Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an ''escape'' step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfAregulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells. M any pathogenic bacteria are able to trigger their uptake by mammalian cells, which is followed by efficient multiplication of the internalized bacteria inside of the host cells. Internalization of these bacteria involves normal phagocytosis when the host cells are professional phagocytes, e.g., macrophages, or triggered phagocytosis in the case of nonprofessional phagocytic host cells, such as epithelial cells, hepatocytes, fibroblasts, and endothelial cells (1, 2). After internalization, most intracellular bacteria reside and replicate inside membrane-bound vacuoles that are specifically modified by the different bacteria (3, 4). Salmonella enterica, Legionella pneumophila, members of the Mycobacterium tuberculosis complex, Mycobacterium leprae, Brucella spp., Chlamydia, Rhodococcus equi, and several others belong to this group of intracellular bacteria. A smaller group of intracellular bacteria, including Shigella spp., the closely related enteroinvasive Escherichia coli (EIEC), Listeria monocytogenes, Listeria ivanovii, and Ricksettia spp., can escape from the primary phagosome into the host cell cytosol where the bacteria proficiently replicate. These latter bacteria synthesize specific proteins that disrupt the phagosomal membrane, thus allowing bacterial entry into the cytosol. In L. monocytogenes, the required proteins are best characterized and comprise the pore-forming lysteriolysin (LLO) and two phospholipases C, PlcA and PlcB (5, 6).It has been reported that the introduction and expression of the listerial hly gene (encoding LLO) in Bacillus subtilis leads to the release of these avirulent bacteria into the cytosol of mammalian cells wher...

During the past 5 years, green fluorescent protein (GFP) has become one of the most widely used in vivo protein markers for studying a number of different molecular processes during development, such as promoter activation, gene expression, protein trafficking and cell lineage determination. GFP fluorescence allows observation of dynamic developmental processes in real time, in both transiently and stably transformed cells, as well as in live embryos. In this review, we include the most up-to-date use of GFP during embryonic development and point out the unique contribution of GFP visualization, which resulted in novel discoveries.

We report the synthesis of a simple pyrene derivative and its application in protamine detection and monitoring of trypsin activity. This assay can be conducted in aqueous solution and features rapid response, visual detection, high sensitivity and selectivity. The limit of detection of protamine was 0.5 μg mL(-1). The IC50 value of a soybean trypsin inhibitor was estimated to be 0.51 U mL(-1).

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