Hpt, a novel virulence factor involved in the intracellular infectious cycle of L. monocytogenes. PrfA-dependent virulence factors acting at each of the steps of the cycle are indicated (reviewed in refs. 2 and 3). Scheme adapted from ref. 40, based on an original drawing in ref. 26.

(A) Growth curves of the L. monocytogenes wt strain P14 (WT) and its Δhpt derivative in brain–heart infusion and LB at 37°C. (B) Growth curves of wt, Δhpt and its hpt-complemented derivative (Δhpt +hpt) in LB-Amberlite (PrfA-activating conditions; see Materials and Methods) supplemented with 10 mM of G6P or Glc. Identical results were obtained in LB without Amberlite by using bacteria of prfA* genotype, in which hpt is constitutively overexpressed (6). When the PrfA system is inactive (bacteria of wt prfA background in normal LB), the growth curves of wt and Δhpt +hpt in G6P (not in Glc) are identical to that of the Δhpt mutant (i.e., 0.5 OD₆₀₀ units maximum growth).

(A) Effect of the Δhpt mutation on the intracellular replication of L. monocytogenes P14 in mammalian cells. Cell monolayers were infected with the following multiplicities of infection: Caco-2, 5:1; HepG2, 10:1; and J774.A1, 1:1. (B) Trans-complementation of L. monocytogenes Δhpt with a plasmid carrying hpt restores rapid intracellular bacterial replication. The experiment shown was performed with strain EGD (multiplicity of infection 10:1) to show that the Δhpt mutation has the same effect independently of the strain used. Results are the mean of at least three independent experiments (each performed in duplicate) ± SE. wt (●); Δhpt mutant (▴), and Δhpt transcomplemented with hpt (■).

Kinetics of L. monocytogenes populations in the cytoplasmic compartment of host cells by using the reporter plasmid pPactA-gfp. Caco-2 cells were infected (multiplicity of infection 10:1) with pPactA-gfp-containing wt and Δhpt bacteria, washed, and incubated in the presence of gentamicin for 2 h (t = 0). (A) Flow cytofluorimetry experiments: (●), wt, (▴) Δhpt mutant. Fluorescence intensity is expressed in arbitrary units. Data are mean values of three experiments ± SE. (B) Representative fluorescence micrographs. With this technique, less sensitive than flow cytofluorimetry, fluorescent bacteria were first detected 2 h after gentamicin addition (i.e., ≈3 h after infection).

L. monocytogenes bacteria lacking hpt are less virulent. Mice were injected by i.v. route with 5 × 10³ bacteria for each strain, and bacterial counts in organs were determined 1 h after infection (t = 0) and at the specified days. Data represent the mean bacterial counts of five animals ± SE; the number of animals with positive cultures is indicated. Gray bars, wt L. monocytogenes; black bars, Δhpt mutant. Similar results were observed after an intragastric inoculation. Note that during the acute phase of infection (days 0–4), no net increase in the load of Δhpt bacteria was observed in the liver, whereas in the spleen there was substantial proliferation of the Δhpt mutant. This finding suggests a more specific involvement of Hpt in colonization of the liver than the spleen by L. monocytogenes. It is tempting to correlate this observation with the fact that hepatocytes are among the mammalian cells that have the highest content of glycogen (up to 8–10% wet weight). Turnover of glycogen is high in hepatocytes to ensure glucose homeostasis, and its breakdown releases G1P, rapidly converted via phosphoglucomutase into G6P, which in turn enters glycolysis after conversion into F6P (21). Thus, the liver constitutes an unlimited source of Hpt-transported substrates, which may account for the more pronounced effect of the hpt mutation in this organ.

(A) Alignment of the sequence of Hpt from L. monocytogenes P14 (EMBL database accession no. AJ315765) with that of the UhpT-related transporters from S. enterica serovar Typhimurium (SwissProt database accession no. P27670), S. flexneri (S. flexneri 2a genome sequence in progress, University of Wisconsin-Madison, http://www.genome.wisc.edu/html/sflex.html), C. trachomatis (SwissProt database accession no. O84548), and human G6PT (SwissProt database accession no. O43826). (B) Phylogenetic tree of bacterial and mammalian HP transporters. The dendrogram was generated with the neighbor-joining algorythm by using the clustalx package (20). The bifurcations are supported by the indicated bootstrap values. The segment above the tree indicates the genetic distance. SwissProt database accession nos.: C. pneumoniae, Q9Z7N9; E. coli, P13408; Rattus norvegicus, Q9Z296; and Mus musculus, Q9D1F9. In parentheses after each species name, the percentage identity/similarity with respect to the L. monocytogenes Hpt (C). Promoter regions of PrfA-dependent virulence genes of L. monocytogenes. PrfA binding sites, centered on position −41.5 relative to the transcription start site, are boxed and −10 motifs are underlined. Promoters with a perfectly symmetric PrfA box (i.e., hly and plcA) are very sensitive to PrfA whereas those with mismatches in their PrfA box require larger amounts of the regulatory protein to become fully activated. The hpt gene has a PrfA box with one terminal mismatch, similar to actA, the promoter of which has a high threshold for PrfA-mediated activation and is therefore inactive extracellularly but fully induced in the host cell cytosol (see text). Regulation by PrfA is less stringent in promoters with PrfA boxes with more than one mismatch (e.g., inlA) (4, 5).