Immunoprotective Analysis of the NFA49590 Protein from Nocardia farcinica IFM 10152 Demonstrates Its Potential as a Vaccine Candidate

Abstract

Nocardia is emerging as a serious and easily neglected pathogen in clinical practice with multidrug resistance that extends the treatment period for months or even years. This has led to the investigation of a vaccine approach to prevent Nocardia infections. However, studies on the protective proteins of Nocardia have not yet been carried out. In the present work, over 500 proteins in the supernatant of N. farcinica IFM10152 were identified by LC−MS/MS. In silico analysis of these proteins with a high content (score > 2000) predicted that NFA49590 was one of the conserved proteins in N. farcinica strains with potential antigenicity. After the rNFA49590 protein was cloned and expressed in E. coli (DE3) and purified using a Ni-NTA column, its good antigenicity was confirmed with sera from mice immunized with different Nocardia species by Western blot. Then we confirmed its ability to activate innate immunity by examining the phosphorylation status of ERK1/2, JNK, p38, and p65 and the cytokine levels of IL-6, TNF-α, and IL-10. Finally, we evaluated its immunoprotective effect in BALB/c mice, and we found that mice immunized with rNFA49590 protein exhibited high antibody titers, enhanced bacterial clearance ability, and generated robust protective effects from the N. farcinica challenge. These results offer strong support for the use of NFA49590 protein as a vaccine candidate and open the possibilities for the exploration of a large array of immunoprotective proteins.

Keywords: NFA49590; Nocardia farcinica; antigenicity; immunoprotection; vaccine.

Figure 1

Expression, purification, and subcellular location of rNFA49590 protein. (A) Screening optimal expression conditions of location, IPTG concentration, temperature (B), and imidazole elution concentration (C) of rNFA49590 protein by SDS–PAGE. (D) Purified rNFA49590 protein. (E) Subcellular location of native NFA49590 protein.

Figure 2

Reactivity of rNFA49590 protein with antiserum antibodies by Western blot. Recognition of rNFA49590 protein with anti-rNFA49590 sera (A) and antisera from mice infected with N. farcinica (B), other Nocardia species, or M. bovis (C).

Figure 3

rNFA49590 protein activated the MAPK and NF-κB pathways in RAW264.7 cells. RAW264.7 cells were incubated with 2 μg/mL rNFA49590 (with or without 100 μg/mL PmB) or 100 ng/mL LPS (with or without 100 μg/mL PmB) for 30 min (A), or 2 μg/mL rNFA49590 protein for 30, 60, or 120 min (B), or 2, 4, or 8 μg/mL rNFA49590 protein for 30 min (C), and the phosphorylation statuses of ERK1/2, JNK, p38, and p65 were analyzed by Western blot. Data are representative of at least three independent experiments.

Figure 4

rNFA49590-mediated cytokine expression was dependent on the phosphorylation statuses of ERK1/2, JNK, p38, and p65. (A) RAW264.7 cells were incubated with 2 μg/mL rNFA49590 protein for 6, 18, and 24 h, or 2 μg/mL rNFA49590 protein (with 100 μg/mL PmB) or 100 ng/mL LPS (with or without 100 μg/mL PmB) for 24 h, and the levels of IL-6, TNF-α, and IL-10 in the supernatants were measured by ELISA. (B) RAW264.7 cells were pretreated for 1 h with inhibitors of 20 µM SB 203580, 20 µM PD 98059, 20 µM SP 600125, or 20 µM BAY11-7082 prior to rNFA49590 protein exposure, and cytokine levels were measured by ELISA. Data are expressed as the means ± SD for three independent experiments. *** p < 0.001 when compared with the control group (A) or rNFA49590 group (B).

Figure 5

Effect of rNFA49590 protein immunization in mice. Female BALB/c mice were randomly divided into two groups and immunized with rNFA49590 protein or PBS three times. (A) Sera rNFA49590-specific IgG, IgG1, IgG2a, and IgG2b antibodies in PBS-immunized (n = 6) and rNFA49590-immunized (n = 6) mice were measured by ELISA. Bacterial survival in whole blood (B) and bone marrow neutrophils (C) after incubation for 2 h. (D–H) PBS-immunized (n = 10) and rNFA49590-immunized (n = 10) mice were intranasally infected with nonlethal N. farcinica, and then body temperature (D), body weight (E), LDH in BALF (F), CFU in lung tissue (G), and TNF-α, IL-10, IFN-γ in lung supernatant (H) were measured 24 h postinfection. Data are expressed as the means ± SD for three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

rNFA49590 immunization enhanced survival of mice after being challenged with N. farcinica. rNFA49590-immunized (n = 10) and PBS-immunized (n = 10) mice were challenged with a lethal dose of N. farcinica intraperitoneally, and mouse survival was monitored daily for a 10-day period (A). The remaining survival mice in two groups were sacrificed, and the lung, liver, and kidney (B) were dissected for histological examination (20×).