The Use of an Organo-Selenium Peptide to Develop New Antimicrobials That Target a Specific Bacteria

This study examines the use of a covalently selenium-bonded peptide and phage that binds to the Yersinia pestis F1 antigen for the targeting and killing of E. coli expressing this surface antigen. Using a Ph.D.-12 phage-display library for affinity selection of the phage which would bind the F1 antigen of Y. pestis, a phage displaying a peptide that binds the F1 antigen with high affinity and specificity was identified. Selenium was then covalently attached to the display phage and the corresponding F1-antigen-binding peptide. Both the phage and peptides with selenium covalently attached retained their binding specificity for the Y. pestis F1 antigen. The phage or peptide not labeled with selenium did not kill the targeted bacteria, while the phage or peptide labeled with selenium did. In addition, the seleno-peptide, expressing the F1 targeting sequence only, killed cells expressing the F1 antigen but not the parent strain that did not express the F1 antigen. Specifically, the seleno-peptide could kill eight logs of bacteria in less than two hours at a 10-µM concentration. These results demonstrate a novel approach for the development of an antibacterial agent that can target a specific bacterial pathogen for destruction through the use of covalently attached selenium and will not affect other bacteria.

Specific Detection of Yersinia pestis Based on Receptor Binding Proteins of Phages

The highly pathogenic bacterium Yersinia pestis is the causative agent of plague, a notorious infectious zoonotic disease. When transmitted from person to person as pneumonic plague via droplets, Y. pestis is highly contagious and in most cases is fatal if left untreated. Thus, when plague is suspected, rapid diagnosis is crucial, as a serious course of the infection is only averted by early antibiotic therapy. The bacterium is easy to cultivate, accessible and has a high potential for nefarious use such as bioterrorism. Highly specific, rapid and easy-to-use confirmatory diagnostic methods are required to reliably identify the pathogen independently from PCR-based methods or F1 antigen-based immunological detection. Yersinia pestis specific phages such as L-413C and ΦA1122 are already used for detection of Y. pestis in bacterial plaque or biosensor assays. Here, we made use of the host specificities conferred by phage receptor binding (or tail fiber/spike) proteins (RBP) for developing a specific, fast and simple fluorescence-microscopy-based detection method for Y. pestis. Genes of putative RBP of phages L-413C (gpH) and ΦA1122 (gp17) were fused with those of fluorescent proteins and recombinant receptor-reporter fusion proteins were produced heterologously in Escherichia coli. When first tested on attenuated Y. pestis strain EV76, RBP-reporters bound to the bacterial cell surface. This assay could be completed within a few minutes using live or formaldehyde-inactivated cells. Specificity tests using cultures of closely related Yersinia species and several inactivated fully virulent Y. pestis strains exhibited high specificities of the RBP-reporters against Y. pestis. The L-413C RBP proved to be especially specific, as it only detected Y. pestis at all temperatures tested, whereas the RBP of ΦA1122 also bound to Y. pseudotuberculosis strains at 37 °C (but not at 28, 20 or 6 °C). Finally, the Y. pestis-specific capsule, produced when grown at 37 °C, significantly reduced binding of phage ΦA1122 RBP, whereas the capsule only slightly diminished binding of L-413C RBP.

Efficiency of Human Plague Vaccination in Tuvinian Natural Plague Focus

Background. Plague is an especially dangerous natural focal infectious disease belonging to a group of quarantine infections. There are eleven natural plague foci in Russian Federation. In Republic Tyva plague endemic territories include Ovyur, Mongun-Taigin and Tes-Hem areas where Y. pestis strains are intermittently isolated from Citellus undulates. Population living at the territory of the natural foci get immunoprophylaxis against plague at complication of epizootic and epidemic conditions.This paper presents the results of monitoring indicators of the immune status of people vaccinated with the plague vaccine living in the territory of the Tuva natural focus.Materials and methods. The study involved 76 volunteers who had not previously been vaccinated. The study included the determination of production IFN-γ, IL-4, TNF-α by blood cells, titers of specific IgG antibodies to the capsule F1 antigen of the Yersinia pestis, and concentrations of immunoglobulins in serum blood, as well as immunophenotyping of blood lymphocytes.Results. In the course of a comprehensive immunological study, features of the development of cellular and humoral reactions in people living in the territory of the Tuva natural plague focus were established in the first months after vaccination. Changes in the concentration dynamics of the main classes of immunoglobulins were accompanied by an increase in the level of specific IgGs to the F1 within 6 months after immunization. In the same period, a significant increase in the production of cytokines, as well as significant changes in terms of the subpopulation composition of the vaccinated blood.Conclusion. It is necessary to note the importance of studying of the human immune status in 1–3 months after plague vaccination as this period coincides with potentially dangerous season from epidemiological point of view. Nevertheless, much important role for improvement of tactics of the specific prevention measures plays the data received after the revaccination.

A microchip developed for detecting antibodies against plaque-derived antigens

Currently available Russia-made preparations intended for serological plague diagnostics are usually aimed at detecting antibodies to single bacterial antigens in the blood serum. To improve reliability of the data obtained, it is rational to use test systems to simultaneously quantify antibodies to several immunodominant Y. pestis antigens. An opportunity of using biochip technology for quantifying specific antibodies to Yersinia pestis antigens was investigated. To do this, 5 commercially available sera, 35 blood sera obtained from individuals vaccinated with live plague vaccine collected 1, 4, 5, 18 months after immunization, as well as 5 sera obtained from healthy donors were analyzed. The objective of this work was to develop a biological microchip (immunochip) for detecting antibodies specific to Y. pestis-derived antigens. In particular, amino-modified slides were sensitized by immunodominant Yersinia pestis-derived antigens: capsule antigen F1, lipopolysaccharide (LPS), main somatic antigen (MSA), fibrinolysin, and pestin PP. Diagnostic specificity and sensitivity of the immunochip were assessed by using the approved homoand heterologous immune-biological preparations and experimental animal sera. It was found that the immunochip demonstrated a 100% diagnostic efficiency. An opportunity of applying this immunochip to determine specific antibody profile in individuals vaccinated with live plague vaccine was estimated. A commercially available ELISA-AB-F1 of Yersinia pestis kit was used for comparison that allowed to detect antibodies to Y. pestis F1 antigen in 77.1% of vaccinated individuals within the examined time period covering between 1 to 18 months post-vaccination, at titer 1:160–1:2560. In contrast, using the immunochip resulted in detecting F1 antigen-specific antibodies in 91.4% of samples post-vaccination at titer 1:320–1:2560. Moreover, such immunochip additionally allowed to detect antibodies specific to the remainder of Y. pestis-derived LPS, MSA, pestin PP in 54.3%, 20%, 42% of vaccinated individuals, respectively. The percentage of positive seroconversion in individuals vaccinated with live plague vaccine was 77.1% based on the ELISA data, 91.4% — to the F1 antigen according to the immunochip data, and 94.3% — by analyzing an extended antigen panel. Combining multiple antigenic markers in our immunochip allowed to identify greater seroconversion among vaccinated people compared to a standard ELISA. Thus, the data obtained suggest that the proposed immunochip technology might be promising in assessing developing humoral immunity.

Synthesis and expression of caf1 gene encoding F1 antigen of Yersinia pestis

Yersinia pestis is the etiologic agent of plague, one of the most deadly infectious diseases described in the history of humanity. It was responsible for millions of deaths all over the world. Yersinia pestis also can be used as a highly lethal biological potential weapon. For plague diagnosis in humans as well as to detect Y. pestis in the environment, fraction 1 capsular antigen (F1) of the bacteria was usually used as a good marker. The aim of this study is to produce Y. pestis F1 antigen to serve as a material for development of immunochromatographic test strips for rapid detection of Y. pestis. Because of the difficulty in Y. pestis culture for DNA extraction as well as F1 antigen production, we artificially synthesized the target caf1 coding for F1 antigen for expression in Escherichia coli. After the codon optimization step, caf1 was synthesized by “gapless” PCR using 22 overlaping oligonucleotides cover the complete sequence of this gene. The sequencing result showed that we successfully synthesized the target gene. In total 6 clones sequence, there are 2 clones sequence which were 100% identity with reference sequence. The target sequence was then introduced into pET-52b(+) vector and expressed in E. coli BL21 (DE3) in the form of (His)10 affinity tag fusion. As the result of SDS-PAGE, the recombinant protein Caf1 of 18 kDa was highly expressed in E. coli as inclusion body form and was purified by His-tag affinity chromatography. The recombinant Caf1 was then confirmed by Western blot with His-tag antibody.