Specific Detection of <i>Yersinia pestis</i> Based on Receptor Binding Proteins of Phages

• Main Authors: Friederike Born, Peter Braun, Holger C. Scholz, Gregor Grass
• Format: Article
• Language: English
• Published: MDPI AG 2020-07-01
• Series: Pathogens
• Subjects: <i>Yersinia pestis</i>; plague; microscopic assay; pathogen detection; receptor binding protein; phage
• Online Access: https://www.mdpi.com/2076-0817/9/8/611

The highly pathogenic bacterium <i>Yersinia pestis</i> is the causative agent of plague, a notorious infectious zoonotic disease. When transmitted from person to person as pneumonic plague via droplets, <i>Y. pestis</i> 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. <i>Yersinia pestis</i> specific phages such as L-413C and ΦA1122 are already used for detection of <i>Y. pestis</i> 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 <i>Y. pestis</i>. Genes of putative RBP of phages L-413C (<i>gpH</i>) and ΦA1122 (<i>gp17</i>) were fused with those of fluorescent proteins and recombinant receptor-reporter fusion proteins were produced heterologously in <i>Escherichia coli</i>. When first tested on attenuated <i>Y. pestis</i> 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 <i>Yersinia</i> species and several inactivated fully virulent <i>Y. pestis</i> strains exhibited high specificities of the RBP-reporters against <i>Y. pestis</i>. The L-413C RBP proved to be especially specific, as it only detected <i>Y. pestis</i> at all temperatures tested, whereas the RBP of ΦA1122 also bound to <i>Y. pseudotuberculosis</i> strains at 37 °C (but not at 28, 20 or 6 °C). Finally, the <i>Y. pestis</i>-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.