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

Abstract

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.

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies

“…Similarly, another commercial LFA (InBios Active Anthrax Detect Rapid Test) reached 82% specificity [ 3 ], however, samples used in that study also included more difficult to test contaminated animal tissues. In contrast to many RBP-based assays [ 12 , 13 ], however, most LFAs are not depending on samples derived from actively growing B. anthracis cells. Among the RBPs previously tested for B. anthracis , we selected RBP λ03 Δ1-120 because it is both host-specific and the least affected by the growth phases of its host cells [ 13 ].…”

“…These phage RBP-based assays are already widely used as versatile tools for pathogen detection [ 9 , 10 ]. Target bacteria include biothreat agents that cause melioidosis ( Burkholderia pseudomallei ) [ 11 ], plague ( Yersinia pestis ) [ 12 ], or anthrax [ 13 ]. For B. anthracis phage Gamma, the GamR protein has been previously identified as the phage’s host cell receptor [ 14 ].…”

Structural and functional characterization of the receptor binding proteins of Escherichia coli O157 phages EP75 and EP335

“…Phage RBPs have evolved to recognize a variety of structures on the surfaces of their bacterial hosts, including protein complexes (e.g., porins and the flagella) and saccharidic components such as teichoic acids, lipopolysaccharides (LPS), and capsular polysaccharides [17] , [18] , [19] . The inherent specificity of RBPs for a given species has led to their implementation in bacterial diagnostics, such as recently developed assays for Listeria [20] , Salmonella [21] , [22] , Pseudomonas [23] , and Yersinia [24] detection. In addition, the ability of certain phage TSPs to enzymatically degrade the protective capsules of certain pathogens, e.g., E. coli [25] , Klebsiella pneumoniae [19] , [26] , and Acinetobacter baumannii [27] , [28] , has led to their investigation as anti-virulence agents to target multidrug resistant bacteria and re-sensitize them to antibiotics.…”

The State of the Art in Biodefense Related Bacterial Pathogen Detection Using Bacteriophages: How It Started and How It’s Going

“…RBP protein fusions as tools for detection: As described before with Bacillus phages, fluorescent protein fusions to the RBP of the Y. pestis phages ϕA1122 (gp17) and L-413C (gpH) have been used to detect the attenuated EV76 strain of Y. pestis species by fluorescent microscopy [29]. This detection capability was dependent on the temperature and time of the culture conditions.…”

“…Finally, the process of inactivation by heat, peracetic acid, ethanol, and paraformaldehyde (PFA) was evaluated in this study. It was determined that 4% PFA was ideal for inactivation and used to demonstrate detection of six different risk group 3 strains of Y. pestis [29].…”