Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl) homoserine lactone induces calcium signaling-dependent crosstalk between autophagy and apoptosis in human macrophages
Graphical abstract
Fig. 8 3oc is a Pseudomonas aeruginosa secreted quorum sensing molecule having pathological significance. This study showed that 3oc elevates [Ca+2]cyt, which leads to mitochondrial dysfunction (MMP drop, mROS generation), and apoptosis in macrophages. In addition, 3oc induces autophagy in a Ca+2-dependent manner. The 3oc induces autophagy by upregulating key autophagy-initiating genes (TFEB, LC3, BECLIN1, STX17, and PINK1). Furthermore, 3oc-induced autophagy plays a pro-death role in apoptosis, as chloroquine (autophagy inhibitor) rescues 3oc-induced cell death, whereas rapamycin synergizes 3oc-induced apoptosis.
Abbreviations: 3oc, N-(3-oxododecanoyl) homoserine lactone; BAPTA-AM, acetyloxymethyl 2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl] amino] phenoxy] ethoxy] anilino] acetate; mROS, mitochondrial ROS generation; MMP, mitochondrial membrane potential; TFEB, transcription factor EB; LC3, microtubule-associated protein 1A/1B-light chain 3; STX17, syntaxin-17; PINK1, PTEN Induced Kinase 1.
Introduction
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen, and it is among one of the most virulent bacteria associated with nosocomial infections [1]. It infects immunocompromised individuals, cystic fibrosis patients, and persons with acute and chronic wound infections and shows higher fatality rates among patients with sepsis infections [2,3]. P. aeruginosa's ability to produce extensive infections stems from an arsenal of cell-associated and released virulence factors driven through the quorum sensing (QS) system [4]. QS is a cell-to-cell communication that regulates biofilm formation and controls various virulence factor genes involved in establishing persistent infections in the host [5,6]. P. aeruginosa employs three major quorum-sensing systems, i.e., LasI, rhlI, and PQS. LasI-LasR circuit controls other QS circuits through autoinducer molecule N-(3-oxododecanoyl) homoserine lactone (3oc) [7]. Apart from regulating QS and controlling virulence factor secretion, 3oc is shown to mediate wide immunological events in different host cells and emerge as a potent immunomodulatory factor that suppresses host immune function [8,9]. 3oc is shown to induce apoptosis in immune cells like macrophages, neutrophils, platelets, and lymphocytes [[10], [11], [12]]. 3oc-induced cell death is not restricted to immune cells; it induces a loss in cell viability in interstitial epithelial cells, airway epithelial cells, corneal epithelial cells, endothelial cells, and intestinal goblet cells [[13], [14], [15], [16], [17], [18]]. 3oc also have been reported to regulate neutrophil chemotaxis, macrophage phagocytosis, impairs antigen presentation in the dendritic cell, modulate dose-dependent pro-inflammatory and anti-inflammatory responses, and negatively regulate nuclear factor-κB (NF-κB) activation [6,[19], [20], [21], [22], [23]]. 3oc mediates its detrimental effect through Ca+2 signaling dysregulation and activating the mitochondrial intrinsic apoptotic pathway [[24], [25], [26]]. Altogether these studies highlight the clinical relevance of 3oc in better understanding of disease pathologies.
Calcium is a universal secondary messenger that maintains several physiological and pathological functions like gene expression, secretion, transcription, metabolism, and cell death [27]. Ca+2 signaling is spatially and temporally maintained by membrane channels and cytosolic organelles like mitochondria and endoplasmic reticulum (ER) [28]. At the same time, Ca+2 overload in these organelles is apoptogenic [29]. ER is a major storehouse of Ca+2, and excessive ER stress can release Ca+2 to the cytoplasm and other organelles to activate the apoptosis cascade [30]. Moreover, cell-permeable Ca+2 chelator BAPTA-AM is shown to mitigate these Ca+2-dependent catastrophic events by controlling intracellular Ca+2 levels [31].
Mitochondria are crucial cellular organelle in maintaining cellular homeostasis and physiology [32]. Mitochondria are primarily responsible for ATP generation and apoptosis [33]. ER-mitochondria tethering is involved in Ca+2 homeostasis across the mitochondria-associated ER membrane (MAM) and mediates transient Ca+2 buffering and regulation of Ca+2 mediated apoptosis during sustained Ca+2 overload [29]. A physiologically transient rise in mitochondrial Ca+2 increases ATP synthesis and respiratory chain activity [34]. In contrast, pathological sustained Ca+2 overload mediates mitochondrial depolarization, mitochondrial ROS (mROS) generation, and mitochondrial transition pore (mPTP) opening, while prolonged mPTP opening causes the release of intermembrane space proteins and cytochrome c to cytosol leading to activation of caspase-mediated apoptosis.
Autophagy is a catabolic pathway that targets cytoplasmic proteins and organelles to lysosomes [35]. It maintains cellular physiology under stress conditions through metabolite recycling and by eliminating damaged organelles [36]. Moreover, excessive autophagy can cause cell death, as increased organelle degradation results in energetic starvation and induction of autophagic cell death [37]. Autophagy and apoptosis commonly coexist in the same cell, with autophagy generally followed by apoptosis [38]. Further, there is a multitude levels of crosstalk between autophagy and apoptosis, with unexplored intricacy between these two pathways [39]. Autophagy-mediated cell death is abrogated through inhibiting autophagy with the late autophagy inhibitor chloroquine, a lysosomotropic agent that raises the pH of the lysosome, thus inhibiting autophagy flux [37]. Whereas rapamycin, induces autophagy through inhibiting the mammalian target of rapamycin (mTOR), rapamycin is shown to potentiate autophagy-dependent cell death [37]. Several studies have proposed that entry of excess Ca+2 induces autophagy through Ca+2 channels [40]. Autophagy is responsive to both Ca+2 overload and depletion [41]. In contrast, aberrant autophagy induction through intracellular Ca+2 mobilization has been linked to apoptosis [42]. However, buffering intracellular Ca+2 with BAPTA-AM impedes autophagy and shows a protective effect against autophagy-induced cell death [43].
This study showed that 3oc induces a multifaceted death signaling by activating autophagy and apoptosis in human macrophages, whereas aberrant autophagy activation flames the apoptosis pathway and exacerbates 3oc-dependent cell death. Concurrently, our study shed comprehensive insight on the molecular mechanism by which 3oc impairs macrophages through the involvement of autophagy and apoptosis.
Section snippets
Reagents, constructs, and antibodies
N-(3-oxododecanoyl)-L-homoserine lactone (3oc), phorbol-12-myristate-13-acetate (PMA), trypan blue, 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate (Hoechst 33342), propidium iodide (PI), 1, 2-bis (2-amino phenoxy) ethane N, N, N′, N′-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), mono-dansyl-cadaverine (MDC) were procured from Sigma Aldrich, USA. Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), Roswell Park Memorial
3oc triggers apoptosis in human macrophages
First, we investigated the cytotoxic effect of 3oc on differentiated macrophage (U937 cells) by MTT assay, as shown in Fig. 1a, b. Macrophages were treated with varying concentrations of 3oc (10–100 μM) for 2 h and 6 h, and at both time frames, we observed considerable dose-dependent cytotoxicity in macrophages from 50 μM 3oc onwards; here, DMSO was used as vehicle control. While 3oc (10–25 μM) did not show cell toxicity at both 2 h and 6 h, on the contrary, 3oc (100 μM) induced a rapid loss in
Discussion
P. aeruginosa is an opportunistic pathogen that causes severe mortality and morbidity in immunocompromised individuals, and it is one of the major causes of nosocomial infections worldwide [4]. P. aeruginosa has an arsenal of virulence factors that rely on the 3oc regulated QS system. In recent studies, 3oc has also been shown to induce immunomodulatory properties as well as cytotoxicity in eukaryotic cells, implying an independent role in disease establishment and pathogenesis [[8], [9], [10],
Conclusion
In conclusion, this study revealed a novel perspective and implications on the involvement of autophagy in 3oc induced apoptosis in macrophages. The findings of the study are summarised in Fig. 8 (graphical abstract). In this study, we have shown that 3oc activates various events associated with calcium dysregulation, including increased mROS generation, mitochondrial depolarization, and induction of apoptosis. Meanwhile, BAPTA-AM rescues these deleterious effects of 3oc on mitochondria and
CRediT authorship contribution statement
Ankit Kushwaha: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. Rama Shanker Verma: Formal analysis, Writing - review & editing. Vishnu Agarwal: Conceptualization, Funding acquisition, Formal analysis, Writing - review & editing.
Acknowledgments
This study was funded by the Indian Council of Medical Research (ICMR) (AMR/ADHOC/184/2019-ECD-II), Government of India; Fund for Improvement of S&T Infrastructure (FIST) of the Department of Science and Technology (DST), Government of India. Ankit Kushwaha thanks the Ministry of Human Resource Development (MHRD) and University Grants Commission (UGC) for providing the fellowship. The authors are thankful to the Central Instrumentation Facility (CIF), Department of Biotechnology, MNNIT
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