Application of fluorescent protein expressing strains to evaluation of anti-Tuberculosis therapeutic efficacy in vitro and in vivo

Ying Kong, Dong Yang, Suat L.G. Cirillo, Shaoji Li, Ali Akin, Kevin P. Francis, Taylor Maloney, Jeffrey D. Cirillo

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-Time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: TdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-TdTomato displayed the highest fluorescence.We used the tdTomatolabeled M. bovis BCG to obtain real-Time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-TdTomato and Hsp60-TdTomato revealed that L5-TdTomato carried four-fold more tdTomato gene copies than Hsp60-TdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-TdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-Treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

Original languageEnglish (US)
Article numbere0149972
JournalPloS one
Volume11
Issue number3
DOIs
StatePublished - Mar 1 2016

Fingerprint

Mycobacterium bovis
tuberculosis
Tuberculosis
Mycobacterium bovis BCG
therapeutics
image analysis
Proteins
Imaging techniques
fluorescence
vaccines
isoniazid
Animals
Therapeutics
Vaccines
Fluorescence
rifampicin
Mycobacterium
Mycobacterium tuberculosis
Density (optical)
Bacterial Load

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Application of fluorescent protein expressing strains to evaluation of anti-Tuberculosis therapeutic efficacy in vitro and in vivo. / Kong, Ying; Yang, Dong; Cirillo, Suat L.G.; Li, Shaoji; Akin, Ali; Francis, Kevin P.; Maloney, Taylor; Cirillo, Jeffrey D.

In: PloS one, Vol. 11, No. 3, e0149972, 01.03.2016.

Research output: Contribution to journalArticle

Kong, Ying ; Yang, Dong ; Cirillo, Suat L.G. ; Li, Shaoji ; Akin, Ali ; Francis, Kevin P. ; Maloney, Taylor ; Cirillo, Jeffrey D. / Application of fluorescent protein expressing strains to evaluation of anti-Tuberculosis therapeutic efficacy in vitro and in vivo. In: PloS one. 2016 ; Vol. 11, No. 3.
@article{3e5bdd9f455a4403b0f83f2fdf8eefa4,
title = "Application of fluorescent protein expressing strains to evaluation of anti-Tuberculosis therapeutic efficacy in vitro and in vivo",
abstract = "The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-Time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: TdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-TdTomato displayed the highest fluorescence.We used the tdTomatolabeled M. bovis BCG to obtain real-Time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-TdTomato and Hsp60-TdTomato revealed that L5-TdTomato carried four-fold more tdTomato gene copies than Hsp60-TdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-TdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-Treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.",
author = "Ying Kong and Dong Yang and Cirillo, {Suat L.G.} and Shaoji Li and Ali Akin and Francis, {Kevin P.} and Taylor Maloney and Cirillo, {Jeffrey D.}",
year = "2016",
month = "3",
day = "1",
doi = "10.1371/journal.pone.0149972",
language = "English (US)",
volume = "11",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "3",

}

TY - JOUR

T1 - Application of fluorescent protein expressing strains to evaluation of anti-Tuberculosis therapeutic efficacy in vitro and in vivo

AU - Kong, Ying

AU - Yang, Dong

AU - Cirillo, Suat L.G.

AU - Li, Shaoji

AU - Akin, Ali

AU - Francis, Kevin P.

AU - Maloney, Taylor

AU - Cirillo, Jeffrey D.

PY - 2016/3/1

Y1 - 2016/3/1

N2 - The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-Time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: TdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-TdTomato displayed the highest fluorescence.We used the tdTomatolabeled M. bovis BCG to obtain real-Time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-TdTomato and Hsp60-TdTomato revealed that L5-TdTomato carried four-fold more tdTomato gene copies than Hsp60-TdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-TdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-Treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

AB - The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-Time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: TdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-TdTomato displayed the highest fluorescence.We used the tdTomatolabeled M. bovis BCG to obtain real-Time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-TdTomato and Hsp60-TdTomato revealed that L5-TdTomato carried four-fold more tdTomato gene copies than Hsp60-TdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-TdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-Treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

UR - http://www.scopus.com/inward/record.url?scp=84961178338&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84961178338&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0149972

DO - 10.1371/journal.pone.0149972

M3 - Article

VL - 11

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 3

M1 - e0149972

ER -