Growth in the Host Cell Cytoplasm

Phagosomal escape is a prerequisite for L. monocytogenes to replicate intracellularly and is hence a critical virulence trait of this species, as it is also for Shigella flexneri and Rickettsia spp. L. monocytogenes starts intracellular multiplication shortly after escape from the vacuole, with intracellular generation times close to those observed in rich broth culture [85]. The host cell cytoplasm hence allows listerial growth with high efficiency. However, the cytosol is poorly characterized as a substrate supporting bacterial growth, and the relative abundance of nutrients is unknown. Whereas various auxotrophic mutants of L. monocytogenes are able to grow intracellularly [86], the expression of several metabolic genes is intracellular^ increased [60], indicating that at least some metabolites may be limiting for growth in the cytosol.

Upon microinjection into the cytoplasm of mammalian cells, only bacteria naturally capable of intracytoplasmic growth like L. monocytogenes and S. flexneri replicated efficiently in these cells [87]. Furthermore, a DprfA mutant of L. monocytogenes multiplied poorly upon microinjection, pointing to the need of specific virulence determinants for efficient intracytoplasmic multiplication. In the search for L. monocytogenes-specific bacterial factors allowing intracellular growth, a gene with high homology to uhpT of E. coli encoding a hexose phosphate transporter which also shows similarity to the mammalian glucose-6-phosphate translocase (this listerial transporter is termed Hpt, encoded by lmo0838) [66] and a gene lplAl (lmo0931) encoding a lipoate protein ligase (LplAl) [88] were identified in the L. monocytogenes genome sequence. Both genes are necessary for efficient intracellular proliferation of L. monocytogenes. Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA. Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence in mice but did not affect bacterial growth in a rich medium [66]. However, Hpt alone is not sufficient for efficient cytoplasmic growth since intracytoplasmic replication of L. innocua expressing Hpt together with LLO is not significantly improved upon infection of macrophages [89].

The lack of LplA1 results in bacteria which cease intracellular replication after about five rounds of replication and which are clearly defective in mouse virulence assays. A major target for LplA is the E2 subunit of the pyruvate dehydrogenase enzyme (PDH) complex, and it was shown that in intracellular^ grown IplAl mutants PDH is no longer lipoylated. Studies of lipoic acid metabolism have furthermore shown that little free lipoic acid exists in the mammalian cytosol. Thus LplA1 may not be important in the replication of L. monocytogenes in culture media where free lipoate is available, but could be required in the host cell where lipoate supply may be limited [88].

0 0

Post a comment