Suppression of mouse skin allograft rejection by protein A-Yersiniae V antigen fusion peptide.

V antigen is an established virulence factor of Yersinia pestis, the causative agent of bubonic plague. Injection of homogenous staphylococcal protein A-V antigen fusion peptide into mice was previously found to suppress tumor necrosis factor-alpha and interferon-gamma necessary for generation of protective granulomas. Here, we show that BALB/c mice receiving daily intraperitoneal injections of 100 microg of control protein A initiated rejection of C57BL/6 mouse tail skin grafts after 6.2+/-1.1 days. This time doubled to 12.2+/-1.4 days upon similar administration of protein A-V antigen fusion peptide (P<0.001); times of total allograft retention remained constant. This finding indicates that V antigen can postpone inflammation known to be associated with recognition and destruction of foreign tissue by T lymphocytes.

Mutagenic properties of cis-plantinum(II)diammino-dichloride in Escherichia coli.

cis-Platinum (II)diamminodichloride (PDD), an anti-tumor agent, induced auxotrophic mutations in Escherichia coli, some of which were reverted to prototrophy by exposure to PDD, 2-aminopurine (2-AP), and N-methyl-N'-nitro-N-mitrosoguanidine (NTG), but not ICR derivatives. Similarly, various 2-AT-, NTG-, and ultraviolet light-induced auxotrophs were reverted to prototrophy by PDD. Some PDD-induced auxotrophs carried nonsense mutations and others could be phenotypically suppressed growth with streptomycin. Although these findings suggest that PDD promotes base substitutions, this mutagen may also cause base subtractions because (like NTG) it induced, at reduced frequency, reversion to prototrophy of certain ICR-induced auxotrophs. Isomeric trans-platinum (II)diamminodichloride, which lacks anti-tumor activity, was an ineffective mutagen. Near-optimal conditions for PDD-induced mutagenesis entailed prolonged cultivation with low levels of mutagen where the frequency of forward mutation to auxotrophy was 10-3 and that of a selected trp isolate to prototrophy was 10-2.

Metabolism of carbohydrates by Pasteurella pseudotuberculosis.

Cell-free extracts of Pasteurella pseudotuberculosis and P. pestis catalyzed a rapid and reversible exchange of electrons between pyridine nucleotides. Although the extent of this exchange approximated that promoted by the soluble nicotinamide adenine dinucleotide (phosphate) transhydrogenase of Pseudomonas fluorescens, the reaction in the pasteurellae was associated with a particulate fraction and was not influenced by adenosine-2'-monophosphate. The ability of P. pseudotuberculosis to utilize this system for the maintenance of a large pool of nicotinamide adenine dinucleotide phosphate could not be correlated with significant participation of the Entner-Doudoroff path or catabolic use of the hexose-monophosphate path during metabolism of glucose. As judged by the distribution of radioactivity in metabolic pyruvate, glucose and gluconate were fermented via the Embden-Meyerhof and Entner-Doudoroff paths, respectively. With the exception of hexosediphosphatase, all enzymes of the three paths were detected, although little or no gluconokinase or phosphogluconate dehydrase was present unless the organisms were cultivated with gluconate. The significance of these findings is discussed with respect to the regulation of carbohydrate metabolism in the pasteurellae, related enteric bacteria, and P. fluorescens.

The Vwa+ virulence factor of yersiniae: the molecular basis of the attendant nutritional requirement for Ca++.

The plague V antigen is a cytoplasmic 90,000-dalton peptide, and the W antigen is a primarily extracellular 145,000-dalton lipoprotein of unknown origin. Wild-type yersiniae undergo restriction of cell division when producing these plasmid-mediated virulence antigens (Vwa+) during cultivation at 37 C in Ca++-deficient media. In Yersinia pestis both restriction and synthesis of V and W antigens are potentiated in this environment by elevated Mg++ and prevented by Ca++ or exogenous nucleoside triphosphates. Restriction occurs by mechanisms that also are involved in nutritional stepdown (shutoff of stable RNA synthesis with reduction of nucleotide pools). Attendant regulatory mechanisms remain undefined; they are independent of MS nucleotides but may involve V antigen. Restriction is similar in Vwa+ Yersinia pseudotuberculosis and Yersinia enterocolitica, which, unlike Y. pestis, produce ancillary outer membrane peptides. Synthesis of these activities is dependent on elevated temperature but may not be influenced by Ca++; their presence is associated with spontaneous autoagglutination in vitro.

Factors promoting acute and chronic diseases caused by yersiniae.

The experimental system constructed with the medically significant yersiniae provides a powerful basic model for comparative study of factors required for expression of acute versus chronic disease. The system exploits the close genetic similarity between Yersinia pestis, the etiological agent of bubonic plague, and enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica. Y. pestis possesses three plasmids, of which one, shared by the enteropathogenic species, mediates a number of virulence factors that directly or indirectly promote survival within macrophages and immunosuppression. The two remaining plasmids are unique and encode functions that promote acute disease by enhancing bacterial dissemination in tissues and resistance to phagocytosis by neutrophils and monocytes. These properties are replaced in the enteropathogenic yersiniae by host cell invasins and an adhesin which promote chronic disease; the latter are cryptic in Y. pestis. Additional distinctions include specific mutational losses in Y. pestis which result in loss of fitness in natural environments plus gain of properties that facilitate transmission and infection via fleabite.

Mechanisms of bacterial virulence.

In this review the nature of prokaryotic parasites was first discussed with emphasis on the evolution of virulence. Subsequently, nonspecific mechanisms of host defense were considered with emphasis on recent findings relating to bacterial killing by serum and professional phagocytes. Based on this background, the nature of virulence factors required for growth of pathogens in the nonimmune host was considered. Strategies used by extracellular and intracellular parasites were compared. It is evident from the resulting overview of experimental findings that knowledge concerning virulence of extracellular parasites outweighs that collected for both facultative and obligate intracellular parasites. Remaining problems regarding extracellular parasitism include precise resolution of the nature of serum resistance, pilus-independent adhesion, tissue invasiveness, and resistance to phagocytosis. Solutions to these questions will probably arise during the course of studies primarily emphasizing bacterial structure and function. Unresolved problems concerning intracellular parasites include definition of regulatory changes involved in adaptation for intra- and extracellular growth, the nature of reactions preventing phagosome-lysosome fusion, mechanisms of survival within phagolysosomes, and explanations for host-cell dependence. These topics provide real problems in cellular and molecular biology, and they will probably be resolved by those familiar with these disciplines. The ability of parasitic prokaryotes to shut off otherwise effective specific immune responses was shown to cross phenotypic lines. Resolution of these somewhat sinister mechanisms of virulence will require an understanding of fundamental immune processes. Further study of bacterial virulence factors will probably provide an understanding of basic cellular processes relevant to other biological disciplines. Indeed, information of this nature may not be obtainable by any other experimental approach.

The recent emergence of plague: a process of felonious evolution.

Yersinia pestis, the causative agent of bubonic plague, evolved from closely related Yersinia pseudotuberculosis within the past 20,000 years, an event that corresponds to the end of the last ice age and distribution of Homo sapiens throughout the world. Y. pseudotuberculosis causes chronic but generally mild enteropathogenic infections whereas plague is the most devastating acute disease experienced by mankind. The very recent evolution of plague from its progenitor assures close genomic homogeneity between the two species and thus high probability that disparities in DNA sequence mediate dramatic differences in symptoms of infection. The purpose of this minireview is to define salient distinctions between the genomes of Y. pestis and Y. pseudotuberculosis and to equate unique functions to respective acute and chronic mechanisms of virulence. The significance of these processes is then related to the procedures the organisms use to survive when between hosts (i.e., the flea vector colonized by Y. pestis and natural environments including soil and water in the case of Y. pseudotuberculosis). Next, an attempt is made to order the various mutational events that caused the recent emergence of Y. pestis as a distinct species. Finally, selective pressures such as predatory soil nematodes are considered that possibly influenced the early evolution of those yersiniae now pathogenic to humans.

Interconversion of Purine Mononucleotides in Pasteurella pestis.

Included among the five established determinants of virulence in Pasteurella pestis are the abilities of cells to accomplish the de novo biosynthesis of purines and to grow as dark pigmented (P(+)) colonies on a solid synthetic medium containing hemin. P(+) isolates of P. pestis strain KIM-10 (mouse intraperitoneal ld(50) < 10 cells) failed to convert exogenous guanine-8-(14)C to adenine residues of ribonucleic acid (RNA) when cultivated in a minimal medium which favored the pigmentation reaction. This conversion occurred in P(+) cells grown in an enriched medium which did not support the pigmentation reaction and was observed in P(-) mutants cultivated in both types of media. Both P(+) and P(-) isolates converted exogenous adenine-8-(14)C but not adenine-2-(14)C at a significant rate to guanosine residues of RNA when grown under a variety of conditions. This difference appeared to reflect a deficiency of adenine deaminase. The mouse intraperitoneal ld(50) of purine-auxotrophs was about 10(2) cells when the metabolic block occurred prior to the de novo formation of inosine monophosphate (IMP). In contrast, the corresponding value for a mutant blocked between IMP and guanine monophosphate was > 10(7) cells in mice and > 10(8) cells in guinea pigs.

Post-translational regulation of Lcr plasmid-mediated peptides in pesticinogenic Yersinia pestis.

The low calcium response of wild type Yersinia pestis, the causative agent of bubonic plague, and of enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica is known to be mediated by a shared Lcr plasmid of about 70 kb. At 37 degrees C in Ca2+-deficient medium, this element promotes restriction of growth with concomitant production of virulence functions including the common V antigen and a set of yersiniae outer membrane peptides termed YOPs (Lcr+). The latter are expressed by the enteropathogenic species but not by wild type Y. pestis which possesses a unique 10 kb Pst plasmid associated with pesticinogeny (Pst+). We show in this report that, after pulse with 35S-methionine, peptides with molecular weights corresponding to YOPs of 78, 47, 45, 44, 36, and 26 kDa are synthesized during the low calcium response by both Lcr+, Pst+ and Lcr+, Pst- cells of Y. pestis. Although stable in the latter, radioactivity in YOPs of wild type was rapidly chased into lower molecular weight degradation products. At least four soluble peptides, including V, were also labeled during starvation for Ca2+; these structures were stable in both Lcr+, Pst+ and Lcr+, Pst- yersiniae. These findings suggest that a product encoded by the Pst plasmid of Y. pestis is required for post-translational regulation of outer membrane but not soluble peptides mediated by a second unrelated Lcr plasmid.

Resistance to pesticin, storage of iron, and invasion of HeLa cells by Yersiniae.

The independent abilities of Yersinia pestis to absorb exogenous pigments including hemin and Congo red (Pgm+) and to produce the bacteriocin pesticin with genetically linked invasive enzymes (Pst+) are established virulence factors of the species. Pst- Pgm+ strains of Y. pestis are sensitive to pesticin (Psts), and mutation of these isolates to pesticin resistance (Pstr) is known to result in concomitant conversion to Pgm-. Wild-type cells of Yersinia pseudotuberculosis and Yersinia enterocolitica are Pgm- but may be Psts; mutation of the latter to Pstr also results in avirulence. In this study, typical Pgm- mutants of Y. pestis exhibited a dramatic nutritional requirement at 37 degrees C but not 26 degrees C for iron which could be fulfilled by either Fe3+ or hemin. Iron privation of Pgm- yersiniae resulted in formation of osmotically stable spheroplasts similar to those previously observed after exposure of Psts bacteria to pesticin. At 37 degrees C, Pgm+ organisms rapidly overgrew initially predominant Pgm- populations in iron-deficient medium. However, Pgm-isolates could undergo a second mutation that permitted successful competition with Pgm+ cells in this environment. The mutation to Pstr in Y. pseudotuberculosis and Y. enterocolitica did not promote a similar requirement for iron but rather prevented these organisms from penetrating HeLa cells. The ability to invade these nonprofessional phagocytes was not shared by Pgm+ or Pgm- cells of Y. pestis.

Outer membrane peptides of Yersinia pestis mediating siderophore-independent assimilation of iron.

It is established that wild-type cells of Yersinia pestis absorb exogenous hemin or Congo red and thus grow as pigmented colonies at 26 degrees C on media containing these chromatophores (Pgm+). Pgm+ isolated are known to possess a siderophore-independent mechanism of iron-transport (required for growth in iron-deficient medium) which is absent in avirulent Pgm- mutants. Production of the bacteriocin pesticin and linked invasins (Pst+) is an additional defined virulence factor of yersiniae; mutation of Pgm+,Pst- organisms to pesticin-resistance (Pstr) results in concomitant conversion to Pgm-. In this study, autoradiograms of two-dimensional gels of [35S]methionine-labeled outer membranes from Pgm- mutants were compared to those of the Pgm+,Pst+ or Pgm+,Pst- parent. An apparently single predominant peptide present in these preparations (greater than 10% of total membrane protein) existed as a family of iron-modifiable 17.9-kDa molecules focusing down to isoelectric points of about 4.6 and up to 5.89. Expression of eight detectable Pst(+)-specific peptides was not significantly influenced by exogenous iron. Pgm+ yersiniae constitutively produced pigmentation-specific peptide F and five iron-repressible peptides termed IrpA to IrpE. Typical spontaneous mutation to Pgm- resulted in loss of peptide F and IrpB-E. A rare Pgm+,Pstr mutant, selected on Congo red agar containing pesticin, also lost IrpB-E but retained peptide F. This isolate, like Pgm- mutants, failed to grow in iron-deficient medium. Regardless of phenotype, all yersiniae utilized hemin, hemopexin, myoglobin, hemoglobin, and ferritin, but not transferrin or lactoferrin, as sole sources of iron.

In vivo comparison of avirulent Vwa- and Pgm- or Pstr phenotypes of yersiniae.

The abilities of Yersinia pestis to undergo restriction in Ca2+-deficient medium with concomitant production of V and W antigens (Vwa+) and to absorb exogenous pigments (Pgm+) are established virulence factors. Mutation of Y. pestis to Pgm- is known to promote resistance to pesticin (Pstr) and reduced lethality by peripheral routes of injection. Vwa+ Pgm- isolates of Y. pestis were shown in this study to retain virulence in mice when injected intravenously. Although Pgm- in appearance, wild-type cells of Yersinia pseudotuberculosis and Yersinia enterocolitica may also be sensitive to pesticin. Pstr mutants of Vwa+ strains of these species were similarly of reduced virulence, especially by peripheral routes of injection. The consequences of mutation to Vwa- and Pgm- or Pstr on growth and persistence in vivo were determined. After intravenous injection, Vwa+ yersiniae of all species exhibited sustained growth in mouse spleen, liver, and lung and accumulated in blood. Septicemia was not observed after similar injection of Vwa- mutants which were unable to maintain comparable rates of net increase in tissues. Mutation to Pgm- or Pstr did not influence proliferation but resulted in enhanced clearance from organs. It is known that reticuloendothelial cells serve as favored sites of replication for all wild-type yersiniae. Our results are consistent with the hypothesis that the Vwa+ phenotype favors growth within macrophages and that the Pgm+ and pesticin-sensitive phenotypes permit long-term, probably extracellular, retention within organs. Virulence in standard animal models (mice, rats, and guinea pigs) was not correlated with resistance to the bactericidal action of serum.

Roles of V antigen in promoting virulence and immunity in yersiniae.

It is established that yersiniae harboring an approximately 45-megadalton Vwa-plasmid can produce V and W antigens (Vwa+), and that sera containing anti-V provides passive protection to mice against Yersinia pestis. This observation was extended by the use of monospecific anti-V prepared by injecting rabbits with partially purified V, absorption of antisera with a Vwa- extract, and then separation of gamma-globulin by traditional processes of fractionation or by affinity chromatography. These preparations provided passive protection against 10 minimum lethal doses of virulent Y. pestis KIM, Yersinia pseudotuberculosis PB1, and Yersinia enterocolitica WA. Kinetics of elimination of these Vwa+ yersiniae from organs and blood of passively immunized mice closely resembled those of avirulent Vwa- mutants from normal mice. Injection into mice of sterile crude extracts of Y. pseudotuberculosis PB1 containing V promoted significant survival and retention of Vwa- mutants of Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. This effect was eliminated by the removal of V before injection by precipitation with monospecific antibody. These results indicate that V antigen per se is the major virulence factor mediated by Vwa-plasmids.

Determination of genome size, macrorestriction pattern polymorphism, and nonpigmentation-specific deletion in Yersinia pestis by pulsed-field gel electrophoresis.

Of 16 restriction endonucleases known to hydrolyze rare 6- or 8-base recognition sequences that were tested, only SpeI, NotI, AscI, and SfiI generated fragments of chromosomal DNA from Yersinia pestis, the causative agent of bubonic plague, of sufficient length to permit physical analysis by pulsed-field gel electrophoresis (PFGE). Of the individual bands detected after single-dimensional PFGE of these digests, the largest sum was obtained with SpeI (3,575.6 +/- 114.6 kb). Of these 41 bands, 3 were found to contain comigrating fragments, as judged by the results of two-dimensional SpeI-ApaI PFGE; addition of these fragments and the three plasmids of the species yielded a refined estimate of 4,397.9 +/- 134.6 kb for the genome. This size was similar for eight strains of diverse geographical origin that exhibited distinct DNA macrorestriction patterns closely related to their biotypes. The high-frequency chromosomal deletion known to exist in nonpigmented mutants (unable to assimilate Fe3+ at 37 degrees C or store hemin at 26 degrees C) was shown by two-dimensional PFGE analysis with SpeI and ApaI or with SfiI and SpeI to be 92.5 and 106 kb in size, respectively. The endpoints of this deletion were precise, and its size was more than sufficient to encode the eight known peptides reported to be absent in nonpigmented mutants. This deletion had not occurred (but was able to do so) in a rare mutant capable of hemin storage but not iron transport.

Mode of action of pesticin: N-acetylglucosaminidase activity.

Homogeneous preparations of pesticin, a bacteriocin produced by Yersinia pestis, neither significantly inhibited net synthesis of deoxyribonucleic acid, ribonucleic acid, or protein in Escherichia coli phi nor caused detectable degradation of deoxyribonucleic acid in vivo. Accordingly, its mode of action does not resemble that of colicin E2 as suggested by others. However, incorporation of cell wall-specific label ([14C]diaminopimelic acid) into trichloroacetic acid-insoluble material of growing cells was inhibited by pesticin which also promoted release of such radioactivity from both resting cells and purified mureinlipoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reaction mixtures containing appropriately labeled mureinlipoprotein showed that [3H]N-acetylglucosamine comigrated either with [14C]diaminopimelic acid in the murein peptide or with [14C]isoleucine of the Braun lipoprotein. As judged by these findings and pesticin-dependent release of reducing equivalents but not 4-hydroxy-2-acetamido sugars, the bacteriocin possesses N-acetylglucosaminidase activity. Hydrolysis of murein-lipoprotein occurred over a broad pH, with an optimum of 4.7. Mureinlipoproteins from a variety of pesticin-sensitive and -resistant organisms were hydrolyzed by the bacteriocin, indicating that its antibacterial specificity resides at the level of absorption.

Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment.

A procedure is described for fractionating Yersinia grown in small cultures into inner and outer membranes and soluble cytoplasmic proteins. The procedure was applied to the three recognized species of the genus grown under conditions simulating mammalian intracellular fluid with respect to Ca2+ and Mg2+. These conditions are known to elicit the production of the plague virulence antigen V. Isolates capable of making this antigen were compared with virulence-antigen-negative derivatives by two-dimensional electrophoresis. The V antigen was localized to the soluble protein fraction as a peptide that comigrates with the major component of a specific immunoprecipitate. This peptide had an apparent molecular weight of 38,000 and was not found in either apparent molecular weight of 38,000 and was not found in either membrane fraction. The comparison of virulence antigen-producers and nonproducers of Y. pseudotuberculosis and Y. enterocolitica revealed large qualitative and quantitative differences in outer membrane protein patterns, whereas the same comparison for Y. pestis showed only minor differences. The complexity of changes in the various protein fractions corroborate data in the literature indicating that extensive physiological changes occur in virulent organisms cultivated under simulated intracellular conditions.

Transport of Ca2+ by Yersinia pestis.

Low-calcium-response, or Lcr, plasmids of yersiniae are known to promote an in vitro nutritional requirement for 2.5 mM Ca2+ at 37 degrees C which, if not fulfilled, results in cessation of growth with induction of virulence functions (Lcr+). The mechanism whereby Ca2+ regulates this metabolic shift is unknown. Radioactive Ca2+ was not actively accumulated by yersiniae but was excluded by an exit reaction analogous to those described for other bacteria. Nevertheless, cultivation at 37 degrees C with 0.1 mM Ca2+, a level insufficient to prevent restriction of cell division, promoted significantly more binding of the cation by Lcr+ organisms than by plasmid-deficient Lcr- mutants. According, Lcr+ yersiniae may possess unique ligands capable of recognizing Ca2+.