Comparative Genomics of Listeria Species Glaser, P.; Frangeul, L.; Buchrieser, C. ...
Science (American Association for the Advancement of Science),
10/2001, Letnik:
294, Številka:
5543
Journal Article
Recenzirano
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.
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.
A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. ...monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 h of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.
Summary
PrfA is the major regulator of Listeria virulence gene expression. This protein is a member of the Crp/Fnr family of transcription regulators. To gain a deeper understanding of the PrfA ...regulon, we constructed a whole‐genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild‐type EGDe and a prfA‐deleted mutant (EGDe ΔprfA). Both strains were grown at 37°C in brain–heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA. We identified three groups of genes that are regulated differently. Group I comprises, in addition to the 10 already known genes, two new genes, lmo2219 and lmo0788, both positively regulated and preceded by a putative PrfA box. Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes (lmo0178–lmo0184) are organized in an operon. Group III comprises 53 genes, of which only two (lmo0596 and lmo2067) are preceded by a putative PrfA box. Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes. In the presence of cellobiose, all the group I genes were downregulated, whereas group III genes remained fully activated. Group II genes were repressed in all conditions tested. A comparison of the expression profiles between a second L. monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14prfA*) and its corresponding prfA‐deleted mutant (P14ΔprfA) and the EGDe strain revealed interesting strain‐specific differences. Sequences strongly similar to a sigma B‐dependent promoter were identified upstream of 22 group III genes. These results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probably expressed from a sigma A‐dependent promoter. In contrast, a second set of PrfA‐regulated genes lack a PrfA box and are expressed from a sigma B‐dependent promoter. This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors.
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.
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.
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