Andreas Bubert

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.

Microinjection and growth of bacteria in the cytosol of mammalian host cells

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an “escape” step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfA-regulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.

Protection Against Murine Listeriosis by Oral Vaccination with Recombinant Salmonella Expressing Hybrid Yersinia Type III Proteins

In the present study, we have investigated the possibility to engage the Yersinia outer protein E (YopE) as a carrier molecule for heterologous Ag delivery by the type III secretion system of Salmonella typhimurium. Defined secretion and translocation domains of YopE were fused to the immunodominant T cell Ags listeriolysin O and p60 of Listeria monocytogenes. In vitro experiments showed that S. typhimurium allows secretion and translocation of large hybrid YopE proteins in a type III-dependent fashion. Translocation and cytosolic delivery of these chimeric proteins into host cells, but not secretion into endosomal compartments, led to efficient MHC class I-restricted Ag presentation of listerial nonamer peptides. Mice orally vaccinated with a single dose of attenuated S. typhimurium expressing translocated hybrid YopE proteins revealed high numbers of IFN-γ-producing cells reactive with listeriolysin O 91–99 or p60 217–225, respectively. This CD8 T cell response protected mice against a challenge with L. monocytogenes. In conclusion, these findings suggest that YopE is a versatile carrier molecule for type III-mediated foreign Ag delivery by Salmonella vaccine strains.

Yersinia enterocolitica‐mediated translocation of defined fusion proteins to the cytosol of mammalian cells results in peptide‐specific MHC class I‐restricted antigen presentation

Yersinia enterocolitica delivers a set of effector proteins [Yersinia outer proteins (Yop)] into the cytosol of target cells to modulate host cell signal transduction pathways required for the extracellular survival of the bacterium. Secretion and subsequent translocation of Yop across the eukaryotic cell membrane are achieved via a type III secretion system. About 50 – 100 amino acids of the N terminus of Yop are required for chaperone‐directed secretion and translocation. In this study, it is demonstrated by immunoblot analysis of Yersinia‐infected cultured epithelial cells that one ot these proteins, YopE, can serve as a molecular carrier to deliver protein fragments of the heterologous p60 antigen of Listeria monocytogenes into the cytosol of target cells. T cell activation assays revealed that the observed type III‐mediated antigen translocation led to a p60 peptide‐specific MHC class I‐restricted antigen presentation. Efficient translocation and antigen presentation were strictly dependent on the co‐localized expression of hybrid YopE‐p60 proteins and the YopE‐specific chaperone SycE. These results suggest that the Yersinia type III secretion system may serve as an attractive tool for antigen delivery in Yersinia‐based live vaccines to induce cellular immune responses.

Nucleic acid‐based, cultivation‐independent detection of Listeria spp. and genotypes of L. monocytogenes

Based on comparative analysis of 16S rRNA gene sequences, two oligonucleotide probes for in situ detection of all members of the genus Listeria were designed. These probes allowed fast and reliable in situ detection of Listeria spp. even in complex samples like raw milk. Almost full-length iap (invasion-associated protein) gene sequences were determined for 69 Listeria monocytogenes strains of all 13 known serotypes. A comparison of these sequences revealed that the L. monocytogenes strains can be grouped into three distinct genotypes. These clusters correlate well with distinct serotypes. Thus, strains of serotypes b and d belong to genotype I, a and c to genotype II, and 4a and 4c, which are rarely isolated from humans, group together within genotype III. These results could be corroborated by further comparative sequence analysis of genes encoding two phospholipases - plcA and plcB. Based on the iap gene sequences, a highly specific and reproducible competitive PCR detection method was developed. Primer pairs targeting genotype-specific regions of the iap gene were designed. The amplification of non-specific PCR products from DNA of non-target strains was prevented by adding competitive primers. By applying this method, the rapid and reliable distinction of the three L. monocytogenes genotypes was possible.

Evaluation of the Duopath Legionella lateral flow assay for identification of Legionella pneumophila and Legionella species culture isolates.

ABSTRACT Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries’ regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.

Colony-blot assay with anti-p60 antibodies as a method for quick identification of Listeria in food

The present study evaluated the ability to isolate Listeria from foods, using shortened procedure of sample enrichment followed by immunomagnetic separation or filtration methods, and serological identification of isolated bacteria by colony-blot and Western blot methods with anti-p60 antibodies. By these rapid methods, identification of Listeria was achieved in much shorter time (40-48 h) than with standard cultivation and biochemical identification procedures. The rapid methods used are easy to perform and, what is most important, their specificity is very high and fulfills the expectations. The possibility to select Listeria colonies growing on non-selective media by blotting with anti-p60 antiserum seems to be particularly valuable in examination of food samples containing/not too many Listeria (1-10 CFU/25 g). However, the blot method using anti-PepD mAb specific to unique region of L. monocytogenes p60 is necessary to distinguish L. monocytogenes from other Listeria species.

Single-Strand Conformation Polymorphisms in the hly Gene and Polymerase Chain Reaction Analysis of a Repeat Region in the iap Gene to Identify and Type Listeria monocytogenes

Two novel methods that allow the powerful identification of Listeria monocytogenes by polymerase chain reaction (PCR) and simultaneous differentiation by special electrophoresis formats are described. The first method involves a PCR-driven single-strand conformation polymorphism (SSCP-PCR) assay using a portion of the noncoding region of the hlv gene. The assay was evaluated with 120 genetically distinct L. monocytogenes strains of either foodborne or clinical origin. Distribution of listerial strains to at least 14 SSCP types was observed. In respect to the panel of strains, 39.7% were assigned to SSCP type 3, and 19% showed SSCP type 5. Further, SSCP type 1 was found in 7.5% of all strains, SSCP type 10 in 6.7%, and 5.8% each for SSCP types 6 and 7. The SSCP types 4, 9, and 11 were infrequently described in 2.55%, 3.3%, and 4.2%, respectively, of all isolates. At least 0.85% represented each of the SSCP types 2, 13, and 14, and 1.7% displayed SSCP types 8 and 12. In the second method, the internal threonine-asparagine repeat portion of the L. monocytogenes p60 protein was used for setting up a PCR-based identification and parallel differentiation assay. Ten different repeat types (RTs), according to different sizes of PCR products, were observed. Of 163 strains tested, 35.58% of samples were assigned to RT 1, 39.26% to RT 2, 3.68% to RT 3, 6.13% to RT 4, 4.29% to RT 5, 2.45% to RT 6, 5.52% to RT 7, 0.61% to RT 8, 0.61% to RT 9, and 1.83% to RT 10. The data suggest that both methods allow the simple identification and differentiation of L. monocytogenes isolates. Therefore, both the SSCP-PCR and the PCR-based identification and parallel differentiation assay could represent single-strand pretyping assays before laborious reference typing methods are applied.

Linkage of Bacterial Protein Synthesis and Presentation of MHC Class I-Restricted Listeria monocytogenes-Derived Antigenic Peptides

The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria monocytogenes is tightly linked to bacterial protein synthesis. We used non-linear regression analysis to fit a mathematical model of bacterial antigen processing to a published experimental data set showing the accumulation and decay of p60-derived antigenic peptides in L. monocytogenes-infected cells. Two alternative models equally describe the experimental data. The simulation accounting for a stable and a hypothetical rapidly degraded form of antigen predicts that the antigenic peptides p60 217–225 and p60 449–457 are derived from a putative instable form of p60 with an average intracellular half-life of approximately 3 minutes accounting for approximately 31% of all p60 molecules synthesized. The alternative model predicts that both antigenic peptides are processed from p60 degraded intracellularly with a half-life of 109 min and that antigen processing only occurs as long as bacterial protein synthesis is not inhibited. In order to decide between both models the intracellular accumulation of p60 in infected cells was studied experimentally and compared with model predictions. Inhibition of p60 degradation by the proteasome inhibitor epoxomicin revealed that during the first 3 h post infection approximately 30% of synthesized p60 molecules were degraded. This value is significantly lower than the approximately 50% degradation of p60 that would be expected in the presence of the predicted putative short-lived state of p60 and also fits precisely with the predictions of the alternative model, indicating that the tight connection of bacterial protein biosynthesis and antigen processing and presentation of L. monocyctogenes-derived antigenic peptides is not caused by the presence of a highly instable antigenic substrate.