Macrophages can transform into smart pathogen detectors
Pathogenic infections are the leading cause of death worldwide and a major threat to global public health. In recent years, the emergence of various drug-resistant strains has become a serious problem of clinically acquired infections. AMR Review, published in the UK, estimates that about 700,000 people worldwide die from drug-resistant bacterial infections every year, and this number will increase to 10 million by 2050, with an estimated economic loss of US$100 trillion. Therefore, effective pathogen recognition methods are urgently needed to control bacterial transmission and facilitate anti-infective therapy.
Currently, plate culture is widely used for bacterial identification; however, the main disadvantages of this method are the long analysis time (often more than 48 h) and the need for specialized culture conditions, which limits its speed and availability. Nucleic acid detection (such as PCR) using nucleic acid primers has excellent sensitivity, but the workflow of these detections is complex (such as cell lysis, nucleic acid extraction, magnetic separation, washing and amplification, etc.), and requires expensive proteases and thermocycling . Immunoassays, such as those based on lateral flow dipsticks, are simple, low cost, and have rapid signal generation. However, sensor design and deployment require screening and optimization. At the same time, it is necessary to know the information of the target bacteria in advance. It is difficult to select suitable bioreceptors to capture and detect bacteria when the pathogen is unknown.
Over millions of years of evolution, innate immune cells, such as macrophages, dendritic cells, and neutrophils, have optimized their ability to recognize a broad spectrum of pathogens and their virulence factors. These immune cells rely on a set of surface receptors, such as toll-like receptors (TLRs), mannose receptors (MR), scavenger receptors (SR), and complement receptors (CR), to bind specific components of pathogens . However, developing immune cells as a smart probe capable of efficiently recognizing, enriching, and reporting specific pathogens remains a great challenge for the following reasons. First, living Møs have low mechanical stability, are fragile to various manipulations, and are easily damaged. Second, cells are not easy to manipulate for routine applications. Finally, immune cells cannot distinguish specific bacterial species, let alone produce detectable signals.
Recently, a research team from China published an article entitled "A smart pathogen detector engineered from intracellular hydrogelation of DNA decorated macrophages" in the journal Nature communications. The authors report a simple strategy，transforming living Møs into smart bacteria detectors by combining intracellular hydrogel technology and DNA nanotechnology. This approach has several benefits: (1) GMøs have a robust gel core and an intact cell membrane that can withstand harsh environments. (2) The bacteria-adsorbed GMøs can be conveniently separated from complex biological media by using magnetic separation. (3) GMøs can efficiently identify, capture and enrich a broad spectrum of bacteria to its surface. (4) GMøs can be modified with responsive DNA elements such as DNAzymes for biosensing of specific bacteria and analysis of bacteria-associated toxins using capture and detection strategies.
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Preparation of DNA-GMøs
The preparation process of DNA-modified gel macrophages (DNA-GMøs) is shown in the figure below. Møs were first incubated with superparamagnetic iron oxide nanoparticles to obtain magnetic cells. Simultaneous use of cytokines such as IL-4 induces upregulation of pathogen-binding receptors such as MR and CD163. Intracellular hydrogelation was then achieved by direct infiltration of photoinitiators and monomers, followed by UV irradiation. Finally, DNA sensing elements (such as DNAzymes) that respond to specific bacteria can be easily modified on gel cells for convenient, rapid and highly sensitive fluorescence readout. Thus, magnetic DNA-GMøs capable of identifying, enriching, and detecting a wide variety of microorganisms were generated. And the authors used different techniques to confirm the successful preparation of DNA-GMøs.
Stability of DNA-GMøs
To assess the stability of DNA-GMøs, the authors subjected the cells to different conditions, such as hypertonicity, repeated freeze-thaw cycles, sonication, high-speed centrifugation, and long-term storage (30 days). In contrast to the vulnerability of living Møs (LMøs) and paraformaldehyde-fixed Møs (FMøs) to the aforementioned environments, DNA-GMøs survived all conditions with negligible changes in cell morphology and number. Collectively, these results confirm the high stability of DNA-GMøs, thereby facilitating their downstream applications such as pathogen capture and detection.
The ability of GMøs to identify and capture different microorganisms
Two representative bacteria, Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus), were used as model pathogens to test the performance of GMøs in binding and separating pathogens. The results showed that after GMøs was incubated with the bacterial suspension for 15 min, the cell particles were collected by a magnet, and bacteria were successfully captured, and the capture ability of GMøs activated by IL-4 was much better than that of non-activated GMøs
GMøs can be used to capture other microorganisms including P. aeruginosa, V. parahemolyticus, S. enteritidis and C. albicans. When three bacteria (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) were mixed in one sample, GMøs could capture all species simultaneously, demonstrating the broad-spectrum capture capability of GMøs. Unlike traditional antibody-modified magnetic beads (ab-mb), the capture capacity of GMøs is not affected by complex media, further emphasizing their advantages.
Specific detection of captured bacteria with dnazyme-modified GMøs
Escherichia coli-targeting DNAzymes (called DzEC-GMøs) modified on GMøs can be used to detect live E. coli. In the absence of E. coli, DzEC-GMøs fluorescence was negligible, indicating a low background signal for this cell-based sensor. Once the bacteria were captured, a strong green fluorescence was observed around the cell membrane. The LOD is approximately 500 CFU/mL and is highly specific, with other bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Streptococcus pyogenes, and Candida albicans failing to produce a detectable signal.
Two DNAzymes targeting Escherichia coli and Staphylococcus aureus (called DzECSA-GMø) were modified on the cell membrane (bottom h). Each enzyme labeled with a fluorophore (FITC or Rh) can bind to the corresponding bacterial-secreted CEMs and cleave its substrate, thereby restoring fluorescence. Introducing one of the two bacteria into the sample restores the specific fluorescent signal, and the two fluorophore signals can only be observed at the same time when both bacteria are present at the same time
Analysis of pore-forming toxins using GMøs
Binding events between the GMø membrane and the target toxin lead to the formation of pores in the cell membrane, facilitating the penetration of propidium iodide. In the absence of Hlα, GMøs remained dark during the experiment. After adding 100 nM Hlα, the red fluorescence gradually appeared and reached a plateau at 25 min. The experimental results showed that the limit of detection (LOD) for the detection of Hlα by PI staining was estimated to be 10 fM.
Real sputum samples from patients with pneumonia caused by Staphylococcus aureus in a local hospital were analyzed using DzSA-GMøs. In this experiment, the presence of S. aureus in the sample activates DNAzymes, producing green fluorescence on the cell membrane, while Hlα causes an increase in PI fluorescence. As shown in panel f below, all patient samples produced a positive signal for S. aureus. At the same time, PI staining experiments showed that not all Staphylococcus aureus secreted H1α (ie, P2 and P5). The results demonstrate the ability of the method to rapidly analyze bacteria and toxins in clinical samples.
