José A. Vázquez-Boland

Listeria Pathogenesis and Molecular Virulence Determinants

SUMMARY The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.

Comparative Genomics of Listeria Species

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated

Evidence for ptelotropic activation of virulence genes in Listeria monocytogenes is presented. A complementation study of a spontaneous prfA‐deletion mutant and analysis of cassette and transposon insertion mutants showed that the gene prfA activates the transcription of four independent genes which code for a phosphatidyl‐inositol‐specific phospholipase C (gene plcA), listeriolysin O (gene hlyA), a metallo‐protease (gene prtA) and a lecithinase (gene prtC). Transcription of prfA is not constitutive. During the growth phase, two peaks of prfA transcript accumulation were observed: the first was during exponential growth, and the second was at the beginning of the stationary phase. In addition, two prf4‐specific transcripts of 2.2 kb and 1 kb are detected. Early in exponential growth, prfA is co‐transcribed with plcA which lies upstream prfA, giving rise to the 2.2 kb plcA‐prfA transcript. In late‐exponential growth and at the beginning of the stationary phase, prfA transcripts of 1 kb are predominantly detected. Our results demonstrate that since prfA controls plcA transcription, it also regulates its own synthesis.

Quantitative Detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly, iap, and lin02483 Targets and AmpliFluor Technology

ABSTRACT We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne 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 R2 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).

Hpt, a bacterial homolog of the microsomal glucose- 6-phosphate translocase, mediates rapid intracellular proliferation in Listeria

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.

Pathogenicity islands and virulence evolution in Listeria

As in other bacterial pathogens, the virulence determinants of Listeria species are clustered in genomic islands scattered along the chromosome. This review summarizes current knowledge about the structure, distribution and role in pathogenesis of Listeria virulence loci. Hypotheses about the mode of acquisition and evolution of these loci in this group of Gram-positive bacteria are presented and discussed.

Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition

A panel of 103 Listeria monocytogenes strains of different origins was examined for haemolysin and lecithinase production in brain-heart infusion (BHI). Three distinct phenotypes were observed. Phenotype 1 was characterized by low to undetectable levels of expression and was exhibited by almost all strains tested. Phenotype 2 expressed high levels of haemolysin and lecithinase and was displayed by five strains: one (P14-A) was a spontaneous mutant derived from a type 1 isolate (P14); the four others (EGD-A, NCTC 7973, SLCC 2373 and CLIP 545) were all laboratory strains kept under in vitro conditions for a long period. Phenotype 3 was intermediate and was exhibited by another laboratory strain (L028). We therefore concluded that phenotype 1 corresponded to the wild type, whereas phenotypes 2 and 3 represented mutant or variant phenotypes. Interestingly, wild-type strains were able to dramatically increase the expression of virulence factors when cultured in BHI treated with activated charcoal (BHIC), up to levels similar to those constitutively expressed by the hyperhaemolytic/lecithinase variants in BHI. Experiments with P14 and P14-A demonstrated that both charcoal and the hyperhaemolytic/lecithinase mutation exerted their effect by inducing (or derepressing) transcription of prfA, the pleiotropic transcriptional activator of the L. monocytogenes virulence regulon. Moreover, P14 and P14-A were equally virulent for mice despite the different levels of virulence factor expression in BHI. Taken together, these observations indicate that L. monocytogenes turns off virulence gene expression when growing in vitro in a rich medium, and suggest that the increased levels of virulence factors in the hyperhaemolytic/lecithinase mutants and in wild-type strains grown in BHIC might represent the levels of expression needed in vivo by L. monocytogenes for infecting host tissues.

Regulation of Listeria virulence: PrfA master and commander

Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection. These bacteria live as soil saprotrophs on decaying plant matter but also as intracellular parasites, using the cell cytosol as a replication niche. PrfA, a regulatory protein, integrates a number of environmental cues that signal the transition between these two contrasting lifestyles, activating a set of key virulence factors during host infection. While a number of details concerning the general mode of action of this virulence master switch have been elucidated, others remain unsolved. Recent work has revealed additional mechanisms that contribute to L. monocytogenes virulence modulation, often via cross-talk with PrfA, or by regulating new genes involved in host colonization.

Regulation of virulence genes in Listeria

As in all pathogenic bacteria, virulence of the facultative intracellular Listeria species is a multifactorial trait. The expression of the bacterial genes involved in the different steps of the infectious process--invasion, intracellular multiplication and spreading--is temporally and spatially controlled, thus ensuring the presence of the respective gene products at the right moment and place. So far, one network which is involved in the regulation of listerial virulence, the PrfA regulon, has been characterized rather well. The key element of this regulon, PrfA, belongs to the Crp/Fnr family of transcriptional regulators. Its synthesis and activity are influenced by a variety of physico-chemical signals outside and inside of eukaryotic host cells. The analysis of virulence gene expression in vivo, i.e. in infected host cells, indicates that yet uncharacterized bacterial factors other than PrfA, and possibly also host factors, modulate the expression of the PrfA regulon. Essentially nothing is known about the signal transduction pathways involved in the observed differential expression of virulence genes. Fermentable carbon sources seem to have a particular role in virulence gene regulation. In addition to the PrfA regulon, the Clp stress proteins have an impact on Listeria virulence. These two regulons interact with each other by an unknown mechanism.

Human Listeriosis Caused by Listeria ivanovii

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.