Type III Secretion Protein, PcrV, Impairs Pseudomonas aeruginosa Biofilm Formation by Increasing M1 Macrophage-Mediated Anti-bacterial Activities

Abstract

Pseudomonas aeruginosa biofilms employ a variety of strategies to hijack the host immune defense system to achieve chronic infection. However, the bacterial components that are involved in this process are not yet fully understood. PcrV, a needle tip protein of the P. aeruginosa type III secretion system (T3SS), was downregulated during P. aeruginosa biofilm infection. The impaired expression of the P. aeruginosa pcrV gene is associated with attenuated immune activation and an increased percentage of M2 macrophages following P. aeruginosa biofilm infection. Treatment with exogenous PcrV produced from Escherichia coli elevated tissue inflammation and the percentage of M1 macrophages, resulting in reduction in the biofilm burden. Further analyses demonstrated that the potential of PcrV to induce classically activated M1 macrophages as evidenced by the increased production of proinflammatory cytokines and anti-bacterial mediators, including inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS), as well as increased phagocytosis of bacteria. Mechanistically, PcrV-mediated promotion of macrophage M1 polarization and phagocytosis occurs through the activation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathways. Collectively, these findings reveal a potential role of PcrV in skewing host immune defense to promote P. aeruginosa biofilm infection and provide new insights into the therapeutic strategies for P. aeruginosa biofilm infection.

Macrophage Related Chronic Inflammation in Non-Healing Wounds

“…During early wound healing, macrophage exhibits an inflammatory phenotype, known as the classically activated M1 macrophage. They act as the first line of defense against pathogen through mainly two ways, namely, recognizing pathogen-associated modifying proteins (PAMPs) on the surfaces of bacteria or fungi to form phagolysosome, and releasing antibacterial mediators, such as reactive oxygen species (ROS) and reactive nitrogen species ( 38 , 39 ). Moreover, they are involved in removal of cellular debris and clearing apoptotic neutrophils ( 40 ).…”

Pseudomonas aeruginosa PcrV Enhances the Nitric Oxide-Mediated Tumoricidal Activity of Tumor-Associated Macrophages via a TLR4/PI3K/AKT/mTOR-Glycolysis-Nitric Oxide Circuit

“…Compared with the control group, PcrV treatment decreased tumor growth ( Figure 1B ) and weight ( Figure 1C ). We previously demonstrated that PcrV significantly increased NO production in normal BMDMs ( 13 ). Considering that NO-mediated cytotoxicity is associated with tissue apoptosis and the inhibition of tumor growth, we then evaluated the levels of tumor cell apoptosis and the expression of the NO-generating enzyme—iNOS—in PcrV-treated tumor tissues.…”

“…These cells showed increased expression of genes related to tumor growth ( Cox2 and Vegfa ), metastasis ( Cox2 , Mmp2 , and Mmp9 ), and immune suppression ( Ido2 ) ( Supplementary Figure 3A ), indicating that TAM generation in vitro had been successful. The levels of tumoricidal M1 polarization markers, including iNos , Cd11c , MhcI , MhcII , Cd86 ( Supplementary Figure 3B ), iNOS ( Supplementary Figure 3C ), NO ( Supplementary Figure 3D ), IL12 p40/70, TNFA ( Supplementary Figure 3E ), MHCII, and CD86 ( Supplementary Figure 3F, G ), were found to be higher in PcrV-primed than mock-treated TAMs, whereas the levels of the protumoral M2 polarization markers c-Myc , Egr2 , Fn1 , Arg1 ( 13 ), and Cd206 were lower ( Supplementary Figure 3B ), demonstrating that PcrV directly reprograms TAMs to an antitumoral M1 profile in vitro .…”

“…Recombinant PcrV protein was expressed from the expression strain Escherichia coli JM109/pQE31-PcrV as described previously ( 13 ). Protein purification was performed using His-Trap HP affinity columns (GE Healthcare, Sweden), and endotoxin was removed using Detoxi-Gel endotoxin removing gel (Thermo Fisher, USA), according to the manufacturer’s instructions.…”

“…However, the mechanism underlying the PcrV-mediated regulation of the host immune response remains unknown. We have previously reported that PcrV reverses host immune suppression elicited following bacterial biofilm infection via the activation of macrophage-mediated immune responses ( 13 ). Considering that TAMs exhibit diverse phenotypes and given their function as tumor-resident macrophages, using PcrV to reeducate TAMs and thereby enhance M1 TAM-mediated antitumoral properties is an attractive possibility that requires further investigation.…”