Annexin A2 Regulates Autophagy in Pseudomonas aeruginosa Infection through the Akt1–mTOR–ULK1/2 Signaling Pathway

Abstract

Earlier studies reported that a cell membrane protein Annexin A2 (AnxA2) plays multiple roles in the development, invasion and metastasis of cancer. Recent studies have demonstrated that AnxA2 also functions in immunity against infection, but the underlying mechanism remains largely elusive. Using a mouse infection model, we now reveal a crucial role of AnxA2 in host defense against Pseudomonas aeruginosa (Pa), as anxa2−/− mice manifested severe lung injury, systemic dissemination, and increased mortality compared to wild-type (WT) littermates. In addition, anxa2−/− mice exhibited elevated inflammatory cytokines (TNF-α, IL-6, IL-1β and IFN-γ), decreased bacterial clearance by macrophages, and increased superoxide release in the lung. We further identified an unexpected molecular interaction between AnxA2 and Fam13A (Family with sequence similarity 13, member A), which activated Rho GTPase. P. aeruginosa infection induced autophagosome formation by inhibiting Akt1 and mTOR. Our results indicate that AnxA2 regulates autophagy and thereby contributing to host immunity against bacteria through Akt1-mTOR-ULK1/2 signaling pathway.

Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity

“…We intranasally inoculated PA14 WT and the mutant strain to C57BL/6J mice at a multiplicity of infection (MOI) of 5 × 10 6 colony forming units (CFU)/mouse [22]. Compared with the PA14 WT group, ∆TCR mutant-, ∆cas3 mutant-and ∆CR1 mutant-infected mice exhibited increased lethality, whereas ∆lasR mutant-infected mice showed decreased lethality ( Figure 3A), and all these changes in mouse survival could be abolished by complementation of each of the corresponding genes ( Figure 3A), suggesting that PA14 CRISPR-Cas system and LasR impact PA14 virulence.…”

“…Various mammalian primary cells or cell lines were changed to antibiotic-free medium and challenged by bacteria in an MOI of 20:1 bacterium-cell ratio (some cases using other MOI). Mice were sedated with 40 mg/kg ketamine and then intranasally inoculated with 5 × 10 6 CFU/mouse of bacteria in 50 µl PBS as described [22]. Mice were monitored for symptoms and killed when they were moribund.…”

“…qPCR was performed using the iTag Universal SYBR Green Supermix (BioRad, Hercules, CA) and gene-specific primers (Supplementary information, Table S3, synthesized in Integrated DNA Technologies, Coralville, IA), in a CFX Connect Real-time PCR Detection System (Bio-Rad). The separate well 2 −∆∆C T cycle threshold method was used to determine relative quantitative levels of individual gene [22]. For bacteria, relative transcript levels were normalized to 16S rRNA, whereas in mammalian cell, relative transcript levels were normalized to GAPDH.…”

“…Lung sections were fixed in 4% formalin for 24 h and then processed to hematoxylin and eosin staining in AML Laboratories (Baltimore, MD), and observed using Nikon 80i Eclipse (upright) microscope [22]. The degree of cellular infiltration was scored using previously described methods [43].…”

“…The dishes were cultured in a 37 °C incubator overnight, and colonies were counted. Triplicates were done for each sample and control [22].…”