Hyperinvasiveness of Listeria monocytogenes sequence type 1 is independent of lineage I‐specific genes encoding internalin‐like proteins

Abstract Listeriosis is a severe disease caused by the opportunistic bacterial pathogen Listeria monocytogenes (L. monocytogenes). Previous studies indicate that of the four phylogenetical lineages known, lineage I strains are significantly more prevalent in clinical infections than in the environment. Among lineage 1, sequence type (ST1) belongs to the most frequent genotypes in clinical infections and behaves hyperinvasive in experimental in vitro infections compared to lineage II strains suggesting that yet uncharacterized virulence genes contribute to high virulence of certain lineage I strains. This study investigated the effect of four specific lineage I genes encoding surface proteins with internalin‐like structures on cellular infection. CNS derived cell lines (fetal bovine brain cells, human microglia cells) and non‐CNS derived cell lines (bovine macrophage cells, human adenocarcinoma cells) that represent the various target cells of L. monocytogenes were infected with the parental ST1 strain and deletion mutants of the four genes. Despite their association with lineage I, deletion of the four genes investigated did not dampen the hyperinvasiveness of the ST1 strain. Similarly, these genes did not contribute to the intracellular survival and intercellular spread of L. monocytogenes ST1, indicating that these genes may have other functions, either during the infection process or outside the host.


| INTRODUC TI ON
Listeria (L.) monocytogenes is an opportunistic bacterial pathogen, which is well adapted to both extracellular dwelling in outdoor and food factory environments (Carpentier & Cerf, 2011;Vivant, Garmyn, & Piveteau, 2013) and intracellular survival when ingested by a host . In the host, L. monocytogenes may pass the intestinal tract without causing disease or may cause selflimiting gastroenteritis (Gahan & Hill, 2014). However, when host barriers are crossed by invasion of cells, L. monocytogenes causes listeriosis, a potentially life-threatening infection associated with septicemia, abortions, and neurological disease (Disson & Lecuit, 2012;Oevermann, Zurbriggen, & Vandevelde, 2010;Siegman-Igra et al., 2002). Despite its low incidence rate, listeriosis is considered as a major public health threat due to the high fatality rate (Maertens de Noordhout et al., 2014;Swaminathan & Gerner-Smidt, 2007).
Studies indicate that strain diversity in L. monocytogenes is relevant in the context of infection and environmental survival. Recent epidemiological studies have shown that the relative prevalence of L. monocytogenes strains differs between clinical infection and environment (Dreyer et al., 2016;Maury et al., 2016;Orsi, Bakker, & Wiedmann, 2011). Of the four phylogenetical lineages known, lineage I strains are significantly more prevalent in clinical infection of both humans and ruminants than in the environment. This is particularly true for strains from specific sequence types (ST, as determined by multilocus sequence typing (MLST)), namely ST1 and 4. TA B L E 1 Primers used for cloning of pMAD and pHoss1 deletion plasmids and PCR analysis in experimental in vivo and in vitro infections (Dreyer et al., 2016;Guldimann et al., 2015;Maury et al., 2016).
Previous studies from our group have shown that a ST1 strain from lineage I isolated from bovine rhombencephalitis (JF5203) behaves hyperinvasive compared to lineage II strains including EGD-e (Dreyer et al., 2016;Rupp, Bartschi, Frey, & Oevermann, 2017) suggesting that, besides to the well-known virulence genes, yet uncharacterized virulence genes may contribute to cellular invasion and virulence of certain strains. Therefore, the aim of this study was to assess the impact of lineage I-specific virulence candidate genes on the cellular infection process. Emphasis was put on four genes of lineage I that encode surface proteins with internalin-like structure. To investigate their effect on cellular infection, CNS-derived cell lines (fetal bovine brain cells, human microglia cells) and non-CNS-derived cell lines (bovine macrophage cells, human adenocarcinoma cells) that represent the various target cells of L. monocytogenes were infected with a ST1 parental strain and deletion mutants derived thereof. their internalin-like protein structure (Appendix 1). All candidate genes specify for potential proteins with a leucine-rich repeat (LRR) binding motif involved in host receptor recognition and interaction, which is found in internalin proteins (Bierne & Cossart, 2007

| Bacterial strains
Listeria monocytogenes strain JF5203 (NCBI Reference Sequence: NZ_ LT985474.1; https://www.ncbi.nlm.nih.gov/nuccore/NZ_LT985474.1) F I G U R E 1 Expression of candidate genes in Listeria monocytogenes parental strain (LMJF5203) and deletion mutants. RNA was extracted from L. monocytogenes strains grown overnight in BHI-broth, and expression of candidate and control genes (gyrA and sigB) was assessed by reverse-transcription PCR. All genes are expressed in the parental strain (a). As expected, the candidate genes are not expressed in the respective deletion mutants (b) g y r A 0 0 3 8 8 0 2 7 6 7 0 1 2 9 1 0 2 5 3 7 g y r A s i g B 500 bp 200 bp 700 bp g y r A g y r A s i g B g y r A g y r A s i g B g y r A g y r A s i g B g y r A g y r A s i g B 0 0 3 8 8 0 2 7 6 7 0 1 2 9 1 0 2 5 3 7 belonging to phylogenetic lineage I, clonal complex 1, sequence type 1, isolated from a rhombencephalitis case in cattle was used as parental strain for cell invasion experiments and generation of the deletion mutants. Listeria monocytogenes strain EGD-e, belonging to lineage II, clonal complex 9, sequence type 35 was used as a reference strain in order to confirm hyperinvasiveness of our parental strain. The deletion mutants LMJF5203_Δ00388 and LMJF5203_Δ02767 were generated using the pHoss1 plasmid, and LMJF5203_Δ01291 and LMJF5203_ Δ02537 using the pMAD plasmid as previously described ( were inserted into the SalI-and XmaI-digested pMAD and pHoss1 plasmids, respectively, by ligation with T4 ligase to create pMad_ Δ01291, pMad_Δ02537, pHOSS_Δ00388, and pHOSS_Δ02767. The parental strain JF5203 was transformed with these deletion plasmids as described (Abdelhamed et al., 2015;Arnaud et al., 2004). Deletion of the genes of interest was confirmed by colony PCR using the flanking region primer pairs as described above and additional primers binding to the ORF regions of the deleted genes (Table 1).

| Whole genome sequencing of deletion mutants
Listeria monocytogenes mutants were grown overnight at 37°C in

| Mammalian cell lines
The bovine macrophage cell line (BoMac), the human microglia cell line (HMC-3), and the human epithelial colorectal adenocarcinoma cell line (Caco-2) were grown in Dulbecco's mod-

| Axenic growth in broth
Single colonies of mutant strains and the parental strain were inoculated into BHI-broth and grown overnight. The following day, fresh broth was inoculated with overnight culture at an OD600 of 0.05, and the OD600 was measured every 30 min for 7 hr. Bacterial growth was quantified in three independent experiments. Growth curves were fitted using a logarithmic scale in base 10, and generation time was calculated.

| Gentamicin protection assay
Cells were grown to confluency in 24-well plates with DMEM medium supplemented with 10% FCS and without penicillin/strepto-

| Immunofluorescence
For microscopical assessment of the gentamicin protection assay, cells were grown on glass coverslips, which were coated with poly-D-lysine hydrobromide for Caco-2 cells. Coverslips were removed from the 24-wells plates at the time points indicated above and  graphpad.com).

| RE SULTS
This study aimed to investigate the putative involvement of

| Deletions do not affect fitness of L. monocytogenes mutants
When grown in BHI medium, the four deletion mutants generated (LMJF5203_Δ00388, LMJF5203_Δ01291, LMJF5203_Δ02537, and LMJF5203_Δ02767) showed growth curves similar to the parental strain JF5203 indicating that the deletions did not exhibit any defect in extracellular growth and fitness (Figure 2).

F I G U R E 5
Infection of FBBC-1 in the gentamicin exclusion assay in three independent experiments performed in triplicates. FBBC-1 were infected with the indicated strains. At the indicated time points, cells were lysed for CFU counting.
Single CFU data are presented as dots, bars indicate the mean, and error bars indicate the standard error of the mean (SEM). Statistical analysis (nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison) did not reveal any significant difference between deletion mutants (LMJF5203_Δ00388, LMJF5203_ Δ02767 (a), LMJF5203_Δ02537, and LMJF5203_Δ01291 (b)) and parental strain

| D ISCUSS I ON
Listeria monocytogenes has a clonal population structure that is organized in four phylogenetic lineages with lineages I and II being the major lineages (Maury et al., 2016;Nightingale, Windham, & Wiedmann, 2005). Within these two lineages, hyper-and hypovirulent clones have been identified (Orsi et al., 2011). Hypovirulent clones of L. monocytogenes generally belong to lineage II (Jacquet et al., 2004;Maury et al., 2016;McLauchlin, 1990;Orsi et al., 2011), while hypervirulent clones have been predominantly found in lineage I, which is also the most prevalent lineage in animal and human infection (Chenal-Francisque et al., 2011;Jacquet et al., 2004;Kim et al., 2018;Maury et al., 2016;Orsi et al., 2011). Of those, strains belonging to sequence type (ST) 1 are particularly prevalent in CNS infection of ruminants and humans (Dreyer et al., 2016;Maury et al., 2016) and behave hyperinvasive in vitro compared to the reference strain EGD-e and other strains from lineage II (Dreyer et al., 2016;Guldimann et al., 2015;Rupp et al., 2017). Differences in genomic gene content suggest that lineage I strains harbor specific genes that may confer hyperinvasion and hypervirulence resulting in the higher prevalence of these strains in clinical infection (Dreyer et al., 2016;Maury et al., 2016). Therefore, we investigated the impact of In this study, we confirmed that compared to strain EGD-e our ST1 strain JF5203 is hyperinvasive in the different cell systems studied (Rupp et al., 2017) and that invasion is dependent on the infected cell line suggesting cell type-specific interactions between L. monocytogenes and the host cell. Our results show that F I G U R E 6 Infection of HMC-3 in the gentamicin exclusion assay in three independent experiments performed in triplicates. HMC-3 were infected with the indicated strains. At the indicated time points, cells were lysed for CFU counting. Single CFU data are presented as dots, bars indicate the mean and error bars indicate the standard error of the mean (SEM). Statistical analysis (nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison) did not reveal any significant difference between deletion mutants (LMJF5203_Δ00388, LMJF5203_Δ02767, LMJF5203_Δ02537, and LMJF5203_Δ01291) and parental strain   Sabet et al., 2008). Therefore, we cannot fully rule out an impact of these four genes on the infectious process either in other cell types, which were not represented in our study, or in more complex physiological systems including the immune system and host barriers where such membrane proteins may exhibit moonlighting functions on the bacterial cell surface (Copley, 2012). The cause for the association of these four internalin-like genes with lineage I remains to be determined.

ACK N OWLED G EM ENT
This work received financial support from the Swiss National

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.