Authors: Hong Xiaoxiao, Geng Pengfei, Tian Na, Li Xueyuan, Gao Mengqiu, Nie Lihui, Sun Zhaogang, Liu Gang
First authors and affiliations: Xiaoxiao Hong, School of Pharmacy, Tsinghua University, Pengfei Geng, School of Pharmacy, Tsinghua University, Na Tian, Beijing Chest Hospital, Capital Medical University
Corresponding authors and affiliations: LIU Gang, School of Pharmacy, Tsinghua University, and SUN Zhaogang, Beijing Chest Hospital, Capital Medical University
From Bench to Clinic: A Nitroreductase Rv3368c-Responsive Cyanine-Based Probe for the Specific Detection of Live Mycobacterium tuberculosis.
Hong X, Geng P, Tian N, Li X, Gao M, Nie L, Sun Z, Liu G.
Analytical chemistry, 2024 Jan 8.
doi:10.1021/acs.analchem.3c04293.
PMID: 38190499.
Background:
Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is characterized by a high mortality rate and a low diagnostic rate. According to the World Health Organization, there are an estimated 10.6 million cases of tuberculosis worldwide in 2021, but only 6.4 million cases have been diagnosed with tuberculosis, and the diagnosis of tuberculosis is the weakest link in tuberculosis control. Sputum smear microscopy with auramine O (AO) or Ziehl-Neelsen is the first-line diagnostic method in countries with a high burden of tuberculosis, however, both stains bind to mycobacterial acid in the cell wall of MTB and cannot distinguish between active and non-replicating MTB and dead bacteria, therefore, they cannot reflect the therapeutic effect of patients with tuberculosis after taking the drug, which can easily lead to the occurrence of drug-resistant tuberculosis. In addition, the cumbersome staining steps of the two methods and the low photostability of AO require rapid quantification, resulting in uneven sensitivity. There is an urgent need to develop a new generation of photostable fluorescent probes for fast and easy labeling of live MTBs. Due to the existence of different bacteria and complex components in clinical sputum samples, there are high requirements for the chemical structure stability, optical stability, specificity and sample processing stability of fluorescent probes.
In this study, a cyano-based probe, Cy3-NO2-tre, was designed and synthesized. Cy3-NO2-tre does not emit light by itself, but it can be reduced by the specific nitroreductase Rv3368c in MTB and then turn on fluorescence, which can quickly track the infection process of MTB, and can also detect MTB in the sputum of tuberculosis patients. Preliminary findings from this study suggest that Cy3-NO2-tre in combination with AO can provide a rapid method for patients with tuberculosis to rapidly assess drug efficacy and optimize drug treatment regimens in the early care phase of patients.
Research Methods:
In this study, the fluorescence fold change (FFC) of bacteria was analyzed by flow cytometry, and the Cy3-NO2-tre with the highest fluorescence intensity was identified as the final fluorescent reagent, and the specificity was verified by labeling the MTB in different species of mixed bacteria with Cy3-NO2-tre. Then, the pull-down assay was used to identify Rv3368c, the target of Cy3-NO2-tre, and the fluorescence intensity of Cy3-NO2-tre in Rv3368c gene knockout, compensation and overexpression of Rv3368c in E. coli DE3 was compared. Then, the cells infected with MTB after co-incubation with Cy3-NO2-tre were imaged by confocal microscopy, and the process of the host cells protruding from the pseudopodia to capture MTB was observed. In addition, the research group also used Cy3-NO2-tre and AO to detect MTB in clinical sputum samples, and compared its fluorescence antifade ability with AO. The research team also combined it with AO to monitor the change of the ratio of live/dead bacteria in the sputum before and after 1 week of anti-drug treatment, preliminarily studied the correlation of CT results after 3 months of drug treatment, and monitored the treatment effect of patients treated with anti-tuberculosis therapy.
Findings:
The results showed that Cy3-NO2-tre could selectively label MTB in mixed bacteria of different species without fluorescent labeling of other bacteria (Figure 1). The research group used the pull-down assay to identify Rv3368c, the target of Cy3-NO2-tre, and verified it by comparing the fluorescence intensity of Cy3-NO2-tre in Rv3368c gene knockout, back-complementing mycobacterial strains, and overexpressing Rv3368c E. coli DE3 (Fig. 2). The results of bioinformatics analysis based on NCBI showed that Rv3368c was mainly expressed in Mycobacteraceae tuberculosis.
Fig.1 Structure and fluorescent labeling ability of cyanine fluorescent probes
Fig.2 Exploration and validation of specific nitroreductase RV3368c
Cy3-NO2-tre can selectively label MTB within host cells without producing non-specific labeling on host cells (Figure 3). Cy3-NO2-tre has the advantages of fast response, no-wash, and high sensitivity, which enables it to detect the whole process of MTB infection of host cells in real time. The research group used Cy3-NO2-tre to successfully observe the process of host cells extending pseudopodia to capture MTB, and also found that some bacteria can successfully escape the "capture" of host cell pseudopodia, which provides a tool molecule for further study of the interaction between host cells and MTB.
Fig.3 Cy3-NO2-tre tracked the interaction between MTB and host cells
Cy3-NO2-tre was used to detect live MTB in clinical sputum samples. Cy3-NO2-tre exhibits significantly stronger fluorescence antifade ability compared to AO (Figure 4). Based on the characteristic that Cy3-NO2-tre only labels live bacteria, the research team combined it with AO to preliminarily study the therapeutic effect of anti-tuberculosis drugs in monitoring tuberculosis patients. Usually, patients with tuberculosis need to take the drug for 3 months and then have a CT scan to determine the patient's sensitivity to the treatment regimen. Cy3-NO2-tre was used to monitor the changes in the ratio of live/dead bacteria in the sputum before and after 1 week of treatment, and the correlation of CT results after 3 months of drug treatment was preliminarily studied (Fig. 5), indicating that Cy3-NO2-tre may be used for early monitoring of the therapeutic effect of anti-tuberculosis drugs.
Fig.4 The fluorescence antifade effect of Cy3-NO2-tre on clinical sputum samples is stronger than that of AO
Fig.5 Cy3-NO2-tre is used to evaluate the efficacy of anti-tuberculosis therapy in patients with tuberculosis in the early stage
Conclusions of the study
In summary, this report reports a novel fluorescent probe, Cy3-NO2-tre, that is highly responsive to the specific nitroreductase Rv3368c. It can be used as a tool to study host-mycobacterial interactions, discover potential targets for tuberculosis treatment, and promote the development of novel anti-tuberculosis drugs by binding to cell wall specific strong labeling of MTB and rapid imaging of phagocycled MTB. In addition, due to the characteristics of high fluorescence intensity, low background fluorescence, good photostability, stable structure and non-washing, Cy3-NO2-tre can also detect live MTB in the sputum of tuberculosis patients with significant specificity and sensitivity. The preliminary results of this study suggest that the combination of Cy3-NO2-tre and AO has the potential to track the efficacy of anti-tuberculosis drug treatment regimens in the early patient care stage, but large-scale prospective clinical investigations are still needed to further investigate the combination of this approach with artificial intelligence technology, and finally develop a rapid and accurate efficacy evaluation technology.