Robert R. Brubaker

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. Images

Interleukin-10 and Inhibition of Innate Immunity to Yersiniae: Roles of Yops and LcrV (V Antigen)

Plague, caused by Yersinia pestis , is recognized as the most devastating acute infectious disease experienced by humankind. This notoriety is based upon the high rate of mortality, the rapid onset, and the appalling pathology associated with both the bubonic and pneumonic forms of the infection.

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.

Pasteurella pestis: Role of Pesticin I and Iron in Experimental Plague

Loss of the genetic determinant for pesticin I in Pasteurella pestis results in concomitant loss of the plague coagulase and fibrinolytic factor. The median lethal dose for mice of an isolate lacking only these activities is increased by factors of about 101, 104, and 107 cells when administered by the intravenous, intraperitoneal, and subcutaneous routes, respectively. Virulence of the aforesaid strain can be enhanced in mice treated with 40 �g of ferrous iron. This response resembles that of Pasteurella pseudotuberculosis, a closely related species that normally lacks pesticin I.

LcrV Plague Vaccine with Altered Immunomodulatory Properties

ABSTRACT 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.

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.

The Genus Yersinia: Biochemistry and Genetics of Virulence With 3 Figures

A highly successful modus operandi in research is to perform an experiment and then decide what problem was solved; the alternative procedure of devising experiments in order to answer preexisting questions may be more difficult. Unfortunately, the latter approach cannot be avoided by those concerned with infectious diseases because the question of why a given microorganism is virulent is automatically framed by its discoverer—in this case, the plague bacillus by Yersin (1894) and the causative agent of pseudotuberculosis by Malassez, and Vignal (1883). The delay of almost a century in obtaining satisfactory answers to these questions simply illustrates that the complexity of the host-parasite relationship is formidable and that experiments relevant to the expression of virulence have not been designed. At the same time, considerable progress towards this end has been made with other pathogens, especially in correlating nutritional requirements and metabolic patterns with the bacterium’s favored habitat in vivo (Moulder, 1962). Nevertheless, the diverse phenomena observed during infection with intracellular parasites cannot always be duplicated in simplified experimental systems. Furthermore, the infectivity of many microorganisms is low or erratic in laboratory animals and the response of the normal host to acute and chronic phases of disease may be quite distinct.

Outer membrane peptides ofYersinia pestis mediating siderophore-independent assimilation of iron

SummaryIt is established that wild-type cells ofYersinia pestis absorb exogenous hemin or Congo red and thus grow as pigmented colonies at 26° C on media containing these chromatophores (Pgm+). Pgm+ isolates 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 (> 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.

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.

Expression of the low calcium response in Yersinia pestis

Pathogenic yersiniae undergo an established low calcium response (LCR) at 37 degrees C in Ca2+-deficient media characterized by restricted growth with synthesis of Lcr plasmid-encoded virulence functions. The latter include outer membrane peptides (Yops) known to undergo Pst plasmid-mediated post-translational degradation in Yersinia pestis but not in enteropathogenic yersiniae lacking this plasmid. Salient Yops of Y. pestis are shown here to be either maintained in the steady state or to exist as a stable degradation product (p24 of Yop E). Processing of plague plasminogen activator (p36 to p33), responsible for hydrolysis of Yops, required 2 h. Avirulence of mutants with inserted Mu dl1 (Apr lac) in yopE was verified and shown to occur independently of introduced fusion-dependent peptides. However, avirulence of such yopE mutants but not that of isolates lacking the Lcr plasmid was phenotypically suppressed in mice injected with iron. Appearance of 20,500 and 40,500 Da heat-shock peptides preceded onset of the LCR. Lcr plasmid mediated V antigen (p38) and p20, Pst plasmid-encoded p36, and chromosomally promoted p56 and p70 were synthesized throughout the LCR. Classical antigen 5 was equated with p70 which was shared by Yersinia pseudotuberculosis but not Yersinia enterocolitica.