Conclusions

Bioinformatic and functional genomic data indicate that L. monocytogenes is a heterotrophic and largely prototrophic bacterium belonging to the group of low G + C Gp bacteria. But its metabolism is also optimally adapted for highly efficient growth within the cytosol of many mammalian cells (Goetz et al., 2001). The metabolism of L. monocytogenes reveals some unusual features which seem to have profound consequences for extra- and intracellular replication of L. monocytogenes and hence for virulence:

1 the inability to use oxidized sulfur and nitrogen sources due to the lack of nitrate and sulfate reductases,

2 the interrupted citrate cycle due to the lack of 2-oxoglutarate dehydrogenase, and

3 the complex PTS-mediated glucose transport.

(1) This feature readily explains the observed auxotrophy of L. monocytogenes for Cys (and in the absence of Cys for Met) and indicates that intracel-lularly replicating L. monocytogenes will entirely depend on the host cell for Cys supply. Cystein is a nonessential amino acid for mammalian cells whereas methionine is an essential one. In L. monocytogenes, the situation is reverse and the two "partners" could therefore provide each other with the necessary sulphur-containing amino acids.

(2) The incomplete citrate cycle renders the synthesis of oxaloacetate by pyruvate carboxylase to be a critical metabolic step when L. monocytogenes grows in environments where glucose (or another carbohydrate) is the sole carbon source. Shortage of this catabolic intermediate (and as a consequence also pyruvate and acetyl-CoA) may be the reason for the unexpected dependency of L. monocytogenes on BCAA (Ile and Leu or Val), Met and to a lesser extent Arg for efficient growth in the minimal media containing glucose as a sole carbon source. Although the biosynthetic pathways for these amino acids are functionally intact, their efficient synthesis depends directly or indirectly on the availability of oxaloacetate. In fact, a B. subtilis mutant deficient in 2-oxoglutarate dehydro-genase required Asp (which derives directly from oxaloacetate and represents an important intermediate for the biosynthesis of these amino acids) for growth at wild-type rates in minimal media due to the inability of the mutant to regenerate oxaloacetate from citrate (Fisher and Magasanik, 1984). The syntheses of these amino acids which are needed for protein synthesis and branched-chain fatty acids (especially Ile) (Nichols et al., 2002) in large amounts depend on oxaloac-etate (via Asp), pyruvate (needed for synthesis of oxaloacetate), or actyl-CoA (deriving from pyruvate). As mentioned above (see discussion on the ilv-leu operon), nitrogen metabolism and carbon metabolism are coregulated by the global regulators CcpA, TnrA, and CodY (Shivers and Sonenshein, 2005; Tojo et al., 2005). This regulatory network (best studied in B. subtilis) guarantees well-balanced intracellular concentrations of the central carbon (especially glucose and its catabolites) and nitrogen (especially Gln, Glu) intermediates essential for the entire cellular metabolism.

The inability of L. monocytogenes to regenerate oxaloacetate from citrate may be overcome within the host cell by the supply of malonate. This intermediate, which is directly converted to oxaloacetate in the citrate cycle, could be provided by the host cell via the oxoglutarate/malate shuttle at the expense of the oxoglutarate produced in excess by L. monocytogenes due to the absence of oxoglutarate dehydrogenase. It has been shown that a B. subtilis mutant deficient in this enzyme secretes considerably larger amounts of OG into the medium than the wild-type strain (Fisher and Magasanik, 1984).

(3) The most important feature is, however, the use of the appropriate carbon source by L. monocytogenes within the host cell. The transport of glucose, a preferred carbon source for L. monocytogenes metabolism, is achieved in a yet unknown way. Although a large number of (in part L. monocytogenes specific) PTS has been identified in the L. monocytogenes genome, a functional PTS-G glucose uptake system (characteristic for many bacteria) is missing and glucose may be cotransported by several other PTS permeases (R. Ecke, personal communication).

Uptake of glucose-1-P generated by degradation of host cell's glycogen (a nonessential storage product of the host cell) by the specific transporter (Hpt) avoids the competition for glucose with the host cell, and at the same time, the inactivation of PrfA by PTS-mediated sugar uptake. The efficiency of intra-cellular replication and virulence may be therefore strongly influenced by the carbon source and its transport.

Phospholipids may act as alternative intracellular carbon source for L. monocy-togenes (and may even become the primary carbon source in mammalian cells lacking glycogen). Phospholipids are probably generated in sufficient amounts by the disruption of the primary phagosome by which L. monocytogenes is internalized. In particular PEA may serve as an important carbon and nitrogen source since it can be converted by cellular lipases of the A-type and especially the listerial PlcB to glycerol, fatty acids, and ethanolamine phosphate, which after dephosphorylation can serve as nitrogen source for L. monocytogenes in presence of glycerol as carbon source (Schaffer et al., unpublished results). UnlikeM. tuberculosis and S. enterica which use the fatty acids as major intracellular carbon sources (Fang et al., 2005; McKinney et al., 2000), L. monocytogenes could utilize mainly glycerol and ethanolamine as intracellular nutrients.

We hypothesize that these three metabolic features, although unfavourable for growth of L. monocytogenes under certain extracellular conditions, are essential for the efficient intracellular replication of L. monocytogenes since they lead to an intimate interference between the metabolism of the bacterium and that of the host cell. This metabolic interference will allow an extended survival of the infected cell, which can then serve as a "host cell" for L. monocytogenes for a longer period of time.

Acknowledgments. The authors thank Stefanie Müller-Altrock for useful discussions and critical reading of the manuscript.

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