Novel internalin P homologs in Listeria

Abstract

Listeria monocytogenes (Lm) is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular lifecycle of Lm and facilitate cell-to-cell spread, allowing it to occupy multiple niches within the host and cross-protective barriers, including the placenta. One family of virulence factors, internalins, contributes to Lm pathogenicity by inducing specific uptake and conferring tissue tropism. Over 25 internalins have been identified thus far, but only a few have been extensively studied. Internalins contain leucine-rich repeat (LRR) domains that enable protein-protein interactions, allowing Lm to bind host proteins. Notably, other Listeria species express internalins but cannot colonize human hosts, prompting questions regarding the evolution of internalins within the genus Listeria. Internalin P (InlP) promotes placental colonization through interaction with the host protein afadin. Although prior studies of InlP have begun to elucidate its role in Lm pathogenesis, there remains a lack of information regarding homologs in other Listeria species. Here, we have used a computational evolutionary approach to identify InlP homologs in additional Listeria species. We found that Listeria ivanovii londoniensis (Liv) and Listeria seeligeri (Ls) encode InlP homologs. We also found InlP-like homologs in Listeria innocua and the recently identified species Listeria costaricensis. All newly identified homologs lack the full-length LRR6 and LRR7 domains found in Lm's InlP. These findings are informative regarding the evolution of one key Lm virulence factor, InlP, and serve as a springboard for future evolutionary studies of Lm pathogenesis as well as mechanistic studies of Listeria internalins.

Keywords: Listeria; inlp; internalin; molecular evolution; phylogeny; placenta.

Fig. 1.

Phylogeny and structure models of InlP and representative Homologs. A phylogenetic tree was generated using the amino acid sequences of InlP homologs in L. monocytogenes (blue), L. ivanovii londoniensis (purple) and L. seeligeri (green). Three-dimensional models were generated using SWISS-MODEL with the crystal structure for InlP _Lm (PDB: 5hl3) as a template and then visualized using ChimeraX. Multiple sequence alignments were generated using MolEvolvR and illustrate the complete LRR6 and LRR7 insertion present in InlP _Lm (inserted motif is highlighted in yellow in the 3D model in a backdrop of a blue protein structure model; also indicated with the arrow). The legend shows the colours of the amino acid residues indicated in the multiple sequence alignment. The height of the MSA (for each of the five sequences) has been increased to show the colours more distinctly, and to highlight the missing motif indicated by the arrow.

Fig. 2.

Phylogeny and domain architectures of putative internalin P Homologs. A multiple sequence alignment and phylogenetic tree were generated using the amino acid sequences of putative InlP homologs identified using MolEvolvR and five Listeria InlP starting points (see the Methods section). The phylogeny of InlP-like proteins (with branch lengths marked using d _N/d _S ratio with the ggtree package) has been overlaid with their domain architectures (generated using MolEvolvR, showing only Pfam domain architectures). The two legends show the colours of the amino acids indicated in the multiple sequence alignment (corresponding to the MSA, right panel) and the Pfam domain annotations (corresponding to the domain architecture, left panel). The arrow lengths and the overall coloured MSA segments correspond to the relative lengths of each of the InlP-like proteins.

Fig. 3.

Percentage similarity and identity of internalin P Homologs in representative Listeria species. Percentage similarity and percentage identity were calculated for the amino acid sequences for representative internalin P homologs from five Listeria species, L. monocytogenes, L. ivanovii, L. seeligeri, L. costaricensis and L. innocua . Matrix showing similarity and identity values was generated using MatGAT2.01 with the BLOSUM 62 matrix and default options were selected.

Fig. 4.

Genomic context of newly identified internalin P gene homologs. Genes homologous to the L. monocytogenes inlP (a) were identified in various other Listeria species. Gene order was maintained in L. monocytogenes , L. seeligeri and L. ivanovii londoniensis . Notably, L. seeligeri encodes three copies of the inlP gene. All homologs categorized as ‘true’ homologs were flanked upstream by an amino acid permease gene (blue) and downstream by an NADPH dehydrogenase gene (orange). L. ivanovii ivanovii contains a pseudogene (purple) and an uncharacterized gene encoding a hypothetical protein (red) in this region. Putative homologous genes in L. costaricensis and L. innocua did not mirror the genomic neighbourhoods seen in the other Listeria species. All inlP homologs are represented in green. Genomic context was determined using RefSeq genomic records and the BioCyc genome browsers for each species (see the Methods section).

Fig. 5.

Multiple sequence alignment of internalin P homologs. Amino acid sequences for identified internalin P homologs in L. monocytogenes , L. ivanovii londoniensis, and L. seeligeri were aligned using Kalign and visualized using Jalview. Below the alignment is the consensus sequence for the four homologs. The red box indicates the insertion present only in the InlP _Lm homolog. The L. monocytogenes homolog is used as the reference InlP protein (for residue numbering).