Mariela Scortti

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.

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.

The genome of a pathogenic rhodococcus : cooptive virulence underpinned by key gene acquisitions

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.

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

ABSTRACT We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.

Negative control of Listeria monocytogenes virulence genes by a diffusible autorepressor

Virulence genes from the facultative intracellular pathogen Listeria monocytogenes are controlled by the transcriptional regulator PrfA. Although PrfA synthesis is activated at 37°C, PrfA‐dependent expression remains low in rich medium. However, a strong induction of the PrfA regulon is observed when L. monocytogenes is cultured in the presence of activated charcoal. Here, we show that the ‘charcoal effect’ results from the adsorption of a diffusible autorepressor substance released by L. monocytogenes during exponential growth. Analyses using an L. monocytogenes strain in which the prfA gene is expressed constitutively at 37°C from a plasmid indicate that the autoregulatory substance represses PrfA‐dependent expression by inhibiting PrfA activity. PrfA presumably functions via an allosteric activation mechanism. The inhibitory effect is bypassed by a PrfA* mutation that locks PrfA in fully active conformation, suggesting that the autorepressor interferes with the allosteric shift of PrfA. Our data indicate that the listerial autorepressor is a low‐molecular‐weight hydrophobic substance. We suggest that this diffusible substance mediates a quorum‐sensing mechanism by which L. monocytogenes restricts the expression of its PrfA virulence regulon. This autoregulatory pathway could serve L. monocytogenes to ensure the silencing of virulence genes during extracellular growth at 37°C. It may also play a role during intracellular infection, by limiting the damage to the host cell caused by an excess production of cytotoxic PrfA‐dependent virulence factors in the PrfA‐activating cytosolic compartment.

New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure-function of the virulence regulator PrfA.

PrfA, a transcription factor structurally related to Crp/Fnr, activates Listeria monocytogenes virulence genes during intracellular infection. We report two new PrfA* mutations causing the constitutive overexpression of the PrfA regulon. Leu‐140Phe lies in αD adjacent to the DNA‐binding motif in the C‐terminal domain, like a previously characterized PrfA* mutation (Gly‐145Ser). Ile‐45Ser, in contrast, maps to the N‐terminal β‐roll, a structure similar to that of the Crp cAMP binding site. The in vitro transcriptional properties of recombinant PrfA*I45S and PrfA*G145S were compared to those of PrfAWT at two differentially regulated PrfA‐dependent promoters, PplcA and PactA. The two PrfA* mutations increased the affinity for the target DNA to a different extent, and the differences in DNA binding (PrfA*G145S > PrfA*I45S >>> PrfAWT) correlated with proportional differences in transcriptional activity. The use of the PrfA* proteins revealed that PplcA had a greater affinity for, and was more sensitive to, PrfA than PactA. RNA polymerase (RNAP) initiated transcription independently of PrfA at PplcA, but not at PactA, consistent with bandshift experiments suggesting that PplcA has a greater affinity for RNAP than PactA. Thus, differences in affinity for both PrfA and RNAP appear to determine the different expression pattern of PrfA‐regulated promoters. Modelling of the PrfA* mutations in the crystal structure of PrfA and comparison with structure–function analyses of Crp, in which similar mutations lead to constitutively active (cAMP‐independent) Crp* proteins, suggested that PrfA shares with Crp an analogous mechanism of cofactor‐mediated allosteric shift. Our data support a regulatory model in which changes in PrfA‐dependent gene expression are primarily accounted for by changes in PrfA activity.

Evolution of the Rhodococcus equi vap Pathogenicity Island Seen through Comparison of Host-Associated vapA and vapB Virulence Plasmids

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 approximately 22-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.

Coexpression of virulence and fosfomycin susceptibility in Listeria : molecular basis of an antimicrobial in vitro – in vivo paradox

Discrepancies between resistance in vitro and therapeutic efficacy in vivo are generally attributed to failure of laboratory susceptibility tests to reflect an antibiotics pharmacokinetic or pharmacodynamic properties. We show here that this phenomenon can result from differential in vitro–in vivo expression of bacterial determinants of antibiotic susceptibility. We found that an in vivo–induced virulence factor, Hpt, also mediates uptake of fosfomycin in Listeria monocytogenes. These bacteria therefore seem resistant to fosfomycin in vitro, although they are in fact susceptible to the antibiotic during infection.

A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins.

Listeria ivanovii differs from the human pathogen Listeria monocytogenes in that it specifically affects ruminants, causing septicaemia and abortion but not meningo‐encephalitis. The genetic characterization of spontaneous L. ivanovii mutants lacking the virulence factor SmcL (sphingomyelinase) led us to identify LIPI‐2, the first species‐specific pathogenicity island from Listeria. Besides SmcL, this 22 kb chromosomal locus encodes 10 internalin (Inl) proteins: i‐InlB1 and ‐B2 are large/surface‐associated Inls similar to L. monocytogenes InlB; i‐InlE to –L are small/excreted (SE)‐Inls, i‐InlG being a tandem fusion of two SE‐Inls. Except i‐inlB1, all LIPI‐2 inl genes are controlled by the virulence regulator, PrfA. LIPI‐2 is inserted into a tRNA locus and is unstable – half of it deleting at ≈10−4 frequency with a portion of contiguous DNA. The spontaneous mutants were attenuated in vivo in mice and lambs and showed impaired intracellular growth and apoptosis induction in bovine MDBK cells. Targeted knock‐out mutations associated the virulence defect with LIPI‐2 genes. The region between the core genome loci ysnB‐tRNAarg and ydeI flanking LIPI‐2 contained different gene complements in the different Listeria spp. and even serovars of L. monocytogenes, including remnants of the PSA bacteriophage int gene in serovar 4b, indicating it is a hot spot for horizontal genome diversification. LIPI‐2 is conserved in L. ivanovii ssp. ivanovii and londoniensis, suggesting an early acquisition during the species’ evolution. LIPI‐2 is likely to play an important role in the pathogenic and host tropism of L. ivanovii.