Zeljka Sokolovic

Surface‐associated, PrfA‐regulated proteins of Listeria monocytogenes synthesized under stress conditions

The expression of the five clustered genes of Listeria monocytogenes: plcA, hly, mpl, actA and plcB is under the control of the positive regulation factor PrfA. Listeriolysin, encoded by the hly gene, is the only prominent PrfA‐controlled gene product observed when L. monocytogenes strain NCTC 7973 is cultured in a rich medium at 37°C to the logarithmic growth phase. Stress conditions such as heat‐shock or stationary culture conditions lead to the induction of additional PrfA‐dependent proteins (PdPs): ActA (92 kDa), a 38kDa protein of unknown function and a 34kDa protein which probably represents PlcA. Under nutrient‐stress conditions PdPs are preferentially synthesized and in addition to the already known PdPs at least five new, not yet functionally identified PdPs are detected. All PdPs are either secreted or are localized at the cell surface. Differences in the amount as well as the sizes of the PdPs are observed in different L. monocytogenes strains.

The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators

The two pathogenic Listeria species, L. ivanovii and L monocytogenes, can be differentiated biochemically and show different host ranges. Virulence of L. monocytogenes is dependent on the integrity of prfA which positively and coordinately regulates transcription of several virulence genes. Until now, a prfA homologue had not been identified in L. ivanovii. We have now cloned a chromosomal region from L. ivanovii comprising two genes with high homology to the picA and prfA genes from L. monocytogenes. Distal from prfA, an open reading frame highly homologous to a phosphorlbosyl pyrophosphate synthetase gene (prs) was newly identified, defining the border of the virulence gene cluster. Transcription of the gene for ivanolysin O and expression of other genes of the virulence gene cluster in L. ivanovii were dependent on PrfA. The pattern of PrfA‐dependent proteins (PdPs) expressed in L ivanovii was similar, but not identical to that of L. monocytogenes. The PrfA proteins, as predicted from nucleotide sequences of both pathogenic Listeria species, are very similar and show significant homology to the Crp‐Fnr family of global transcription regulators.

Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron

The PrfA protein, which is a member of the Crp/Fnr family of prokaryotic transcription activators, regulates the virulence genes of Listeria monocytogenes. In this work, specific binding of PrfA to its target DNA was determined by electrophoretic mobility‐shift assays (EMSAs) using cell‐free extracts from the two L. monocytogenes strains EGD and NCTC 7973. PrfA‐specific binding differs between the two strains, even when the concentration of PrfA was adjusted to similar levels. Both strains exhibited increased PrfA‐specific binding after a shift into minimal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in brain–heart infusion (BHI). The purified PrfA protein from strain EGD produced in Escherichia coli did not exhibit specific binding to the target DNA but did so upon addition of PrfA‐free extracts from various Listeria species and Bacillus subtilis. The observed activation of PrfA seems to be caused by a PrfA‐activating factor (Paf), which is probably a protein since elevated temperature, but not RNase treatment, destroyed the activation potential of such PrfA‐free extracts. Moreover, fractionation of these extracts by sucrose gradient centrifugation yielded the Paf activity in a fraction sedimenting at 3.2 S. Specific binding of PrfA‐containing extracts from strain EGD to the hly and actA promoter sequences was strongly inhibited by iron, whereas that of extracts from strain NCTC 7973 was only slightly reduced. The iron effect seems to be mediated by Paf rather than by PrfA itself.

Transcriptional regulation of prfA and PrfA-regulated virulence genes in Listeria monocytogenes.

The ActA protein, the lecithinase PlcB and listeriolysin are the major PrfA‐dependant proteins synthesized when brain‐heart infusion (BHI)‐cultured Listeria monocytogenes is shifted to minimum essential medium (MEM) In the presence of the transcriptional inhibitor rifampicin. Enhanced synthesis of all three proteins under these conditions depends, however, on a short incubation (about 5 min) of the bacteria in MEM without rifampicin, suggesting that induction of these proteins in MEM requires de novo transcription. The enhanced synthesis of these three proteins is observed in the L. monocytogenes wild‐type strains EGD and NCTC 7973, both of which belong to the serotype 1/2a. A significant induction of the bicistronic mRNA for ActA and PlcB is observed in both strains shortly after shifting the bacteria from BHI to MEM. This mRNA as well as the monocistronic listeriolysin (hly)‐specific mRNA is highly stable in L. monocytogenes NCTC 7973 shifted to MEM. In contrast to the actA‐plcB mRNA, no enhanced transcription in MEM is observed for the regulatory prfA gene or for the PrfA‐controlled virulence genes hlyA and plcA in strain NCTC 7973. However, transcription of these genes is induced in strain EGD. Transcriptional induction of the mpl gene is observed in neither strain NCTC 7973 nor in strain EGD. The life‐time of the prfA, plcA, and mpl transcripts is short. ActA was also found to be the most abundant newly synthesized surface protein when the two wild‐type strains of L. monocytogenes replicated within the phagocytic cell line J774. ActA synthesis seemed to be induced in the cytoplasm since the non‐haemolytic mutant M3 did not induce ActA when taken up by J774 cells.

Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA

The two Listeria monocytogenes strains EGD and NCTC 7973 display a different regulation pattern of their PrfA‐dependent genes. All PrfA‐dependent genes from L. monocytogenes NCTC 7973 are much more efficiently transcribed in brain‐heart infusion medium than those from strain EGD. Transcription of these genes in EGD is, however, induced after shift into Minimal Essential Medium (MEM) to a level that is comparable to that of strain NCTC 7973. Expression of the internalin gene (inIA) is also influenced by PrfA, but only one (P2) out of three mapped promoters is strictly dependent on PrfA. In contrast to the other PrfA‐regulated genes, transcription of inIA even from the P2 promoter is reduced in both strains after shift to MEM. The prfA deletion mutant SLCC 53 complemented with multiple copies of prfA synthesizes large amounts of monocistronic prfA transcript, but there is no concomitant increase in the transcripts of the PrfA‐dependent genes. However, upon a shift into MEM, transcription of the PrfA‐dependent genes (with the exception of the inIA gene) is highly induced even in the absence of de novo protein synthesis. The PrfA proteins of the two studied L. monocytogenes strains differ in their ability to activate PrfA‐dependent genes. This difference is probably the result of amino acid exchange(s) in the C‐terminal part of these proteins. Strain EGD supplemented with multiple copies of prfA‐7973 shows a similar regulation of the PrfA‐dependent genes as strain NCTC 7973, whereas multiple copies of prfA‐EGD introduced into strain EGD hardly change the rate of transcription of the PrfA‐dependent genes. Our data thus suggest that PrfA‐mediated gene expression depends not only on the amount of the PrfA protein and the ‘quality’ of the putative PrfA‐binding sites, but also on the ‘quality’ of the PrfA protein which seems to be influenced by its amino acid composition and by environmental parameters.

DIFFERENTIAL INTERACTION OF THE TRANSCRIPTION FACTOR PRFA AND THE PRFA-ACTIVATING FACTOR (PAF) OF LISTERIA MONOCYTOGENES WITH TARGET SEQUENCES

The interaction of the purified PrfA transcription factor with the regulatory sequences located upstream of the PrfA‐dependent listeriolysin (hly) and internalin (inlA) genes was studied in the presence and in the absence of Paf (PrfA‐activating factor)‐containing extracts. It is shown that PrfA protein is able to bind, independently of additional factors, to a 109 bp DNA fragment including the entire hly promoter sequence with the anticipated PrfA binding site (‘PrfA‐box’). PrfA alone, but not in combination with Paf, can also bind to a shorter target sequence of 28 bp comprising essentially the PrfA‐box of the hly promoter. The addition of a Paf‐containing extract does not lead to significant protein binding to these two hly target sequences in the absence of PrfA but converts the complex (CIII) consisting of PrfA and the 109 bp hly DNA fragment to a slower migrating PrfA–Paf–DNA complex (CI). Incubation of cell‐free extracts of wild‐type Listeria monocytogenes with the 109 bp DNA fragment leads to the formation of CI. The addition of polyclonal PrfA antibodies causes a supershift of CIII. Purified PrfA and PrfA–Paf also bind to a DNA fragment containing the PrfA‐dependent promoter P2 of inlA, albeit at a lower rate when compared with the corresponding hly sequence. In contrast to the hly target DNA, the inlA promoter sequence efficiently binds Paf alone, and this Paf binding reduces that of PrfA and PrfA–Paf to the inlA target DNA. DNase I footprint experiments show that purified PrfA protects sequences of dyad symmetry previously proposed as PrfA binding sites in the hly and in the inlA promoter regions.

Control of Listeria Monocytogenes Virulence by the Transcriptional Regulator PrfA

Listeria monocytogenes is the causative agent of severe infections in humans and animals. These Gram-positive, rod-shaped and nonsporulating bacteria belong to the genus Listeria which comprises next to L. monocytogenes, the species L. ivanovii which is pathogenic for animals but not for humans and the four apathogenic species L. seeligeri, L. innocua, L. grayi and L. welshimeri which are frequently encountered in the natural environment.1 Risk groups among humans that are primarily affected by L. monocytogenes are immunocompromised people and pregnant women. The major symptoms of listeriosis are septicemia, stillbirth and meningitis.2 The bacteria seem to be taken up by contaminated food and the intestine appears to be the major route of entry in humans and animals.3,4 After crossing the intestinal barrier these facultative intracellular bacteria are able to replicate in the cytoplasm of the infected epithelial cells after disrupting the endosomal membrane.5 At least in established mammalian cell lines, L. monocytogenes spreads from cell to cell without leaving the intracellular environment. In these cell cultures it has been shown that the intra- and intercellular movement is brought about by the formation of polymerized actin filaments. These filaments are first formed on the surface of the listerial cell.

Hemolysin from listeria — Biochemistry, genetics and function in pathogenesis

SummaryThiol-activated hemolysins (listeriolysins) fromListeria monocytogenes (Sv4b) andListeria ivanovii were purified to homogeneity. The N-terminal amino acid sequences of the 58 kDa listeriolysin ofL. ivanovii and of a 24 kDa protein which may represent the CAMP-factor ofL. ivanovii were determined. Antibodies raised against theL. ivanovii listeriolysin and anti-streptolysin O antibodies were used in Western blot analyses to detect listeriolysin(s) in virulent and avirulent Listeria strains. It was found that all virulent strains ofL. monocytogenes synthesize and secrete listeriolysin (Mr 58–59 kDa), albeit in significantly variable quantities. No protein cross-reaction with anti-listeriolysin antibodies or anti-streptolysin O-antibodies was present in the supernatant ofListeria innocua, Listeria welshimeri, Listeria grayi andListeria murrayi strains. Furthermore, the avirulent but hemolyticListeria seeligeri did not cross-react with these antibodies. In aL. monocytogenes (strain EGD) gene bank constructed inEscherichia coli two types of hemolytic clones were identified. The first type carried recombinant plasmids with a common 2.0 kb fragment coding for a 23 kDa protein. This hemolytic activity was not activated by DTT and the 23 kDa protein did not cross react with anti-listeriolysin or anti-streptolysin antibodies. The other type of hemolytic clones was detected by using anti-streptolysin O antibodies to screen the gene bank. Some of these clones synthesized a protein of 61 kDa which cross reacted with anti-streptolysin O (or anti-listeriolysin) antibodies. By transposon Tn916 mutagenesis ofL. monocytogenes two types of nonhemolytic mutants were obtained. Type I produced no extracellular protein that cross reacted with anti-listeriolysin (or anti SLO) antibodies. Instead of the 58 kDa listeriolysin protein, type II mutants released proteins which were smaller in size than listeriolysin and cross reacted with anti-SLO. These proteins probably represent truncated listeriolysins. Virulence tests in a mouse model indicated that non-hemolytic mutants ofL. monocytogenes were avirulent. Furthermore, it was shown that these nonhemolytic mutants were unable to survive in mouse peritoneal macrophages but were still capable of entering mouse embryo fibroblast (3T6) cells. All virulentL. monocytogenes produced a quantitatively abundant extracellular protein of Mr 60 kDa. We isolated mutants which were still hemolytic but produced significantly reduced amounts of this protein. The ability of these mutants to enter 3T6 cells was severely impaired.ZusammenfassungSH-aktivierbare Hämolysine (Cytolysine) ausListeria monocytogenes (Sv4b) undListeria ivanovii wurden zur Homogenität gereinigt. Die N-terminalen Aminosäuresequenzen des 58 kDa großen Listeriolysins ausL. ivanovii und eines 24 kDa Protein, das vermutlich der CAMP-Faktor vonL. ivanovii ist, wurden bestimmt. Mit Hilfe von Antikörpern gegen Listeriolysin ausL. ivanovii und Streptolysin O wurden im Western Blot virulente und avirulente Listerienstämme auf ihre Fähigkeit, Listeriolysin zu bilden, getestet. Danach synthetisieren und scheiden alle virulenten Stämme vonL. monocytogenes Listeriolysin (Mr 58–59 kDa) aus, allerdings in sehr unterschiedlicher Menge. In den Kulturüberständen vonListeria innocua, Listeria welshimeri, Listeria grayi undListeria murrayi konnte kein mit Listeriolysin- oder Streptolysin-O-Antikörpern kreuzreagierendes Protein nachgewiesen werden. Die avirulente, aber hämolytische ArtListeria seeligeri zeigte ebenfalls keine Kreuzreaktion mit diesen Antikörpern. Zwei Typen von hämolytischenEscherichia coli Klonen wurden in einer Genbank vonL. monocytogenes (Stamm EGD) nachgewiesen. Der erste Typ besaß rekombinante Plasmide, die ein gemeinsames Fragment von 2 kb trugen. Dieses kodierte für ein Protein von 23 kDa, das für die hämolytische Aktivität verantwortlich ist. Diese Aktivität wurde weder mit DTT aktiviert, noch kreuzreagierte das 23-kDa-Protein mit Antikörpern gegen Listeriolysin oder Streptolysin O. Der andere Typ von hämolytischen Klonen wurde in der Genbank mit Hilfe von Streptolysin-O-Antikörpern identifiziert. Einige dieser Klone synthetisierten ein Protein von 61 kDa, das mit Antikörpern gegen Streptolysin O (oder Listeriolysin) kreuzreagierte. Durch Transposonmutagenese vonL. monocytogenes mit Tn916 wurden 2 Typen von nichthämolytischen Mutanten erhalten. Mutanten des Typs I produzierten kein extrazelluläres Protein, das mit Antikörpern gegen Listeriolysin kreuzreagierte. Typ-II-Mutanten schieden anstelle des 58 kDa Listeriolysins Proteine mit geringerer Größe als Listeriolysin aus, die noch mit diesen Antikörpern reagierten und somit wahrscheinlich verkürzte Listeriolysinproteine darstellen. Tests auf Virulenz in einem Mausmodell zeigten, daß beide Typen von nichthämolytischen Mutanten avirulent sind. Weiterhin konnte gezeigt werden, daß diese nichthämolytischen Mutanten nicht mehr in der Lage sind, in peritonealen Mausmakrophagen zu überleben, jedoch noch in embryonale Mäusefibroblasten (3T6-Zellen) eindringen können. Alle virulenten Stämme vonL. monocytogenes synthetisierten in relativ großer Menge ein extrazelluläres Protein von 60 kDa. Mutanten konnten isoliert werden, die zwar noch hämolytisch sind, das 60-kDa-Protein jedoch nur noch in geringer Menge produzieren. Die Mutanten haben die Fähigkeit, in 3T6-Zellen einzudringen, weitgehend verloren.

Studies on the pathogeneicity of Listeria monocytogenes

SummaryThe characterization of mutants ofListeria monocytogenes with reduced virulence properties is described. Reduction in the amount of the extracellular protein p60 (encoded by the ipa gene) leads to cell filaments with impaired invasiveness. Mutants which cannot synthesize listeriolysin are still invasive but unable to survive within phagocytic cells. One type of listeriolysin-negative mutants is defective in the synthesis of a positive regulatory element PrfA which coordinately regulates the listeriolysin gene (lisA) together with several other genes, including those for a phosphatidylinositol-specific phospholipase and a metalloprotease.ZusammenfassungDie Charakterisierung vonL. monocytogenes Mutanten mit verminderter Virulenz wird beschrieben. Mutanten mit reduzierter Synthese des extrazellulären Proteins p60 (kodiert von dem iap Gen) bilden lange septierte Zellfilamente, die erheblich weniger invasiv für nichtprofessionell phagozytische Animalzellen sind als der Wildstamm. Mutanten, die kein Listeriolysin bilden, sind zwar noch invasiv, können aber in phagozytischen Zellen nicht mehr überleben. Ein Typ dieser Listeriolysin-negativen Mutanten weist eine Mutation in einem Gen (prfA) auf, das einen positiv wirkenden Transkriptionsregulatur (PrfA) kodiert, der das Listeriolysingen (lisA) und mehrere andere Gene koordiniert reguliert. Dazu gehören Gene für eine Phosphatidylinositol-spezifische Phospholipase und eine Metalloprotease.