No abstract

Efficient replication in vivo is essential for a microparasite to colonize its host and the understanding of the molecular mechanisms by which microbial pathogens grow within host tissues can lead to the discovery of novel therapies to treat infection. Here we present evidence that the foodborne bacterial pathogen Listeria monocytogenes, a facultative intracellular parasite, exploits hexose phosphates (HP) from the host cell as a source of carbon and energy to fuel fast intracellular growth. HP uptake is mediated by Hpt, a bacterial homolog of the mammalian translocase that transports glucose-6-phosphate from the cytosol into the endoplasmic reticulum in the final step of gluconeogenesis and glycogenolysis. Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA, which upon entry into host cells induces a set of virulence factors required for listerial intracellular parasitism. Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence in mice. Hpt is the first virulence factor to be identified as specifically involved in the replication phase of a facultative intracellular pathogen. It is also a clear example of how adaptation to intracellular parasitism by microbial pathogens involves mimicry of physiological mechanisms of their eukaryotic host cells.

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB ؉ plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA ؉ plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA ؉ plasmid but differed over an Ϸ22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA ؉ plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.Rhodococcus equi is a member of the mycolic acid-containing group of actinobacteria, or mycolata, which also includes the Corynebacterium, Gordonia, Mycobacterium, and Nocardia genera (18). Like many other actinomycetes, R. equi is ubiquitous in nature and lives as a saprophyte in soil (25,35,41). The genus Rhodococcus is a genetically diverse taxon that may be empirically classified into two groups (4, 23): the nonpathogenic, or environmental, rhodococci, exemplified by Rhodococcus erythropolis, which includes metabolically versatile bacteria of industrial interest (32), and the pathogenic rhodococci, with two species, the plant pathogen Rhodococcus fascians (22) and the animal pathogen R. equi (25,35,41). All rhodococci typically harbor large conjugative plasmids encoding nicheadaptive functions, such as various primary and secondary metabolic processes in the environmental species and host colonization (virulence) factors in the pathogenic ones. Some of these extrachromosomal replicons are linear megaplasmids of up to 1 Mb in size, whereas others are circular plasmids of Ϸ100 kb (34, 55).R. equi can be isolated from pulmonary and extrapulmonary pyogranulomatous infections in various mammalian hosts. It causes equine purulent bronchopneumonia, or rattles, a severe respiratory disease of foals characterized by extensive abscessation of the lung parenchyma, lymphadenitis, and a high mortality rate. R. equi is also an opportunistic human pathogen associated with AIDS and immunosuppression. Human rho...

dWe report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted "bovine-type" allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species.

Two species of Listeria are pathogenic; L. monocytogenes infects humans and animals, and L. ivanovii has been considered to infect ruminants only. We report L. ivanovii–associated gastroenteritis and bacteremia in a man. This isolate was indistinguishable from prototypic ruminant strains. L. ivanovii is thus an enteric opportunistic human pathogen.

Molecular typing of the actinomycete Rhodococcus equi is insufficiently developed, and little is known about the epidemiology and transmission of this multihost pathogen. We report a simple, reliable polymerase chain reaction typing system for R. equi based on 3 plasmid gene markers: traA from the conserved conjugal transfer machinery and vapA and vapB, found in 2 different plasmid subpopulations. This "TRAVAP" typing scheme classifies R. equi into 4 categories: traA(+)/vapA(+)B(-), traA(+)/vapA(-)B(+), traA(+)/vapAB(-), and traA(-)/vapAB(-) (plasmidless). A TRAVAP survey of 215 R. equi strains confirmed the strong link between vapA (traA(+)/vapA(+)B(-) plasmids) and horse isolates and revealed other host-related plasmid associations: between traA(+)/vapA(-)B(+) and pigs and between traA(+)/vapAB(-)--a new type of R. equi plasmid--and cattle. Plasmidless strains were more frequent among isolates from nonpathological specimens. All plasmid categories were common in human isolates, which possibly reflects the predominantly opportunistic nature of R. equi infection in this host and a zoonotic origin.

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the foodborne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R 2 values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules). Bacteria of the facultative anaerobic gram-positive genusListeria are widely distributed in the environment, particularly the closely related species Listeria monocytogenes and L. innocua. Both of these Listeria spp. are frequently found in food products, where they can grow over a pH range of 4.39 to 9.40, even at refrigeration temperatures. Ingestion of foods contaminated with L. monocytogenes can result in listeriosis, a severe infectious disease characterized by meningoencephalitis, abortion, septicemia, and a high fatality rate (30%). Listeriosis predominantly affects certain risk groups, including pregnant women, newborns, elderly people, and immunocompromised patients. L. innocua, in contrast, is nonpathogenic, and its presence in foods is no hazard to human health (20,25,35,37,41). Human listeriosis outbreaks are most often associated with ready-to-eat food products that are consumed without prior cooking (8, 36). To err on the side of caution, food safety regulations have tended to adopt a zero-tolerance attitude for L. monocytogenes in these products (9). However, as clinical cases of listeriosis are usually associated with high loads of L. monocytogenes (6,8) and as it is difficult to eradicate listeriae from the environment of food-processing plants (11), the International Commission on Microbiological Specification for Foods concluded that 100 CFU of L. monocytogenes per g of food is accepta...