Download - Pathogenicity islands and virulence evolution in Listeria

Pathogenicity islands and virulence evolutionin Listeria

José A. Vázquez-Bolanda*, Gustavo Domínguez-Bernala, Bruno González-Zorna, Jürgen Kreftb, Werner Goebelb

aGrupo de Patogénesis Molecular Bacteriana, Unidad de Microbiología e Inmunología, Departamento de Patología Animal I, Facultad de Veterinaria,Universidad Complutense, 28040 Madrid, Spain

bLehrstuhl für Mikrobiologie, Biozentrum, Universität Würzburg, 97074 Würzburg, Germany

ABSTRACT – As in other bacterial pathogens, the virulence determinants of Listeria species areclustered in genomic islands scattered along the chromosome. This review summarizes currentknowledge 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-positivebacteria are presented and discussed. © 2001 Éditions scientifiques et médicales Elsevier SAS

Listeria / Listeria monocytogenes / Listeria innocua / Listeria ivanovii / pathogenicity islands / LIPI-1 / LIPI-2 /LRR proteins / virulence evolution / microbial pathogenesis

1. Introduction

The bacterial genus Listeria consists of a group offacultatively anaerobic, asporogenous Gram-positive rodsclosely related phylogenetically to Bacillus, Streptococ-cus, Enterococcus, Staphylococcus and Clostridium [1, 2].Only two of the six species currently recognized in thisgenus are pathogenic: L. monocytogenes and L. ivanovii.They cause listeriosis, an opportunistic infection of humansand animals involving severe clinical manifestations suchas meningoencephalitis, abortion and septicemia. Thenatural habitat of Listeria is thought to be the surface layerof soil rich in decaying plant matter. From this habitat, theygain access to the vertebrate host via the oral route usingcontaminated food as a vehicle. L. monocytogenes caninfect a wide range of animal species, including mammalsand birds. L. ivanovii has a narrower host range, beingpathogenic mostly for ruminants [3–5]. Both L. monocy-togenes and L. ivanovii are typical facultative intracellularparasites. They are able to proliferate within macrophagesand a variety of normally nonphagocytic cells, such asepithelial and endothelial cells and hepatocytes. In allthese cell types, pathogenic Listeria develop a character-istic intracellular life cycle with the following steps: (i)early escape from the phagocytic vacuole, (ii) multiplica-tion in the host cell cytoplasm, (iii) directional intracyto-solic motility by induction of actin polymerization at onepole of the bacterial cell, (iv) protrusion of centrifugallymoving bacteria within cytoplasmic evaginations and (v)

phagocytosis of the pseudopod-like structures by neigh-boring cells, in which the cycle reinitiates (figure 1). Sixvirulence factors responsible for key steps in this intracel-lular parasitic life cycle have been identified and charac-terized at the molecular level during the last 15 years.Their genes are physically linked in a 9-kb chromosomalisland [5–8].

*Correspondence and reprints.E-mail address: [email protected] (J.A. Vázquez-Boland).

Figure 1. Schema of the intracellular life cycle of pathogenicListeria. 1, entry into host cells; 2, survival within the phagocyticvacuole; 3, disruption of phagosomal membranes and escape intothe cytosol; 4, replication in the cytosol; 5, actin-based motility;6, direct cell-to-cell spread; 7, survival in secondary (doublemembrane) phagosomes; 8, escape from secondary phagosomesand reinitiation of the cycle. Adapted and modified from anoriginal drawing in [128].

Microbes and Infection, 3, 2001, 571−584© 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

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2. A virulence gene clusteressential for intracellular parasitism2.1. The genes of the clusterand their roles in the intracellular life cycle

This virulence locus consists of three transcriptionalunits (figure 2). The central position is occupied by the hlymonocistron, encoding a pore-forming toxin of thecholesterol-binding, ’thiol-activated’ family. Its product,Hly (called listeriolysin O in L. monocytogenes), isrequired for disruption of the phagocytic vacuole and therelease of bacteria into the cytoplasm, a prerequisite fortheir intracellular proliferation (figure 1). Hly is thereforean essential virulence factor and its absence leads to totalavirulence [5, 6, 9, 10].

Downstream from hly and transcribed in the sameorientation is a 5.7-kb operon comprising three genes:mpl, actA and plcB [11] (figure 2). actA encodes thesurface protein ActA, the factor responsible for actin-based motility and cell-to-cell spread. Like Hly, ActA isindispensable for L. monocytogenes pathogenicity[12–14]. ActA can recruit actin monomers either bydirectly interacting with G-actin [15] or via the G-actin-binding protein profilin, which binds to the actin

cytoskeleton-associated ActA ligand proteins VASP andMena [16, 17]. However, actin-based bacterial motilityitself depends on activation of the Arp2/3 complex nucle-ation activity by ActA. This listerial protein has evolvedfunctional domains that mimic the natural role of theArp2/3 complex-activating proteins of the WASP/Scar fam-ily, which are downstream effectors of the signaling path-ways involved in dynamic remodeling of the actin cytosk-eleton [18–21]. Experimental evidence has been obtainedthat ActA is also involved in internalization by host cells[22]. plcB encodes a zinc-dependent, broad-substraterange phospholipase C, PlcB, homologous to the clostridialphospholipase C (α-toxin) [11]. The enzyme is secreted asan inactive propeptide, which is extracellularly processedby the Mpl protease, the product of the first gene of theoperon. Mpl is, like PlcB, a zinc-metalloenzyme. PlcBcooperates with Hly (and PlcA, see following paragraph)in the disruption of the primary vacuoles formed after thephagocytosis of extracellular Listeria. However, the prin-cipal function of PlcB is to mediate dissolution of thedouble-membrane secondary phagosomes formed aftercell-to-cell spread [11, 23, 24] (figure 1). Thus, the threeprotein products of the mpl-actA-plcB operon are allinvolved in one function that is essential in Listeria patho

Figure 2. Genetic structure of the chromosomal region of the hly virulence gene cluster (LIPI-1) in Listeria spp. Genes belonging to LIPI-1are in green (the more divergent actA gene is hatched). LIPI-1 is inserted in a region flanked by the prs and ldh loci, encoding, respectively,the housekeeping enzymes phosphoribosyl-pyrophosphate synthase and lactate dehydrogenase. In the plcB-ldh intergenic region, twoORFs, orfA and orfB, are found in all Listeria spp., indicating that the insertion point of LIPI-1 is between the prs and orfB loci. The orfAproduct (224 amino acids, accession no. P33381) shows marginal similarity (47% in 83 amino acids overlap) to the transcriptiontermination factor Rho of Borrelia burgdorferi. The orfB product (110 amino acids, accession no. P33382) has orthologs in many bacteria(Eschericia coli, Bacillus anthracis, Enterococcus faecalis) and archaea, all of unknown function. In the intergenic region between plcB and orfBin L. monocytogenes, there are two small ORFs, orfX (in yellow) and orfZ (in red), which delimit the putative excision point of LIPI-1 inL. innocua. orfX contains an overlapping ORF, orfY, encoding a small hypothetical protein without homologs in the protein databases [11].In the plcB-orfB intergenic region of L. ivanovii, two small ORFs, orfX (in yellow; accession no. AJ409322) and orfL (in blue; accession no.AJ409323), are also present. As in L. monocytogenes, these two ORFs delimit the deletion point of the virulence gene cluster in thenonpathogenic species L. welshimeri. orfX encodes a homolog of orfX from L. monocytogenes, whereas orfL codes for a polypeptide unrelatedto the L. monocytogenes orfZ product. orfZ and orfL encode polypeptides showing significant similarity with bacteriophage proteins (see figure4). The additional ORFs found in the L. seeligeri virulence gene cluster are in orange (orfC, D and E). Those hatched in orange and green (orfKand dplcB) encode polypeptides showing similarities to LIPI-1 products (the orfK product shows 39% similarity with the phosphatidyli-nositol phospholipase PlcA and dplcB is a truncated duplicate of the plcB gene). The figure has been constructed using data from [5, 8, 11,38, 45] and unpublished results from J. Kreft (L. seeligeri) and B. González-Zorn and J.A. Vázquez-Boland (L. ivanovii).

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genesis: the direct passage of bacteria from one cell toanother. This function enables bacteria to avoid the extra-cellular compartment and, hence, the humoral effectors ofthe immune system during their dissemination in hosttissues [25]. Therefore, Listeria infection can only beresolved by the mounting of an adequate cellular immuneresponse involving cytotoxic T cells and activated mac-rophages, which accumulate around the infectious foci,forming typical granulomas [26].

Upstream and divergent from hly lies the plcA-prfAoperon (figure 2). The first gene of this bicistron encodesPlcA, a phosphatidylinositol-specific phospholipase C,which synergizes with Hly and PlcB in the destabilizationof primary phagosomes [24]. The second gene codes forthe PrfA protein, a transcription factor structurally andfunctionally related to the Crp (CAP) protein of enterobac-teria. PrfA is required for the transcriptional activation ofall the genes of the cluster (including prfA itself) andtherefore, like Hly and ActA, it is absolutely indispensablefor pathogenicity. PrfA is the only virulence regulatoridentified to date in Listeria and is the main switch of aregulon comprising virulence-associated loci scatteredthroughout the listerial chromosome, including membersof the internalin multigene family [27] (see section 3). Anumber of environmental and growth-phase dependentsignals modulate expression of the virulence regulon viaPrfA. The activating signals include high temperature(37 °C) [28], stress conditions [29], sequestration of extra-cellular growth medium components by activated char-coal [30], contact with host cells [31] and the eukaryoticcytoplasmic environment [31–34]. The current model pre-dicts a regulatory mechanism involving allosteric activa-tion of PrfA by a putative low-molecular-weight cofactor,the levels of which would depend on the environmentalconditions sensed [35, 36]. PrfA activation leads to thesynthesis of more PrfA protein by positive feedback, medi-ated by a PrfA-dependent promoter, which governs thesynthesis of a bicistronic plcA-prfA mRNA [36, 37] (figure2).

2.2. Distribution and structure of the virulence genecluster in the genus Listeria: evolutionary implications

The genetic structure and transcriptional organizationof the hly virulence gene cluster are identical in L. mono-cytogenes and L. ivanovii [8, 38] (figure 2). The corre-sponding gene homologs, however, show a significantdegree of divergence (73–78% identity at the DNA level[39, 40]), compatible with the genetic distance separatingthe two pathogenic Listeria spp. This divergence is evenmore pronounced for the actA genes, the encoded pro-teins being only 34% identical in the two species despitehaving the same function in actin-based motility [41–43].These data suggest that a virulence gene cluster withessentially the same structure was already present in acommon ancestor of the currently known Listeria spp.Supporting this notion, the chromosome of a third Listeriaspp., L. seeligeri, also carries the virulence gene cluster. Inthis nonpathogenic species, however, this gene cluster isnonfunctional due to an insertion between the plcA andprfA genes (the divergently transcribed orfE locus) (figure2), interrupting the positive autoregulatory loop necessary

for appropriate expression of prfA and PrfA-dependentvirulence genes [8, 38, 44]. The L. seeligeri hly locuscarries additional open reading frames (ORFs), includingpartial duplications of plcB and, possibly also, of the plcAgene (orfK) (figure 2). The significance of these additionalputative genes is unknown and it is unclear if theL. seeligeri hly region is a corrupted form of a chromo-somal fragment with a function that is no longer required(thus tolerating gene insertions), or represents an ancestralform of the gene cluster in its process of depuration.

In the three species, the virulence gene cluster is stablyinserted at the same chromosomal position (figure 2) andthere are no obvious traces of mobility genes, insertionsequences (IS), direct repeats and target sequences nor-mally used for the integration of mobile genetic elements.In addition, its G+C content, codon usage and relativedinucleotide frequency do not differ significantly fromthose of the rest of the chromosome in the correspondingspecies [8]. All this evidence makes it indeed extremelyunlikely that the hly virulence gene cluster is present in thethree Listeria spp. as a result of independent events ofhorizontal gene transfer that occurred after the speciationprocess.

The virulence gene cluster is, however, totally absentfrom the other three described listerial species, L. innocua,L. welshimeri and L. grayi, which are all nonpathogenic[8, 38, 45]. The case of L. innocua is of particular rel-evance because this species is very closely related toL. monocytogenes (as described later in the text). Thus, ifthe hly virulence gene cluster was indeed present in acommon listerial ancestor, the only reasonable explana-tion for its absence in L. innocua is that it excised from thechromosome of this species at a later stage in the evolutionof the genus Listeria. The virulence gene cluster confershighly specialized functions in intracellular parasitism andseems to be totally unnecessary outside the vertebratehost, as shown by the abundance of L. innocua in theenvironment [46–48]. Its deletion may even be advanta-geous, as it can alleviate the physiological burden thatrepresents the expression of a relatively large piece ofDNA, which in turn controls the expression of a number ofadditional loci (i.e. the members of the PrfA-dependentregulon). Indeed, many studies show a clear predomi-nance of L. innocua over L. monocytogenes in raw foodand environmental samples [49–55], indicating that thenonpathogenic species may be better adapted tosaprophytic life.

It is likely that the soil was also the primary habitat ofthe common ancestor of modern-day Listeria. It is there-fore possible that the virulence gene cluster evolved origi-nally in the listerial ancestor as a means of defense againstphagocytosis by soil protists and that, as a result of thecontinuous interaction with the tissues of the vertebratehost after passive intestinal translocation, it further adaptedlater in evolution to fulfil its current functions in intracel-lular parasitism [8]. However, L. innocua lacks the viru-lence gene cluster, but successfully survives in the envi-ronment, suggesting that the impact of phagocytosis byprotist on the survival of Listeria bacteria in the naturalhabitat is negligible. Alternatively, therefore, the virulencegene cluster may have been involved primarily in the

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colonization of animal tissues, with the listerial ancestorcarrying it already being a parasite of higher eukaryotes.

The virulence gene cluster confers a clear advantage onbacteria, as it expands their possibilities for survival and

multiplication into animal tissues. In this nutrient-richniche, devoid of competing microflora but highly hostiledue to the innate and acquired defence mechanisms of thehost, bacteria are clearly subject to potent selection pres-sures, different from those encountered in soil. Conse-quently, the acquisition (and subsequent loss by certainclones) of the cluster must necessarily have had a majorimpact on the evolution of the genus Listeria and in thedetermination of its current structure. Phylogenetic analy-ses based on the sequence of the 16S and 23S rRNAindicate that the genus Listeria comprises two sublines ofdescent, one corresponding to L. grayi and the otherembracing the other Listeria spp. [1, 2]. The latter, in turn,are divided in two distinct evolutionary branches, oneincluding L. monocytogenes and L. innocua, and the other,L. ivanovii, L. seeligeri and L. welshimeri. This structuresuggests an evolutionary history for the genus in whichL. grayi corresponds to the outcome of the listerial ances-tor never having acquired a virulence gene cluster, whereasthe other two branches correspond to clonal diversifica-tion of the parasitic ancestor of Listeria after the acquisi-tion of the gene cluster and its stabilization (L. monocyto-genes and L. ivanovii), deletion (L. innocua andL. welshimeri) or functional inactivation (L. seeligeri) dur-ing evolution (figure 3).

The origin of this virulence gene cluster remains amatter of speculation. From its genetic composition, itseems clear that its source was in the phylogenetic divisionof Gram-positive bacteria in which the genus Listeria is

Figure 3. Phylogeny of the genus Listeria and hypotheticalmodel for the evolution of the intracellular life cassette, LIPI-1.The path followed by LIPI-1 after its acquisition by a line ofdescent of the common listerial ancestor is indicated by arrows.Thick red lines indicate conservation of LIPI-1 function. Thesolid circles indicate loss of LIPI-1, leading to the nonpathogenicspecies L. innocua and L. welshimeri. The empty circle indicatescorruption of LIPI-1, leading to a nonfunctional version of thegene cluster (empty arrowhead) in the nonpathogenic speciesL. seeligeri. The dendrogram is a schematic reconstruction of thephylogeny of Listeria according to [1, 2]. The dimensions of thebranches do not reflect the actual genetic distances.

Figure 4. BLASTP alignments of the polypeptides encoded by orfX (accession no. P33383) and orfZ (accession no. P33385) fromL. monocytogenes and orfL from L. ivanovii (accession no. AJ409323) with viral proteins (respectively: gp160 of HIV1, accession no.AF015921; Orf6 of the A511 bacteriophage, accession no. X91069 [129]; serine/threonine protein phosphatase from λ phage; accessionno. P03772). The orfX product has no homologs in the bacterial protein database. The orfZ product shows similarity to the Orbivirus outercapsid protein VP5 in searches of the protein BLOCKS database and has a homolog in E. coli (a 17.2-kDa hypothetical protein encoded inthe molR-bglX intergenic region; accession no. B644980). The orfL product has homologs in archaea and eubacteria, which belong to afamily of conserved hypothetical proteins with probable phosphoesterase-serine/threonine protein phosphatase activity.



located. Thus, Hly is a member of a broad family ofpore-forming toxins (as described previously), the distribu-tion of which is restricted to low-G+C Gram-positivebacteria [56]. Similarly, PlcB belongs to a family of zinc-dependent phospholipases, the members of which arepresent only in the genera Bacillus and Clostridium [11].Homologs of the PlcA phospholipase and of thethermolysin-related zinc-metalloprotease, Mpl, are alsocommon in low G+C-content Gram-positive bacteriaclosely related to Listeria [57, 58]. The only exceptions arePrfA, which is a member of the Crp/Fnr superfamily oftranscription factors, widespread in both Gram-negativeand Gram-positive bacteria [27, 40], and ActA, for whichno obvious structural homologs have been identified inprokaryotes. The ActA domains that mimic functionalmotifs present in eukaryotic proteins, such as thoseinvolved in the binding of the Arp2/3 complex and of thecytoskeletal adaptor and regulator proteins of the Ena/VASP family [17, 18, 43], may result from convergentevolution. However, an attractive alternative hypothesis isthat they were acquired via horizontal transfer of eukary-otic DNA during intracellular parasitism.

It is also unclear whether the virulence gene cluster wasoriginally acquired by the ancestor as a cassette in a singlerecombination event or whether it is the product of a longstep-by-step assembly process that occurred specificallyduring evolution in the genus Listeria. Its entire deletionfrom the L. innocua (and L. welshimeri) chromosome (fig-ure 2) is consistent with the gene cluster (or a substantialpart of it) belonging to a horizontally transferable element.This implies the intervention of a specific mobility mecha-nism, of which, as mentioned earlier, no obvious traceswere found thus far. However, at the extreme right-end ofthe gene cluster (plcB-orfB intergenic region) of L. mono-cytogenes, there are two small, contiguous ORFs (orfX andorfZ) encoding polypeptides of unknown function andwith no reported protein homologs in the sequence data-bases [11]. orfZ is present in both L. monocytogenes andL. innocua, whereas orfX is absent from L. innocua (figure2), indicating that the two ORFs delimit the putative exci-sion point of the virulence gene cluster in this species. Twosmall ORFs are also present at the same position in theplcB-orfB intergenic region of L. ivanovii (B. González-Zorn and J.A. Vázquez-Boland, unpublished). The firstcodes for a polypeptide homologous (54% similarity) tothat encoded by orfX, whereas the second, here desig-nated orfL, encodes a 228-amino acid protein unrelated tothe L. monocytogenes orfZ. Sequence analysis of the prs-ldh intergenic region of L. welshimeri (i.e. the L. innocuacounterpart in the evolutionary branch to which L. ivanoviibelongs; see above), has revealed the absence of orfX andthe presence of an orfL homolog contiguous to the 3’ endof orfB [45] (figure 2). This indicates that, as for L. innocua,the right end of the deleted fragment in L. welshimeri islocated immediately downstream from orfX (figure 2).More careful similarity searches for these ORFs, restrictedto the database of viral proteins, have now provided cluesas to how the virulence gene cluster was mobilized to thegenome of the Listeria ancestor. Thus, the 153-amino acidproduct of orfZ is significantly similar to the polypeptideencoded by orf6 in the major capsid and sheath protein

gene region of the A511 Listeria bacteriophage (41%similarity in an overlap of 114 amino acids) (figure 4).Likewise, the orfL product, although unrelated to the orfZ-encoded polypeptide, also exhibits significant similarity toa phage protein, a serine/threonine protein phosphatasefrom λ (figure 4). This λ polypeptide has 221 residues, i.e.a length very similar to that of the orfL product. The107-residue polypeptide encoded by orfX from L. mono-cytogenes also shows significant similarity to a viral pro-tein (46% similarity in an overlap of 99 amino acids withthe N-terminal end of glycoprotein gp160 of HIV type 1)(figure 4). Indeed, these sequence similarities suggest thatthe small ORFs in the extreme right-end of the virulencegene cluster may be remnants of a transducing bacterioph-age.

3. The internalin islets3.1. Internalins, a family of leucine-rich repeat proteins

The first internalin locus to be identified, inlAB, wasdiscovered by screening a bank of transposon mutants fordefective internalization in epithelial cell monolayers(hence the name internalins) [59]. Since then, a number ofother internalin loci have been found in L. monocytoge-nes and L. ivanovii (figure 5), and there is evidence thatthey are also present in nonpathogenic species such asL. innocua [59]. All these loci form a multigene familyexclusive to Listeria, encoding proteins with a character-istic domain containing a variable number of leucine-richrepeats (LRRs). The LRR unit of internalins consists of a22-amino acid oligopeptide with leucine or isoleucineresidues at positions 3, 6, 9, 11, 16 and 22 [59, 60]. Thissequence defines a novel spatial structure, a right-handedhelix called parallel �-helix, first identified in the pectatelyase of Erwinia chrysamthemi [61, 62] and subsequentlyfound in a large number of proteins, now known as theLRR protein superfamily [63, 64]. LRR domains are thoughtto mediate protein–protein interactions and are mostlypresent in eukaryotes, in a diversity of proteins with differ-ent functions, such as components of signal transductionpathways, extracellular matrix proteoglycans and the prod-ucts of the plant disease resistance (R) genes [63–65]. Inprokaryotes, they are less abundant, usually being presentin virulence-associated proteins, for example IpaH of Shi-gella flexneri, YopM of Yersinia, the SspH1 and SspH2proteins of Salmonella typhimurium and the filamentoushemagglutinin of Bordetella pertussis [66, 67]. Most of theprokaryotic LRR motifs described to date belong to pro-teins of the listerial internalin family. The presence of alarge multigene family encoding highly homologous LRRproteins is a characteristic unique to the genus Listeria.

There are two subfamilies of internalins. One consistsof large proteins (70–80 kDa) which are attached via theirC-terminal regions to the bacterial cell wall. This group isexemplified by the inlAB-encoded InlA and InlB polypep-tides and includes at least six other members (inlC2, inlD,inlE, inlF, inlG and inlH), all found in L. monocytogenes[59, 68, 69]. The other group includes proteins generallymuch smaller in size (25–30 kDa), which lack theC-terminal cell-wall anchor region and are released into



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the extracellular medium. The prototype is InlC (or IrpA)from L. monocytogenes [70, 71] but the remaining mem-bers of this group (i-InlC, i-InlD, i-InlE, i-InlF and i-InlG)have all been identified in L. ivanovii [8, 72–74] (figure 5).

The known internalin loci usually comprise from twoup to several inl genes, forming ’internalin islets’. The onlyexceptions are the inlC and inlF genes, which lie alone inthe L. monocytogenes chromosome. Due to the presenceof highly conserved repeated sequences (the LRR domaincommon to all internalins and the B or C repeats present inthe C-terminal region of the large, cell wall-associatedinternalins; see [68, 75] for more details about structuralfeatures of internalins), the inl genes exhibit a very highdegree of sequence similarity. This characteristic makesthem naturally prone to recombination and may be at theorigin of their diversity, as postulated for the R geneclusters of plants [76]. Indeed, it is possible that theinternalin multigene family arose from a single internalindeterminant that originally evolved in the Listeria ances-tor, by gene duplication and further diversification bysequence exchanges via intra- or intergenic homologousrecombination events. It is clear that such rearrangementshave occurred, for example, in i-inlG, an internalin genefound in L. ivanovii, which presumably has resulted fromthe tail-to-head, tandem fusion of two secreted internalingenes (figure 5) ([5] and G. Domínguez-Bernal, J.A.Vázquez-Boland et al., in preparation). Evidence that theserecombination events are an ongoing process is provided

by the observation that the same internalin islet has adifferent complement of inl genes in two different isolatesof a serovar 1/2a strain of L. monocytogenes (EGD) (inlC2,inlD and inlE in one isolate and inlG, inlH and inlE in theother) [8, 68, 69] (figure 5). Internalin loci also have adifferent distribution, gene composition and location inL. monocytogenes and L. ivanovii. For example, theinlG(C2D)/HE internalin islet of L. monocytogenes ispresent between two housekeeping genes (ascB and dapE)that are contiguous in L. ivanovii (and all the other Listeriaspp.) [8]. Another example: whereas the inlC gene ofL. monocytogenes is inserted alone between the rplS andinfC genes, its counterpart in L. ivanovii, i-inlC (nucleotidesequence 92% identical to that of inlC), is adjacent toanother, highly similar internalin gene (i-inlD) and bothare inserted at a different position, between an rRNA geneand a gene coding for a putative TetR-like regulator [72](figure 5).

The i-inlCD locus L. ivanovii also offers a clue to theway in which internalin islets may have arisen, becausethere is a tRNA gene at its insertion point. tRNA genes areknown targets for the integration of lysogenic phages andare frequently found at the insertion sites of pathogenicityislands (PAIs) in bacterial genomes [77, 78]. A tRNA genewas also found at the insertion site of a recently discoveredvirulence gene cluster in L. ivanovii carrying a large num-ber of internalin genes (see below).

Figure 5. The internalin multigene family of L. monocytogenes and L. ivanovii. The direction of transcription is from left to right except forthe smcL gene (indicated by an arrow within the gene box). Relevant structural features of the encoded proteins are indicated. The large, cellwall-associated internalins attach to the bacterial surface by two different mechanisms: (i) via an LPXTG anchor, present in many surfaceproteins of Gram-positive bacteria and consisting of a pentapeptide Leu-Pro-X-Thr-Gly which covalently links the Inl protein to thepeptidoglycan, followed by a hydrophobic membrane-spanning region of approximately 20 amino acids [130]; (ii) via a novel mechanisminvolving a C domain, described in InlB and consisting of tandemly arranged 80-amino acids repeats starting with the sequence GW, whichuses lipoteichoic acid as ligand and mediates a loose attachment of the protein to the bacterial surface [131]. The schema shows the differentgene arrangements found in the same inl locus in two isolates of serovar 1/2a L. monocytogenes strain EGD. Sequence comparisons betweenthe two inl gene clusters suggest that inlH was generated by recombination of the 5’-terminal part of inlC2 and the 3’-terminal part of inlD[8, 69].



3.2. Role in virulence

Internalins are currently believed to be important viru-lence factors for host cell invasion, but their actual role inListeria pathogenesis is still far from being fully under-stood. Using a number of experimental in vitro approaches,the cell wall-associated internalins InlA and InlB havebeen shown to be necessary and sufficient to trigger inter-nalization by susceptible normally nonphagocytic cells[59, 79, 80]. In vitro experimental data also show that InlAand InlB have different host cell specificities. Thus, InlAappears to promote invasion only in cells possessing itsreceptor, human E-cadherin (i.e. human epithelial cells)[81, 82], whereas InlB seems to mediate entry into a widerrange of cell types (epithelial cells, fibroblasts, hepato-cytes and endothelial cells) from various animal species[68, 83–86]. These observations led to the suggestion thatthe various cell wall-associated internalins found in patho-genic Listeria might be involved in cell and host tropism[87]. However, despite their proven involvement in cellinvasion in vitro, no clear role in pathogenesis has yetbeen demonstrated for InlA and InlB in in vivo animalmodels of infection. In mice, inlAB mutants are onlyslightly less persistent (if any) in organs, invade hepato-cytes in vivo at rates similar to those of the wild type andshow no impairment in brain tissue colonization in anexperimental meningoencephalitis model [68, 83, 88–91].Similarly, inlAB mutants show no impairment in intestinalinvasion and translocation in a rat ligated ileal loop model[92]. Analyses with deletion mutants in other InlA- andInlB-like surface-associated internalin genes of L. mono-cytogenes (the inlGHE, inlGC2DE and inlF loci) (figure 5)have also been disappointing, as no role in internalizationcould even be demonstrated for these loci in vitro usingcell monolayers [68, 69]. In vivo, a deletant of one of theseloci, inlGHE, was moderately impaired in the capacity tosurvive in mouse organs, suggesting that the correspond-ing internalins play at least some role in host tissue colo-nization [69].

Evidence of an involvement in the infectious process invivo is stronger for the members of the small, secretedinternalin subfamily. A significant increase in mouse LD50

is observed following the deletion of inlC in L. monocyto-genes [71]. Similarly, deletion of the i-inlE and i-inlF genesleads to a significant increase in the LD50 of L. ivanovii[73]. There is an important difference in the expressioncharacteristics between the small and large internalins thatis possibly relevant to pathogenesis. The genes encodingthe latter type of internalin are generally not controlled (atleast not tightly) by the virulence gene transcriptionalactivator, PrfA [34, 69]. As shown for inlAB, they aresignificantly expressed extracellularly in rich brothmedium (e.g., brain heart infusion) but poorly expressedwithin cells [34, 35, 93], consistent with the role postu-lated for InlA and InlB as invasins mediating internaliza-tion from the extracellular space. In contrast, all the small,secreted internalin genes characterized to date are PrfA-regulated [8, 70–74]. The genes of the PrfA-dependentregulon are downregulated in broth medium and at highiron concentrations (extracellular conditions), but are pref-erentially activated within host cells [30, 31, 33, 34, 94,

95]. This is true for the genes of the hly virulence genecluster, reflecting the key role of their products in theintracellular life cycle. This suggests that small internalinsmay play a role in the listerial intracellular life cycle.Preferential intracellular expression has indeed beenexperimentally confirmed for the inlC gene of L. monocy-togenes, especially at late stages of infection, when bacte-ria are in the process of active intercellular spread [34, 71].However, deletion of inlC has been shown to affect neitherintracellular proliferation nor cell-to-cell spread. Thismutation was also shown not to affect internalization bycell monolayers in vitro, suggesting that the small interna-lins are not involved in cell invasion [70, 71, 84]. Nointracellular target has been identified for InlC and theprecise role of the small, secreted internalins in virulenceremains to be determined.

4. A large virulence gene clusterspecific to L. ivanovii

In L. ivanovii, a large (22 kb) virulence gene cluster hasrecently been identified that contains a sphingomyelinasegene, smcL, surrounded by a large number of inl genes.Most of these inl genes encode small secreted internalinsand are PrfA-dependent ([74, 96]; G. Domínguez-Bernal,J.A.. Vázquez-Boland, et al., in preparation). smcL, incontrast, is expressed independently of PrfA and is tran-scribed in the opposite orientation to the surrounding inlgenes [96] (figure 5). The smcL gene is specific toL. ivanovii and its protein product, the SmcL enzyme, ishighly similar (> 50% sequence identity) to the sphingo-myelinases C of Staphylococcus aureus (�-toxin) and Bacil-lus cereus [96]. These data suggest that smcL inserted intoa pre-existing internalin locus at relatively recent stages ofvirulence evolution in L. ivanovii after its acquisition dur-ing a genetic exchange with either of the above twophylogenetically very close bacteria. This large virulencegene cluster is absent from L. monocytogenes, its insertionis targeted to a tRNA locus and it is spontaneously deletedat low frequency. Such deletion mutants have significantlyimpaired virulence in mice and sheep. This L. ivanovii-specific virulence gene cluster is one of the few examplesof an unstable virulence-associated chromosomal locus ina Gram-positive bacterium. As discussed earlier, a role incell and host tropism has been demonstrated in vitro formembers of the internalin multigene family. In vitro experi-mental data also suggest that SmcL, which selectively lysesspingomyelin-rich membranes (for example those of sheeperythrocytes), may be also involved in host tropism [74,96]. It is therefore possible that this novel virulence genecluster plays a significant role in the pathogenic tropism ofL. ivanovii for ruminants.

5. Conclusions and perspectiveAlthough plasmids are not uncommon in Listeria

[97–100], all the virulence determinants identified to datein these bacteria are chromosome encoded. As in otherbacterial pathogens, listerial virulence genes are orga nized



577

into discrete genetic islands. One of these islands plays acentral role in Listeria pathogenesis, as it carries virulencegenes essential for intracellular parasitism. This 9-kb ’intra-cellular life’ genetic cassette is stably inserted at the sameposition in the chromosome of species belonging to twodifferent evolutionary branches of the genus Listeria, has aDNA composition similar to that of the listerial coregenome, lacks any obvious trace of mobility factors (inte-grases, transposases, IS), and its insertion point is devoid ofthe usual integration signals (direct repeats, tRNAs). Thisgene cluster appears thus to be an ancient PAI that wasacquired by a common Listeria ancestor and that, as aresult of a long evolution (and, probably also, a strongselective pressure for conservation for it is indispensablefor animal host tissue colonization), became stabilized inthe core genome of pathogenic Listeria spp. Two smallORFs at the right end of the gene cluster exhibit significantsimilarities with bacteriophage and other viral proteins,suggesting that it was originally mobilized via bacterioph-age transduction. This ancient PAI has been given variousnames, such as hly- or prfA-virulence gene cluster and,more recently, vcl (for virulence cluster of Listeria) or vgc(for virulence gene cluster) locus [5, 8, 38, 45]. Here wepropose for this primordial Listeria PAI the name LIPI-1 (forListeria pathogenicity island 1), using a unified nomencla-ture for the designation of any large, genetically heteroge-neous (i.e. with at least two different classes of virulencedeterminants) PAI identified in Listeria.

Besides LIPI-1, pathogenic Listeria contain other chro-mosomal loci associated with virulence. Examples are theloci encoding the Clp stress tolerance mediators involvedin intraphagosomal survival [101–103], the Ami proteininvolved in attachment to host cells [104] and the PrfA-dependent Hpt hexose phosphate transporter required forefficient intracellular proliferation ([105]; I. Chico, J.A.Vázquez-Boland, et al., submitted). Of these other viru-lence loci, the members of the internalin multigene familyare particularly interesting from the evolutionary point ofview. The presence of inl genes in various Listeria spp.seems to indicate that they were already present in acommon listerial ancestor and, therefore, that internalinislets are at least as old as LIPI-1. However, the persistencein some of these islets of clear traces of their possiblemechanism of mobilization (tRNA genes at their insertionpoint) and, in particular, the genetic instability of LIPI-2(name given to the newly described large virulence genecluster of L. ivanovii according to the proposed nomen-clature), point to the fact that inl loci are more recent thanLIPI-1 in the evolutionary history of the genus Listeria. Thisis also suggested by the expressional subordination of asubset of inl genes to LIPI-1, which contains the geneencoding the PrfA regulator. Indeed, the LRR motifs char-acteristically carried by Inl proteins are widespread amongeukaryotic proteins but are rare in bacterial proteins, prob-ably reflecting that they are a recent development inprokaryotes. The different gene complements and loca-tions of internalin islets in the various Listeria spp. suggestthat these loci have been horizontally transferred withinthe genus, possibly carried by phages, which are commonin Listeria, have interspecies infectivity and are capable ofDNA transduction [106, 107].

Such a large repertoire of LRR-containing inl genes inthe genus Listeria is intriguing. Variability in the exposedface of the internalin LRR motifs is thought to be importantfor the creation of a variety of specific protein–proteininteractions [60]. Indeed, LRR domains of the L. monocy-togenes surface proteins InlA and InlB are required forinvasion of permissive host cells [108, 109]. It is thereforelikely that the immune response and the huge diversity ofligands present in the infected host exert a positive diver-sifying selection over internalin LRR motifs, leading tofunctional polymorphisms involved in evasion or in celland tissue tropism. Such a mechanism of sequence diver-sification has been shown to operate in the LRR motifs ofplant R proteins during their coevolution with avirulencedeterminants of bacterial phytopathogens [65, 110]. Theexistence of inl genes in nonpathogenic Listeria speciesindicates that internalins also fulfill biological functionsnot associated with virulence and pathogenesis.

Listeria spp. have a remarkable genomic stability, asreflects the clonal structure of their bacterial populations.There is, however, significant genetic heterogeneity withinListeria spp. [111–113], indicating that horizontal genetransfer events are relatively common in these bacteria.Bacteriophages are probably major players in the genomicplasticity of Listeria, but other mechanisms, such as theconjugal transfer of plasmids and, in particular, DNAtransformation (homologs of the competence network ofBacillus subtilis have been identified in the L. monocyto-genes genome [114]), followed by intergration via IS,transposons, integrative replicons and homologous recom-bination, may operate as well. Studies on antibiotic resis-tance in Listeria show clearly that genetic material involvedin short-term evolutionary adaptation can be successfullytransferred between Listeria spp., and even between List-eria and related Gram-positive bacteria, such as entero-cocci [98, 115–117]. Similar mechanisms as those respon-sible for the transfer of antibiotic resistance might also beinvolved in the transfer of virulence genes and be at theorigin of the observed virulence heterogeneity amongstrains of pathogenic Listeria spp. [118–120].

The best example of virulence heterogeneity in L. mono-cytogenes is the well-known association between antigencomposition and pathogenicity. Thus, only three of the 12known serovars of L. monocytogenes, 1/2a, 1/2b and 4b,account for more than 90% of human and animal cases oflisteriosis [3, 5, 121]. Of these, 4b strains cause over 50%of the cases of listeriosis worldwide, although strains ofsomatic antigen group 1/2 (1/2a, 1/2b and 1/2c) predomi-nate in food isolates [3, 5, 121, 122]. This associationbetween antigen composition and virulence is particularlyinteresting because infectivity of Listeria bacteriophages isserovar/serogroup-specific, indicating that antigenic sur-face structures are used as phage receptors [106, 123]. Inaddition, a genomically similar group of serovar 4b iso-lates, characterized by exhibiting the same phage type,has been found to be responsible for major outbreaks offoodborne human listeriosis worldwide [112, 124]. All thisevidence suggests that serovar 4b strains may not only bebetter adapted for colonizing mammalian host tissues, butthat certain clones of this serovar may be particularlypathogenic.



A number of observations also suggest the existence ofstrain-to-strain differences in pathogenic tropism inL. monocytogenes. In humans, serovar 4b strains havebeen reported to occur more frequently in feto–maternalcases than in other clinical conditions [125]. In two out-breaks of listeriosis in France in 1992 and 1993, eachassociated with a different phage type of serovar 4b, thepercentage of feto–maternal cases differed significantly(33% versus 80%) despite the similar structure of the targetpopulations [126]. In sheep, the two major clinical formsof L. monocytogenes infection, meningoencephalitis andabortion, tend not to occur simultaneously in the sameflock during an outbreak [5]. A recent study has providedmolecular epidemiological evidence for such pathogenictropism in L. monocytogenes [127]. The isolates of thisspecies can be grouped into three distinct serovar-relatedevolutionary branches or lineages [111–113]. In the study,lineage I (serovar 1/2b and 4b strains) contained all iso-lates from human foodborne epidemics and isolates fromsporadic cases in humans and animals, lineage II (serovars1/2a, 1/2c and 3a) contained both human and animalcases but no isolates from human foodborne epidemics,and lineage III (serovar 4a strains) contained exclusivelystrains isolated from animals. A particular ribotype inlineage I included all the serovar 4b strains associated withhuman foodborne epidemics but less than 10% of theruminant isolates, indicating a possible pathogenic tro-pism for humans. In addition, the animal strains of thisribotype were all from encephalitis cases, suggesting anassociation with this clinical form of the disease in rumi-nants [127].

According to current knowledge on the molecular evo-lution of bacterial virulence, differences in virulence andpathogenic potential probably depend on the presence ofspecific PAIs in the corresponding strains. The genomicera of Listeria research has recently begun with thesequencing of the genomes of L. monocytogenes and L. in-nocua (European Listeria Consortium, manuscript in prepa-ration). Comparisons of these genomes with genomesfrom other L. monocytogenes strains and Listeria spp. willcertainly lead to the identification of novel listerial PAIsand will provide interesting insights into the mechanismsof their acquisition and the evolution of pathogenicity inthe genus Listeria.

Acknowledgments

We thank P. Garrido for her contribution in the sequenc-ing and analysis of the plcB-orfB intergenic region ofL. ivanovii. This work was supported by the EuropeanCommission (BIOMED 2 contract BMH4-CT96-0659), theDirección General de Enseñanza Superior e InvestigaciónCientífica of the Spanish Ministry for Education and Sci-ence (grant DGESIC PB97-0327-C03-01) and the Deut-sche Forschungsgemeinschaft (grant Kr 1203). J.-A.V.-B.wishes to thank the Universidad Complutense de Madridfor the sabbatical leave awarded during the year 2000,and the Lehrstuhl für Mikrobiologie of the University ofWürzburg for their kind hospitality and support during thissabbatical.

References

[1] Collins M.D., Wallbanks S., Lane D.J., Shah J.,Nietupski R., Smida J., Dorsch M., Stackebrandt E.,Phylogenetic analysis of the genus Listeria based on reversetranscriptase sequencing of 16S rRNA, Int. J. Syst. Bac-teriol. 41 (1991) 240–246.

[2] Sallen B., Rajoharison S., Desverenne S., Quinn F., Mabi-lat C., Comparative analysis of 16S and 23S rRNAsequences of Listeria species, Int. J. Syst. Bacteriol. 46(1996) 669–674.

[3] Farber J.M., Peterkin P.I., Listeria monocytogenes, a food-borne pathogen, Microbiol. Rev. 55 (1991) 476–511.

[4] Fenlon D.R., in: Ryser E.T., Marth E.H. (Eds.), Listeriamonocytogenes in the natural environment, Listeria, Listerio-sis, and Food Safety, 2nd ed., Marcel Dekker Inc., NewYork, 1999, pp. 21–37.

[5] Vázquez-Boland J.A., Kuhn M., Berche P.,Chakraborty T., Domínguez-Bernal G., Goebel W.,González-Zorn B., Wehland J., Kreft J., Listeria patho-genesis and molecular virulence determinants, Clin.Microbiol. Rev. (2001) (in press).

[6] Cossart P., Portnoy D., in: Fischetti V.A., Novick R.P.,Ferretti J.J., Portnoy D.A., Rood J.I. (Eds.), Gram-PositivePathogens, American Society for Microbiology, Washing-ton D.C., 2001, pp. 507–515.

[7] Kuhn M., Goebel W., Internalization of Listeria monocyto-genes by nonprofessional and professional phagocytes, Sub-cell. Biochem. 33 (2000) 411–436.

[8] Kreft J., Vazquez-Boland J.A., Ng E., Goebel W., in:Kaper J., Hacker J. (Eds.), Pathogenicity Islands andOther Mobile Virulence Elements, American Society forMicrobiology, Washington, DC, 1999, pp. 219–232.

[9] Cossart P., Vincente M.F., Mengaud J., Baquero F., Perez-Diaz J.C., Berche P., Listeriolysin O is essential for viru-lence of Listeria monocytogenes: Direct evidence obtained bygene complementation, Infect. Immun. 57 (1989)3629–3636.

[10] Bielecki J., Youngman P., Connelly P., Portnoy D.A.,Bacillus subtilis expressing a haemolysin gene from Listeriamonocytogenes can grow in mammalian cells, Nature 345(1990) 175–176.

[11] Vázquez-Boland J.A., Kocks C., Dramsi S., Ohayon H.,Geoffroy C., Mengaud J., Cossart P., Nucleotide sequenceof the lecithinase operon of Listeria monocytogenes and pos-sible role of lecithinase in cell-to-cell spread, Infect.Immun. 60 (1992) 219–230.

[12] Kocks C., Gouin E., Tabouret M., Berche P., Ohayon H.,Cossart P., L. monocytogenes-induced actin assembly requiresthe actA gene product, a surface protein, Cell. 68 (1992)521–531.

[13] Smith G.A., Portnoy D.A., How the Listeria monocytogenesActA protein converts actin polymerization into a motileforce, Trends Microbiol. (1997) 272–276.

[14] Lasa I., Dehoux P., Cossart P., Actin polymerization andbacterial movement, Biochim. Biophys. Acta 1402 (1998)217–228.

[15] Cicchetti G., Maurer P., Wagener P., Kocks C., Actin andphosphoinositide binding by the ActA protein of thebacterial pathogen Listeria monocytogenes, J. Biol. Chem.274 (1999) 33616–33626.



579

[16] Chakraborty T., Ebel F., Domann E., Gerstel B., Pistor S.,Temm-Grove C.J., Jockusch B.M., Reinhard M.,Walter U., Wehland J., A novel focal adhesion factordirectly linking intracellular motile Listeria monocytogenesand Listeria ivanovii to the actin-based cytoskeleton ofmammalian cells, EMBO J. 14 (1995) 1314–1321.

[17] Niebuhr K., Ebel F., Frank R., Reinhard M., Domann E.,Carl U.D., Walter U., Gertler F.B., Wehland J.,Chakraborty T., A novel proline-rich motif present inActA of Listeria monocytogenes and cytoskeletal proteins isthe ligand for the EVH1 domain, a protein module presentin the Ena/VASP family, EMBO J. 16 (1997) 5433–5444.

[18] Welch M.D., Rosenblatt J., Skoble J., Portnoy D.A.,Mitchison T.J., Interaction of human Arp2/3 complex andthe Listeria monocytogenes ActA protein in actin filamentnucleation, Science 281 (1998) 105–108.

[19] Yarar D., To W., Abo A., Welch M.D., The Wiskott-Aldrich syndrome protein directs actin-based motility bystimulating actin nucleation with the Arp2/3 complex,Curr. Biol. 9 (1999) 555–558.

[20] Machesky L.M., Mullins R.D., Higgs H.N., Kaiser D.A.,Blanchoin L., May R.C., Hall M.E., Pollard T.D., Scar, aWASp-related protein, activates nucleation of actin fila-ments by the Arp2/3 complex, Proc. Natl. Acad. Sci. USA96 (1999) 3739–3744.

[21] Rohatgi R., Ma L., Miki H., Lopez M., Kirchhausen T.,Takenawa T., Kirschner M.W., The interaction betweenN-WASP and the Arp2/3 complex links Cdc42-dependentsignals to actin assembly, Cell 97 (1999) 221–231.

[22] Alvarez-Domínguez C., Vázquez-Boland J.A., Carrasco-Marín E., López-Mato P., Leyva-Cobián F., Host cell hepa-ran sulfate proteoglycans mediate attachment and entry ofListeria monocytogenes, and the listerial surface protein ActAis involved in heparan sulfate receptor recognition, Infect.Immun. 65 (1997) 78–88.

[23] Marquis H., Doshi V., Portnoy D.A., The broad-rangephospholipase C and a metalloprotease mediate listeriol-ysin O-independent escape of Listeria monocytogenes from aprimary vacuole in human epithelial cells, Infect. Immun.63 (1995) 4531–4534.

[24] Smith G.A., Marquis H., Jones S., Johnston N.C., Port-noy D.A., Goldfine H., The two distinct phospholipases Cof Listeria monocytogenes have overlapping roles in escapefrom a vacuole and cell-to-cell spread, Infect. Immun. 63(1995) 4231–4237.

[25] Portnoy D.A., Innate immunity to a facultative intracel-lular bacterial pathogen, Curr. Opin. Immunol. 4 (1992)20–24.

[26] Kaufmann S.H.E., Immunity to intracellular bacteria,Annu. Rev. Immunol. 11 (1993) 129–163.

[27] Goebel W., Kreft J., Böckmann R., in: Fischetti V.A.,Novick R.P., Ferretti J.J., Portnoy D.A., Rood J.I. (Eds.),Gram-Positive Pathogens, American Society for Microbi-ology, Washington DC, 2000, pp. 499–506.

[28] Leimeister-Wächter M., Domann E., Chakraborty T., Theexpression of virulence genes in Listeria monocytogenes isthermoregulated, J. Bacteriol. 174 (1992) 947–952.

[29] Sokolovic Z., Fuchs A., Wuenscher M., Goebel W., Syn-thesis of species-specific stress proteins by virulent strainsof Listeria monocytogenes, Infect. Immun. 58 (1990)3582–3587.

[30] Ripio M.T., Domínquez-Bernal G., Suárez M., Brehm K.,Berche P., Vázquez-Boland J.A., Transcriptional activa-tion of virulence genes in wild-type strains of Listeriamonocytogenes in response to a change in the extracellularmedium composition, Res. Microbiol. 147 (1996)311–384.

[31] Renzoni A., Cossart P., Dramsi S., PrfA, the transcrip-tional activator of virulence genes, is upregulated duringinteraction of Listeria monocytogenes with mammalian cellsand in eukaryotic cell extracts, Mol. Microbiol. 34 (1999)552–561.

[32] Freitag N.E., Jacobs K.E., Examination of Listeria monocy-togenes intracellular gene expression by using the greenfluorescent protein of Aquorea victoria, Infect. Immun. 67(1999) 1844–1852.

[33] Moors M.A., Levitt B., Youngman P., Portnoy D.A.,Expression of listeriolysin O and ActA by intracellular andextracellular Listeria monocytogenes, Infect. Immun. 67(1999) 131–139.

[34] Bubert A., Sokolovic Z., Chun S.K., Papatheodorou L.,Simm A., Goebel W., Differential expression of Listeriamonocytogenes virulence genes in mammalian host cells,Mol. Gen. Genet. 261 (1999) 323–336.

[35] Ripio M.T., Dominquez-Bernal G., Lara M., Suárez M.,Vázquez-Boland J.A., A Gly145Ser substitution in thetranscriptional activator PrfA causes constitutive overex-pression of virulence factors in Listeria monocytogenes, J. Bac-teriol. 179 (1997) 1533–1540.

[36] Vega Y., Dickneite C., Ripio M.T., Böckmann R.,González-Zorn B., Novella S., Domínguez-Bernal G.,Goebel W., Vázquez-Boland J.A., Functional similaritiesbetween the Listeria monocytogenes virulence regulator PrfAand cyclic AMP receptor protein: the PrfA* (Gly145Ser)mutation increases binding affinity for target DNA, J. Bac-teriol. 180 (1998) 6655–6660.

[37] Mengaud J., Dramsi S., Gouin E., Vazquez-Boland J.A.,Milon G., Cossart P., Pleiotropic control of Listeria mono-cytogenes virulence factors by a gene that is autoregulated,Mol. Microbiol. 5 (1991) 2273–2283.

[38] Gouin E., Mengaud J., Cossart P., The virulence genecluster of Listeria monocytogenes is also present in Listeriaivanoii, an animal pathogen, and Listeria seeligeri, a non-pathogenic species, Infect. Immun. 62 (1994) 3550–3553.

[39] Haas A., Dumbsky M., Kreft J., Listeriolysin genes: com-plete sequence of ilo from Listeria ivanovii and of lso fromListeria seeligeri, Biochim. Biophys. Acta 1130 (1992)81–84.

[40] Lampidis R., Gross R., Sokolovic Z., Goebel W., Kreft J.,The virulence regulator protein of Listeria ivanovii is highlyhomologous to PrfA from Listeria monocytogenes and bothbelong to the Crp-Fnr family of transcription regulators,Mol. Microbiol. 13 (1994) 141–151.

[41] Kreft J., Dumbsky M., Theiss S., The actin-polymerizationprotein from Listeria ivanovii is a large repeat proteinwhich shows only limited amino acid sequence homologyto ActA from Listeria monocytogenes, FEMS Microbiol. Lett.126 (1995) 113–122.



[42] Gouin E., Dehoux P., Mengaud J., Kocks C., Cossart P.,iactA of Listeria ivanovii, although distantly related toListeria monocytogenes actA, restores actin tail formation inan L. monocytogenes actA mutant, Infect. Immun. 63 (1995)2729–2737.

[43] Gerstel B., Gröbe L., Pistor S., Chakraborty T., Weh-land J., The ActA polypeptides of Listeria ivanovii andListeria monocytogenes harbor related binding sites for hostmicrofilament proteins, Infect. Immun. 64 (1996)1929–1936.

[44] Karunasagar I., Lampidis R., Goebel W., Kreft J., Comple-mentation of Listeria seeligeri with the plcA-prfA genesfrom L. monocytogenes activates transcription of seeligerol-ysin and leads to bacterial escape from the phagosome ofinfected mammalian cells, FEMS Microbiol. Lett. 146(1997) 303–310.

[45] Chakraborty T., Hain T., Domann E., Genome organiza-tion and the evolution of the virulence gene locus inListeria species, Int. J. Med. Microbol. 290 (2000)167–174.

[46] Rocourt J., Seeliger H.P.R., Distribution des espèces dugenre Listeria, Zbl. Bakt. Hyg. A259 (1985) 317–330.

[47] Ryser T., Marth E. (Eds.), Listeria, Listeriosis and FoodSafety, 2nd ed., Marcel Dekker, Inc., New York, 1999.

[48] MacGowan A.P., Bowker K., McLauchlin J., Ben-nett P.M., Reeves D.S., The occurrence and seasonalchanges in the isolation of Listeria spp. in shop boughtfood stuffs, human faeces, sewage and soil from urbansources, Int. J. Food Microbiol. 21 (1994) 325–334.

[49] Farber J.M., Sanders G.W., Malcolm S.A., The presence ofListeria spp. in raw milk in Ontario, Can. J. Microbiol. 34(1988) 95–100.

[50] Luppi A., Bucci G., Maini P., Rocourt J., Ecologicalsurvey of Listeria in the Ferrara area (northern Italy), Zbl.Bakt. Mikrobiol. Hyg. A269 (1988) 266–275.

[51] Pritchard T.J., Flanders K.J., Donnelly C.W., Comparisonof the incidence of Listeria on equipment versus environ-mental sites within dairy processing plants, Int. J. FoodMicrobiol. 26 (1995) 375–384.

[52] Jeyasekaran G., Karunasagar I., Karunasagar I., Incidenceof Listeria spp. in tropical fish, Int. J. Food Microbiol. 31(1996) 333–340.

[53] Beumer R.R., Te Giffel M.C., Spoorenberg E., RomboutsF.M., Listeria species in domestic environments, Epide-miol. Infect. 117 (1996) 437–442.

[54] Sergelidis D., Abrahim A., Sarimvei A., Panoulis C.,Karaiannoglou P., Genigiorgis C., Temperature distribu-tion and prevalence of Listeria spp. in domestic, retail andindustrial refrigerators in Greece, Int. J. Food Microbiol.34 (1997) 171–177.

[55] Ryser E.T., Arimi S.M., Donnelly C.W., Effects of pH ondistribution of Listeria ribotypes in corn, hay and grasssilage, Appl. Environ. Microbiol. 63 (1997) 3695–3697.

[56] Alouf J.E., in: Alouf J.E., Freer J.H. (Eds.), The Compre-hensive Sourcebook of Bacterial Protein Toxins, 2nd ed.,Academic Press, London, 1999, pp. 443–456.

[57] Mengaud J., Braun-Breton C., Cossart P., Identification ofphosphatidylinositol-specific phospholipase C activity inListeria monocytogenes: a novel type of virulence factor? Mol.Microbiol. 5 (1991) 367–372.

[58] Mengaud J., Geoffroy C., Cossart P., Identification of anew operon involved in Listeria monocytogenes virulence: Itsfirst gene encodes a protein homologous to bacterial met-alloproteases, Infect. Immun. 59 (1991) 1043–1049.

[59] Gaillard J.L., Berche P., Frehel C., Gouin E., Cossart P.,Entry of Listeria monocytogenes into cells is mediated byinternalin, a repeat protein reminiscent of surface antigensfrom gram-positive cocci, Cell 65 (1991) 1127–1141.

[60] Marino M., Braun L., Cossart P., Ghosh P., A frameworkfor interpreting the leucine-rich repeats of Listeria inter-nalins, Proc. Natl. Acad. Sci. USA 97 (2000) 9784–9788.

[61] Yoder M.D., Keen N.T., Jurnak F., New domain motif:the structure of the pectate lyase C, a secreted plantvirulence factor, Science 260 (1993) 1503–1507.

[62] Heffron S., Moe G.R., Sieber V., Mengaud J., Cossart P.,Vitali J., Jurnak F., Sequence profile of the parallel betahelix in the pectate lyasesuperfamily, J. Struct. Biol. 122(1998) 223–235.

[63] Kobe B., Deisenhofer J., The leucine-rich repeat: A versa-tile binding motif, Trends Biochem. Sci. 19 (1994)415–421.

[64] Kajava A.V., Structural diversity of leucine-rich repeatproteins, J. Mol. Biol. 277 (1998) 519–527.

[65] Jones D.A., Jones J.D.G., The roles of leucine rich repeatsin plant defences, Adv. Bot. Res. Adv. Plant. Pathol. 24(1996) 90–167.

[66] Miao E.A., Scherer C.A., Tsolis R.M., Kingsley R.A.,Adams L.G., Bäumler A.J., Miller S.I., Salmonella typhimu-rium leucine-rich repeat proteins are targeted to the SPI1and SPI2 type secretion systems, Mol. Microbiol. 34 (1999)850–864.

[67] Makhov A.M., Hannah J.H., Brennan M.J., Trus B.L.,Kocsis E., Conway J.F., Wingfield P.T., Simon M.N.,Steven A.C., Filamentous hemagglutinin of Bordetella per-tussis. A bacterial adhesin formed as a 50-nm monomericrigid rod based on a 19-residue repeat motif rich inbeta-strands and turns, J. Mol. Biol. 241 (1994) 110–124.

[68] Dramsi S., Dehoux P., Lebrun M., Goossens P.L., Cos-sart P., Identification of four new members of the interna-lin multigene family of Listeria monocytogenes EGD, Infect.Immun. 65 (1997) 1615–1625.

[69] Raffelsbauer D., Bubert A., Engelbrecht F., Scheinp-flug J., Simm A., Hess J., Kaufmann S.H.E., Goebel W.,The gene cluster inlC2DE of Listeria monocytogenes containsadditional new internalin genes and is important for viru-lence in mice, Mol. Gen. Genet. 260 (1998) 144–158.

[70] Domann E., Zechel S., Lingnau A., Hain T., Darji A.,Nichterlein T., Wehland J., Chakraborty T., Identificationand characterization of a novel PrfA-regulated gene inListeria monocytogenes whose product, IrpA, is highlyhomologous to internalin proteins, which contain leucine-rich repeats, Infect. Immun. 65 (1997) 101–109.

[71] Engelbrecht F., Chun S.K., Ochs C., Hess J., Lottspeich F.,Goebel W., Sokolovic Z., A new PrfA-regulated gene ofListeria monocytogenes encoding a small, secreted proteinwhich belongs to the family of internalins, Mol. Micro-biol. 21 (1996) 823–837.



581

[72] Engelbrecht F., Dickneite C., Lampidis R., Götz M.,DasGupta U., Goebel W., Sequence comparison of thechromosomal regions encompassing the internalin C genes(inlC) of Listeria monocytogenes and Listeria ivanovii, Mol.Gen. Genet. 257 (1998) 186–197.

[73] Engelbrecht F., Dominguez-Bernal G., Dickneite C.,Hess J., Greiffenberg L., Lampidis R., Raffelsbauer D.,Kaufmann S.H.E., Kreft J., Vazquez-Boland J.A.,Goebel W., A novel PrfA-regulated chromosomal locus ofListeria ivanovii encoding two small, secreted internalins isessential for virulence in mice, Mol. Microbiol. 30 (1998)405–417.

[74] González-Zorn B., Domínguez-Bernal G., Suárez M.,Vega Y., Novella S., Rodríguez A., Chico-Calero I., Tier-rez A., Vázquez-Boland J.A., SmcL, a novel membrane-damaging virulence factor in Listeria, Int. J. Med. Micro-biol. 13 (2000) 369–374.

[75] Braun L., Dramsi S., Dehoux P., Bierne H., Lindahl G.,Cossart P., InlB: an invasion protein of Listeria monocytoge-nes with a novel type of surface association, Mol. Micro-biol. 25 (1997) 285–294.

[76] McDowell J.M., Dhandaydham M., Long T.A.,Aarts M.G.M., Goff S., Holub E.B., Dangl J.L., Intragenicrecombination and diversifying selection contribute to theevolution of downy mildew resistance at the RPP8 locus ofArabidopsis, Plant Cell 10 (1998) 1861–1874.

[77] Hacker J., Blum-Oehler G., Mühldorfer I., Tschäpe H.,Pathogenicity islands of virulent bacteria: structure, func-tion and impact on microbial evolution, Mol. Microbiol.23 (1997) 1089–1097.

[78] Hacker J., Kaper J., The concept of pathogenicity islands,in: Kaper J., Hacker J. (Eds.), Pathogenicity Islands andOther Mobile Virulence Elements, American Society forMicrobiology, Washington, DC, 1999, pp. 1–12.

[79] Lecuit M., Ohayon H., Braun L., Mengaud J., Cossart P.,Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization,Infect. Immun. 65 (1997) 5309–5319.

[80] Braun L., Ohayon H., Cossart P., The InlB protein ofListeria monocytogenes is sufficient to promote entry intomammalian cells, Mol. Microbiol. 27 (1998) 1077–1087.

[81] Mengaud J., Ohayon H., Gounon P., Mege R.M., Cos-sart P., E-cadherin is the receptor for internalin, a surfaceprotein required for entry of Listeria monocytogenes intoepithelial cells, Cell 84 (1996) 923–932.

[82] Lecuit M., Dramsi S., Gottardi C., Fedor-Chaiken M.,Gumbiner B., Cossart P., A single amino acid in E-cadherinresponsible for host specificity towards the human patho-gen Listeria monocytogenes, EMBO J. 18 (1999) 3956–3963.

[83] Dramsi S., Biswas I., Maguin E., Braun L., Mastroeni P.,Cossart P., Entry of Listeria monocytogenes into hepatocytesrequires the expression of InlB, a surface protein of theinternalin multigene family, Mol. Microbiol. 16 (1995)251–261.

[84] Greiffenberg L., Goebel W., Kim K.S., Weiglein I.,Bubert A., Engelbrecht F., Stins M., Kuhn M., Interactionof Listeria monocytogenes with human brain microvascularendothelial cells: InlB-dependent invasion, long-termintracellular growth and spread from macrophages toendothelial cells, Infect. Immun. 66 (1998) 5260–5267.

[85] Müller S., Hain T., Pashalidis P., Lingnau A., Domann E.,Chakraborty T., Wehland J., Purification of the inlB geneproduct of Listeria monocytogenes and demonstration of itsbiological activity, Infect. Immun. 66 (1998) 3128–3133.

[86] Parida S.K., Domann E., Rohde M., Müller S., Darji A.,Hain T., Wehland J., Chakraborty T., Internalin B isessential for adhesion and mediates the invasion of Listeriamonocytogenes into human endothelial cells, Mol. Micro-biol. 28 (1998) 81–93.

[87] Sheehan B., Kocks C., Dramsi S., Gouin E., Klars-feld A.D., Mengaud J., Cossart P., Molecular and geneticdeterminants of the Listeria monocytogenes infectious pro-cess, Curr. Topics Microbiol. Immunol. 192 (1994)187–216.

[88] Lingnau A., Domann E., Hudel M., Bock M., Nichter-lein T., Wehland J., Chakraborty T., Expression of Listeriamonocytogenes EGD inlA and inlB genes, whose productsmediate bacterial entry into tissue culture cell lines, byPrfA-dependent and -independent mechanisms, Infect.Immun. 64 (1995) 1002–1006.

[89] Gregory S.H., Sagnimeni A.J., Wing E.J., Expression ofthe inlAB operon by Listeria monocytogenes is not requiredfor entry into hepatic cells in vivo, Infect. Immun. 64(1996) 3983–3986.

[90] Schlüter D., Domann E., Buck C., Hain T., Hof H.,Chakraborty T., Deckert-Schlüter M., Phospha-tidylcholine-specific phospholipase C from Listeria mono-cytogenes is an important virulence factor in murine cere-bral listeriosis, Infect. Immun. 66 (1998) 5930–5938.

[91] Appelberg R., Leal I.S., Mutants of Listeria monocytogenesdefective in in vitro invasion and cell-to-cell spreadingstill invade and proliferate in hepatocytes of neutropenicmice, Infect. Immun. 68 (2000) 912–914.

[92] Pron B., Boumaila C., Jaubert F., Sarnacki S., Monnet J.P.,Berche P., Gaillard J.L., Comprehensive study of the intes-tinal stage of listeriosis in a rat ligated ileal loop system,Infect. Immun. 66 (1998) 747–755.

[93] Bohne J., Kestler H., Uebele C., Sokolovic Z., Goebel W.,Differential regulation of the virulence genes of Listeriamonocytogenes, Mol. Microbiol. 20 (1996) 1189–1198.

[94] Conte M.P., Longhi C., Polidoro M., Petrone G., Buon-figlio V., Di Santo S., Papi E., Seganti L., Visca P., Val-enti P., Iron availability affects entry of Listeria monocytoge-nes into enterocytelike cell line Caco-2, Infect. Immun. 64(1996) 3925–3929.

[95] Böckmann R., Dickneite C., Middendorf B., Goebel W.,Sokolovic Z., Specific binding of the Listeria monocytogenestranscriptional regulator protein PrfA to target sequencesrequires additional factor(s) and is influenced by iron, Mol.Microbiol. 22 (1996) 643–653.

[96] González-Zorn B., Domínguez-Bernal G., Suarez M.,Ripio M.T., Vega Y., Novella S., Vázquez-Boland J.A.,The smcL gene of Listeria ivanovii encodes a sphingomyeli-nase C that mediates bacterial escape from the phagocyticvacuole, Mol. Microbiol. 33 (1999) 510–523.

[97] Pérez-Díaz J.C., Vicente M.F., Baquero F., Plasmids inListeria, Plasmid 8 (1982) 112–118.

[98] Poyart-Salmeron C., Carlier C., Trieu-Cuot P., Court-ieu A.L., Courvalin P., Transferable plasmid-mediatedantibiotic resistance in Listeria monocytogenes, Lancet 335(1990) 1422–1426.



[99] Lebrun M., Audurier A., Cossart P., Plasmid-borne cad-mium resistance genes in Listeria moncytogenes are presenton Tn5422, a novel transposon closely related to Tn917,J. Bacteriol. 176 (1994) 3049–3061.

[100] Lemaitre J.P., Echchannaoui H., Michaut G., Divies C.,Rousset A., Plasmid-mediated resistance to antimicrobialagents among listeriae, J. Food. Prot. 61 (1998)1459–1464.

[101] Rouquette C., Ripio M.T., Pellegrini E., Bolla J.M., Tas-con R.I., Vazquez-Boland J.A., Berche P., Identification ofa ClpC ATPase required for stress tolerance and in vivosurvival of Listeria monocytogenes, Mol. Microbiol. 21 (1996)977–987.

[102] Nair S., Frehel C., Nguyen L., Escuyer V., Berche P., ClpE,a novel member of the HSP100 family, is involved in celldivision and virulence of Listeria monocytogenes, Mol. Micro-biol. 31 (1999) 185–196.

[103] Gaillot O., Pellegrini E., Bregenholt S., Nair S., Berche P.,The serine-protease ClpP is essential for the intracellularparasitism of the pathogen Listeria monocytogenes, Mol.Microbiol. 35 (2000) 1286–1294.

[104] Milohanic E., Pron B., The European Listeria GenomeConsortium, Berche P., Gaillard J.-L., Identification ofnew loci involved in adhesion of Listeria monocytogenes toeukaryotic cells, Microbiol. 146 (2000) 731–739.

[105] Ripio M.T., Brehm K., Lara M., Suárez M., Vázquez-Boland J.A., Glucose-1-phosphate utilization by Listeriamonocytogenes is PrfA dependent and coordinately expressedwith virulence factors, J. Bacteriol. 197 (1997)7174–7180.

[106] Loessner M.J., Busse M., Bacteriophage typing of Listeriaspecies, Appl. Environ. Microbiol. 56 (1990) 1912–1918.

[107] Hodgson D.A., Generalized transduction of serotype 1/2and serotype 4b strains of Listeria monocytogenes, Mol. Micro-biol. 35 (2000) 312–323.

[108] Lecuit M., Ohayon H., Braun L., Mengaud J., Cossart P.,Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization,Infect. Immun. 65 (1997) 5309–5319.

[109] Braun L., Nato F., Payrastre B., Mazié J.C., Cossart P., The213-amino-acid leucine-rich repeat region of the Listeriamonocytogenes InlB protein is sufficient for entry into mam-malian cells, stimulation of PI 3-kinase and membraneruffling, Mol. Microbiol. 34 (1999) 10–23.

[110] Keen N.T., Gene-for-gene complementarity in plant-pathogen interactions, Annu. Rev. Genet. 24 (1990)447–463.

[111] Piffaretti J.C., Kressebuch H., Aeschbacher M., Bille J.,Bannermann E., Musser J.M., Selander R.K., Rocourt J.,Genetic characterization of clones of the bacterium Listeriamonocytogenes causing epidemic disease, Proc. Natl. Acad.Sci. USA 86 (1989) 3818–3822.

[112] Bibb W.F., Gellin B.G., Weaver R., Schwartz B., Plikay-tis B.D., Reeves M.W., Pinner R.W., Broome C.V., Analy-sis of clinical and food-borne isolates of Listeria monocyto-genes in the United States by multilocus enzymeelectrophoresis and application of the method to epide-miological investigations, Appl. Environ. Microbiol. 56(1990) 2133–2141.

[113] Rasmussen O.F., Skouboe P., Dons L., Rossen L., OlsenJ.E., Listeria monocytogenes exists in at least three evolution-ary lines: evidence from flagellin, invasive associated pro-tein and listeriolysin O genes, Microbiology 141 (1995)2053–2061.

[114] Buchrieser C., Glaser P., Frangeul L., Rusniok C., Dussur-ger O., Taourit S., Chetouani F., Couvé E., Dehoux P.,Fsishi H., The Listeria Genome Consortium, Kunst F.,Cossart P., Evolution of Listeria monocytogenes as revealed bycomparative genomics with Listeria innocua and Bacillussubtilis, Abstracts book of the 3rd Conference Louis Pas-teur on Evolution of Pathogens and Their Hosts, Paris,5–7 October 2000, p. 3.

[115] Doucet-Populaire F., Trieu-Cuot P., Dosbaa I.,Andremont A., Courvalin P., Inducible transfer of conju-gative transposon Tn1545 from Enterococcus faecalis to List-eria moncytogenes in the digestive tracts of gnotobiotic mice,Antimicrob. Agents Chermother. 35 (1991) 185–187.

[116] Roberts M.C., Facinelli B., Giovanetti E., Varaldo P.E.,Transferable erythromycin resistance in Listeria spp. iso-lated from food, Appl. Environ. Microbiol. 62 (1996)269–270.

[117] Biavasco F., Giovanetti E., Miele A., Vignaroli C.,Facinelli B., Varaldo P.E., In vitro conjugative transfer ofVanA vancomycin resistance between enterococci and list-eriae of different species, Eur. J. Clin. Microbiol. Infect.Dis. 15 (1996) 50–59.

[118] Brosch R., Catimel B., Milon G., Buchrieser C., Vindel E.,Rocourt J., Virulence heterogeneity of Listeria monocytoge-nes strains from various sources (food, human, animal) inimmunocompetent mice and its association with typingcharacteristics, J. Food. Protect. 56 (1993) 296–301.

[119] Barbour A.H., Rampling A., Hormaeche C.E., Compari-son of the infectivity of isolates of Listeria monocytogenesfollowing intragastric and intravenous inoculation in mice,Microb. Pathogen. 20 (1996) 247–253.

[120] Van Langendonck N., Bottreau E., Bailly S., Tabouret M.,Marly J., Pardon P., Velge P., Tissue culture assays usingCaco-2 cell line differentiate virulent from non-virulentListeria monocytogenes strains, J. Appl. Microbiol. 85 (1998)337–346.

[121] Schuchat A., Swaminathan B., Broome C.V., Epidemiol-ogy of human listeriosis, Clin. Microbiol. Rev. 4 (1991)169–183.

[122] Rocourt J., Brosch R., Human listeriosis 1990. DocumentWHO/HPP/FOS/92.4. World Health Organization,Geneva.

[123] Promadej N., Fiedler F., Cossart P., Dramsi S.,Kathariou S., Cell wall teichoic acid glycosylation inListeria monocytogenes serotype 4b requires gtcA, a novel,serogroup-specific gene, J. Bacteriol. 181 (1999) 418–425.

[124] Jacquet C., Catimel B., Brosch R., Buchrieser C., Dehau-mont P., Goulet V., Lepoutre A., Veit P., Rocourt J.,Investigations related to the epidemic strain involved inthe French listeriosis outbreak in 1992, Appl. Environ.Microbiol. 61 (1995) 2242–2246.

[125] McLauchlin J., Distribution of serovars of Listeria monocy-togenes isolated from different categories of patients withlisteriosis, Eur. J. Clin. Microbiol. Infect. Dis. 9 (1990)210–213.



583

[126] Rocourt J., Jacquet C., Epidémiologie des infectionshumaines à Listeria monocytogenes en 1994: certitudes etinterrogations, Ann. Inst. Pasteur/Actualités 5 (1994)168–174.

[127] Wiedmann M., Bruce J.L., Keating C., Johnson A.E.,McDonough P.L., Batt C.A., Ribotypes and virulencegene polymorphisms suggest three distinct Listeria mono-cytogenes lineages with differences in pathogenic potential,Infect. Immun. 65 (1997) 2707–2716.

[128] Tilney L.G., Portnoy D.A., Actin filaments and thegrowth, movement, and spread of the intracellular bacte-rial parasite, Listeria monocytogenes, J. Cell Biol. 109 (1989)1597–1608.

[129] Loessner M.J., Scherer S., Organization and transcrip-tional analysis of the Listeria phage A511 late gene regioncomprising the major capsil and tail sheath protein genescps and tsh, J. Bacteriol. 177 (1995) 6601–6609.

[130] Dhar G., Faull K.F., Schneewind O., Anchor structure ofcell wall surface proteins in Listeria monocytogenes, Bio-chem. 39 (2000) 3725–3733.

[131] Jonquières R., Bierne H., Fiedler F., Gounon P., Cossart P.,Interaction between the protein InlB of Listeria monocyto-genes and lipotheicoic acid: a novel mechanism of proteinassociation at the surface of Gram-positive bacteria, Mol.Microbiol. 34 (1999) 902–914.

