Let There Be Light! Bioluminescent Imaging to Study Bacterial Pathogenesis in Live Animals and Plants

Abstract

: Bioluminescence imaging (BLI) of bacteria was primarily designed to permit real-time, sensitive, and noninvasive monitoring of the progression of infection in live animals. Generally, BLI relies on the construction of bacterial strains that possess the lux operon. The lux operon is composed of a set of genes that encode the luciferase enzyme and its cognate substrate, which interact to produce light-a phenomenon that is referred to as bioluminescence. Bioluminescence emitted by the bacteria can then be detected and imaged within a living host using sensitive charge-coupled device (CCD) cameras. In comparison to traditional host-pathogen studies, BLI offers the opportunity for extended monitoring of infected animals without resorting to euthanasia and extensive tissue processing at each time point. Therefore, BLI can reduce the number of animals required to generate meaningful data, while significantly contributing to the understanding of pathogenesis in the host and, subsequently, the development and evaluation of adequate vaccines and therapeutics. BLI is also useful in characterizing the interactions of pathogens with plants and the para-host environment. In this chapter, we demonstrate the broad application of BLI for studying bacterial pathogens in different niches. Furthermore, we will specifically focus on the use of BLI to characterize the following: (1) the pathogenesis of Brucella melitensis in mice (animal host), and (2) the progression of infection of Clavibacter michiganensis subsp. michiganensis in tomatoes (plant host). These studies will provide an overview of the wide potential of BLI and its role in enhancing the study of unique-and sometimes difficult-to-characterize-bacterial pathogens.

Discovery of New Substrates for LuxAB Bacterial Bioluminescence

“…The LuxAB system has been previously used in whole animal bioluminescent imaging . The new substrates 5a and 5b could also be applied to bioluminescent imaging in vivo (Figure ).…”

“…The bacterial lux operon is an important bioreporter system that has been expanded for the detection of chemical signal molecules and imaging within a living host using cooled charge‐coupled device (CCD) camera. Moreover, either full or parts of them ( luxAB ) could express in alternative host organisms such as Escherichia coli . After several decades of research, great contribution has been made to the development of modified lux gene cassette expressing in various prokaryotic cell lines even the eukaryotic cell line .…”

Thein vitroandin vivoeffects of constitutive light expression on a bioluminescent strain of the mouse enteropathogenCitrobacter rodentium

“…Bioluminescently-labelled bacteria have gained popularity as a powerful tool for investigating microbial pathogenicity in vivo , and for preclinical drug and vaccine development ( Steinhuber et al , 2008 ; Massey et al , 2011 ; Sun et al , 2012 ; Kassem et al , 2016 ). Individual infected and/or treated animals can be followed over time, in contrast to the large numbers of animals that are euthanised at specific time points of interest for quantifying bacterial loads using labour-intensive plate count methods.…”

Sensor-based phenotyping of above-ground plant-pathogen interactions

“…Transgenic pathogen strains that express detectable markers such as fluorescent proteins or bioluminescence conferring enzymes are another alternative to track and quantify pathogen growth directly [ 24 , 25 ]. Fluorescent proteins have mainly been used to study the infection process in vivo at the cell level using epi-fluorescence or fluorescence confocal laser scanning microscopy.…”