Listeria monocytogenes Infection of Bat Pipistrellus nathusii Epithelial cells Depends on the Invasion Factors InlA and InlB

L. monocytogenes is a widespread facultative intracellular pathogen. The range of natural hosts that supporting L. monocytogenes persistence in the environment has not been fully established yet. In this study, we were interested in the potential of L. monocytogenes to infect cells of bats, which are being increasingly recognized as a reservoir for microorganisms that are pathogenic to humans and domestic animals. A stable epithelial cell line was developed from the kidneys of Pipistrellus nathusii, a small bat widely distributed across Europe. The wild-type L. monocytogenes strain EGDe infected this cell line with an invasion efficiency of 0.0078 ± 0.0009%. Once it entered bat cells, L. monocytogenes doubled within about 70 min. When L. monocytogenes lacked either of the major invasion factors, InlA and InlB, invasion efficiency decreased by a factor of 10 and 25 respectively (p < 0.000001). The obtained results suggest that bat epithelial cells are susceptible to L. monocytogenes infection and that L. monocytogenes invasion of bat cells depends on the major invasion factors InlA and InlB. These results constitute the first report on in vitro studies of L. monocytogenes infection in bats.


Introduction
Bats are increasingly being recognized as reservoir hosts of microorganisms that can affect humans and domestic animals [1]. In the last several years, attention has primarily focused on highly pathogenic viruses, such as Marburg virus, Nipah virus, Hendra virus, Rabies virus, and coronaviruses hosted by bats [2][3][4][5][6][7]. Much less attention has been paid to pathogenic bacteria in bats. Nevertheless, available information supports the assumption that bats can host representatives of both novel pathogenic genera such as Bartonella, Borrelia, Leptospira, Pseudomonas, and Acinetobacter and well-known pathogens such as Salmonella enterica, Escherichia coli, Yersinia pseudotuberculosis and Listeria monocytogenes [8][9][10][11][12][13][14].
The majority of the listed bacteria were detected in the feces and gastrointestinal tracts of bats. The question of whether bats are just carriers of pathogenic bacteria or are themselves susceptible to infectious diseases needs to be elucidated for each causative agent [1,15]. There are only a few reports of serious diseases in bats caused by human pathogens. Lethal outbreaks caused by Y. pseudotuberculosis in bats has not been conducted to date. Moreover, there are no data on whether both major L. monocytogenes invasion factors-InlA and InlB-are necessary for Listeria invasion of bat cells. In this work, we address these questions and assess a new cell model for studying the mechanisms of pathogenicity of L. monocytogenes.

Primary Cell Cultures and Their Characteristics
Nathusius' pipistrelle (forest bat, Pipistrellus nathusii) is a small bat widely distributed across Europe [56]. In particular, it is a typical inhabitant of forests in the European regions of Russia. The length of this bat (head and body) reaches 60 mm with a wingspan of about 220-250 mm. Its weight is about 12-16 g [57]. Two typical male individuals captured in the Volgograd region, Russia, were used as donors of kidney epithelial cells (Figure 1). nathusii is a small bat from the family of Vespertilionidae bats, with adults reaching a length of 6 cm and body weight of 11 grams; (B) The photo shows a dense coat of medium length; the base of the hairs is dark, the color of the back is reddish-brown, and the abdomen is paler and grayish-yellow; the ear is relatively large, pointed, with tragus elongated with a rounded tip; the thumb is longer than the width of the wrist on the folded wing; (C) The photo shows that the outer upper incisor is slightly shorter than the inner one; (D) The wingspan of an adult is 25 cm. All of this corresponds to this type. Primary culture obtained from trypsinized kidney tissue was represented by morphologically heterogeneous cellular subpopulations with the predominance of epithelial-like cells (Figure 2A). After the first re-plating, cells showed islet growth at 72-and 96-hour post-plating. On the 5th day, islands merged to form a confluent monolayer ( Figure 2B-D). Upon the formation of a confluent monolayer of primary cells, subsequent re-plating steps were performed 2-3 times a week. At a seeding concentration of 1.5 × 10 5 cells/mL, the formation of a confluent monolayer took 72 h, and the proliferation index was 3.3. The cell monolayer showed no signs of cell degeneration, and the cytopathic effect was preserved without changing the medium for 45 days (observation period).
Morphological research of subcultures showed that starting from the fourth passage, the monolayer consisted of epithelial-like cells with a polygonal shape. Their nuclei were oval or ellipsoid, less often spherical, with 1-3 (sometimes more) spherical nucleoli varying in size. The nuclear matrix was uniform. When passaging continued, the cell monolayer retained its characteristic morphology. The cell culture was designated as bat kidney epithelial cells (bKEC). The viability of bKEC cells before cryopreservation was 98%, and recovery after thawing ranged from 79% to 85%. bKEC cells were stable and able to survive in low-temperature storage for more than a year (data not shown).
Cytogenetic analysis was performed to confirm the species identity of cells and to assess the stability of their chromosome set under long-term cultivation conditions. The cytogenetic analysis of 50 metaphase plates of bKEC subcultures at the second and third passages revealed a diploid modal number of 2n = 44 and a total number of chromosomal arms of FN = 50 before and after thawing ( Figure 3). Cytogenetic analysis of the bKEC subculture at the 36th passage demonstrated the stability of the karyotype, which preserved both the diploid set of chromosomes and the absence of chromosomal rearrangements and the formation of marker chromosomes. Thus, our results confirm that the obtained culture of kidney cells of the bat P. nathusii retains the diploid set of chromosomes for at least 36 passages (the observation period).

L. monocytogenes Invades and Reproduces in Bat Kidney Cells
The bKEC subculture taken at the 4th passage was infected with the wild-type L. monocytogenes strain EGDe. We used a standard gentamicin assay to infect 70% bKEC monolayer with L. monocytogenes at a multiplicity of infection (MOI) of 1:100 (cell/bacteria). Two hours post-infection, the average number of intracellular bacteria was 301 ± 11 CFU per well ( Figure 4). The invasion efficiency measured as the percentage of intracellular bacteria relative to applied bacteria was 0.0078 ± 0.0009%. Despite the relatively low invasion efficiency, once inside bat cells, L. monocytogenes multiplied effectively, with a doubling time of 70 min. Thus, the number of intracellular bacteria increased more than 32-fold within 8 hours post-infection ( Figure 4). The obtained results suggested that bat primary cells are susceptible to L. monocytogenes infection.

L. monocytogenes Cell-to-Cell Spread
To further investigate the process of intracellular parasitism of L. monocytogenes we studied bacterial spreading. The effectiveness of spreading from cell to cell was evaluated by the ability to form plaques of lysis in the monolayer of bKEC. Bacteria formed visible plaques in the confluent monolayer of cells three days after the infection that suggested their effective spreading from the initially infected cell to neighbors ( Figure 5A).
Intracytoplasmic L. monocytogenes utilizes host actin filaments to mediate its movement within a cell and from cell to cell [58][59][60]. Actin filaments associated with the bacteria can be visualized by fluorescence microscopy with Alexa Fluor ® 555 phalloidin (Thermo Fisher Scientific, Waltham, MA, USA), a reagent that binds to a high-affinity F-actin and not to G-actin. We investigated the ability of L. monocytogenes to participate in cytoskeletal rearrangement in bKEC. After 8-h post-infection, we visualized intracellular bacteria and actin microfilaments with fluorescent microscopy. Staining of non-infected cells showed that most of F-actin was concentrated around the nucleus. Cells infected with bacteria also showed a concentric arrangement of F-actin, but it was possible to notice cytoskeleton rearrangements to the "comet-tail" structures associated with bacteria ( Figure 6G,H).

Invasion Factors InlA and InlB Are Required for L. monocytogenes Invasion into bKEC
Host cell invasion is mediated by the L. monocytogenes surface proteins of the internalin family, InlA and InlB [41,61,62]. The cognitive eukaryotic receptors for InlA and InlB are E-cadherin and c-Met, respectively, which are conserved receptors found on the surface of the epithelial cell [63][64][65][66].
Mechanisms of interactions of InlA and InlB and their target receptors are well-studied. InlA recognizes the human N-terminal immunoglobulin (Ig)-like domain EC1 (amino acid residues 1-95), which is the first of five extracellular Ig-like domains of E-cadherin. InlB interacts with the c-Met PSI-(for plexin-semaphorin-integrin) and Ig1-domains, which are located consecutively in the human c-Met (amino acid residues 519-656) [67,68].
To understand the potential of InlA and InlB to interact with bat receptors, we compared internalin-binding domains in human, bat, mouse and guinea pig receptors. The genome of P. nathusii is not yet available, so we took sequences of related bat species Myotis lucifugus (accession numbers of XP_023611752 and XP_006100243 for E-cadherin and c-Met, respectively) and Myotis brandtii (accession numbers of XP_014384634 and XP_005877763 for E-cadherin and c-Met, respectively) belonging to the same family of Vespertilionidae available in GenBank. Only E-cadherin EC1-domain and c-Met PSI-Ig1-domains, which are directly involved in interactions with bacterial invasion factors, were compared.
The maximum likelihood dendrogram and the multiple alignment sequence scores revealed that the bat E-cadherin EC1-domain is less similar to human E-cadherin than mouse and guinea pig proteins ( Figure 7A,B). EC1-domain interacts with InlA with the highest value being proline at position 16 of EC1-domain [67,69]. The alignment analysis showed that in bats, as well as in humans and guinea pigs, proline is located in the 16 position. This is in contrast to mouse E-cadherin, for which proline 16 is replaced by glutamine that prevents interactions of InlA with mouse E-cadherin ( Figure 7A) [69]. Other residues important for E-cadherin and InlA interactions, including Phe17, Pro18, Asn27, and Gly54 [67], were conserved among sequences of Homo sapiens, Cavia porcellus, Mus musculus and M. brandtii. For M. lucifugus, the substitution Asn27Ser was revealed. However, this substitution can be considered as synonymous as both asparagine and serine are hydrophilic and have a similar charge. Therefore, despite the relatively low similarity of the bat and human EC1-domains ( Figure 7C), the bat E-cadherins carries key amino acid residues required for interactions with InlA ( Figure 7A).
To test this assumption, we compared invasion efficiency of the wild type strain EGDe and its isogenic deviation, EGDe∆inlA, with the deletion of the inlA gene. Invasion efficiency of EGDe∆inlA was 0.00077 ± 0.00002%, i.e., 10-fold less than the invasion efficiency of EGDe (p < 0.000001, Figure 7D). These data support our suggestions that L. monocytogenes invasion in bKEC depends on InlA.
Next, we analyzed the importance of another invasion factor, InlB, for L. monocytogenes invasion into bKEC cells. The concave face of the InlB, leucine-rich repeat region, interacts tightly with the PSI-Ig1-domains of the c-Met stalk [68]. The partial sequence of bat c-Met was aligned with the human, guinea pig, and mouse c-Met proteins ( Figure 8A). The amino acids in the Ig1-domain involved in interactions of human c-Met with InlB, including lysines Lys599 and Lys600, and glycines Gly643 and Gly645 ( Figure 8A) [68], were conserved in bats. In the guinea pig, Gly643 was missing that might explain the inability of InlB to interact with guinea pig c-Met. Taken together, these results showed that bat c-Met carries all key amino acid residues and seemed to be able to interact with InlB.
The maximum likelihood dendrogram showed that the PSI-Ig1-domains of the bat species M. lucifugus and M. brandtii formed the cluster together with human c-Met ( Figure 8B). The similarity of bat and human proteins was 84 % that was higher than for human and mouse, and for human and guinea pig sequences (82 % and 74 %, respectively) ( Figure 8C). The similarity between bat proteins was 98% that was higher than its similarity to c-Met proteins of other species ( Figure 8C).
To determine the role of InlB in interactions of L. monocytogenes with bat cells, bKEC was infected with the isogenic strains EGDe and EGDe∆inlB ( Figure 8D). Invasion efficiency of EGDe∆inlB was 0.00031 ± 0.000011%, i.e., 25-fold less than the invasion efficiency of EGDe (p < 0.000001). The lack of InlB resulted in a very low invasion by single bacterial cells. Therefore, both InlA and InlB are involved in L. monocytogenes invasion into bat cells.

Natural InlB Variants Differently Restored Invasion of the Strain EGDe∆inlB.
The deletion of the inlB gene almost completely disrupted L. monocytogenes invasion into bKEC cells, providing a higher effect than the deletion of the inlA gene (25-fold and 10-fold respectively). To get a better understanding of the role of InlB in bat cell invasion, we supplemented the inlB deletion with two distinct inlB alleles and compared the invasion of the recombinant strains into bKEC cells. One inlB allele designated as inlB EGDe was cloned from the strain EGDe and was expected to restore invasion. Another allele designated as inlB 081 was from the L. monocytogenes strain VIMVR081, isolated from the liver of the grey red-backed vole (Myodes rufocanus) captured in the pristine environment in the far eastern part of Eurasia [26]. The strain VIMVR081 (accession number NZ_CP018148.1) belongs to the clonal complex CC2 [30]. Results of MLST typing are available from the L. monocytogenes Institut Pasteur MLST database at http://www.pasteur.fr/mlst and GeneBank database at https://www.ncbi.nlm.nih.gov/assembly/GCF_001889585.1. The same inlB allele was found in wild rodent isolates belonging to the clonal complexes CC2 and CC315 [26,30]. The inlB alleles were cloned into the pTRKH2 vector as described in Materials and Methods. The c-Met binding domains of the recombinant proteins differed by 14 amino acid substitutions ( Figure 9A). The obtained strains were used to infect bKEC cells as described above. The allele inlB EGDe as it was supposed, restored invasion ( Figure 9B). The bacterial strain carrying the inlB 081 allele had low efficiency in invasion. The invasion efficiency of the strain carrying inlB 081 was only two-fold higher than that of the strain EGDe∆inlB (p < 0.05). The level of InlB expression in the EGDe strain and recombinant strains was at the same level ( Figure 9C). Obtained data supported the view that InlB is important for L. monocytogenes invasion into bat cells and suggested the importance of the InlB isoform for bat cell invasion.

Discussion
Chiroptera is the most numerous (majority) group of mammals that includes up to a quarter of all known mammalian species representing one of the most abundant animal species on the planet [18]. Bats can actively occupy anthropogenic environments, increasing risks of infection transmission to human populations. The Covid19 epidemic is an example of the potential role of bats as a source of dangerous infections for humans. [3,70].
In this study, we performed in vitro testing of bat susceptibility to the pathogenic bacterium L. monocytogenes belonging to the group of infectious agents with a sapronotic origin, also known as soilborne pathogens [71,72]. This large group of microorganisms includes human pathogens such as L. monocytogenes, Y. pseudotuberculosis, Legionella pneumophila, Francisella tularensis, and Pseudomonas aeruginosa [8]. These pathogenic bacteria circulate in natural environments and can be transmitted to anthropogenic environments, usually by wild carriers. The typical feature of such pathogens is polyhostality, i.e., the ability to cause disease in a wide range of susceptible hosts. Polyhostality can be well illustrated by L. monocytogenes, which is a proven pathogen of humans and domestic animals [24].
To prevent the transition of new pathogens from natural hosts to humans, understanding the mechanisms that underlie pathogen polyhostality is required, but this is impossible without experimental studies on interactions of the pathogen with different groups of hosts. However, the use of laboratory animal models can be difficult, both from the ethical point of view and because of the specific physiology of the object under study: most bats live in colonies including enormous numbers of individuals, and their body temperatures may physiologically vary between less than 20 • C during hibernation and more than 40 • C during flight [73]. To solve the ethical issues and accumulate primary data on host-pathogen interactions on a molecular basis, it is beneficial to use cellular models. To be useful, a cellular model should demonstrate the reliable stability of basic cellular characteristics.
In our study, a finite culture of kidney cells (bKEC) was obtained for the first time from the widespread small bat Nathusius' pipistrelle. Cytogenetic analysis of bKEC cell culture revealed a diploid modal number of 2n = 44 and a total number of chromosomal arms of FN = 50; these findings are consistent with the data obtained for some other representatives of the genus Pipistrellus, such as P. savii koreensis and P. kuhli [74][75][76]. The bKEC culture demonstrated high stability, and its morphological and karyotypic characteristics remained stable from the 3rd up to the 36th consecutive re-plating steps. This stability provided reliable results on interactions between bKEC cells and the facultative intracellular pathogen L. monocytogenes.
Our data demonstrate the ability of wild-type L. monocytogenes once inside bat cells. The observed level of invasion was 0.0078 ± 0.0009% or about 301 ± 11 bacterial cells per well. This relatively low invasion efficiency is consistent with the results of other authors that used primary or finite cell lines of human, mouse and pig origin [40,41]. Immortalization of finite cell lines results in a noticeable increase in the L. monocytogenes invasion [77]. Finite pig and mouse cells showed a level of invasion by L. monocytogenes that was approximately two orders magnitude less than that of continuous cell lines obtained from the finite cells by transformation with the SV40 virus [78].
Entrance to and multiplication within epithelial cells are critical steps of L. monocytogenes infection in mammals, particularly required for crossing the intestinal barrier, followed by entering the lamina propria and intestinal lymph nodes [53]. The obtained data on the successful interactions of L. monocytogenes with bat epithelial cells are in line with the previously reported L. monocytogenes isolation from bat intestinal lymph nodes [14].
The data obtained in this study suggest that the initial stages of listeriosis in humans and bats are similar, at least when it comes to the need for InlA and InlB [41,82]. For comparison, in mice and guinea pigs, the invasion of epithelial cells depends on only one of the two invasion factors, either InlB or InlA, respectively [54,69]. Our previous findings have demonstrated that naturally occurring variants of InlB domain differ from each other on their ability to support intragastric infection in mice [50]. InlB variant of CC2 was found in 13 phylogenetically distant L. monocytogenes isolated from wild small rodents in remote parts of Russia in different years [30]. However, the distribution of this variant was not restricted to rodent isolates [30]. We used CC2 variant InlB to compare invasion of L. monocytogenes in mouse cells and bat cells. Interestingly, the InlB variant found in L. monocytogenes CC2 strains isolated from wild small rodents was less effective in bat cells than the InlB variant from the strain EGDe ( Figure 9B). To explain this effect, further investigations are needed.
Taken together, the obtained data suggest that bats represent another phylum of widespread small mammals that support L. monocytogenes cell invasion via mechanisms similar to those active in humans. We believe that the cell model developed in this work will be useful for in vitro studies of intracellular bacteria and will help to provide insight into the mechanisms of the polyhostality of L. monocytogenes and other sapronotic pathogens.

Animals
All the experiments on animals were performed in accordance with Russian legislation on animal welfare, which is in line with the Directive of the European Parliament 2010/63/EU, Code of ethics for the conduct of biomedical research with the use of animals, documents of the International UNESCO bioethics Committee, intergovernmental Committee UNESCO bioethics, and Steering Committee on bioethics of the Council of Europe, with the approval of FRCVM Bioethics commission control (Protocol #2020-01/1).
Nathusius's pipistrelle bats (P. nathusii) were caught using a fog net in the Volgograd region, Russia. Two clinically healthy male individuals weighing 15 and 12 grams were selected as kidney donors. Bats were euthanized using an intraperitoneal injection of 500 mL of 70% ethanol and dislocation of the cervical vertebrae after two minutes of post-cardiac arrest.

Tissue Trypsinization and Cell Isolation
In the first step, we removed the renal pelvis and the medulla from the kidneys. Then, the cortical layer of the kidney was mechanically ground to fragments with sizes of about 2-3 mm 3 in 50 mL sterile tubes (Eppendorf, Hamburg, Germany). Pieces of the tissue were washed five times with MEM (HyClone, Marlborough, MA, USA) to remove blood. Then, fragments of bat kidneys were transferred to a flat-bottomed flask with a volume of 1.0 dm 3 filled with a dispersing solution of 0.25% trypsin (PANEKO, Moscow, Russia) and 0.02% versene (PANEKO, Moscow, Russia) in the proportion of 2:1 and pre-warmed to 37 • C. The tissues were mixed on a magnetic stirrer for 15 min. Then, the flask was removed from the magnetic stirrer, non-disrupted tissue pieces were allowed to settle for 2 min, and the cell suspension was transferred into a 0.5 L vial. Then, FBS (HyClone, Marlborough, MA, USA) was added to the cells in a dispersing solution to neutralize trypsin up to 10%. The non-disrupted tissue pieces were reprocessed in a new cycle of enzymatic degradation with stirring. The procedure was repeated three times until complete tissue depletion. Bat cells were harvested by centrifugation at 1000 rpm for 10 min. The sediment of cells was resuspended in 25 mL of the MEM medium with the addition of 10% of FBS. The derived cell suspension was filtered through a gauze filter designed for the mechanical elimination of non-trypsinized fragments of renal tissue. Estimation of the viability of cells performed in a trypan blue (Thermofisher Scientific, Waltham, MA, USA) vital staining test revealed that it was 78%.
In the second step, the obtained cell suspension was diluted with DMEM (HyClone, Marlborough, MA, USA) supplemented by 10% FBS, 10 µg/mL ciprofloxacin (Sintez, Kurgan, Russia), and 5 µg/mL amphotericin B (Sintez, Kurgan, Russia) to achieve a seeding density of 4 × 10 5 cells/mL. This cell suspension was transferred into plastic cell culture flasks (Corning, USA) and incubated for 3 h to allow cells to attach. The cells were incubated at 37 • C with 5% CO 2 . Finally, the flasks were carefully washed with the DMEM medium to remove non-attached cells and further cultivated in the same medium.
The growth medium was changed after the first day of cultivation and then every 2-3 days. The growth intensity and cytomorphological characteristics of cells were evaluated using an inverted microscope (Olympus CK2, Tokyo, Japan). The homogenous culture of epithelial cells obtained at the second passage was named the bKEC culture.

Cell Culture and Subculture
bKEC cells were inoculated into a 24-well plate (Corning, Corning, NY, USA) and incubated at 37 • C in a 5% CO 2 atmosphere. After adhesion, the DMEM medium was changed, and the plates were examined daily under an inverted microscope (Olympus CK2, Tokyo, Japan) to estimate the cell growth. Subculturing was performed by successive passages in DMEM medium.

Cryopreservation and Thawing of Cells
A cryobank of cell subcultures was created. Subcultures obtained at the 4th, 10th, 13th, 18th, 20th, 22nd, 28th and 33rd passages were cryopreserved in liquid nitrogen. Cryopreservation of cells was performed in sterile DMEM medium with the addition of 10% DMSO (Sigma-Aldrich, St. Louis, MO, USA) as a cryoprotector after equilibrating at 4 • C for 60 min, followed by rapid freezing at −70 • C [83]. After 14 days of storage, frozen cells were deep-frozen in liquid nitrogen (−196 • C). Immediately before the experiment, cryovials with cells were thawed at 37 • C in a water bath. Then the contents of the cryovials were added to a sterile Falcon tube containing DMEM medium, warmed up to 37 • C, and centrifuged at 1000 rpm for 10 min. The cell pellet was resuspended in DMEM medium, transferred into new vials, and incubated at 37 • C in a 5% CO 2 atmosphere [84].

Chromosome Preparations and Karyological Analysis
The daily culture of bKEC cells was used for chromosome preparations. Karyological analysis was carried out according to the standard technique [85,86].

Bacterial Strains and Growth Conditions
The wild-type L. monocytogenes strain EGDe and its derivatives EGDe∆inlA and EGDe∆inlB, lacking the inlA and inlB genes, respectively, were used [87]. The strains with inlA and inlB deletions were generously provided by Prof. J. Vazquez-Boland, Univ. Edinburgh, UK, and have been used in previous studies [50,88]. Complementation of the inlB deletion with the InlB-expressing plasmid was described earlier [50]. Briefly, the inlB gene was expressed from the promoter of the inlAB operon cloned into the shuttle vector pTRKH2 [50,89]. To maintain the plasmid, erythromycin (Sigma-Aldrich, St. Louis, MO, USA) was added to the medium to a concentration of 10 µg mL −1 .
L. monocytogenes was cultivated in the BHI medium (Becton, Dickinson and Company, East Rutherford, NJ, USA) and grown at 37 • C with agitation at 180 rpm. Plasmid-bearing strains were grown in the presence of 10 µg/mL erythromycin. To prepare a culture for infection, bacteria were grown to the mid-exponential phase, washed with PBS (Amresco, Solon, OH, USA) three times, aliquoted, and frozen in the presence of 10% glycerol (Sigma-Aldrich, St. Louis, MO, USA). The use of pre-frozen cultures allows for more accurate standardization of infection doses between the compared strains. The concentration of bacterial cells was determined by plating serial dilutions from the frozen culture the day before the experiment.

In Vitro Invasion and Proliferation Assay
bKEC cells from the fourth passage of subcultures were used in the experiment. Before the analysis of invasion and proliferation, bKEC cells were cultured without antibiotics for 5 days. An invasion assay was performed according to Suarez et al. [90]. Bacteria frozen in 10% glycerol were thawed immediately before the experiment and resuspended in PBS to a volume of 1 mL. The bacterial suspension was diluted in DMEM with 10% FBS to achieve final concentrations with a multiplicity of infection (MOI) of 100 CFU per cell and added to the cells reached 70-80% confluence in a 24-well. After 1 h incubation at 37 • C in a 5% CO 2 atmosphere, cells were washed with PBS three times, and fresh DMEM supplemented with 100 µg mL −1 gentamicin (Sigma-Aldrich, St. Louis, MO, USA) was added to kill extracellular bacteria. After 1 h of contact with gentamicin, cells were washed with PBS and incubated with 200 µL of the mixture of 0.25% trypsin and 0.02% EDTA (Sigma-Aldrich, St. Louis, MO, USA) in the proportion of 2:1 for 10 min. Then, 800 µL of PBS was added to each well, and cells were collected in centrifuge tubes (SSIbio, Lodi, CA, USA) and treated with ultrasound at 18 kHz for 10 s in an ice bath. The number of bacteria that successfully entered cells was assessed by plating the serial dilutions of cell lysates on a solid BHI medium. Plates were incubated at 37 • C for 24 h, and then colonies were quantified. The efficiency of invasion was evaluated by the ratio of the number of entered bacteria to that of bacteria used for cell infection.
To assess intracellular proliferation, infected cells were incubated in the gentamicin-containing medium at 37 • C. Cells were lysed at 2, 4, 6 and 8 hpi (hours post-infection), and bacteria were plated from lysates as described above. The effectiveness of intracellular proliferation was evaluated by determining the number of CFU at this time to the number of introduced bacteria. All the experiments were performed in triplicate and repeated at least three times.

Immunoblotting
The restoration of InlB expression was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Membrane-bound protein samples were prepared from overnight L. monocytogenes cultures grown in BHI broth supplemented with 0.2% charcoal to activate the PrfA regulon as described previously [93]. SDS-PAGE was performed in accordance with the generally accepted techniques. Cell lysates of L. monocytogenes were boiled for 10 min, separated on 10% SDS-PAGE and transferred onto PVDF membrane. InlB was visualized with polyclonal primary anti-InlB antibodies (were obtained as described in the next section) and donkey anti-rabbit IgG HRP-labeled antibodies (Abcam, London, UK).

Primary Antibody Development and Purification
Two male rabbits (weighing 2.5 kg) were immunized with the recombinant protein InlB321/15 according to the scheme (Table 1). InlB321/15 was purified from the recombinant E. coli BL21::pET28b::inlBallele9 strain as described previously [94]. Briefly, after the first immunization with the protein applied intravenously, subcutaneously and intramuscularly injections were performed on the 4th, 8th and 10th weeks. Blood samples were taken after two weeks post the last immunization and onwards every two weeks for 3 months long. Blood samples were collected in vacuum tubes Improvacuter ® with coagulation activator (SiO 2 ) and gel (Guangzhou Improve Medical Instruments CO., LTD., Guangzhou, China). After coagulation, the blood was centrifuged at 3000 rpm for 15 min. Sera were stored at −20 • C. Anti-InlB 321 antibodies were purified from the antiserum using an immunosorbent. The immunosorbent developed on the basis of BrCN-activated sepharose 4B (Pharmacia, Srockholm, Sweden) conjugated with purified InlB 321 according to the manufacturer's instructions. Immunoadsorption was carried out in phosphate buffer (pH 7.0) with 0.15 M NaCl. After applying the antiserum, the column was washed with a phosphate buffer (pH 7.0) with 0.3 M NaCl. Antibody elution was performed with 4.5 M MgCl 2 followed by a dialysis step. Antibodies were stored in 50% glycerol at −80 • C. Anti-GAPDH-antibodies were obtained analogically.

Plaque Forming Assay
Bacteria for the assay were prepared and used for the infection of the confluent monolayer of bKEC cells cultured in six well plates. The assay was performed in general as described in [95]. After 1 h incubation, the cells were washed three times with PBS, and non-penetrated bacteria were killed by 1 h incubation with gentamicin (100 µg mL −1 ). The infected monolayers were then overlayed with 10 mL of the soft agar, prepared by mixing equal volumes of 2 × DMEM supplemented with 20 µg mL −1 gentamicin and 2% agar. Cells were further incubated under 5% CO 2 at 37 • C for up to three days following infection, and plaques were visualized by staining monolayers with 1 mL Neutral Red Solution (Sigma Aldrich, St. Louis, MO, USA) diluted 1:10 in PBS for 3 h. A culture of non-infected cells was used as a control.

Determination of Cytoskeleton Rearrangements and Bacterial Cells Visualization in bKEC
The bKEC cells were streaked on cover glasses at a concentration of 10,000 cells/cover glass and incubated in the DMEM medium with 10% FBS for 18 h. Next, we did the infection as described in the "Materials and Methods" section. The negative control was the non-infected cells. After 8-h incubation, the cells were washed two times in PBS and fixed with 3.7% formalin for 10 min. The cells were then permeabilized with 0.1% Triton X-100 (Panreac, Barcelona, Catalonia, Spain) for 10 min and stained with L. monocytogenes antibody-fluorescein isothiocyanate conjugate (FITC) (LifeSpan BioSciences, Seattle, WA, USA) Inc. for 1 h. Then cells washed three times with PBS and stained with Phalloidin Alexa Fluor 555 (Thermo FisherScientific, Waltham, MA, USA) for 20 min as described by the producer. All samples were assessed and captured thrice with Axio Scope A1 fluorescence microscope at 1000× magnification.

Statistics
Student's unpaired t-test, included in the Excel software package (Microsoft, Redmond, WA, USA), was used, and p-values of less than 0.05 were considered statistically significant.