Yersinia pestis undergoes an obligate flea-rodent-flea enzootic life cycle. The rapidly fatal properties of Y. pestis are responsible for the organism's sustained survival in natural plague foci. Lipopolysaccharide (LPS) plays several roles in Y. pestis pathogenesis, prominent among them being resistance to host immune effectors and induction of a septic-shock state during the terminal phases of infection. LPS is acylated with 4-6 fatty acids, the number varying with growth temperature and affecting the molecule's toxic properties. Y. pestis mutants were constructed with a deletion insertion in the lpxM gene in both virulent and attenuated strains, preventing the organisms from synthesizing the most toxic hexa-acylated lipid A molecule when grown at 25 6C. The virulence and/or protective potency of pathogenic and attenuated Y. pestis DlpxM mutants were then examined in a mouse model. The DlpxM mutation in a virulent strain led to no change in the LD 50 value compared to that of the parental strain, while the DlpxM mutation in attenuated strains led to a modest 2.5-16-fold reduction in virulence. LPS preparations containing fully hexa-acylated lipid A were ten times more toxic in actinomycin D-treated mice then preparations lacking this lipid A isoform, although this was not significant (P.0.05). The DlpxM mutation in vaccine strain EV caused a significant increase in its protective potency. These studies suggest there is little impact from lipid A modifications on the virulence of Y. pestis strains but there are potential improvements in the protective properties in attenuated vaccine strains.

A library of monoclonal antibodies (MAbs) which recognised different epitopes of Yersinia pestis fibrinolysin (Fib) was developed. These MAbs were species-specific and demonstrated no cross-reaction in indirect immunofluorescence tests (IIFT) with other gram-negative bacteria possessing plasminogen activator activity. All the MAbs provided equally high levels of immunofluorescence with pPst þ Y. pestis strains cultivated at 378C and at 288C. In all cases, the MAbs inhibited both fibrinolytic and coagulase (Coag) activities of Y. pestis in Fib-activity inhibition and coagulase-activity inhibition reactions, and reacted with 35-and 37-kDa proteins of Y. pestis in immunoblotting, demonstrating bifunctional activity possibly similar to the properties of MAbs produced by hybrid hybridomas. On the basis of these and earlier studies, the immunochemical identity of Fib and Coag, two distinct subunits of a bifunctional fusion protein whose specific functional activity depends upon the temperature factor, was established. A new rapid, cheap, strictly specific and safe dot-ELISA based on the use of MAb against Y. pestis Fib (MAb-Fib) for reliable identification of Y. pestis strains was developed. This technique has great advantages over monoclonal diagnostic kits based on the use of MAb against Y. pestis fraction I (FI) because it allows detection of plague bacilli grown at 378C as well as at 288C. This dot-ELISA will be valuable as a clinical diagnostic tool and might be applicable to field studies and plague surveillance.

Growth medium simulating the phagolysosomal environment in which Yersinia pestis resides during its intracellular growth in vivo was made by acidi®cation of Ca 2 -de®cient medium. When used for cultivation of Y. pestis EV-76 (pLCR ; pPst ;pFra ) and its isogenic derivatives ± KM-217 (pLCR ;pPst 2 ;pFra 2 ) and KM-218 (pLCR 2 ;Ppst 2 ; pFra 2 ) ± this medium permitted survival and proliferation of viable bacteria without any growth restriction. Moreover, a correlation between the pH of growth medium and bacterial yield was established. Acidi®cation completely inhibited ®brinolytic (pla protease) activity (PAA) of Y. pestis carrying pPst and allowed synthesis of speci®c outer-membrane proteins (Yops) without any degradation by the pla protease.Comparison of whole-cell lysates of the strains tested in PAAG-SDS showed that, in addition to previously described Yops, Y. pestis synthesised new acidic proteins which appeared only under acidic conditions and were encoded by pLCR or chromosomally. Some changes in O-speci®c polysaccharide chains of Y. pestis LPS that were dependent on cultivation temperature and pH of the medium were also demonstrated.

Antigenic and immunochemical properties of Yersinia pestis fraction I (FI) preparations extracted by different methods were studied with polyclonal and monoclonal antibodies. The existence of mature FI in a form of a complex antigen whose subunits have different genetic control was demonstrated. Galactolipid was shown, with caf1 product, to be the second species-speci®c component of the FI complex molecule and is probably encoded by chromosomal genes. It, like caf1 product, was expressed in higher quantities at 378C than at 288C. Among FI subunits there were at least two proteins of 28 6 2 kDa and 43 6 2 kDa which were not speci®c for Y. pestis but were found also in all Yersinia spp. and some other bacteria. These proteins were synthesised independently of the incubation temperature (48±408C) and are possibly encoded chromosomally but outside the caf operon and galactolipid-encoding genes. Both proteins together with galactolipid comprise an envelope antigen found in pFra 2 or plasmidless Y. pestis strains. Organisation of Y. pestis FI (mature capsular antigen) in the form of a complex of the envelope antigen and the caf1 product is discussed.

Human and murine erythrocytes (RBC) were invaded by Yersinia pestis in vivo and in vitro during a short period and were probably used as an essential source of iron and porphyrin for survival, effective gross multiplication and rapid spread of these bacteria in the bloodstream of mammals. Both iron and porphyrin were extracted by Y. pestis from the RBC through oxidase-catalase activity which produced oxidation of the RBC glucose with generation of H 2 O 2 in large concentration leading to oxidative transformation of haemoglobin into haemin. Furthermore, some mainly chromosomally encoded effector proteins were implicated in this process because all were synthesised by Y. pestis grown in media simulating the intracellular conditions of mammalian RBC. Damaged RBC lost the ability to transport O 2 in the mammalian organism. As a result, signi®cant oxygen de®ciency developed in host tissues providing speci®c clinical disease features of plague which are similar to characteristic symptoms of poisoning with haemotoxic substances when the transformation of the haemoglobin Fe 2 to Fe 3 occurs.