This chapter discusses the inhibitors of cytoskeletal function and signal transduction to study bacterial invasion. Cytochalasins are a class of drugs that inhibit actin polymerization, thereby blocking microfilament function; however, these drugs also have several other significant effects on cells. Uptake of most, if not all, bacteria into eukaryotic cells (including nonphagocytic cells such as those of epithelial origin) can be blocked by the use of cytochalasins. Cytochalasin D is considered to be the most specific and potent of the cytochalasins. To determine if this drug affects bacterial uptake, cultured cells are preincubated in 1μg/ml cytochalasin D (Sigma) for 30 min prior to infecting with bacteria and measuring invasion levels. It is becoming apparent that invasive bacteria have developed mechanisms with which they can exploit existing host signal transduction pathways to trigger cytoskeletal rearrangement and bacterial uptake. Many of these pathways use protein kinases to transmit signals, including kinases that phosphorylate serine and threonine residues, such as protein kinase C (PKC), and those that phosphorylate tyrosine residues, such as tyrosine protein kinases (TPKs). Agents are becoming available that inhibit these various enzymes, and thus they can be used to probe whether bacterial invasion involves a host kinase.
Bibliographical noteFunding Information:
Work in B.B.F.'s laboratory is supported by operating grants from the British Columbia Health Care Research Foundation, the Medical Research Council of Canada, the Canadian Bacterial Diseases Center of Excellence, and a Howard Hughes International Research Scholars Award. I.R. is a recipient of a long-term fellowship from the European Molecular Biology Organization (EMBO) and a Canadian Association of Gastroenterology Industry Fellowship.