New targets to combat bacterial infections: Confocal microscopy to investigate bacterial invasion?

The search for innovative antibacterial natural products has pinpointed new targets. Inhibition of host-pathogen interaction has been shown to reduce the infection risk and the progression of the infection [1], [2], [3], [4]. Also, invasion into host cell can be influenced by natural products. The standard method to investigate bacterial invasion is the gentamicin protection assay, which require great workload and material requirements. The statistical significance is highly dependent on the cultivability of the bacteria including whether VBNC forms are present.

More specific and effective can be the monitoring of bacterial invasion by use of confocal laser scanning microscopy (CLSM). It can be determined at which position of the cell the bacteria are located, thus providing a direct indication of both adhesion and invasion. Also, time-dependent and quantitative evaluations can be performed easily by CSM. Using Campylobacter jejuni as model organism and Caco-2 cells, an easy-to-use CSM-protocol has been developed.

Host cells are stained fluorescently with DAPI for detection of intracellular compartments and Alexa-WGA-594, a lectin that binds to the cell membrane, to localize adherent bacteria. The bacteria themselves are fluorescently labelled with CFDA-SE. Invasion is performed as in the standard method. After the images are acquired, it can be determined to what degree the bacteria are adhered and/or invaded, and qualitatively, it can be assessed where they prefer to reside. Depending on laboratory equipment and experimental conditions, the adhesion and invasion of bacteria to and into the cell can be recorded as a time-lapse in a live cell imaging chamber and evaluated ([Fig. 1]).

Fig. 1 Time dependency of adhesion and invasion of bacteria.

Publication History

Article published online:
12 December 2022

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