Min Li

Mycoplasma pneumoniae (M. pneumoniae, Mp) is a cell wall-deficient microorganism known to cause chronic respiratory infections in both children and adults. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor primarily responsible for identifying muramyl dipeptide (MDP) found in bacterial cell walls. Previous experiments have demonstrated that Mycoplasma ovipneumoniae induces macrophage autophagy through NOD2. In this study, we conducted RNA-seq analysis on macrophages infected with M. pneumoniae and observed an up-regulation in the expression of genes associated with the NOD2 signaling pathway. Mechanistic investigations further revealed the involvement of the NOD2 signaling pathway in the inflammatory response of macrophages activated by M. pneumoniae. We utilized GST pull-down technology in conjunction with liquid chromatography–tandem mass spectrometry (LC–MS/MS) to pinpoint the M. pneumoniae proteins that interact with NOD2. Additionally, co-immunoprecipitation (Co-IP) and immunofluorescence co-localization techniques were used to confirm the interaction between DUF16 protein and NOD2. We found that DUF16 protein can enter macrophages and induce macrophage inflammatory response through the NOD2/RIP2/NF-κB pathway. Notably, the region spanning amino acids 13–90 was identified as a critical region necessary for DUF16-induced inflammation. This research not only broadens our comprehension of the recognition process of the intracellular receptor NOD2, but also deepens our understanding of the development of M. pneumoniae infection.

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a species of Mycoplasma bacteria that specifically infects sheep and goat, causing ovine infectious pleuropneumonia. However, the mechanism underlying the pathogen-host interaction between M. ovipneumoniae and airway epithelial cells is unknown. A primary air-liquid interface (ALI) epithelial culture model generated from the bronchial epithelial cells of Ningxia Tan sheep (ovis aries) was employed to explore the potential mechanism of M. ovipneumoniae-induced cell apoptosis by characterizing the production of reactive oxygen species (ROS), methane dicarboxylic aldehyde (MDA) and anti-oxidative enzymes, as well as the mitochondrial membrane potentials, cytochrome C release, and activities of ERK and caspase signalling pathways. Increased ROS production and MDA concentration with mitochondrial membrane dysfunction and apoptotic cell death but decreased expression of the antioxidant enzymes catalase (CAT), glutathione synthetase (GSS), total superoxide dismutaes (T-SOD) and Mn-SOD were observed in sheep airway epithelial cells infected with M. ovipneumoniae. Mechanistically, the M. ovipneumoniae-induced cell apoptosis and disruption of mitochondrial integrity reflected mechanisms by which pathogen-activated mitogen-activated protein kinase (MAPK) signalling sequentially led to mitochondrial damage and release of Cyt-C into the cytoplasm, which in turn triggered the activation of caspase signalling cascade, resulting in the apoptosis of host cells. These results suggest that M. ovipneumoniae-induced ROS and MAPK signalling-mediated mitochondrial apoptotic pathways might play key roles in the pathogenesis of M. ovipneumoniae infection in sheep lungs.

Necrosis of alveolar macrophages following Mycobacterium tuberculosis infection has been demonstrated to play a vital role in the pathogenesis of tuberculosis. Our previous study demonstrated that Wnt/β-catenin signaling was able to promote mycobacteria-infected cell apoptosis by a caspase-dependent pathway. However, the functionality of this signaling in the necrosis of macrophage following mycobacterial infection remains largely unknown. Murine macrophage RAW264.7 cells were infected with Bacillus Calmette-Guerin (BCG) in the presence of Wnt/β-catenin signaling. The necrotic cell death was determined by cytometric assay and electronic microscopy; the productions of reactive oxygen species (ROS) and reduced glutathione (GSH) were measured by a cytometric analysis and an enzyme-linked immunosorbent assay, respectively; and the activity of poly (ADP-ribose) polymerase 1 (PARP-1)/apoptosis inhibition factor (AIF) signaling was examined by an immunoblotting assay. The BCG can induce RAW264.7 macrophage cells necrosis in a dose- and time-dependent manner along with an accumulation of reactive oxygen species (ROS). Intriguingly, an enhancement of Wnt/β-catenin signaling shows an ability to reduce the mycobacteria-induced macrophage necrosis. Mechanistically, the activation of Wnt/β-catenin signaling is capable of inhibiting the necrotic cell death in BCG-infected RAW264.7 cells through a mechanism by which the Wnt signaling scavenges intracellular ROS accumulation and increases cellular GSH concentration. In addition, immunoblotting analysis further reveals that Wnt/β-catenin signaling is capable of inhibiting the ROS-mediated cell necrosis in part through a PARP-1/AIF- dependent pathway. An activation of Wnt/β-catenin signaling can inhibit BCG-induced macrophage necrosis by increasing the production of GSH and scavenging ROS in part through a mechanism of repression of PARP-1/AIF signaling pathway. This finding may thus provide an insight into the underlying mechanism of alveolar macrophage cell death in response to mycobacterial infection.