Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters

Abstract

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the PmtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.

Synthesis, structure, antimycobacterial and anticancer evaluation of new pyrrolo-phenanthroline derivatives

“…The data from Table 1 illustrate that all tested indolizine with phenanthroline skeleton have a good solubility in microbiological medium, which is quite promising from the pharmacological properties point of view. Then, the IC 50 , IC 90 and MIC of compound were determined by measuring bacterial growth after 5 days in the presence of test compounds [36][37][38]49 . The obtained results are listed in Table 1.…”

Hybrid imidazole (benzimidazole)/pyridine (quinoline) derivatives and evaluation of their anticancer and antimycobacterial activity

“…Then, IC 50 , IC 90 , and MIC were determined by measuring bacterial growth after five days in the presence of test compounds [31][32][33][34] . As can be seen, from the seven tested compounds, only one 9 had activity against M. tuberculosis H37Rv under aerobic conditions, with an IC 90 of 77 mg/mL and a MIC4100 mM.…”

Reporter systems forin vivotracking of lactic acid bacteria in animal model studies

“…The most popular include bioluminescence with bacterial lux genes (lux), firefly and click beetle luciferase (luc), and fluorescence recorded in the case of green and red proteins. 22,31,32 Other well established imaging systems include magnetic resonance imaging (MRI) and positron emission tomography. 25,33 In vivo Fluorescence…”

“…32 The mCherry fluorophore is also highly resistant to photo bleaching, which means that the fluorophore will not lose its ability to fluoresce during continuous illumination. 50 The mCherry gene has been codon-optimized for optimal expression in various organisms, 32,34,35,51,52 including probiotic lactic acid bacteria (Fig. 1).…”

“…20,29,30 Supporting this notion, an absence of bioluminescence production as a result of bacteria being in a phase of dormancy has been reported for lux-expressing Leishmania amazonesis 73 and Mycobacterium smegmatis. 32 Absorption of light by mammalian tissues is one of the main factors influencing the sensitivity and detection limits of optical imaging ( Table 5). Hemoglobin serves as one of the most significant causes of light absorption in animals.…”