Microinjection and growth of bacteria in the cytosol of mammalian host cells

Abstract

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...

Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery

“…This suggests that bacterial cell disruption and subsequent plasmid release is an autocatalyzing process probably initiated by the spontaneous lysis of a single bacterial cell which releases phage lysin (in the case of pSP118) and other peptidoglycan-hydrolyzing enzymes subsequently disrupting most other carrier bacteria present in the target cell thus resulting in the release of sufficient plasmid DNA to warrant expression of the plasmid-encoded gene by the target cell. The observation that microinjection of S. typhimurium carrying the same plasmid (pSP118) yields considerably fewer EGFP-expressing cells than even the L. monocytogenes DtrpS strain with pSP0 supports this assumption; S. typhimurium is virtually unable to replicate in Caco-2 cell cytosol after microinjection 23 and hence the amount of plasmid DNA released by spontaneous lysis of these carrier bacteria will be rather low and hence EGFP expression less efficient.…”

“…23 By coinjection of Texas red-labelled dextran together with the bacterial carrier strains (Figure 7, right panel) we observed that EGFP expression occurred in the microinjected cell 24 h after microinjection (indicated by the yellow fluorescence caused by the overlap of the green EGFP and the red fluorescence of Texas red). As summarized in Figure 7, about 5% of the cells microinjected with the L. monocytogenes D(trpS actA) strain harboring pSP0 and more than 13% of those harboring pSP118 expressed EGFP.…”

“…23 Additionally, bacteria were microinjected after resuspension with PBS buffer containing 0.5 mg/ml Texas red-labelled dextran (70 000 MW, Molecular Probes).…”

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…This metabolic process depends on the synthesis of Hpt, a hexose phosphate transporter protein that enables bacterial intracellular multiplication and is necessary for proliferation in mouse organs. 145,146 The lipoate ligase LplA1 is among other bacterial proteins required for growth in host cells. This enzyme is essential to perform a critical lipoyl modification in the E2 subunit of pyruvate dehydrogenase, in the presence of limiting concentrations of available host lipoyl substrates, which suggests that abortive growth is due to loss of pyruvate dehydrogenase function.…”

Growth and killing of a Salmonella enterica serovar Typhimurium sifA mutant strain in the cytosol of different host cell lines

“…subtilis and non-pathogenic Escherichia coli (Bielecki et al, 1990 ;Gentschev et al, 1995 ;Goebel & Kuhn, 2000 ;Goetz et al, 2001).…”