necessary for the acute infection caused by the organism and that a single protein can influence the life cycle of the organism at multiple stages.The largest extrachromosomal element present in Y. pestis was commonly called the cryptic plasmid until 1983. Protsenko et al. (73) demonstrated that the capsular protein fraction 1 (F1) and the murine toxin (MT) were both encoded by the ∼100-kb element now called pMT1. The genes for each of the proteins have been cloned from Y. pestis EV76 and sequenced previously (36, 37, 49). Data addressing the involvement of these proteins in plague pathogenesis are open to interpretation since the effect that mutational loss has on the 50% lethal dose depends on the animal model used in the study as well as the route of infection (8, 9). However, pMT1 does appear to contribute to the acute phase of plague infection, as evidenced by the fact that strains lacking the 100-kb plasmid demonstrate reduced morbidity (27, 80, 96).Information pertaining to the genetic characterization of the pMT1 molecule is limited. The size of the plasmid has been found to vary from approximately 90 to 288 kb in size (31). Furthermore, pMT1 has been found to integrate at multiple sites into the chromosome of Y. pestis at high frequency (74), with speculation that the observed integration of pMT1 into the chromosome may have been due to IS100 homology between the two molecules. Both F1 and MT gene activation have been characterized in relation to environmental cues such as temperature and calcium (28). F1 capsule synthesis is maximal at 37°C in the absence of extracellular calcium while murine toxin expression is induced at 26°C. F1 expression is therefore maximum under conditions similar to those that induce the expression of one of the major virulence determinants of Y. pestis (91–93). In contrast, MT production is induced in an environment similar to that which Y. pestis would be expected to encounter in the flea vector. The presence of genes induced under these widely different conditions indicates the presence of at least two networks regulating expression of virulence determinants operating in plague.DNA-sequencing technology has progressed to the point that large amounts of genetic material can be sequenced in a relatively short time. Several facts make pMT1 a good candidate for large-scale DNA sequencing. First, the plasmid is unique to Y. pestis. Second, some derivative of the ∼100-kb plasmid is always present in clinical isolates (31). Third, we already know that genes regulated by two different environmental stimuli that mimic different environments encountered during the life cycle of plague are present on this molecule. Here, we report and annotate the entire DNA sequence of the pMT1 plasmid derived from the Y. pestis laboratory strain KIM.     

Role of Yops in inhibition of phagocytosis and killing of opsonized Yersinia enterocolitica by human granulocytes.

The virulence plasmid of Yersinia enterocolitica codes for the production of the outer membrane protein YadA and the secretion of several proteins, called Yops, which may play a role in the interaction between granulocytes and this bacterium. We investigated whether the expression of YadA or the secretion of Yops affected the phagocytosis and killing of opsonized Y. enterocolitica by human granulocytes. The rates of phagocytosis and killing of Y. enterocolitica by granulocytes in suspension in the presence of rabbit Yersinia antibodies and complement were determined by microbiological assays. In addition, noningested cell-adherent bacteria were differentiated from ingested yersiniae by immunofluorescence microscopy. Plasmid-bearing opsonized Y. enterocolitica was able to inhibit phagocytosis and killing by human granulocytes. The inhibition of phagocytosis was specific for the plasmid-bearing strain of Y. enterocolitica, since granulocytes were still able to phagocytose and kill Staphylococcus aureus in the presence of Y. enterocolitica. Plasmid-cured Y. enterocolitica was readily phagocytosed and killed by these cells. To investigate the role of YadA or Yops in the inhibition of phagocytosis by granulocytes, the phagocytosis of mutant strains unable to express YadA or to secrete Yops was studied. A Y. enterocolitica mutant unable to secrete Yops lost its ability to inhibit phagocytosis; a mutant expressing only YadA was readily ingested by granulocytes. These results indicate that after attachment of opsonized Y. enterocolitica to granulocytes, Yops play an important role in inhibiting the ingestion of Y. enterocolitica by human granulocytes.     

DNA Sequencing and Analysis of the Low-Ca2+-Response Plasmid pCD1 of Yersinia pestis KIM5

The low-Ca²⁺-response (LCR) plasmid pCD1 of the plague agent Yersinia pestis KIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenic Yersinia spp., that had been identified previously in one or more of Y. pestis or the enteropathogenic yersiniae Yersinia enterocolitica and Yersinia pseudotuberculosis. Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS100 and two new IS elements, IS1616 and IS1617) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case for Y. pestis EV76, and the gene for the lipoprotein YlpA found in Y. enterocolitica likely is a pseudogene in Y. pestis. The yopM gene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream of ypkA-yopJ is discussed as a potential Syc gene, and a previously undescribed ORF downstream of yopE was identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.     

Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis

Yersinia pestis, the causative agent of plague, expresses a capsule-like antigen, fraction 1 (F1), at 37 degrees C. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is unique to Y. pestis. F1 is a surface polymer composed of a protein subunit, Caf1, with a molecular mass of 15.5 kDa. The secretion and assembly of F1 require the caf1M and caf1A genes, which are homologous to the chaperone and usher protein families required for biogenesis of pili. F1 has been implicated to be involved in the ability of Y. pestis to prevent uptake by macrophages. In this study we addressed the role of F1 antigen in inhibition of phagocytosis by the macrophage-like cell line J774. The Y. pestis strain EV76 was found to be highly resistant to uptake by J774 cells. An in-frame deletion of the caf1M gene of the Y. pestis strain EV76 was constructed and found to be unable to express F1 polymer on the bacterial surface. This strain had a somewhat lowered ability to prevent uptake by J774 cells. Strain EV76C, which is cured for the virulence plasmid common to the pathogenic Yersinia species, was, as expected, much reduced in its ability to resist uptake. A strain lacking both the virulence plasmid and caf1M was even further hampered in the ability to prevent uptake and, in this case, essentially all bacteria (95%) were phagocytosed. Thus, F1 and the virulence plasmid-encoded type III system act in concert to make Y. pestis highly resistant to uptake by phagocytes. In contrast to the type III effector proteins YopE and YopH, F1 did not have any influence on the general phagocytic ability of J774 cells. Expression of F1 also reduced the number of bacteria that interacted with the macrophages. This suggests that F1 prevents uptake by interfering at the level of receptor interaction in the phagocytosis process.     

Resistance to phagocytosis by Yersinia

Enteropathogenic species of the genus Yersinia penetrate the intestinal epithelium and then spread to the lymphatic system, where they proliferate extracellularly. At this location, most other bacteria are effectively ingested and destroyed by the resident phagocytes. Yersinia, on the other hand binds to receptors on the external surface of phagocytes, and from this location it blocks the capacity of these cells to exert their phagocytic function via different receptors. The mechanism behind the resistance to phagocytosis involves the essential virulence factor YopH, a protein tyrosine phosphatase that is translocated into interacting target cells via a type III secretion machinery. YopH disrupts peripheral focal complexes of host cells, seen as a rounding up of infected cells. The focal complex proteins that are dephosphorylated by YopH are focal adhesion kinase and Crk-associated substrate, the latter of which is a common substrate in both professional and non-professional phagocytes. In macrophages additional substrates have been found, the Fyn-binding/SLP-76-associated protein and SKAP-HOM. Phagocytosis is a rapid process that is activated when the bacterium interacts with the phagocyte. Consequently, the effect exerted by a microbe to block this process has to be rapid and precise. This review deals with the mechanisms involved in impeding uptake as well as with the role of the YopH substrates and focal complex structures in normal cell function.     

Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ

Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1beta (IL-1beta) in na?ve macrophages infected with Yersinia enterocolitica. Na?ve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1beta was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1beta following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1beta were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages.     

Diagnosis of Yersinia pestis

There is no plaque case report in Taiwan since 1952. However, it is necessary to set up a laboratory system to investigate the distribution of Yersinia pestis in the natural environment to implement the public policy for preventing plague. Besides the traditional methods; e.g. culture, microscopic observation, biochemical characteristics, anti-F1 antigen detection by slide agglutination, immunofluorescence, and phage lytic assay, PCR was used as rapid screening test in our study. These laboratory methods were used to examine whether the flea samples harvested in King-Men island carry Y. pestis. The results showed that the flea index per mouse was high but no Y. pestis was detected in the fleas.     

Yops of Yersinia enterocolitica inhibit receptor-dependent superoxide anion production by human granulocytes

The virulence plasmid-borne genes encoding Yersinia adhesin A (YadA) and several Yersinia secreted proteins (Yops) are involved in the inhibition of phagocytosis and killing of Yersinia enterocolitica by human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis of Y. enterocolitica by human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing (pYV+) Y. enterocolitica inhibits O2- production by human granulocytes in response to various stimuli and whether YopH is involved. Granulocytes were preincubated with mutant strains unable to express YadA or to secrete Yops or YopH. O2- production by granulocytes during stimulation was assessed by measuring the reduction of ferricytochrome c. PYV+ Y. enterocolitica inhibited O2- production by granulocytes incubated with opsonized Y. enterocolitica or N-formyl-Met-Leu-Phe (f-MLP). This inhibitory effect mediated by pYV did not affect receptor-independent O2- production by granulocytes in response to phorbol myristate acetate, indicating that NADPH activity remained unaffected after activation of protein kinase C. The inhibition of f-MLP-induced O2- production by granulocytes depends on the secretion of Yops and not on the expression of YadA. Insertional inactivation of the yopH gene abrogated the inhibition of phagocytosis of antibody- and complement-opsonized Y. enterocolitica by human granulocytes but not of the f-MLP-induced O2- production by granulocytes or tyrosine phosphorylation of granulocyte proteins. These findings suggest that the specific targets for YopH are not present in f-MLP receptor-linked signal transduction and that other Yop-mediated mechanisms are involved.     

鼠疫耶尔森菌与表面抗体相互作用的原子力显微镜观测研究

目的 以原子力显微镜(atomic force microscopy,AFM)观察比较鼠疫耶尔森菌EV76株与正常兔血清以及兔抗F1抗体反应后微观形态变化,探讨以原子力显微镜为工具的鼠疫耶尔森菌的免疫检测方法.方法 用兔抗F1抗体及正常兔血清处理鼠疫耶尔森菌菌液,并与对照菌一起制样后在原子力显微镜下观察鼠疫耶尔森菌的表面结构的改变,并对其主要指标,包括Ra、Rq改变进行测量比较.结果 正常菌体组细胞呈椭圆形,两端钝圆,长为1.1～1.3 μm,宽为0.8～1.0 μm,阶高为0.04～0.06 μm,细胞形状规则,表面相对比较光滑;对照抗体及F1抗体加入组,细菌阶高均明显增高;F1抗体结合株菌体形状不规则,表面粗糙度明显增加.结论 获得原子力显微镜下的鼠疫耶尔森菌形态特征;表面抗体与鼠疫耶尔森菌结合后,对鼠疫耶尔森菌的表面超微结构有显著的影响,其中粗糙度可以作为原子力显微镜免疫检测的指标.     

Yersinia pseudotuberculosis inhibits Fc receptor-mediated phagocytosis in J774 cells.

Nonopsonized as well as immunoglobulin-G (IgG)-opsonized Yersinia pseudotuberculosis resists phagocytic uptake by the macrophage-like cell line J774 by a mechanism involving the plasmid-encoded proteins Yops. The tyrosine phosphatase YopH was of great importance for the antiphagocytic effect of the bacteria. YopH-negative mutants did not induce antiphagocytosis; instead, they were readily ingested, almost to the same extent as that of the translocation mutants YopB and YopD and the plasmid-cured strain. The bacterial determinant invasin was demonstrated to mediate phagocytosis of nonopsonized bacteria by these cells. In addition to inhibiting uptake of itself, Y. pseudotuberculosis also interfered with the phagocytic uptake of other types of prey: J774 cells that had been exposed to virulent Y. pseudotuberculosis exhibited a reduced capacity to ingest IgG-opsonized yeast particles. This effect was impaired when the bacterium-phagocyte interaction occurred in the presence of gentamicin, indicating a requirement for in situ bacterial protein synthesis. The Yersinia-mediated antiphagocytic effect on J774 cells was reversible: after 18 h in the presence of gentamicin, the phagocytic capacity of Yersinia-exposed J774 cells was completely restored. Inhibition of the uptake of IgG-opsonized yeast particles was dependent on the Yops in a manner similar to that seen for blockage of Yersinia phagocytosis. This similarity suggests that the pathogen affected a general phagocytic mechanism. Despite a marked reduction in the capacity to ingest IgG-opsonized yeast particles, no effect was observed on the binding of the prey. Taken together, these results demonstrate that Yop-mediated antiphagocytosis by Y. pseudotuberculosis affects regulatory functions downstream of the phagocytic receptor and thereby extends to other types of phagocytosis.     

Molecular typing of Yersinia pestis

The present review is focused on methods of Yersinia pestis genotyping that are reproducible in different laboratories and allow for differentiation of individual bacterial isolates into intraspecies groups corresponding to subspecies, biovars, and natural foci. A variant of the intraspecies classification of Y. pestis compliant with the rules of the International Code of Nomenclature of Bacteria is presented.     

5′ Nuclease PCR Assay To Detect Yersinia pestis

The 5′ nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5′ nuclease PCR assay targeting the plasminogen activator gene (pla) of Yersinia pestis. The assay is species specific, with a detection threshold of 2.1 × 10⁵ copies of the pla target or 1.6 pg of total cell DNA. The assay detected Y. pestis in experimentally infected Xenopsylla cheopis fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.     

Characterization of chromosomal regions conserved in Yersinia pseudotuberculosis and lost by Yersinia pestis

The transformation of the enteropathogenic bacterium Yersinia pseudotuberculosis into the plague bacillus, Yersinia pestis, has been accompanied by extensive genetic loss. This study focused on chromosomal regions conserved in Y. pseudotuberculosis and lost during its transformation into Y. pestis. An extensive PCR screening of 78 strains of the two species identified five regions (R1 to R5) and four open reading frames (ORFs; orf1 to orf4) that were conserved in Y. pseudotuberculosis and absent from Y. pestis. Their conservation in Y. pseudotuberculosis suggests a positive selective pressure and a role during the life cycle of this species. Attempts to delete two ORFs (orf3 and orf4) from the chromosome of strain IP32953 were unsuccessful, indicating that they are essential for its viability. The seven remaining loci were individually deleted from the IP32953 chromosome, and the ability of each mutant to grow in vitro and to kill mice upon intragastric infection was evaluated. Four loci (orf1, R2, R4, and R5) were not required for optimal growth or virulence of Y. pseudotuberculosis. In contrast, orf2, encoding a putative pseudouridylate synthase involved in RNA stability, was necessary for the optimal growth of IP32953 at 37°C in a chemically defined medium (M63S). Deletion of R1, a region predicted to encode the methionine salvage pathway, altered the mutant pathogenicity, suggesting that the availability of free methionine is severely restricted in vivo. R3, a region composed mostly of genes of unknown functions, was necessary for both optimal growth of Y. pseudotuberculosis at 37°C in M63S and for virulence. Therefore, despite their loss in Y. pestis, five of the nine Y. pseudotuberculosis-specific chromosomal loci studied play a role in the survival, growth, or virulence of this species.     

Detection of Yersinia pestis in sputum by real-time PCR

A 5' nuclease PCR assay for detection of the Yersinia pestis plasminogen activator (pla) gene in human respiratory specimens with simulated Y. pestis infection was developed. An internal positive control was added to the reaction mixture in order to detect the presence of PCR inhibitors that are often found in biological samples. The assay was 100% specific for Y. pestis. In the absence of inhibitors, a sensitivity of 10(2) CFU/ml of respiratory fluid was obtained. When inhibitors were present, detection of Y. pestis DNA required a longer sample treatment time and an initial concentration of bacteria of at least 10(4) CFU/ml. The test's total turnaround time was less than 5 h. The assay described here is well suited to the rapid diagnosis of pneumonic plague, the form of plague most likely to result from a bioterrorist attack.     

Mutations Influencing the Assimilation of Nitrogen by Yersinia pestis

Cells of 20 isolates of Yersinia (Pasteurella) pestis exhibited an unusual nutritional requirement which could be fulfilled by glycine or l-threonine. Meiotrophic mutants which required neither of these amino acids (Gly/Thr(+)) were isolated from cultures of all 20 strains at a frequency of 10(-7). Wild-type and Gly/Thr(+) cells of 14 strains failed to utilize l-amino acids or urea (0.01 m) as primary sources of nitrogen and grew slowly in the presence of low concentrations of NH(4) (+) (<== 5 mm). Cells of six strains (termed N(+)) utilized certain l-amino acids and urea (0.01 m) as primary sources of nitrogen and grew rapidly in the presence of <== 5 mm NH(4) (+). N(+) but not N(-) organisms cultivated with NH(4) (+) (0.01 m) as a primary source of nitrogen excreted a complete spectrum of naturally occurring amino acids; under this condition of growth the aspartase and particulate nicotinamide adenine dinucleotide phosphate transhydrogenase activities of N(+) and N(-) cells were repressed. N(+) meiotrophs arose at a frequency of 10(-6) in cultures of all 14 N(-) isolates, and urease-positive meiotrophs could be selected at a frequency of 10(-7) from N(+) but not N(-) cells of all 20 strains on a medium containing urea (0.01 m) as a primary source of nitrogen. These findings illustrate a reversible loss of genetic potential which has occurred during the evolution of Y. pestis as an obligate parasite and suggest that this organism is unable to efficiently remove dispensable deoxyribonucleic acid from its chromosome.