1. Introduction =============== Human skin is inhabited by abundant microorganisms. The colonization of a human\'s skin by bacteria plays an important role in the skin health. Some bacteria may cause diseases, such as impetigo, pyoderma, and rosacea, or even threaten the health of some skin by producing toxins. Some bacteria in our skin are considered to be beneficial; they can produce antimicrobial agents against pathogens that may cause infection. The antimicrobial peptides (AMP) of bacteria in the skin might be one of the factors to maintain homeostasis and are crucial for host defense \[[@B1-ijms-16-03148]\]. Besides the innate immune cells, including dendritic cells and macrophages in our skin, T cells are an important part of the cellular components of the skin and also a key factor for the defense system against pathogens, and T cell differentiation can be stimulated by both self and non-self peptides. Therefore, understanding how bacteria produce different types of AMPs and other peptides, and how the peptides influence the functions of T cells and regulate the immune system are very interesting topics. *Corynebacterium parvum* is a Gram-positive, rod-shaped bacterium that causes severe and often fatal infection in immunocompromised individuals. *C. parvum* produces various extracellular virulence factors such as lipases, phospholipases, leukotoxins, phospholipases, lipopolysaccharides, and surface proteins to increase their invasiveness and infectivity. The major outer membrane proteins (MOPs) are one of the surface proteins expressed by *C. parvum*. MOPs are highly conserved proteins with an immunodominant N-terminal extracellular domain (BspA), a tandem-repeated domain in the cell wall-anchoring sequence, and a cytoplasmic domain \[[@B2-ijms-16-03148],[@B3-ijms-16-03148]\]. The MOPs of *C. parvum* might have diverse functions, because there are several genes encoding MOPs in the genome. Some MOPs were found to possess the functions of *C. parvum* virulence factors \[[@B3-ijms-16-03148],[@B4-ijms-16-03148]\]. A recombinant protein containing the BspA extracellular domain and lipopolysaccharide (LPS) core also possessed the functions of the virulence factors such as adhesion and invasiveness \[[@B5-ijms-16-03148],[@B6-ijms-16-03148],[@B7-ijms-16-03148]\]. Another MOP, the P30 protein of *C. parvum* could activate the host immune system through toll-like receptor 4 \[[@B8-ijms-16-03148],[@B9-ijms-16-03148]\]. To date, MOPs of *C. parvum* have been found to be capable of playing a role in bacteria-host interaction and causing the host disease; thus MOPs are considered to be useful as an antigen and a vaccine candidate. In the present study, the recombinant MOP of *C. parvum* was purified. Then, the effect of MOP on the functions of mouse CD4^+^ T cells was examined. Furthermore, we conducted experiments to identify the active peptide of MOP in the induction of cytokines from T cells. 2. Results and Discussion ========================= 2.1. The Recombinant MOP Was Expressed in Mammalian Expression Vectors ----------------------------------------------------------------------- The nucleotide sequence of the *opag* gene encoding the MOP of *C. parvum* was amplified by PCR, cloned, and sequenced as previously described \[[@B5-ijms-16-03148]\]. This product was then subcloned into the mammalian expression vector pcDNA3.1(-) in *E. coli*. Recombinant MOPs were expressed in mammalian cells after transfection with pDNA3.1(+)-MOP ([Figure 1](#ijms-16-03148-f001){ref-type="fig"}). ![The recombinant MOP was expressed in mammalian cells. The pcDNA3.1(+)-MOP plasmid was transfected into CHO cells for 24 h and then detected by western blotting with an anti-His tag monoclonal antibody. Lysates from non-transfected CHO cells and CHO cells transfected with the pcDNA3.1(+) plasmid were used as controls.](ijms-16-03148-g001){#ijms-16-03148-f001} 2.2. The MOP Expressed in Transfected CHO Cells Could Stimulate Proliferation of Splenocytes and Lymphocytes ------------------------------------------------------------------------------------------------------------ MOPs are abundant proteins in the outer membrane of the bacterium *C. parvum*; the antigenicity of MOPs is similar to those of lipopolysaccharides (LPS), so MOPs have been considered to be potent mitogens and activators of cell-mediated immunity. The present study was designed to evaluate the effect of the recombinant MOP in the proliferation of murine splenocytes and murine lymphocytes. We used murine spleen cell suspension, mouse peripheral blood lymphocytes (PBLs), and CHO-K1 cells as a negative control, PBLs stimulated with ConA as a positive control, and CHO-K1 cells as a control, respectively. The results showed that the recombinant MOP could stimulate the proliferation of murine splenocytes and lymphocytes, but not CHO-K1 cells ([Figure 2](#ijms-16-03148-f002){ref-type="fig"}). These results indicate that the recombinant MOP could stimulate proliferation of murine T cells. ![The recombinant MOP could stimulate the proliferation of murine splenocytes and lymphocytes. Splenocytes, peripheral blood lymphocytes (PBLs), CHO-K1 cells and CHO cells transfected with pcDNA3.1(+) vector were used. Cells were stimulated with the recombinant MOP (20 µg/mL), ConA (1 µg/mL), and lipopolysaccharide (LPS) (1 µg/mL). The results of PBLs from five mice per group were averaged. Means and standard deviations (error bars) are shown (*p* \< 0.05).](ijms-16-03148-g002){#ijms-16-03148-f002} 2.3. MOP Induced IL-2 Production from CD4^+^ T Cells ---------------------------------------------------- The MOP of *C. parvum* plays a role in activating the immune system; it is expected that MOP will interact with T cells to promote the function of T cells. T helper (Th) cells are divided into different subgroups depending on the cytokines secreted by T cells. To further determine the interaction between MOP and T cells, CD4^+^ T cells were cultured with or without recombinant MOP, and then IFN-γ and IL-2 were measured by ELISA. The results showed that MOP induced the production of IFN-γ and IL-2 in a dose-dependent manner ([Figure 3](#ijms-16-03148-f003){ref-type="fig"}). However, MOP did not affect the production of IL-4, IL-5 and IL-17. These results indicate that MOP can directly interact with CD4^+^ T cells and induce cytokine production by CD4^+^ T cells. ![MOP induced IL-2 production from CD4^+^ T cells. Splenic CD4^+^ T cells were cultured with 0, 1, 5, 10, and 20 μg/mL of MOP for 72 h at 37 °C in 5% CO~2~. Then IFN-γ and IL-2 were measured by ELISA. The control group was cultured with the same concentration of BSA. \* *p* \< 0.05 *vs.* the control group; \# *p* \< 0.05 *vs.* the MOP group. All experiments were repeated at least three times.](ijms-16-03148-g003){#ijms-16-03148-f003} 2.4. Purification of Recombinant MOP ------------------------------------ The recombinant MOP was purified by Nickel-NTA Superflow Columns. MOP in a concentration of 0.6 mg/mL was loaded into a 2 mL purification column that had been equilibrated with T buffer (20 mM Tris, 1 mM EDTA, 150 mM NaCl, pH 8.0). The column was washed with T buffer plus 10 mM imidazole and then the protein was eluted with a linear imidazole gradient from 10 to 400 mM. The chromatogram of recombinant MOP purification is shown in [Figure 4](#ijms-16-03148-f004){ref-type="fig"}. The purified protein concentration was determined by measuring the absorbance at 280 nm, and the final concentration of the recombinant MOP was determined to be 0.6 mg/mL. This recombinant MOP had a single band on SDS-PAGE and the purity of the recombinant MOP was estimated to be about 95%. ![Recombinant MOP purification chromatogram. Purified MOP was run on a SDS-PAGE gel and stained with Coomassie Brilliant Blue. Lane 1, recombinant MOP; lane 2, recombinant MOP and BSA; lane 3, recombinant MOP and purified recombinant MOP. Lane 4, recombinant MOP.](ijms-16-03148-g004){#ijms-16-03148-f004} 2.5. MOP Induced the Proliferation of CD4^+^ T Cells ---------------------------------------------------- ### 2.5.1. CD4^+^ T Cells Stimulated with ConA Could Be Subjected to the Second