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

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bact eria escape the phagosomal compartment with the help of cytolytic (pore-for ming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumve nt the requirement of an "escape" step, we developed a procedure allowing t he efficient direct injection of bacteria into the cytosol of mammalian cel ls. With this technique, we show that most bacteria, including extracellula r bacteria and intracellular pathogens that normally reside in a vacuole, a re unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogene s, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Fu rther L. monocytogenes with deletion in its PrfA-regulated hpt gene was fou nd to be impaired in replication when injected into the cytosol. Complement ation of the hpt mutation with a plasmid carrying the wild-type hpt gene re stored the replication ability in the cytosol. These data indicate that cyt osolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.