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Attenuated Yersinia pestis strain EV NIIEG is a licensed live plague vaccine (LPV) for human use in Russia that can elicit protection against both the bubonic and pneumonic forms of disease. However, little is known about the mechanisms underlying the formation of immunity to Y. pestis provided by this vaccine. Our recent study reported a prevalence of specific IgG antibodies to the capsular antigen F1 and the type three secretion system (T3SS) structural subunit YscF in humans immunized with live plague vaccine. In this study, IgG subclasses of antibodies to the plague antigens F1 and YscF in the sera of vaccinees were determined by using an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. IgG subclass antibodies to F1 antigen were predominantly IgG1, although IgG2 and IgG4 subclasses were detectable. The IgG1 titers were markedly higher in donors who received multiple annual injections, while the IgG2 and IgG4 titers showed no increase and stayed low in all donors. The IgG1 anti-F1 remained detectable even 20 years post-vaccination, suggesting a long-term immunity. Sera of YscF-positive donors also contained predominantly IgG1-specific antibodies. The IgG2 and IgG4 titers to YscF were undetectable in all donors, including those who received multiple vaccinations. In conclusion, we describe for the first time that IgG1 is a dominating isotype for both F1 and YscF antigens in humans immunized with live plague vaccine. [ABSTRACT FROM AUTHOR]

Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here, we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons with available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveal 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, to our knowledge, represent the only new genetic material in Y. pestis acquired since the the divergence from Y. pseudotuberculosis. In contrast, 149 other pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive insertion sequence-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of preexisting gene expression pathways, appear to be more important than acquisition of genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is the causative agent of a variety of chlamydial infections in humans with a predominantly (up to 80%) asymptomatic course of disease. In this study, a potentially novel C. trachomatis sequence type (ST) was detected in an asymptomatic man who has sex with a man among the nine STs revealed in urogenital swabs from individuals with chlamydia ( n  = 18). Phylogenetically this ST270 clustered separately as a single clade to an ST13-founded group of C. trachomatis strains and differed from the latter by a single allele, hflX . This finding emphasizes the importance of careful investigation of individuals with asymptomatic chlamydia infections.

Deletion mutants in the lpxM gene in two Yersinia pestis strains, the live Russian vaccine strain EV NIIEG and a fully virulent strain, 231, synthesise a less toxic penta-acylated lipopolysaccharide (LPS). Analysis of these mutants revealed they possessed marked reductions in expression and immunoreactivity of numerous major proteins and carbohydrate antigens, including F1, Pla, Ymt, V antigen, LPS, and ECA. Moreover, both mutants demonstrated altered epitope specificities of the antigens as determined in immunodot-ELISAs and immunoblotting analyses using a panel of monoclonal antibodies. The strains also differed in their susceptibility to the diagnostic plague bacteriophage L-413C. These findings indicate that the effects of the lpxM mutation on reduced virulence and enhanced immunity of the Y. pestis EV DeltalpxM is also associated with these pleiotropic changes and not just to changes in the lipid A acylation.

The lpxM mutant of the live vaccine Yersinia pestis EV NIIEG strain synthesising a less toxic penta-acylated lipopolysaccharide was found to be avirulent in mice and guinea pigs, notably showing no measurable virulence in Balb/c mice which do retain some susceptibility to the parental strain itself. Twenty-one days after a single injection of the lpxM-mutant, 85-100% protection was achieved in outbred mice and guinea pigs, whereas a 43% protection rate was achieved in Balb/c mice given single low doses (10(3) to 2.5 x 10(4) CFU) of this vaccine. A subcutaneous challenge with 2000 median lethal doses (equal to 20,000 CFU) of fully virulent Y. pestis 231 strain, is a 6-10-fold higher dose than that which the EV NIIEG itself can protect against.

A rapid method for the detection, purification, and identification of proteins in bacterial extracts was developed using surface enhanced laser desorption/ionization (SELDI) ProteinChip technology. The effectiveness of this technique for monitoring the expression and identification of temperature- and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mammalian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degrees C (the temperature of the flea vector) to 37 degrees C (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degrees C and 37 degrees C in a Ca(2+)-deficient medium. Through an initial protein profiling of the crude bacterial extract on strong anion exchange and copper affinity, ProteinChip arrays detected five proteins that were up-regulated and three proteins that were down-regulated at 37 degrees C. Two of the proteins predominately expressed at 37 degrees C were semi-purified in less than two days. The two proteins were identified as catalase-peroxidase and Antigen 4. Aside from its speed, a salient feature of the SELDI technique is the microgram amounts of crude sample required for analysis.

C3H (H-2(k)) mice are susceptible to a vaginal challenge with human strains of Chlamydia trachomatis and thus are a useful strain for testing potential Chlamydia vaccine candidates. However, C3H mice are fairly poor responders in terms of the level of antibody resulting from immunization with potential protective peptides representing variable domains (VDs) of the major outer membrane protein (MOMP). C57BL/6 (H-2(b)) mice, on the other hand, are moderately resistant to a vaginal challenge but are good responders to the chlamydial MOMP VDs. Peptides representing universal T-cell helper epitopes were employed to determine whether the antibody response to a peptide representing VD4 of the MOMP, which has been shown to contain neutralizing epitopes, could be enhanced in C3H and C57 mice. Universal T-cell helper peptides from tetanus toxin, the pre-S2 region of hepatitis B virus, and the mouse heat shock protein 60, as well as the corresponding segment of the Chlamydia heat shock protein 60 (hspct), were coadministered with the VD4 peptide. Peptides were coencapsulated in liposomes containing the adjuvant monophosphoryl lipid A and administered by using a combination of mucosal and intramuscular injection. The only T-cell helper peptide that improved the immune response as judged by antibody level, in vitro neutralization assays, and T-cell proliferation was hspct. The response in the C57BL/6 strain was not significantly enhanced with hspct over levels achieved with VD4 alone; however, in C3H mice the levels of serum antibody to C. trachomatis increased to that seen in C57 mice. However, the molecular specificity and immunoglobulin subclass distribution differed from those of the C57 response, and the neutralizing titers and T-cell proliferation responses were lower. In both strains of mice, titers of vaginal antibody to C. trachomatis were low. In summary, of the T-helper peptides used, only hspct significantly enhanced the immune response of C3H mice to the VD4 peptide, but it had only a modest effect on the immune response of C57 mice.

The first temperature-dependent proteins (expressed at 37 degrees C, but not 26 degrees C) to be identified in Yersinia pestis were antigens 3 (fraction 1), 4 (pH 6 antigen), and 5 (hereafter termed KatY). Antigens 3 and 4 are now established virulence factors, whereas little is known about KatY, except that it is encoded chromosomally, produced in abundance, possesses modest catalase activity, and is shared by Yersinia pseudotuberculosis, but not Yersinia enterocolitica. We report here an improved chromatographic method (DEAE-cellulose, calcium hydroxylapatite, and Sephadex G-150) that yields enzymatically active KatY (2,423 U/mg of protein). Corresponding mouse monoclonal antibody 1B70.1 detected plasminogen activator-mediated hydrolysis of KatY, and a polyclonal rabbit antiserum raised against outer membranes of Y. pestis was enriched for anti-KatY. A sequenced approximately 16-kb Y. pestis DNA insert of a positive pLG338 clone indicated that katY encodes an 81.4-kDa protein (pI 6.98) containing a leader sequence of 2.6 kDa; the deduced molecular mass and pI of processed KatY were 78.8 kDa and 6. 43, respectively. A minor truncated variant (predicted molecular mass of 53.6 kDa) was also expressed. KatY is similar (39 to 59% identity) to vegetative bacterial catalase-peroxidases (KatG in Escherichia coli) and is closely related to plasmid-encoded KatP of enterohemorrhagic E. coli O157:H7 (75% identity). katY encoded a putative Ca2+-binding site, and its promoter contained three homologues to the consensus recognition sequence of the pCD-encoded transcriptional activator LcrF. rbsA was located upstream of katY, and cybB, cybC, dmsABC, and araD were mapped downstream. These genes are not linked to katG or katP in E. coli.

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.

The Escherichia coli strains (75) isolated from patients suffering from diarrhea were screened for ability to produce the temperature-labile or stable toxins (ST or LT) by the different techniques (the hybridization with DNA probes, biological, enzyme immunoassay). The majority of tested strains was shown to harbor the tox-genes controlling the synthesis of ST, LT or both enterotoxins. However, the phenotypic expression of the genes was registered in only some of the strains. The hybridization with the DNA probes is noted to be most perspective in the mass screening of toxigenic strains. The DNA probe used contained the fused estA-eltB genes that makes one able to detect the genes for both enterotoxins.

A non-isotopic amplification system was used to identify and indicate Brucella. The terminal sequences of a protein gene fragment in Brucella outer membrane were identified and direct and reverse primers were chosen for a polymerase chain reaction. (PCR). PCR amplifies a specific DNA fragment, 700 kb in size, only in representatives of the Brucella genus. A probe was design, which is the central part of the amplified DNA fragment, 550 kb in size. Single Brucella cells were detectable with an unlabelled probe in the analyzed samples during hybridization reactions. The system can be recommended for a rapid and reliable analysis in medical and veterinary practice.

We have sequenced the lcrGVH operon from Y. pseudotuberculosis plasmid pYV995 and compared its sequence with that of Y. pestis. The sequences were highly homological, however, six base pair substitutions were found in one short 14 bp region termed variable sequence. Two oligonucleotides corresponding to variable sequence of Y. pestis (pes-V) or Y. pseudotuberculosis (ptb-V) were synthesized and were used as molecular probes in hybridization experiments with sets of Y. pestis and Y. pseudotuberculosis strains. All 17 Y. pestis strains tested were positive only with the pes-V probe, 18 of 21 Y. pseudotuberculosis strains were positive with the ptb-V probe, while three Y. pseudotuberculosis strains reacted with the pes-V probe but not the ptb-V probe. The 200 bp fragment including variable sequence was sequenced in seven Y. pseudotuberculosis strains. The Y. pseudotuberculosis strains which were positive with the pes-V probe possessed the 200 bp fragment sequence almost identical with that from Y. pestis. No correlation between the Y. pestis-like lcrV sequence and virulence was found for these strains. Moreover, the Y. pseudotuberculosis strains with Y. pestis-like sequences in contrast to Y. pestis possessed unaltered yadA gene. However, we have found the yadA frameshift mutation characteristic for Y. pestis in one Y. pseudotuberculosis strain 312.

A gene bank of Rickettsia prowazekii strain E constructed in the phage vector lambda EMBL4 was screened for antigen production with anti-R. prowazekii serum. One of the immunoreactive clones, grown at 37 degrees C exhibited the expression of at least two antigens of molecular weight (M(r)) 37 kD and 14 kD. Subcloning and further analysis revealed that the antigens (polypeptides) of Mr 37, 14, and/or 16 kD apparently represent structural units of the 138 kD complex antigen. Assembly of the above mentioned polypeptides was found to be thermosensitive as it took place at 30 degrees C but not at 37 degrees C and resulted in an oligomeric structure of M(r) 138 kD. The nucleotide sequence of the gene coding for a precursor of the mature polypeptides of Mr 14 and/or 16 kD was determined.

The phenotypic properties conferred to Yersinia pseudotuberculosis cells by the genetical determinants of a 25Md fragment of the plasmid pVM82 coding for the modified cellular immune response in the infected organism. The fragment was shown to determine the conjugative properties of the plasmid, the resistance of bacterial cells to a number of hydrophobic agents and cellular ability to absorb the Congo red dye. The latter confirms the presence of additional structural components in the cell wall of the strain harbouring the plasmid pVM82. The increased resistance of the plasmid-containing strain to bactericidal effect of the blood plasma was demonstrated as compared with the resistance of the strains harbouring the p57 plasmid lacking the 25Md fragment or no plasmid at all.

It has been the purpose of this paper to study molecular-biological features of the Bordetella bacteriophage interaction with the host cell during lysogeny and conversion as well as to determine the degree of homology between genomes of homologous and heterologous bacteriophages. Genomes of bacteriophages from B. pertussis 134, 41405 and B. bronchiseptica 214 were studied. Heteroduplex and restriction analyses revealed a heterogeneity of bacteriophage populations, and their DNAs were found to differ in size and position of inserts. As shown by blot hybridization, the bacteriophage genome is not inserted into the chromosome of the lysogenic cell but apparently exists as an autonomous plasmid replicon. It has been established that during conversion only a part of the phage genome is inserted into the chromosome of the recipient cell.

Plasmid pNM1, the derivative of R100.1, has been constructed by insertion of transposon Tn5 into structural tet genet (Tn10) of the parental plasmid. The frequency of precise excision of Tn5 from plasmidic genome is 10(-5). The high frequency of precise excision obtained in this system permits one, to use it for isolation of mutants having low frequencies of precise excision. Two mutants were isolated in which the frequencies of precise excision of Tn5 were decreased for two orders. The pex1 and pex2 mutations responsible for the effect decrease the precise excision of Tn5 from R100.1 as well as from RP4 genomes.

The data of genetic mapping of the cholera toxin regulatory gene by conjugation mating of Vibrio cholerae eltor donor strain with V. cholerae classica recipients are presented. The close genetic linkage of tox locus to pur-63 is shown. The gene order asp - cys - nal - pur-61 - trp - his - pur-63 - tox - ile of the chromosomal region examined is established.