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Applied and Environmental Microbiology, March 2007, p. 1928-1939, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.01796-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Presence of the Internalin Gene in Natural Atypically Hemolytic Listeria innocua Strains Suggests Descent from L. monocytogenes{triangledown} ,{dagger}

Dmitriy V. Volokhov,1 Sandra Duperrier,2 Alexander A. Neverov,1 Joseph George,1 Carmen Buchrieser,2 and Anthony D. Hitchins3*

Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20895,1 Laboratoire de Génomique des Microorganismes Pathogènes, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France,2 Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland 207403

Received 28 July 2006/ Accepted 28 December 2006


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ABSTRACT
 
The atypical hemolytic Listeria innocua strains PRL/NW 15B95 and J1-023 were previously shown to contain gene clusters analogous to the pathogenicity island (LIPI-1) present in the related foodborne gram-positive facultative intracellular pathogen Listeria monocytogenes, which causes listeriosis. LIPI-1 includes the hemolysin gene, thus explaining the hemolytic activity of the atypical L. innocua strains. No other L. monocytogenes-specific virulence genes were found to be present. In order to investigate whether any other specific L. monocytogenes genes could be identified, a global approach using a Listeria biodiversity DNA array was applied. According to the hybridization results, the isolates were defined as L. innocua strains containing LIPI-1. Surprisingly, evidence for the presence of the L. monocytogenes-specific inlA gene, previously thought to be absent, was obtained. The inlA gene codes for the InlA protein which enables bacterial entry into some nonprofessional phagocytic cells. PCR and sequence analysis of this region revealed that the flanking genes of the inlA gene at the upstream, 5'-end region were similar to genes found in L. monocytogenes serotype 4b isolates, whereas the organization of the downstream, 3'-end region was similar to that typical of L. innocua. Sequencing of the inlA region identified a small stretch reminiscent of the inlB gene of L. monocytogenes. The presence of two clusters of L. monocytogenes-specific genes makes it unlikely that PRL/NW 15B95 and J1-023 are L. innocua strains altered by horizontal transfer. It is more likely that they are distinct relics of the evolution of L. innocua from an ancestral L. monocytogenes, as postulated by others.


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INTRODUCTION
 
Listeria innocua is a species that is ubiquitously distributed in the natural environment, and unlike L. monocytogenes, represents an example of a nonharmful, nonhemolytic saprophytic Listeria sp. L. innocua has been isolated from a variety of environmental sources, including surface water, soil, sewage, vegetation, and food-processing plants. According to current knowledge, L. innocua does not carry the virulence-associated genes or clusters present and described in the genomes of the pathogenic Listeria species, L. monocytogenes and L. ivanovii. However, several unusual L. monocytogenes-like hemolytic L. innocua strains were isolated and phenotypically and genetically characterized (19). An example is the food isolate PRL/NW 15B95, a naturally but atypically hemolytic L. innocua strain (19). We have shown previously that this L. innocua strain contains the L. monocytogenes PrfA-regulated pathogenicity island 1 (LIPI-1) gene cluster. LIPI-1 of strain PRL/NW 15B95 is inserted in a background in which L. innocua genes preponderate. The initial characterization of this strain did not allow a definitive conclusion about how it evolved, although the results indicated horizontal transfer of this island, as other, non-LIPI-1 genes of L. monocytogenes were absent. Thus, importation of the cluster into an ancestral L. innocua strain rather than vertical evolution of the strain from a common ancestor of L. monocytogenes and L. innocua was suggested.

An earlier comparison of the LIPI-1 and flanking regions among L. monocytogenes, classic L. innocua, and Bacillus subtilis indicated that this gene cluster was probably acquired by a common ancestor of Listeria and that L. innocua subsequently lost most of it (7, 13). Furthermore, phylogenetic analysis of the 16S rRNA gene and 16S-23S rRNA intergenic spacer region, as well as multilocus enzyme electrophoresis analysis, indicate that L. innocua and L. monocytogenes form one branch on the phylogenetic tree of the genus Listeria (33, 38). However, in the case that other non-LIPI-1-specific L. monocytogenes genes are found to be present in some L. innocua strains, this could mean that they represent "inherent fossils" of a common ancestor of Listeria, and thus, further genome comparisons may improve our knowledge about the genetic basis and evolution of pathogenicity in Listeria (10, 37, 38). In the search for other non-LIPI-1-specific L. monocytogenes virulence genes that probably are present in such intermediate strains, like PRL/NW 15B95, the analysis of the family of internalin-coding genes is of particular interest.

Internalins, containing leucine-rich repeat (LRR) domains, are an important protein family in Listeria, containing many members (6, 16, 37). At least two, InlA and InlB, have been shown to be involved in the internalization of L. monocytogenes by various mammalian nonphagocytic cells (12, 36, 37). The origin of the inl genes is not known. LRR domains are widespread in eukaryotic proteins but rare in bacterial proteins, suggesting that they are a relatively recent acquisition by prokaryotes. inl genes are also found in the nonpathogenic species 7L. innocua (13, 33, 38). The presence of members of the internalin multigene family in Listeria spp. representing distinct phylogenetic branches of the genus suggests that inl genes were already present in the common listerial ancestor and, consequently, that evolutionarily they are probably as old as LIPI-1 (38). However, inl loci have different distributions, gene organizations, and locations in the different Listeria spp. Independently of this study, it was recently predicted, on the basis of an analysis of the internalin A and B gene regions of a variety of strains, that there must have been an evolutionary intermediate with an inlA and B region bearing a strong resemblance to that studied here in PRL/NW 15B95 (C. Buchrieser, presented at the Leopoldina symposium Life Strategies of Microorganisms in the Environment and in Host Organisms, Bremen, Germany, 5 to 8 April 2006).

Therefore, analysis of such atypically hemolytic L. innocua strains as PRL/NW 15B95 by using comparative genomics and whole-Listeria genome DNA arrays (10, 15, 42) may help us to understand new aspects of the evolution of the members of the L. innocua-L. monocytogenes phylogenetic branch as well as of the taxonomic status of such intermediate Listeria isolates. Thus, the goal of this study was to try to understand the evolutionary changes leading to the genome structure and content of this natural atypically hemolytic L. innocua strain. Our results suggest that a common ancestor of L. monocytogenes and L. innocua existed and that L. innocua strain PRL/NW 15B95 is evidence for this concept. The identification of additional, non-LIPI-1 L. monocytogenes-specific loci in the genome of natural atypically hemolytic L. innocua PRL/NW 15B95 will be an interesting question for the future.


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MATERIALS AND METHODS
 
Bacterial strains.
Listeria innocua strain PRL/NW 15B95, the main subject of this study, was isolated from imported Korean clams by using selective enrichment and isolation (17). L. innocua type strain F4078 and L. monocytogenes type strain KC1778 are Centers for Disease Control and Prevention strains. Listeria innocua strain J1-023 was from the Listeria strain collection of the Department of Food Science, Cornell University, Ithaca, NY. The strains used for macroarray hybridization are listed in Table 1.


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  		TABLE 1. Origins and descriptions of reference strains used in this study

Culturing the strains.
Strain stock cultures were preserved at –80°C in Trypticase soy broth with 0.6% (wt/vol) yeast extract and 15% (vol/vol) glycerol. Working cultures were maintained on slants of Trypticase soy agar with 0.6% (wt/vol) yeast extract and stored at 5°C. Cultures for DNA array studies were grown in brain heart infusion broth.

Comparative genomic DNA array hybridization and analysis.
The Listeria DNA array used in this study contained 704 probes specific for genes of four different Listeria isolates for which the genome was sequenced: L. monocytogenes strain EGD-e (serovar 1/2a), L. innocua strain CLIP11262 (serovar 6a), L. monocytogenes strain CLIP80459 (serovar 4b), and L. monocytogenes strain F2365 (serovar 4b) (13, 24). The 704 probes selected are described in Hong et al. (18), and the corresponding target genes are listed in Table S1 in the supplemental material. Primer design, PCR amplification conditions, array preparation, genomic DNA extraction, DNA labeling and hybridization, data generation, and analysis were done as described previously (10). This analysis generated a binary score for the chromosomal DNA of each strain analyzed, indicating whether a gene was absent or present. The binary matrices were analyzed using the J-Express program and compared to the results obtained for 300 L. monocytogenes strains from our in-house database.

Detection of L. monocytogenes-specific genes.
By using PCR, the presence of different internalin genes (listed below) was studied. The sizes of amplicons and their entire sequences were compared among the test strains and control strains. The genes searched for were inlA, inlB, inlC, inlG, inlH, inlE, inlD, inlF, and inlC2 of L. monocytogenes, which do not have orthologues in L. innocua CLIP11262 (GenBank accession no. NC_003212) (13). The primers used for this and the previous study (19) are defined in Table S2 in the supplemental material.

PCR amplification.
The standard PCR mixture (50 µl) contained 1.5 U of HotStar Taq DNA polymerase, 1x reaction buffer supplemented with 2.5 mM MgCl2 (QIAGEN, Chatsworth, CA), 600 nM (each) forward and reverse primers, a 200-µM concentration of each deoxynucleoside triphosphate (dATP, dGTP, dCTP, and dTTP), and 1 to 2 µl of DNA template (approximately 0.2 µg of bacterial DNA). PCR was performed with a Gene Amp PCR system 9700 thermocycler (Applied Biosystems, Foster City, CA) with the following conditions: initial activation at 95°C for 15 min, 40 cycles at 94°C for 40 s, 50°C for 40 s, and 72°C for 1 min per 1 kb and a final extension at 72°C for 10 min. The PCR products were separated by electrophoresis in 1% agarose gels containing 1x Tris-acetate-EDTA (TAE) buffer and visualized by staining with ethidium bromide.

Inverse PCR for inlA flanking regions.
An inverse PCR protocol (26) was used to map the flanking regions of the inlA genes of the L. innocua strains studied. Listeria cells were harvested from 24-h cultures (5 ml), and genomic DNA was isolated using a DNeasy kit (QIAGEN) according to the manufacturer's recommendations. Genomic DNA was examined for purity on 1% agarose-TAE gels and quantified by measuring optical density. For amplification of the DNA flanking regions of the inlA gene, MspI, NheI, and PstI digestion, ligation, and inverse PCR were performed. These restriction enzymes were chosen because their cut sites are present in the inlA genes of L. innocua PRL/NW 15B95 and J1-023 and they were likely to cut near the site of insertion due to the large number of cut sites in the L. innocua genome. Between 250 and 500 ng of genomic DNA was digested overnight with 5 U of MspI, NheI, or PstI (NEB) (20-µl reaction mixture volume) and heat inactivated at 85°C for 1 h. T4 DNA ligase (2 U) (NEB) and its corresponding buffer were added to the digest mixture to bring the volume to 25 µl. The mixture was incubated overnight at room temperature. The ligation product was used as the template for an inverse PCR with inlA-specific primers. The PCR products were electrophoretically separated in 1% agarose-TAE gels, and all samples yielding a single major band were selected for cleanup and sequencing.

DNA sequencing.
DNA sequencing was conducted by using an ABI PRISM BigDye Terminator v3.1 cycle sequencing kit. The cycle sequencing reaction was conducted according to the ABI PRISM BigDye Terminator v3.1 cycle sequencing kit's protocol. Reaction samples were then purified with a Centrisep spin column (Princeton Separations, Adelphia, NJ), and dried under vacuum. The samples were sequenced using an Applied Biosystems 3730xl DNA analyzer.

Phylogenetic analysis of sequences and construction of phylogenetic trees.
The genes in the inlA region and the LIPI-1 cluster were compared to the GenBank nucleotide and protein databases using the BLASTN and BLASTP algorithms at GenBank (2). The nucleotide and deduced amino acid sequences for each gene were initially aligned in ClustalX (1). Inter- and intraspecies similarity score matrices for each gene were generated using MEGA version 2.1 and BioEdit software, at http://www.megasoftware.net and http://www.mbio.ncsu.edu/BioEdit/bioedit.html, respectively. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1. Genetic distances were calculated by the Kimura two-parameter and Tamura-Nei models (21). Phylogenetic trees were constructed and compared using neighbor-joining, maximum parsimony, and minimum evolution algorithms, and bootstrap analyses were performed with 1,000 replicates.

Lecithinase assay.
Lecithinase (PlcB) activity was detected by the formation of phospholipolysis opacification zones around colonies growing on egg yolk (Difco, Detroit, MI) brain heart infusion (BBL, Baltimore, MD) agar overlays. This method has been validated (R. E. Duvall and A. D. Hitchins, presented at the AOAC International Annual Meeting, St. Louis, MO, 19 to 23 September 2004) with 82 food and clinical isolates of known species of Listeria, including some Hly-negative L. seeligeri (39).

Nucleotide sequence accession numbers.
DNA sequences from this study were deposited in GenBank under accession numbers EF071911, EF0719112, EF093136, and EF110559 to EF110565.


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RESULTS AND DISCUSSION
 
Multiple genome typing array.
PRL/NW 15B95 and J1-023 chromosomal DNA was hybridized with a "Listeria biodiversity" DNA array (see Table S1 in the supplemental material) to see whether these strains clustered hierarchically more with L. monocytogenes strains or with L. innocua strains. The isolates' DNA clustered with all 15 different L. innocua strains tested, showing that it is most similar to L. innocua strains (Fig. 1). PRL/NW 15B95 was most closely related to L. innocua serovar 6a (formerly designated 4f), and this is consistent with the previous finding of O-antigenic factor 5 and the absence of factors 6, 7, 8, and 9 (19). PRL/NW 15B95 and J1-023 did not cluster with any of the L. monocytogenes strains tested, which included all its serovars except serovar 7, and neither did they cluster with the representatives of all other Listeria species.


Figure 1
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  		FIG. 1. Cluster analysis of DNA macroarray data. Red and black denote the presence and absence of genes, respectively. The dendrogram shows the estimates of the genomic relationships of the different strains obtained by hierarchical cluster analysis with the J-Express program. The strains depicted are listed in Table 1. Abbreviations: L. m, L. monocytogenes; L. wel, L. welshimeri; L. seel, L. seeligeri; L. iva/iva, L. ivanovii subsp. ivanovii; L. iva/lon, L. ivanovii subsp. londiniensis; L. ina, L. innocua; L. gra, L. grayi.

LIPI-1 virulence genes.
Hybridization with sequences corresponding to the LIPI-1 virulence genes, prfA, plcA, hly, mpl, actA, and plcB, was positive for PRL/NW 15B95 but negative for 15 other strains of L. innocua (Table 1), as expected from previous results (19). Previously, we showed by specific PCRs that strain PRL/NW 15B95 and the Cornell isolate J1-023 were hly positive and that they phenotypically produced hemolysin. Since this gene is actively expressed, the strains can be reasonably assumed to contain their intact expression-controlling gene, prfA (positive regulatory factor), which is part of the six-gene cluster, and to be able to produce its biologically active product, PrfA (19).

In our earlier publication, we demonstrated by overlapping PCR with primers derived from sequences of the LIPI-1 genes in L. monocytogenes and hybridization of resulting amplicons with gene-specific oligoprobes on a DNA microarray that all the LIPI-1 genes are present in the atypical L. innocua strains. In addition, PCR-based data suggested the same organization of the genes in the virulence gene cluster as in classical L. monocytogenes. Nevertheless, the multiple sequence alignment and phylogenetic analysis of the LIPI-1 flanking regions (gcaD-prs and orfB-orfA-ldh-ctc) demonstrated their L. innocua-specific origin. The phylogenetic trees, constructed using the minimum evolution algorithm, are shown in Fig. 2A and B. These trees clearly demonstrate the presence of a well-defined L. innocua cluster containing the atypical L. innocua strains. Additionally, the phylogenetic analysis of the housekeeping phosphoribosyl-pyrophosphate synthetase (prs) genes, which are flanking genes at the 5' ends of LIPI-1, of the atypical L. innocua strains and other Listeria species available in GenBank showed that the prs genes from these atypical strains were of L. innocua origin (Fig. 3).


Figure 2
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  		FIG. 2. Dendrograms showing the phylogenetic relationships among classical L. innocua strains and the atypical hemolytic L. innocua strains based on the nucleotide sequence data of the entire gcaD-prs (A) and orfB-orfA-ldh-ctc (B) regions. These regions flank LIPI-1 in the L. monocytogenes (L. monocyt) and the atypical hemolytic L. innocua strain genomes. Two L. monocytogenes strains and an L. welshimeri strain are included as references. These trees were constructed by the minimum evolution method (Kimura two-parameter model) in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 1,000 replications. The bar scales show substitutions per site.


Figure 3
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  		FIG. 3. Dendrogram showing the phylogenetic relationships among Listeria species and the atypical hemolytic L. innocua strains based on the nucleotide sequence data of the entire phosphoribosyl pyrophosphate synthetase (prs) gene. This tree was constructed by the minimum evolution method (Kimura two-parameter model) in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 1,000 replications. GenBank accession numbers are indicated for each strain used in creating this dendrogram. The bar scale shows substitutions per site L. monocyt, L. monocytogenes.

During the current study, the whole LIPI-1 region (gcaD-prs-prfA-plcA-hly-mpl-actA-plcB-orfX-orfZ-orfB-orfA-ldh-ctc) from each of the atypical strains PRL/NW 15B95 and J1-023 was sequenced. Direct sequencing and phylogenetic analysis of the whole LIPI-1 region from the two atypical hemolytic L. innocua strains were carried out to identify their phylogenetic position relative to other published LIPI-1 region sequences of classical L. monocytogenes with different serotypes and lineages (40). The prfA virulence gene cluster sequences from 113 L. monocytogenes strains, available in GenBank, were used for this phylogenetic analysis. The results clearly show that the LIPI-1 regions from these L. innocua strains are closely related to those of L. monocytogenes; however, they form a distinctive branch, regardless of the phylogenetic methods used, and do not match any of the classical L. monocytogenes lineages (Fig. 4). The LIPI-1 nucleotide homology was 96.8% between the analyzed atypical L. innocua strains and 84.8 to 93% among all 113 L. monocytogenes strains. That these two atypical L. innocua strains form a group that is phylogenetically distant from all presently known L. monocytogenes serotypes and lineages by LIPI-1 analysis renders less likely the possible acquisition of this locus by horizontal transmission from an existing L. monocytogenes strain in recent times.


Figure 4
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  		FIG. 4. Dendrogram showing the phylogenetic relationships among L. monocytogenes (L. monocyt) strains and the atypical hemolytic L. innocua strains based on the nucleotide sequence data of the entire LIPI-1 cluster. This tree was constructed by the minimum evolution method (Kimura two-parameter model) in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 1,000 replications. GenBank accession numbers are indicated for each strain used in creating this dendrogram. The bar scale shows substitutions per site.

The possibility of horizontal transfer of the LIPI-1 island to these atypical L. innocua was recognized previously (19). However, the current experiments with comparative genomic Listeria DNA array hybridization, the complete sequencing of the LIPI-1 clusters from these strains, and the presence of the inlAB cluster, albeit modified, make it less likely that PRL/NW 15B95 and J1-023 are simply L. innocua strains that have been altered recently by horizontal gene transfer, but suggest that the atypical isolates represent a stage in the evolution of L. innocua from a common ancestor of L. monocytogenes and L. innocua, a process that has been postulated by others (13).

Non-LIPI-1 virulence genes (inlA, inlB, inlC, inlG, inlH, inlE, inlD, inlF, inlC2, hpt, and bsh).
Macroarray hybridization results confirmed that the sequence of the non-LIPI-1 virulence gene inlB was absent from L. innocua PRL/NW 15B95 and J1-023, as expected from previous results (19). PRL/NW 15B95 and J1-023 and all the control L. innocua strains were confirmed to be negative for the gene by using two different sets of PCR primers in two studies covering about 75% of inlB. Unexpectedly from the previous results, macroarray hybridization with sequences of the non-LIPI-1 virulence genes inlA and inlC was positive for PRL/NW 15B95 and negative for the control L. innocua strains (CLIP11262, CLIP74915, CLIP74916, CLIP94201, and CM) except for one control L. innocua strain, CLIP88566, which was positive for inlC. The apparent presence of "inlC" in PRL/NW 15B95 was confirmed with PCR primers inlC3F and inlC3R (see Table S2 in the supplemental material). The presence of an inlC sequence in a typical L. innocua strain like CLIP88566 is so unusual that it is reasonable to ascribe this to the presence of a paralogue sequence rather than an inlC sequence per se. It is possible that the same explanation applies to the atypical L. innocua strain PRL/NW 15B95. Sequencing analysis of these amplified "inlC" products from both PRL/NW 15B95 and CLIP88566 indicated non-inlC derivations consistent with the paralogue explanation.

In addition, PCR experiments showed that other L. monocytogenes-specific internalins (inlG, inlH, inlE, inlD, inlF, and inlC2) were absent in L. innocua strain PRL/NW 15B95 and the PRL/NW 15B95-like strain, J1-023. Furthermore, the L. monocytogenes-specific PrfA-regulated non-LIPI-1 gene, hpt, which codes for a hexose phosphate transport protein, was found to be absent in PRL/NW 15B95 in this study. Hpt is a homolog of the mammalian hexose phosphate translocase that was shown to be important for the cytosolic replication of L. monocytogenes and for in vivo sensitivity to fosfomycin (8, 34). The bile salt hydrolase gene, bsh, which is important for intestinal and hepatic colonization, was also absent in the PRL/NW 15B95 strain (11).

Presence of the inlAB operon in the atypical hemolytic L. innocua strain.
In this study, different primer sets (see Table S2 in the supplemental material) were used to detect the inlA and inlB genes. The primer sets inlAF/T7-inlAR and inlAF/inlAR that were used previously did not detect, respectively, the whole inlA gene or part of it, but the primer set inlAF1/inlAR1 used in this study did amplify a fragment of 516 bp. Finally, using primers covering 85% of the complete inlA sequence (see Table S2 in the supplemental material) the complete inlA genes of L. innocua PRL/NW 15B95 and J1-023 were amplified. Sequence analysis of the two L. innocua inlA genes revealed an average nucleotide similarity of 96.7% for inlA of PRL/NW 15B95 and 94.3% for inlA of J1-023 to the different L. monocytogenes inlA sequences present in the GenBank database (Table 2). The InlA gene encodes a major virulence factor of L. monocytogenes. It codes for the internalin protein that allows the invasion of L. monocytogenes into different nonphagocytic cells (4, 13, 35, 38). For further analysis, a phylogenetic study of the inlA genes from these atypical hemolytic L. innocua strains, PRL/NW 15B95 and J1-023, was carried out by comparing them with the complete sequences of 19 different L. monocytogenes inlA sequences currently available in GenBank to identify the most-closely related L. monocytogenes inlA sequence. The average percentage of similarity among the complete amino acid sequences deduced from the inlA genes in all the Listeria strains examined was 97.6% (Table 2). The data show that the inlA genes of the atypical L. innocua are very similar to the L. monocytogenes inlA genes. However, it is not possible to state definitively that they are most closely related to particular serotypes, e.g., the 4a and c group, to which L. innocua was hypothesized to be most closely related (10). The conceptually translated InlA proteins from the PRL/NW 15B95 and J1-023 strains are 94.7% similar to each other and have 799 amino acids each. However, in contrast to currently available GenBank InlA sequences, the InlA of PRL/NW 15B95 contains strain-specific and unique amino acid substitutions, as does the InlA of J1-023 (Table 3).


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  		TABLE 2. Percent identity among nucleotides (below diagonal formed by boldface entries) and percent similarity among deduced amino acids (above diagonal) for complete sequences of inlA genes between different L. monocytogenes and atypically hemolytic L. innocua strains


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  		TABLE 3. Amino acid substitutions in the InlA proteins of PRL/NW 15B95 and J1-023

However, because these amino acid substitutions in the InlA proteins of PRL/NW 15B95 and J1-023 strains are unique or strain specific, we do not know how they may affect InlA function and interaction with their human receptor, E-cadherin. No information was found in the literature about experiments on saturation mutagenesis of InlA and the functionality of such mutants. The transcription activator PrfA is an essential, but not the only, factor which regulates the transcription of the LIPI-1 virulence genes (22, 35). It also activates the expression of the inlA-inlB operon genes which are located outside the LIPI-1 gene cluster (9). Recent studies have shown that the PrfA DNA-binding domain, which has a typical helix-turn-helix structure, interacts with a conserved 14-bp-or-less DNA sequence (the "PrfA box") having dyadic symmetry. The PrfA box occurs in front of all known PrfA-dependent genes and operons (9, 41), being centered at about position –40 from the transcriptional start sites. Indeed, recent studies have shown that this site is essential for the binding of PrfA to DNA fragments derived from the hly and inlA promoters (9, 22, 35, 41). Sequencing analysis of the upstream 5' regions (promoter regions) of the inlA genes in PRL/NW 15B95 and J1-023 showed the presence of coding for intact PrfA boxes (ATAACATAAGTTAA) at 429 and 419 bp upstream, respectively, of the GTG start codon for inlA transcription. Nevertheless, strain PRL/NW 15B95 does not express immunologically detectable InlA (S. Pilgrim and W. Goebel, personal communication).

Phylogenetic analysis of the inlA genes from these two atypical hemolytic L. innocua strains was carried out to identify their positions on the inlA tree by comparison with the complete inlA sequences of the 19 different strains of L. monocytogenes currently available in GenBank that have known serovars and lineages (Fig. 5). The results, which were the same regardless of the construction method used, clearly show that the inlA genes from these two L. innocua strains were most closely phylogenetically related to those of the lineage 3 L. monocytogenes strains (Fig. 5). The division of L. monocytogenes strains into different lineages, which correlate with serovars, is based on many different studies using different typing methods (3, 5, 14, 28) as well as on multilocus sequence typing based on the polymorphisms of several genes (23, 25, 30, 32). Interestingly, even on this single inlA gene polymorphism tree, there is fairly tight clustering of strains according to lineages, especially for the lineage 1 and 2 isolates. The atypical L. innocua strains are clearly related, although quite distantly, to the much more loosely linked lineage 3 cluster.


Figure 5
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  		FIG. 5. Dendrogram showing the phylogenetic relationships among L. monocytogenes strains and the atypical hemolytic L. innocua strains based on the nucleotide sequence data of the entire inlA gene. This tree was constructed by the minimum evolution method (Kimura two-parameter model) in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 1,000 replications. GenBank accession numbers are indicated for each strain used in creating this dendrogram. The bar scale shows substitutions per site. The lineage-serovar associations are as follows: lineage 1, serovars 1/2b, 4b, and 3b; lineage 2, serovars 1/2a, 1/2c, 3a, and 3c; and lineage 3, serovars 4a, 4c, and some 4b (6a).

Next, attention was focused on the flanking regions and the juxtapositional extragenic regions of inlA. An inverse PCR protocol (26) was applied, which involved the isolation of genomic DNA, digestion with restriction enzymes that cut once within the inlA gene but numerous times within the genome, and ligation to circularize all linear genomic fragments, followed by PCR using two outward-facing, inlA-specific primers. All genomic DNA samples were MspI, NheI, or PstI digested and processed through the inverse PCR protocol. All PCR-positive products were purified, sequenced, and compared to the published L. monocytogenes, L. innocua, and L. welshimeri genomes (L. monocytogenes serovar 4b, strain F2365, GenBank accession no. NC_002973; L. innocua serovar 6a, strain CLIP11262, GenBank accession no. NC_003212; L. monocytogenes serovar 1/2a, strain EGD-e, GenBank accession no. NC_003210; L. monocytogenes serovar 1/2a, strain F6854, GenBank accession no. NZ_AADQ00000000; L. monocytogenes serovar 4b, strain H7858, GenBank accession no. NZ_AADR00000000; and L. welshimeri serovar 6b, strain SLCC5334, GenBank accession no. NC_008555). The sequencing and inlA site mapping results are summarized in Fig. 6. By a combination of inverse and overlapping PCRs, the inlA flanks (approximately 2 kb upstream and more than 25 kb downstream) were amplified and sequenced. Surprisingly, the Basic Local Alignment Search Tool (BLAST) analysis and the nucleotide substitution patterns of the inlA flanks revealed that these L. innocua inlA genes were embedded within genes similar to genes found in genomes of L. monocytogenes serotype 4b isolates at the upstream 5'-end region and within genes shared in common by L. innocua and L. monocytogenes serovar 1/2a, strain EGD-e, at the downstream 3'-end region.


Figure 6
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  		FIG. 6. Genetic organization of the inlAB operon and its flanking genes in the atypical strains and in the various L. monocytogenes strains with known genomes. ORFs shown in the same colors are orthologues. Arrows indicate the inlA-inlB genes and their flanking genes (open reading frames) as well as their transcription directions. The L. innocua CLIP11262 region that has an organization similar to that of the inlAB operon in L. monocytogenes EGD-e and the atypical L. innocua strain is also presented. An asterisk indicates a gene inactivation in CLIP80489 and F2365 due to a frameshift mutation. The hypothetical gene upstream of inlA is only putatively translated and is inactive although its sequence is similar to that of an intact gene which is present in L. innocua. +, these two putative lin0456-like ORFs are partially similar to lin0456 (encoding a hypothetical protein) of L. innocua CLIP11262 (NP_469800), having 76% and 71% similarity. The red inlB blocks in PRL/NW 15B95 and J1-023 indicate possible traces of inlB genes that are represented by approximately 75-nucleotide-long sequences at the beginning of the inlA-lin0457 spacer, with 89 to 91% similarity to the inlB sequences of L. monocytogenes available in GenBank.

Upstream of inlA.
The flanking regions of PRL/NW 15B95 and J1-023 are distinct from the inlA upstream region of L. monocytogenes serovar 1/2a, strain EGD-e. On the left (upstream 5'-end region), three putative open reading frames (ORFs) were found; these ORFs are conceptually transcribed in the same direction as inlA and were found to be homologous to hypothetical proteins LMOh7858_0493 (GenBank accession no. ZP_00230339), LMOh7858_0494 (GenBank accession no. ZP_00230345), and LMOh7858_0495 (GenBank accession no. ZP_00230346) of L. monocytogenes serovar 4b, strain H7858, with 98%, 100%, and 83% similarity, respectively. The result for the corresponding upstream 5'-end region in the PRL/NW 15B95-like strain J1-023 was different, and two ORFs were found; these ORFs are conceptually transcribed in the same direction as inlA and have been found to be homologous to hypothetical protein LMOf2365_0466 (GenBank accession no. YP_013073) and phycobilisome lyase Huntington's disease-like repeat domain protein LMOf2365_0464 (GenBank accession no. YP_013075) of the L. monocytogenes serovar 4b strain F2365, with 93% and 92% similarity, respectively. Therefore, the inlA genes of PRL/NW 15B95 and J1-023 have different near-neighborhood upstream 5'-end regions. The upstream 5' region of inlA in PRL/NW15B95 was shown to be similar to the genome organization in L. monocytogenes serotype 4b, strain H7858 (NZ_AADR00000000), a meat isolate sequenced by the Institute for Genomic Research, Rockville, MD (24), whereas the upstream 5' region of inlA in strain J1-023 was comparable to the genome organization in L. monocytogenes serotype 4b, strain F2365 (NC_002973). Despite having the same serotype, these two L. monocytogenes strains show differences in gene organization upstream of their inlA genes (13, 24). Interestingly, the upstream regions of inlA in both L. innocua strains were not comparable to the upstream 5'-region organization of inlA in L. monocytogenes serotype 1/2a, strain EGD (NC_003210), which has upstream 5'-region organization that is similar to the same region in L. innocua CLIP11262, except that L. innocua CLIP11262 has no inlAB cluster inserted in the region. However, so far the analysis has not reached an L. innocua-specific gene in the upstream neighborhood region of inlA in either L. innocua strain. As shown in Fig. 6, the wapA-like gene is present in several L. monocytogenes strains as the flanking gene of the inlAB cluster in the upstream region. The gene is also present in normal L. innocua strains (see Table S1 in the supplemental material). The wapA-like gene was found in PRL/NW 15B95 and J1-023 by macroarray hybridization and by using PCR with gene-specific primers. However, the use of PCR (with two different polymerases, HotStar Taq and a highly processive enzyme, AccuPrime Pfx) with forward wapA-specific primers plus reverse inlA cluster-specific primers failed to amplify the intergenic region between the wapA-like and inlA genes, showing that it is not the closest gene to the inlA cluster in these isolates. Similarly, in L. monocytogenes F2365 and H7858, the wapA-like genes are also distant from the inlA genes, about 10.3 and 17.4 kb, respectively.

Downstream of inlA.
The downstream regions of inlA in the Cornell strain, J1-023, and in PRL/NW 15B95 were found to be similar to that in the CLIP11262 reference strain of L. innocua (NC_003212). We then walked more than 25 kb in the downstream 3' region of inlA in both strain PRL/NW 15B95 and strain J1-023. In both strains, the 3' region downstream of inlA includes a cluster of ORFs similar to lin0457 through lin0473, thus conservatively reflecting the organization of L. innocua CLIP11262. This region is also quite conserved in the L. monocytogenes strains, particularly in L. monocytogenes serotype 1/2a, strain EGD (NC_003210). However, the first gene, lin0457, encoding a putative peptidoglycan-bound protein (LPXTG motif), of the ORF clusters in both PRL/NW 15B95 and J1-023 is separated from inlA by spacer regions that differ in the two strains.

The inlA-lin0457 spacer region in PRL/NW 15B95 was found to be 997 bp long and does not have putative ORFs. A BLASTN search of this noncoding spacer in PRL/NW 15B95 and J1-023 showed that an approximately 75-nucleotide-long sequence at the beginning of the spacer was partially (89 to 91%) similar to inlB sequences of L. monocytogenes available in GenBank. Hypothetically, we assume that this intergenic region is most likely a relict sequence of a functional inlB, rather than a precursor sequence of a functional inlB.

The inlA-lin0457 spacer region in J1-023 was found to be 1,102 bp long and to have two putative ORFs which have 76% and 71% similarity to lin0456, encoding a hypothetical protein, of L. innocua CLIP1262 (NP_469800). However, the BLASTN search did not reveal any homology of the noncoding parts of the inlA-lin0457 spacer regions of either strain with any sequences in other bacteria. Nor did it provide evidence for any transposon insertion site or transposon-related repeat structures in the region. The inlA gene operon organization in both the strains is unique and, in contrast to the L. monocytogenes EGD inlAB operon, they probably contained only traces of inlB. No indication of horizontal transfer of inlA (or the inlAB operon) into the L. innocua genome or loss of inlB from the operon before or after putative horizontal transfer was found. The possibility that there was vertical transmission of inlA (or inlAB) into PRL/NW 15B95 and J1-023 from the genus Listeria ancestor thus seems increasingly more probable.

Conclusions.
Doumith et al. (10) have postulated that L. innocua devolved from L. monocytogenes, the respective serovars 4a and 6a having a common ancestor. The hemolytic L. innocua isolate PRL/NW 15B95 appears to provide tangible evidence in support of their hypothesis. Thus, it can be considered as a link in the devolutionary chain (33). It still has the LIPI-1 virulence cluster but is apparently starting to lose the inlAB operon: inlA, while present, is accumulating point mutations and only a small residue of inlB remains. Nevertheless, a polyclonal antiserum raised against L. monocytogenes EGD-e did not reveal the InlA of PRL/NW 19B95 (S. Pilgrim, personal communication). In contrast, at least some of the LIPI-1 cluster genes are functional. Thus, PlcA and Hly activities are present (19). However, in this study, no PlcB (lecithinase) activity was detectable with the egg yolk test in either PRL/NW 15B95 or J1-023. Neither was it observed in the two other reported atypical hemolytic isolates of L. innocua (19). The Mpl activity necessary to convert prolecithinase to lecithinase has not been tested. The Hly and PlcA activities implied that PrfA, the expression regulator for LIPI-1 genes, is active (19). The expression of inlAB operon genes is partially dependent on functional PrfA (35), and indeed, it has been shown that an imported plasmid-encoded inlA can express an anchored InlA product in PRL/NW 15B95 (S. Pilgrim, personal communication). In cell culture studies, up-regulation of actA mRNA has also been demonstrated and this and the production of spreading foci (29) indicate that PRL/NW 15B95 produces a functional ActA. Since ActA is processed by Mpl (20, 31), ActA functionality implies that the mpl of PRL/NW 15B95 is expressed in a functional Mpl and that, therefore, failure to detect PlcB activity (see above) is not due to lack of an active Mpl gene. Thus, the LIPI-1 gene cluster is largely functional compared to the inlAB operon.

Over the past two decades, numerous studies have provided insight into the mechanisms of L. monocytogenes cellular pathogenesis through the identification and characterization of the virulence genes located in the major virulence gene cluster (i.e., prfA, plcA, hly, mpl, actA, and plcB). The prfA product regulates the expression of the other genes within the cluster, as well as the expression of additional virulence genes elsewhere on the chromosome (e.g., inlA, inlB, inlC, hpt, and bsh). A complex of functionally active virulence genes is required for host cell invasion, vacuolar escape, and cell-to-cell spreading (38). In contrast to our knowledge of the mechanisms used by L. monocytogenes to survive during the intracellular invasion and to persist in the environment, such knowledge is absent for ancestral strains, like PRL/NW 15B95, which have nevertheless apparently survived a long time. However, the possession of the LIPI-1 virulence cluster and part of the inlAB operon by this L. innocua strain may explain its apparent ability to intracellularly replicate in mammalian cells, as evidenced by the production of spreading foci (S. Pilgrim, personal communication). These considerations are especially interesting because a case of fatal bacteremia was recently reported to be caused by an L. innocua strain, a species not described in association with human disease before (27). Natural heterogeneity in an avirulent Listeria species, reflected in its ability to acquire or retain virulence-associated genes, may permit some of its strains to be causative agents of disease, especially in immunocompromised mammals. Also, such strains may constitute a reservoir of transferable virulence genes.


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ACKNOWLEDGMENTS
 
We thank Sabine Pilgrim from W. Goebel's laboratory, Department of Microbiology, University of Würzburg Biocenter, Germany, for sharing results with us and Robert E. Duvall of the Center for Food Safety and Applied Nutrition, Food and Drug Administration, for performing the lecithinase assay.

We thank Vladimir E. Chizhikov (Center for Biologics Evaluation and Research, Food and Drug Administration) for support of this study. The Institut Pasteur provided C.B. with financial support (GPH 9).


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FOOTNOTES
 
* Corresponding author. Mailing address: FDA, 5100 Paint Branch Pkwy., College Park, MD 20740. Phone: (301) 436-1649. Fax: (301) 436-1649. E-mail: Anthony.Hitchins{at}fda.hhs.gov. Back

{triangledown} Published ahead of print on 12 January 2007. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, March 2007, p. 1928-1939, Vol. 73, No. 6
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