Physiology of Yersinia pestis.

This chapter outlines the physiology of Yersinia pestis with emphasis on identifying unique functions required for tissue invasion and acute disease. These activities are opposed to often incompatible processes expressed by very closely related Yersinia pseudotuberculosis, which causes localized gastrointestinal infection. Gain of new information in Y. pestis entailed lateral transfer of plasminogen activator and anti-phagocytic capsular antigen via the plasmids pPCP and pMT, respectively, and derepression of the pigmentation locus facilitating colonization of the flea vector. The ability to survive in austere natural environments became unnecessary following mastery of the closed flea-rodent-flea life cycle permitting concomitant chromosomal degeneration (large and small deletions, additions, inversions, translocations, transposon inserts, and single base substitutions causing nonsense and missense mutations). Consequently, modern Y. pestis lacks a functional pentose-phosphate pathway, glyoxylate bypass, and is unable to directly catabolize L-aspartate and close metabolic derivatives directly via the tricarboxylic acid cycle. The missing gene products accounting for these and numerous other metabolic lesions are now well-established. This group includes formyltetrahydrofolate deformylase (PurU) required for synthesis of glycine. This deficiency is associated with a dramatic ability of Y. pestis to catabolize L-serine, required by the host to initiate methylation of DNA (necessary in turn to initiate successful innate immune processes leading to delayed-type hypersensitivity).

Recent findings regarding maintenance of enzootic variants of Yersinia pestis in sylvatic reservoirs and their significance in the evolution of epidemic plague.

Despite the widespread presence of bubonic plague in sylvatic reservoirs throughout the world, the causative agent (Yersinia pestis) evolved in its present form within the last 20,000 years from enteropathogenic Yersinia pseudotuberculosis. Comparison of the genomes from the two species revealed that Y. pestis possesses only a few unique plasmid-encoded genes that contribute to acute disease, whereas this organism has lost about 13% of the chromosomal genes that remain active in Y. pseudotuberculosis. These losses reflect readily detectable additions, deletions, transpositions, inversions, and acquisition of about 70 insertion sequence (IS) inserts, none of which are likely to promote increased virulence. In contrast, major enzymes of intermediary metabolism, including glucose 6-phosphate dehydrogenase (Zwf ) and aspartase, are present but not catalytically functional due to the presence of missense mutations. The latter are generally not detectable by the technology of bioinformatics and, in the case of Y. pestis, result in radical changes in the metabolic flow of carbon. As an important consequence, plague bacilli exhibit a stringent low-calcium response characterized by conversion of L-glutamate (and metabolically related amino acids) to L-aspartate with secretion of the latter into supernatant fluid at 37 degrees C in culture media containing Na(+) but lacking added Ca(2+). This phenomenon also occurs in vivo and likely adversely affects the bioenergetics of host amino acid pools. Curiously, aspartase is functional in all tested enzootic (pestoides) strains of Y. pestis. These isolates are typically restricted to the ancient plague reservoirs of Central Asia and Africa and are fully virulent in members of the rodent Superfamily Muroidea but avirulent in guinea pigs and man. The implications of these findings for the distribution and ecology of Y. pestis could be significant.

Growth of calcium-blind mutants of Yersinia pestis at 37 degrees C in permissive Ca2+-deficient environments.

Cells of wild-type Yersinia pestis exhibit a low-calcium response (LCR) defined as bacteriostasis with expression of a pCD-encoded type III secretion system (T3SS) during cultivation at 37 degrees C without added Ca(2+) versus vegetative growth with downregulation of the T3SS with Ca(2+) (>or=2.5 mM). Bacteriostasis is known to reflect cumulative toxicity of Na(+), l-glutamic acid and culture pH; control of these variables enables full-scale growth ('rescue') in the absence of Ca(2+). Several T3SS regulatory proteins modulate the LCR, because their absence promotes a Ca(2+)-blind phenotype in which growth at 37 degrees C ceases and the T3SS is constitutive even with added Ca(2+). This study analysed the connection between the LCR and Ca(2+) by determining the response of selected Ca(2+)-blind mutants grown in Ca(2+)-deficient rescue media containing Na(+) plus l-glutamate (pH 5.5), where the T3SS is not expressed, l-glutamate alone (pH 6.5), where l-aspartate is fully catabolized, and Na(+) alone (pH 9.0), where the electrogenic sodium pump NADH : ubiquinone oxidoreductase becomes activated. All three conditions supported essentially full-scale Ca(2+)-independent growth at 37 degrees C of wild-type Y. pestis as well as lcrG and yopN mutants (possessing a complete but dysregulated T3SS), indicating that bacteriostasis reflects a Na(+)-dependent lesion in bioenergetics. In contrast, mutants lacking the negative regulator YopD or the YopD chaperone (LcrH) failed to grow in any rescue medium and are therefore truly temperature-sensitive. The Ca(2+)-blind yopD phenotype was fully suppressed in a Ca(2+)-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD.

Attenuated enzootic (pestoides) isolates of Yersinia pestis express active aspartase.

It is established that Yersinia pestis, the causative agent of bubonic plague, recently evolved from enteropathogenic Yersinia pseudotuberculosis by undergoing chromosomal degeneration while acquiring two unique plasmids that facilitate tissue invasion (pPCP) and dissemination by fleabite (pMT). Thereafter, plague bacilli spread from central Asia to sylvatic foci throughout the world. These epidemic isolates exhibit a broad host range including man as opposed to enzootic (pestoides) variants that remain in ancient reservoirs where infection is limited to muroid rodents. Cells of Y. pseudotuberculosis are known to express glucose-6-phosphate dehydrogenase (Zwf) and aspartase (AspA); these activities are not detectable in epidemic Y. pestis due to missense mutations (substitution of proline for serine at amino position 155 of Zwf and leucine for valine at position 363 of AspA). In this study, functional Zwf was found in pestoides strains E, F and G but not seven other enzootic isolates; enzymic activity was associated with retention of serine at amino acid position 155. Essentially, full AspA activity occurred in pestoides isolates where valine (pestoides A, B, C and D) or serine (pestoides E, F, G and I) occupied position 363. Reduced activity occurred in strains Angola and A16, which contained phenylalanine at this position. The kcat but not Km of purified AspA from strain Angola was significantly reduced. In this context, aspA of the recently described attenuated enzootic microtus biovar encodes active valine at position 363, further indicating that functional AspA is a biomarker for avirulence of Y. pestis in man.

Pneumonic plague pathogenesis and immunity in Brown Norway rats.

The Brown Norway rat was recently described as a bubonic plague model that closely mimics human disease. We therefore evaluated the Brown Norway rat as an alternative small animal model for pneumonic plague and characterized both the efficacy and potency of vaccine candidates. When infected by intranasal instillation, these rats rapidly developed fatal pneumonic plague within 2 to 4 days of infection. Plague disease was characterized by severe alveolar edema and vascular hemorrhage in the lung in addition to fulminant necrotizing pneumonia caused by massive bacterial replication and inflammation. Twenty-four hours before death, animals developed systemic disease with an apparent delayed inflammatory response. We evaluated the ability of the protective antigen, LcrV, and a mutant derivative, V10, to protect these rats from pneumonic plague. Both were highly effective vaccines because complete protection was observed at challenge doses of 7500 LD(50). Antibody analyses suggested stronger potency of V10 immune sera compared with LcrV in the passive transfer of immunity to bubonic plague, with multiple neutralizing epitopes in LcrV. Taken together, these data demonstrate the effectiveness of inhibiting type III secretion in the prevention of pneumonic plague in rats and reveal critical contributions from both the cellular and humoral immune systems. Thus, the Brown Norway rat is an appealing alternative small animal model for the study of pneumonic plague pathogenesis and immunity.

A missense mutation causes aspartase deficiency in Yersinia pestis.

It is established that cells of Yersinia pestis, the causative agent of bubonic plague, excrete l-aspartic acid at the expense of exogenous l-glutamic acid during expression of the low-calcium response. Results of enzymic analysis provided here suggest that a previously defined deficiency of aspartase (AspA) accounts for this phenomenon rather than an elevated oxaloacetate pool. The only known distinction between most sequenced isolates of aspA from Y. pestis and the active gene in Yersinia pseudotuberculosis (the immediate progenitor of Y. pestis) is a single base transversion (G.C-->T.A) causing replacement of leucine (encoded by UUG) for valine (encoded by GUG) at amino acid position 363. The gene from Y. pestis KIM possesses a unique second transversion (G.C-->T.A) at amino acid 146 causing substitution of aspartic acid (encoded by GAU) with tyrosine (encoded by UAU). We show in this study that Y. pestis expresses aspA as cross-reacting immunological material (CRIM). Functional and inactive aspA of Y. pseudotuberculosis PB1 and Y. pestis KIM, respectively, were then cloned and expressed in AspA-deficient Escherichia coli. After purification to near homogeneity, the products were subjected to biochemical analysis and found to exhibit similar secondary, tertiary and quaternary (tetrameric) structures as well as comparable Michaelis constants for l-aspartic acid. However, the k(cat) of the Y. pestis CRIM of strain KIM is only about 0.1 % of that determined for the active AspA of Y. pseudotuberculosis. Return of valine for leucine at position 363 of the Y. pestis enzyme restored normal turnover (k(cat) 86+/-2 s(-1)) provided that the amino acid substitution at position 146 was also reversed. These observations have important implications for understanding the nature of the stringent low-calcium response of Y. pestis and its role in promoting acute disease.

Intermediary metabolism, Na+, the low calcium-response, and acute disease.

The variables carriage of pCD, CO2 tension, exogenous ATP, L-glutamate, Mg2+, Na+, pH, source of energy, and temperature are known to modulate the low calcium response of Yersinia pestis in vitro. The role of these effectors and the basis of their interactions are defined here with emphasis on known Y. pestis-specific missense mutations in glucose 6-phosphate dehydrogenase and aspartase, which preclude use of the hexose monophosphate pathway and prevent efficient catabolism of L-glutamic acid, respectively. A physiological Ca2+-deficient rescue scenario is provided that permits essentially full-scale growth of virulent Y. pestis (<0.1 mM Na+ and 25 mM L-glutamate at pH 6.5) with expression of pCD-encoded virulence effectors and their attendant type III secretion system. Multiplication in this environment indicates that Ca2+ prevents innate toxicity of Na+. However, Na+ actually promotes growth in Ca2+-deficient medium at pH 9.0 due to the evident action of Na+-translocating NADH-ubiquinone oxidoreductase. Another Ca2+-deficient rescue scenario (100 mM Na+ and 25 mM L-glutamate at pH 5.5) permitted growth while downregulating pCD-encoded functions. A consequence of the abrupt Na+-mediated bacteriostasis typical of aspartase-deficient Y. pestis is conversion of L-glutamate to L-aspartate with release of the latter into culture supernatant fluids. Occurrence of this event in vivo would radically alter the equilibrium of host amino acid pools thereby contributing to enhanced lethality.

How the structural gene products of Yersinia pestis relate to virulence.

Bubonic plague is the most devastating acute infectious disease known to man. The causative agent, Yersinia pestis, is now more firmly entrenched in sylvatic reservoirs throughout the world than at any time in the past. Consequently, the organism increasingly causes casual human disease and is readily available for use as a bioweapon. Recent attempts to understand the severe nature of plague have focused upon its very recent divergence from Yersinia pseudotuberculosis, an etiological instrument of chronic enteropathogenic infection. This review emphasizes that the invasive nature of plague and its dissemination by fleabite is mediated by plasmids not shared by enteropathogenic yersiniae. The basis for high lethality is considered within the context of chromosomal degeneration causing loss of normal metabolic functions and modification of virulence factors, permitting a terminal anti-inflammatory phase associated with pronounced septicemia.

Attachment of LcrV from Yersinia pestis at dual binding sites to human TLR-2 and human IFN-gamma receptor.

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-gamma or a synthetic C-terminal fragment (hIFN-gamma132-143). The latter, but not mouse IFN-gamma (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-alpha in human target cells. The ability of LcrV to utilize human IFN-gamma (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.

Influence of Na(+), dicarboxylic amino acids, and pH in modulating the low-calcium response of Yersinia pestis.

The virulence of yersiniae is promoted in part by shared approximately 70-kb plasmids (pCD in Yersinia pestis and pYV in enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica) that mediate a low-calcium response. This phenotype is characterized at 37 degrees C by either bacteriostasis in Ca(2+)-deficient medium with expression of pCD/pYV-encoded virulence effectors (Yops and LcrV) or vegetative growth and repression of Yops and LcrV with > or =2.5 mM Ca(2+) (Lcr(+)). Regulation of Yops and LcrV is well defined but little is known about bacteriostasis other than that Na(+) plus l-glutamate promotes prompt restriction of Y. pestis. As shown here, l-aspartate substituted for l-glutamate in this context but only Na(+) exacerbated the nutritional requirement for Ca(2+). Bacteriostasis of Y. pestis (but not enteropathogenic yersiniae) was abrupt in Ca(2+)-deficient medium at neutral to slightly alkaline pH (7.0 to 8.0), although increasing the pH to 8.5 or 9.0, especially with added Na(+) (but not l-glutamate), facilitated full-scale growth. Added l-glutamate (but not Na(+)) favored Ca(2+)-independent growth at acidic pH (5.0 to 6.5). Yops and LcrV were produced in Ca(2+)-deficient media at pH 6.5 to 9.0 regardless of the presence of added Na(+) or l-glutamate, although their expression at alkaline pH was minimal. Resting Ca(2+)-starved Lcr(+) cells of Y. pestis supplied with l-glutamate first excreted and then destroyed l-aspartate. These findings indicate that expression of Yops and LcrV is necessary but not sufficient for bacteriostasis of Ca(2+)-starved yersiniae and suggest that abrupt restriction of Y. pestis requires Na(+) and the known absence of aspartate ammonia-lyase in this species.

LcrV plague vaccine with altered immunomodulatory properties.

Yersinia pestis, the causative agent of plague, secretes LcrV (low-calcium-response V or V antigen) during infection. LcrV triggers the release of interleukin 10 (IL-10) by host immune cells and suppresses proinflammatory cytokines such as tumor necrosis factor alpha and gamma interferon as well as innate defense mechanisms required to combat the pathogenesis of plague. Although immunization of animals with LcrV elicits protective immunity, the associated suppression of host defense mechanisms may preclude the use of LcrV as a human vaccine. Here we show that short deletions within LcrV can reduce its immune modulatory properties. An LcrV variant lacking amino acid residues 271 to 300 (rV10) elicited immune responses that protected mice against a lethal challenge with Y. pestis. Compared to full-length LcrV, rV10 displayed a reduced ability to release IL-10 from mouse and human macrophages. Furthermore, the lipopolysaccharide-stimulated release of proinflammatory cytokines by human or mouse macrophages was inhibited by full-length LcrV but not by the rV10 variant. Thus, it appears that LcrV variants with reduced immune modulatory properties could be used as a human vaccine to generate protective immunity against plague.

Temporal global changes in gene expression during temperature transition in Yersinia pestis.

DNA microarrays encompassing the entire genome of Yersinia pestis were used to characterize global regulatory changes during steady-state vegetative growth occurring after shift from 26 to 37 degrees C in the presence and absence of Ca2+. Transcriptional profiles revealed that 51, 4, and 13 respective genes and open reading frames (ORFs) on pCD, pPCP, and pMT were thermoinduced and that the majority of these genes carried by pCD were downregulated by Ca2+. In contrast, Ca2+ had little effect on chromosomal genes and ORFs, of which 235 were thermally upregulated and 274 were thermally downregulated. The primary consequence of these regulatory events is profligate catabolism of numerous metabolites available in the mammalian host.

Evolutionary genetics: CCR5 mutation and plague protection.

A recent and prevalent mutation in the chemokine receptor CCR5 in humans of northern European ancestry has been proposed to provide protection against bubonic plague. Here we infect both normal and CCR5-deficient mice with the bacterium Yersinia pestis, the cause of the plague epidemics that wiped out one-third of Europeans in the Middle Ages, and find no difference in either bacterial growth or survival time between the two groups. Unless the pathogenesis of Yersinia infection differs markedly between mice and humans, our results indicate that CCR5 deficiency in people is unlikely to protect against plague.

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.

Genetic variability of Yersinia pestis isolates as predicted by PCR-based IS100 genotyping and analysis of structural genes encoding glycerol-3-phosphate dehydrogenase (glpD).

A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed.