The Characteristics and Function of Internalin G in Listeria monocytogenes

The reference strain of LM ATCC^®19111 (1/2a) and human colorectal adenocarcinoma (Caco-2) cells were purchased from the China Culture Collection Center. Escherichia coli competent cells BL21 (DE3), prokaryotic expression plasmid pET-28a (+), and LM shuttle plasmid pKSV7 were preserved in our laboratory. The primers used for cloning inlG and the construction of the deletion strain LM-AinlG are shown in Table I.

To grow LM ATCC^®19111 strains, overnight cultures were diluted 100-fold in Brain Heart Infusion medium (BHI, China) and shaken at 37°C. The recombinant plasmid pET-28a (+)-InlG was constructed, which was transformed into the expression strain BL21 (DE3). We carried out IPTG induction to positive recombinant plasmid pET-28a (+)-InlG and optimization of the expression conditions, SDS-PAGE analysis of the expression product size, and western blotting to verify the antigenicity of the expressed protein.

Thirty-two 7-week-old male BALB/c mice were obtained from the Experimental Animal Center of the Chinese Academy Agricultural of Sciences (Lanzhou, China) and maintained in cages with food and water ad libitum. They were divided into two groups of 16 mice, respectively. Groups were immunized with 100 pg of recombinant inlG mixed with complete Freund’s adjuvant (Sigma, USA) for priming and incomplete Freund’s adjuvant (Sigma, USA) for boosting. Immunizations were carried out on days 1, 15, and 30. Blood was collected by tail vein bleeding before immunization. The antibody titer was measured after each immunization by indirect ELISA. When the titer increased more than 1:52,000, mice were intraperitoneally infected with LM at twice LD₅₀, and the immunoprotective effect of inlG on mice was evaluated by measuring the survival rate of mice.

Mice in the three groups (unchallenged, unimmunized-challenged, and immunized-challenged) (n = 3, respectively) were dissected after death, and brain and liver tissues were removed and prepared in paraffin sections. After hematoxylin-eosin staining, pathological changes were observed under an optical microscope (Olympus, Japan).

The primers were designed to amplify the upstream and downstream homologous arms of the inlG gene. The homologous arm of AinlG was amplified with gene splicing by overlap extension PCR (SOE-PCR). After sequencing, the homologous arm of AinlG was linked to the shuttle plasmid pKSV7 to form the recombinant plasmid pKSV7-AinlG. The pKSV7-AinlG was transferred into LM competent cells by Gene Pulser MXcell^TM (BioRad, USA), and the positive bacteria were screened. Homologous recombination was carried out at 41°C in the presence of chloramphenicol. After complete recombination, the pKSV7 plasmid was lost under 30°C and no-chloramphenicol conditions. The genetic stability of the gene deletion strain was tested by PCR.

The growth of wild-type LM and LM-inlG was evaluated by measuring OD₆₀₀ at 30°C, 37°C, and 41°C and pH 5, 7, and 9. The adhesion and invasion rates of the two strains were calculated as described previously (Medeiros et al. 2021; Pereira et al. 2021). Human colon carcinoma cells (Caco-2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, China) with high glucose, supplemented with10% fetal bovine serum (FBS) (Gibco, China). They were then seeded in 12-well plates at a density of 1 × 10⁵ cells/well and incubated at 37°C in the presence of 5% CO₂. For the adhesion assay, overnight (18 h)-grown bacterial cultures were washed thrice with PBS, adjusted to OD₆₀₀ = 0.12, and suspended in DMEM to a final concentration of 1 × 10⁸ CFU/ml. The Caco-2 cell monolayer was washed three times using DMEM, then exposed separately to the LM and LM-inlG and incubated for 2 h at 37°C under the atmosphere with 5% CO₂. After incubation, cells were washed three times with PBS and lysed using 500 pl of cold 0.5% Triton X-100. For the invasion assay, the monolayers were exposed to LM and LM-inlG, washed as performed in the adhesion assay, and treated with gentamicin (100 µg/ml, 1 h) and with 0.5% Triton X-100 (37°C, 10 min). The lysed cell suspensions from both adhesion and invasion experiments were serially diluted in PBS, and the bacterial concentration in the samples was determined by the number of colony-forming units (CFU). The adhesion/invasion efficiency (%) for each strain was calculated as the number of bacteria attached/invaded to the cells compared with the total number of CFU provided in the inoculation samples multiplied by 100. However, the LD₅₀ of mice for the deletion strain was determined the same as the wild strain.

Total RNA was extracted from LM and LM-AinlG samples using the RNAprep Pure Kit (Tiangen, China), following the manufacturer’s instructions. In total, 5 pg RNA/sample was sent to Beijing Novogene Technology Co., Ltd., China for RNA sequencing. According to the instructions, the Library Prep Kit (Tiangen, China) was used to construct the chain-specific library for RNA samples. The library was sequenced using the Illumina sequencing platform (HiSeq 4000). Raw data (raw reads) of FASTQ format were firstly processed through in-house Perl scripts. The LM reference genome (NC_003210) and associated gene annotation information were downloaded from the NCBI database. The sequence alignment software, Bowtie2 (2.3.4.3) was used for genome location analysis of clean reads.

The number of gene counts per sample was standardized using DEGSeq2 (1.20) software. We then performed hypothesis testing and set the threshold as |log2 Fold Chang e| > 1, p_adj value (corrected p-value) < 0.05 to screen out DEGs between LM and LM-AinlG. To systematically analyze gene biological function and genome information, the clusterProfiler (3.8.1) software was used for Gene Ontology (GO) function enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for DEGs. p-Values were calculated and selected p_adj values were set to < 0.05 to determine the significant gene enrichment (Liao et al. 2019).

The molecular weight of the inlG protein was estimated to be 53 ku, but the size of the inlG protein was about 70 ku according to SDS-PAGE. Therefore, the expressed protein was identified by mass spectrometry. The mass spectrometry results matched the LM reference sequence to the highest degree, and the protein was confirmed to be an expression product of the inlG gene. LM-positive serum was used as the primary antibody, and sheep anti-rabbit IgG was used as the secondary antibody. Western blotting showed that the induced protein had potential reactogenicity (Fig. 1).

Fig. 1

The mortality of mice infected with LM is shown in Table II and was calculated according to the formula: logLD₅₀ = [Xm-i (XP-0.5)]. Finally, the LD₅₀ of LM was 1.0 × 10^6.6 CFU. The antibody titer of inlG was more than 1:52,000 in mice by indirect ELISA. After intraperitoneal infection of mice with LM, the control group had rough hair and were huddled and listless. Individual mice gradually developed tremors, increased secretion at the canthus, and accelerated respiratory rate. Fifteen mice died within 72 h, and one survived. However, eight mice in the test group developed similar symptoms and died. The remaining eight mice became normal at 48 h after the challenge (Table II).

Compared with the brain tissue of the normal group (Fig. 2a), the challenge group after immunization with saline was filled with red blood cells, and a large number of lymphocytes were infiltrated in the pons (Fig. 2b). After protein immunization, the interface between the cortex and medulla in the brain tissue of mice in the challenge group was clear, and there were fissure vacuoles in the medulla (Fig. 2c). Brain tissue damage in the immunoprotein group post-challenge was milder than that in the normal saline group.

Fig. 2

Compared with liver tissue in the normal group (Fig. 2d), mice in the challenge group after immunization with saline (Fig. 2e) suffered a severe liver injury, irregular arrangement of hepatic lobules, and dilated central veins. The hepatic cord of mice in the challenge group disappeared after protein immunization (Fig. 2f). As in the brain, liver injury in the protein challenge group was milder than in the normal saline challenge group.

The amplified upstream and downstream homologous arms of inlG were 633 and 596 bp, respectively. The homologous arms of the inlG were fused by SOE-PCR, and a fragment of 1,229 bp was obtained, which was named AinlG. The pKSV7 plasmid was ligated with AinlG by BamHI and Xbal double digestion, and the product was transformed into DH5a competent cells. The recombinant plasmid pKSV7-AinlG was transformed into LM competent cells by electric shock and cultured at 30°C with chloramphenicol. The positive transformants were screened and amplified as 1,229 bp. At the 70 th generation, the homologous recombination was completed. The shuttle plasmid was lost successfully at the 30 th generation under the condition of 30°C and no antibiotics. The recombinant strain was cultured at 37°C for 20 generations, and a genetically stable deletion strain was finally obtained.

There was no significant difference in growth characteristics and biochemical and hemolysis test results between wild-type LM and LM-AinlG. The LD₅₀ of the deleted strain LM was 1.0 × 10^6.9 CFU, and the wild strain LM was 1.0 × 10^6.6 CFU. The adhesion rate of LM-AinlG to Caco-2 cells was 1.3 times higher than the wild-type strain, and the invasion rate was about 2.45 times higher than the wild-type strain (Fig. 3).

Fig. 3

The transcribed genes were classified into gene ontology (GO) categories: 45 genes were classified into biological processes, 19 to cellular components, and 29 to molecular functions. Compared to the wild strain, 18 genes were up-regulated and 24 genes were down-regulated in the LM-AinlG (Fig. 4). The DEGs were compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg) and GO databases (http://www.geneontology.org). In Fig. 5, the abscissa represents the enrichment factor; the vertical coordinate represents the enriched function of the KEGG term. The larger the circle, the more differential genes are enriched to this function. Twenty metabolic pathways were enriched in the LM-AinlG (Fig. 5). Quorum sensing, Propanoate metabolism, Valine, leucine, and isoleucine degradation, Glycerophospholipid metabolism, Butanoate metabolism are the five significant KEGG enriched pathways from LM-AinlG compared LM (Table III).

Fig. 4

Fig. 5