NOD2/c-Jun NH ₂ -Terminal Kinase Triggers Mycoplasma ovipneumoniae-Induced Macrophage Autophagy

Abstract

Mycoplasma ovipneumoniae (M. ovipneumoniae) belongs to Mycoplasma, a genus containing the smallest self-replicating microorganisms, and causes infectious pleuropneumonia in goats and sheep. Nucleotide-binding oligomerization domain-containing protein (NOD2), an intracellular pattern recognition receptor, interacts with muramyl dipeptide (MDP) to recognize bacterial peptidoglycans and is involved in autophagy induction. However, there have been no reports about NOD recognition of mycoplasmas or about M. ovipneumoniae-induced autophagy. In this study, we sought to determine the role of NOD2 in M. ovipneumoniae-induced autophagy using western blot, immunofluorescence, real-time polymerase chain reaction (RT-PCR), and color change unit (CCU) analysis. M. ovipneumoniae infection markedly increased NOD2, but not increased NOD1 expression in Raw264.7. Treating Raw264.7 cells with MDP significantly increased colocalization of M. ovipneumoniae and LC3, whereas treatment with NOD inhibitor, NOD-IN-1, decreased colocalization of M. ovipneumoniae and LC3. Furthermore, suppressing NOD2 expression with siRNA-NOD2 failed to trigger M. ovipneumoniae-induced autophagy by detecting autophagy markers Atg5, Beclin1, and LC3-II. In addition, M. ovipneumoniae infection significantly increased the p-JNK/JNK, p-Bcl-2/Bcl-2, Beclin1, Atg5, and LC3-II ratios in Raw264.7 cells. Treatment with JNK inhibitor, SP600126, or siRNA-NOD2 did not increase this reaction. These findings suggested that M. ovipneumoniae infection activated NOD2, and both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy. This study provides new insight into the NOD2 reorganization mechanism and the pathogenesis of M. ovipneumoniae infection. Important Mycoplasma ovipneumoniae, which lacks a cell wall, causes infectious pleuropneumonia in goats and sheep. In the present study, we focused on the interaction between NOD and M. ovipneumoniae, as well as its association with autophagy. We showed for the first time that NOD2 was activated by M. ovipneumoniae even when peptidoglycans were not present. We also observed that both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy.

Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro

“…For example, a type III secretion system enables Shigella to avoid being detected by the host autophagic machinery [ 12 ]; Legionella pneumophila inhibits autophagy in mammalian cells [ 30 ]; and Staphylococcus aureus impairs autophagy for replication [ 28 , 31 ]. Similarly, Mycoplasma pneumoniae induced autophagy in mice [ 14 ], whereas Mycoplasma ovipneumoniae activated autophagy in RAW 264.7 cells [ 13 ]. In this study, to investigate autophagy, the ultramicrostructure of intracellular M. bovis was investigated.…”

“…For Mycoplasma species, which are the smallest prokaryotes capable of self-replication, autophagy has an important role in host–pathogen interactions. For example, Mycoplasma ovipneumoniae induced autophagy in macrophages in vitro [ 13 ]. Furthermore, autophagy had a critical role in stimulating the inflammatory response to Mycoplasma pneumoniae infections in mice [ 14 ].…”

Genomic regions associated withMycoplasma ovipneumoniaepresence in nasal secretions of domestic sheep

“…Also, PTPN13 (PTP-BL) interacts with the Rho effector kinase protein kinase C-related kinase 2 ( PRK2 ) [53] and PRK2 has been demonstrated to have a role in cilia signaling [54] as well as regulation of the formation of apical junctions in bronchial epithelial cells [55]. Infection of a murine macrophage cell line with M. ovipneumoniae increases levels of nucleotide-binding oligomerization domain-containing 2 ( NOD2 ) and NOD2 along with activation of c -Jun NH2-terminal protein kinase ( JNK ) induces autophagy [56]. NOD2 interacts with RAC family small GPTase 1 ( Rac1 ) in membrane ruffles [57] and it has been suggested that Rho GTPases such as Rac1 regulate PRK2 activity during control of the cell cycle [58].…”

Genes involved in immune, gene translation and chromatin organization pathways associated with Mycoplasma ovipneumoniae presence in nasal secretions of domestic sheep

“…Infection of a murine macrophage cell line with M . ovipneumoniae increases levels of nucleotide-binding oligomerization domain-containing 2 ( NOD2 ) and NOD2 along with activation of c -Jun NH2-terminal protein kinase ( JNK ) induces autophagy [ 64 ]. NOD2 interacts with RAC family small GPTase 1 ( Rac1 ) in membrane ruffles [ 65 ] and it has been suggested that Rho GTPases such as Rac1 regulate PRK2 activity during control of the cell cycle [ 66 ].…”

“…Also, PTPN13 (PTP-BL) interacts with the Rho effector kinase protein kinase C-related kinase 2 (PRK2) [61] and PRK2 has been demonstrated to have a role in cilia signaling [62] as well as regulation of the formation of apical junctions in bronchial epithelial cells [63]. Infection of a murine macrophage cell line with M. ovipneumoniae increases levels of nucleotide-binding oligomerization domain-containing 2 (NOD2) and NOD2 along with activation of c-Jun NH2-terminal protein kinase (JNK) induces autophagy [64]. NOD2 interacts with RAC family small GPTase 1 (Rac1) in membrane ruffles [65] and it has been suggested that Rho GTPases such as Rac1 regulate PRK2 activity during control of the cell cycle [66].…”