Rapid Microscopic Detection of Bacillus anthracis by Fluorescent Receptor Binding Proteins of Bacteriophages.

Peter Braun,Ana Clara Jacinto,Carolina Tocantins,Johannes Poppe,Leonie Reetz,Lilia Bachstein,Immanuel Wolfschläger,Gregor Grass

doi:10.3390/microorganisms8060934

Abstract

Bacillus anthracis, the etiological agent of anthrax disease, is typically diagnosed by immunological and molecular methods such as polymerase chain reaction (PCR). Alternatively, mass spectrometry techniques may aid in confirming the presence of the pathogen or its toxins. However, because of the close genetic relationship between B. anthracis and other members of the Bacillus cereus sensu lato group (such as Bacillus cereus or Bacillus thuringiensis) mis- or questionable identification occurs frequently. Also, bacteriophages such as phage gamma (which is highly specific for B. anthracis) have been in use for anthrax diagnostics for many decades. Here we employed host cell-specific receptor binding proteins (RBP) of (pro)-phages, also known as tail or head fibers, to develop a microscopy-based approach for the facile, rapid and unambiguous detection of B. anthracis cells. For this, the genes of (putative) RBP from Bacillus phages gamma, Wip1, AP50c and from lambdoid prophage 03 located on the chromosome of B. anthracis were selected. Respective phage genes were heterologously expressed in Escherichia coli and purified as fusions with fluorescent proteins. B. anthracis cells incubated with either of the reporter fusion proteins were successfully surface-labeled. Binding specificity was confirmed as RBP fusion proteins did not bind to most isolates of a panel of other B. cereus s.l. species or to more distantly related bacteria. Remarkably, RBP fusions detected encapsulated B. anthracis cells, thus RBP were able to penetrate the poly-γ-d-glutamate capsule of B. anthracis. From these results we anticipate this RBP-reporter assay may be useful for rapid confirmative identification of B. anthracis.

Highlights

Bacillus anthracis causing the zoonotic infectious disease anthrax in mammals and humans phylogenetically belongs to the Bacillus cereus sensu lato group of very closely related Firmicutes bacteria
We recognized that a hypothetical protein, BA4079, very similar to RBPγ was encoded on the B. anthracis chromosome located within previously identified prophage LambdaBa03 [24]
The confirmatory specific identification of B. anthracis is often achieved by means of antigen–antibody interaction, be it in the form of enzyme-linked immunosorbent assays (ELISA) [38], lateral flow assays [39,40] or by the use of fluorescently labeled antibodies in microscopy [41]

Summary

Introduction

Bacillus anthracis causing the zoonotic infectious disease anthrax in mammals and humans phylogenetically belongs to the Bacillus cereus sensu lato group of very closely related Firmicutes bacteria. Some species carry characteristic virulence plasmids on which the genetic information for certain toxins is encoded. These include megaplasmid pCER270 for production of cereulide toxin in a clade of B. cereus sensu stricto strains [3] or plasmid pXO1 encoding a three-partite AB toxin from B. anthracis better known as lethal and edema toxin, respectively [4]. These phenotypic characteristics facilitate clinical differentiation, but do not always constitute reliable criteria for rapid identification of individual species. Virulence plasmids typical for B. anthracis (pXO1 and pXO2) can be found in certain B. cereus isolates [1]

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Discussion

Conclusion