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Abstract Purpose Nocardia is emerging as a common and easily neglected cause of both healthcare- and occupation-associated infections worldwide, however, human vaccines for Nocardia prevention are not yet available. In this study, we aimed to evaluate the immunoprotective effect of the NFA47630 protein, a secreted protein abundant in the N. farcinica IFM10152 supernatant. Methods Conservation and characteristics of nfa47630 were analyzed by PCR and bioinformatics. Then recombinant NFA47630 protein was cloned, expressed and purified for further antigenicity analysis. Subsequently, the ability to activate innate immunity was evaluated by examining the phosphorylation status of the MAPK signaling pathway and cytokine levels. Finally, the protective effect was evaluated on rNFA47630-immunized mice. Results nfa47630 was conserved in N. farcinica strains with good antigenicity. The rNFA47630 protein was expressed under the optimal conditions of 0.2 mM IPTG, 28 °C, and it can be recognized by anti-N. farcinica and anti-N. cyriacigeorgica sera, but not anti-N. asteroids, anti-N. brasiliensis, anti-N. nova and anti-Mycobacterium bovis sera. It can upregulate the phosphorylation status of ERK, JNK, P38 and the cytokine levels of TNF-α, IL-10, IL-12, and IFN-γ. In addition, mice immunized with rNFA47630 protein exhibited higher antibody titers, greater bacterial clearance ability, milder organ infection, and higher survival rates than PBS-immunized mice. Conclusions Our data demonstrate that NFA47630 is a potential vaccine candidate for defending against N. farcinica infection.

Abstract Background Nocardia, a rare but potentially fatal pathogen, can induce systemic infections with diverse manifestations. This study aimed to investigate the tissue and organ damage caused by Nocardia farcinica (N. farcinica) in mice via different infection routes, evaluate the resulting host immune responses, and assess its invasiveness in brain tissue. Methods BALB/c mice were infected with N. farcinica through intranasal, intraperitoneal, and intravenous routes (doses: 1 × 10^8, 1 × 10^7, 1 × 10^7 CFU in 50 µl PBS). Over a 7-day period, body temperature, weight, and mortality were monitored, and samples were collected for histopathological analysis and bacterial load assessment. Serum was isolated for cytokine detection via ELISA. For RNA-seq analysis, mice were infected with 1 × 107 CFU through three infection routes, after which brain tissue was harvested. Results Intraperitoneal and intravenous N. farcinica infections caused significant clinical symptoms, mortality, and neural disruption in mice, resulting in severe systemic infection. Conversely, intranasal infection primarily affected the lungs without causing significant damage to other organs. Intraperitoneal and intravenous infections significantly increased serum cytokines, particularly TNF-α and IFN-γ. RNA-seq analysis of brains from intravenously infected mice revealed significant differential gene expression, whereas the intranasal and intraperitoneal routes showed limited differences (only three genes). The enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the intravenous group were primarily related to immune processes. Conclusion The study demonstrated that intravenous N. farcinica infection induces significant clinical symptoms, triggers an inflammatory response, damages multiple organs, and leads to systemic infections.

ABSTRACT Nocardia farcinica can cause a rare, yet potentially fatal, central nervous system infection. NbtS protein may be a key virulence factor in N. farcinica infection of the brain. In this study, we investigated the function of the virulence-associated factor NbtS in microglial cells in vitro and in infected mice in vivo. We explored the interactions between NbtS and microglial cells (BV2 and human microglial clone 3), revealing that NbtS activates the toll-like receptor 4-dependent MyD88-IRAK4-IRAK1 and MAPK/nuclear factor kappa B (NF-κB) pathways, significantly enhancing pro-inflammatory responses as indicated by increased levels of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), as measured by ELISA and quantitative PCR. Apoptosis was elevated in these cells, as shown by increased expression of Bax and caspase-3 and decreased Bcl-2 levels. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay also confirmed the occurrence of apoptosis. In vivo, mice infected with an RS03155-deficient strain of N. farcinica exhibited higher survival rates and reduced brain inflammation, suggesting a pivotal role for the NbtS protein in the pathogenesis of Nocardia. Conservation of the RS03155 gene across Nocardia spp. was verified by PCR, and the immunogenic potential of NbtS was confirmed by Western blot analysis using sera from infected mice. These findings suggest that targeting NbtS may offer a novel therapeutic strategy against Nocardia infection.IMPORTANCEThe study presented in this article delves into the molecular underpinnings of Nocardia farcinica-induced neuroinflammation. By focusing on the salicylate synthase gene, RS03155, and its encoded protein, NbtS, we uncover a pivotal virulence factor that triggers a cascade of immunological responses leading to apoptosis in microglial cells. This research not only enhances our comprehension of the pathogenesis of Nocardia infections but also provides a potential therapeutic target. Given the rising importance of understanding host-microbe interactions within the context of the central nervous system, especially in immunocompromised individuals, the findings are of significant relevance to the field of microbiology and could inform future diagnostic and treatment modalities for Nocardia-associated neurological disorders. Our work emphasizes the need for continued research into the intricate mechanisms of microbial pathogenesis and the development of novel strategies to combat life-threatening infections.