Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using wholegenome microarray and mutant analyses. We found that ϳ17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses.Listeriosis is a food-borne disease with high mortality rates. Listeria monocytogenes, the causative agent, can survive in diverse habitats, including extracellular abiotic and intracellular environments, and infect a large number of vertebrate and invertebrate hosts. All of the identified virulence genes in L. monocytogenes are known to be under the direct or partial control of positive regulatory factor A (PrfA) (9, 34). Entry into nonprofessional phagocytes is mediated by surface-associated gene products internalin A and B (12, 31). Early after internalization, the bacteria disrupt the phagosomal membrane of the host by expressing a pore-forming toxin, listeriolysin (Hly), and a phospholipase (PlcA) to access the cytoplasm of the host cell. Intracellular movement of the bacteria inside the host cell is mediated by ActA, which polymerizes the host actin molecules and propels itself inside the cytosol of the host cell. Spreading from one cell to another is dependent on hemolysin (Hly) and another phospholipase (PlcB) (39). Mutations have been introduced into virulence genes of L. monocytogenes that lead to debilitating phenotypes in the mouse model of infection. In addition, a hexose phosphate transporter, UhpT, that is required for efficacious intracellular growth has recently been described (11). Rece...

Background: The opportunistic food-borne gram-positive pathogen Listeria monocytogenes can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, B , has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine B expression during growth of L. monocytogenes EGD-e. Whole genome-based transcriptional profiling was used to identify B -dependent genes at different growth phases.

The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP 4 ). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultracentrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP 4 sites have roughly equivalent affinities with dissociation constants of about 4 M. Mutational analysis of the FP 4 motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.

Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC؉vgc1 and pUvBBAC؉vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics.With the rapid increase in the number of completed microbial genome sequences, interest in the manipulation and functional characterization of whole genomes is being revolutionized. Methods and technologies that permit the cloning and manipulation of large chromosomal regions, such as the genomic islands which impart either virulence or novel metabolic properties to pathogenic and other microorganisms, are rapidly becoming valuable tools to study in detail properties encoded within these regions (41).The development of bacterial artificial chromosomes (BACs) has provided an important genetic tool for the cloning and mapping of complex genomes. Different cloning vectors based on low-copy-number replicons either from the bacterial F plasmid (45) or bacteriophage P1 (23, 49) have been developed. These provide a powerful resource in molecular biology, because they allow the cloning of several tens to hundreds of kilobases of contiguous DNA sequences (55) and are stable and easy to handle (31). Their particular use is in the study of functional genome segments that are otherwise too large to be cloned into other more conventional vectors. BAC-based libraries of genomic DNA from numerous viruses (1, 4, 8, 29-31, 42, 43) and plant (9, 15, 27, 32, 47, 53), animal (10, 34, 50, 55), and fungal (20, 39, 54) species have been generated and are now established technologies in large-scale sequencing projects. Classical and molecular genetic techniques are being used in conjunction with BAC recombinants for the introduction of reporter systems into mammalian organisms, the in vivo complementation of mutations, and in vivo and in vitro reverse genetic technologies that introduce point mutations (25), targeted deletions, or new sequence elements into BAC vectors (21). More recently, in vivo recombination ("recombineering") (37) technology employing either the bacterial recA mutant or the bacteriophage red gam mutant recombination has been used for the dir...

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