Laryngeal squamous cell carcinoma (LSCC) is the second most common malignant tumor in head and neck. Autophagy and circular RNAs (circRNAs) play critical roles in cancer progression and chemoresistance. However, the function and mechanism of circRNA in autophagy regulation of LSCC remain unclear. The autophagy-suppressive circRNA circPARD3 was identified via RNA sequencing of 107 LSCC tissues and paired adjacent normal mucosal (ANM) tissues and high-content screening. RT-PCR, Sanger sequencing, qPCR and fluorescence in situ hybridization were performed to detect circPARD3 expression and subcellular localization. Biological functions of circPARD3 were assessed by proliferation, migration, invasion, autophagic flux, and chemoresistance assays using in vitro and in vivo models. The mechanism of circPARD3 was investigated by RNA immunoprecipitation, RNA pulldown, luciferase reporter assays, western blotting and immunohistochemical staining. Autophagy was inhibited in LSCC, and circPARD3 was upregulated in the LSCC tissues (n = 100, p < 0.001). High circPARD3 level was associated with advanced T stages (p < 0.05), N stages (p = 0.001), clinical stages (p < 0.001), poor differentiation degree (p = 0.025), and poor prognosis (p = 0.002) of LSCC patients (n = 100). Functionally, circPARD3 inhibited autophagy and promoted LSCC cell proliferation, migration, invasion and chemoresistance. We further revealed that activation of the PRKCI-Akt-mTOR pathway through sponging miR-145-5p was the main mechanism of circPARD3 inhibited autophagy, promoting LSCC progression and chemoresistance. Our study reveals that the novel autophagy-suppressive circPARD3 promotes LSCC progression and chemoresistance through the PRKCI-Akt-mTOR pathway, providing new insights into circRNA-mediated autophagy regulation and potential biomarker and target for LSCC treatment.

/pdf/circpard3-drives-malignant-progression-and-chemoresistance-1mojsijfrc.pdf

circPARD3 drives malignant progression and chemoresistance of laryngeal squamous cell carcinoma by inhibiting autophagy through the PRKCI-Akt-mTOR pathway

Autophagy is a fundamental and highly conserved eukaryotic process, responsible for maintaining cellular homeostasis and releasing nutrients during times of starvation. An increasingly important function of autophagy is its role in the cell autonomous immune response; a process known as xenophagy. Intracellular pathogens are engulfed by autophagosomes and targeted to lysosomes to eliminate the threat to the host cell. To counteract this, many intracellular bacterial pathogens have developed unique approaches to overcome, evade, or co-opt host autophagy to facilitate a successful infection. The intracellular bacteria Legionella pneumophila and Coxiella burnetii are able to avoid destruction by the cell, causing Legionnaires' disease and Q fever, respectively. Despite being related and employing homologous Dot/Icm type 4 secretion systems (T4SS) to translocate effector proteins into the host cell, these pathogens have developed their own unique intracellular niches. L. pneumophila evades the host endocytic pathway and instead forms an ER-derived vacuole, while C. burnetii requires delivery to mature, acidified endosomes which it remodels into a large, replicative vacuole. Throughout infection, L. pneumophila effectors act at multiple points to inhibit recognition by xenophagy receptors and disrupt host autophagy, ensuring it avoids fusion with destructive lysosomes. In contrast, C. burnetii employs its effector cohort to control autophagy, hypothesized to facilitate the delivery of nutrients and membrane to support the growing vacuole and replicating bacteria. In this review we explore the effector proteins that these two organisms utilize to modulate the host autophagy pathway in order to survive and replicate. By better understanding how these pathogens manipulate this highly conserved pathway, we can not only develop better treatments for these important human diseases, but also better understand and control autophagy in the context of human health and disease.

/pdf/interfering-with-autophagy-the-opposing-strategies-deployed-j0y3tlm1vh.pdf

Interfering with Autophagy: The Opposing Strategies Deployed by Legionella pneumophila and Coxiella burnetii Effector Proteins.

The Q fever agent Coxiella burnetii uses a defect in organelle trafficking/intracellular multiplication (Dot/Icm) type 4b secretion system (T4SS) to silence the host innate immune response during infection. By investigating C. burnetii effector proteins containing eukaryotic-like domains, here we identify NopA (nucleolar protein A), which displays four regulator of chromosome condensation (RCC) repeats, homologous to those found in the eukaryotic Ras-related nuclear protein (Ran) guanine nucleotide exchange factor (GEF) RCC1. Accordingly, NopA is found associated with the chromatin nuclear fraction of cells and uses the RCC-like domain to interact with Ran. Interestingly, NopA triggers an accumulation of Ran-GTP, which accumulates at nucleoli of transfected or infected cells, thus perturbing the nuclear import of transcription factors of the innate immune signaling pathway. Accordingly, qRT-PCR analysis on a panel of cytokines shows that cells exposed to the C. burnetii nopA::Tn or a Dot/Icm-defective dotA::Tn mutant strain present a functional innate immune response, as opposed to cells exposed to wild-type C. burnetii or the corresponding nopA complemented strain. Thus, NopA is an important regulator of the innate immune response allowing Coxiella to behave as a stealth pathogen.

https://www.pnas.org/content/pnas/early/2020/05/28/1914892117.full.pdf

Modulation of innate immune signaling by a Coxiella burnetii eukaryotic-like effector protein

Autophagy is a highly conserved and fundamental cellular process to maintain cellular homeostasis through recycling of defective organelles or proteins. In a response to intracellular pathogens, autophagy further acts as an innate immune response mechanism to eliminate pathogens. This review will discuss recent findings on autophagy as a reaction to intracellular pathogens, such as Salmonella typhimurium, Listeria monocytogenes, Mycobacterium tuberculosis, Staphylococcus aureus, and pathogenic Escherichia coli. Interestingly, while some of these bacteria have developed methods to use autophagy for their own benefit within the cell, others have developed fascinating mechanisms to evade recognition, to subvert the autophagic pathway, or to escape from autophagy.

https://www.mdpi.com/2076-0817/10/2/110/pdf

Autophagy-A Story of Bacteria Interfering with the Host Cell Degradation Machinery.

Both symbiotic and pathogenic bacteria in the family Coxiellaceae cause morbidity and mortality in humans and animals. For instance, Coxiella-like endosymbionts (CLEs) improve the reproductive success of ticks - a major disease vector, while Coxiella burnetii causes human Q fever, and uncharacterized coxiellae infect both animals and humans. To better understand the evolution of pathogenesis and symbiosis in this group of intracellular bacteria, we sequenced the genome of a CLE present in the soft tick Ornithodoros amblus (CLEOA) and compared it to the genomes of other bacteria in the order Legionellales. Our analyses confirmed that CLEOA is more closely related to C. burnetii, the human pathogen, than to CLEs in hard ticks, and showed that most clades of CLEs contain both endosymbionts and pathogens, indicating that several CLE lineages have evolved independently from pathogenic Coxiella. We also determined that the last common ancestor of CLEOA and C. burnetii was equipped to infect macrophages, and that even though horizontal gene transfer (HGT) contributed significantly to the evolution of C. burnetii, most acquisition events occurred primarily in ancestors predating the CLEOA-C. burnetii divergence. These discoveries clarify the evolution of C. burnetii, which previously was assumed to have emerged when an avirulent tick endosymbiont recently gained virulence factors via HGT. Finally, we identified several metabolic pathways, including heme biosynthesis, that are likely critical to the intracellular growth of the human pathogen but not the tick symbiont, and show that the use of heme analog is a promising approach to controlling C. burnetii infections.

/pdf/coxiella-burnetii-and-related-tick-endosymbionts-evolved-4rvfd8kupd.pdf

Coxiella burnetii and related tick endosymbionts evolved from pathogenic ancestors.

Coxiella burnetii, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a Coxiella transposon mutant library, we observed that transposon insertions in cbu0626 led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the Coxiella vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as Coxiella-containing vacuoles (CCVs). We thus renamed this effector CvpF for Coxiella vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, cvpF::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for Coxiella virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal C. burnetii vacuole biogenesis.Abbreviations: ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: Coxiella-containing vacuole; Cvp: Coxiella vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.

Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence.

https://hal.umontpellier.fr/hal-02322148/document

The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for severe worldwide outbreaks of the zoonosis Q fever. The remarkable resistance to environmental stress, extremely low infectious dose and ease of dissemination, contributed to the classification of C. burnetii as a class B biothreat. Unique among intracellular pathogens, C. burnetii escapes immune surveillance and replicates within large autophagolysosome-like compartments called Coxiella-containing vacuoles (CCVs). The biogenesis of these compartments depends on the subversion of several host signalling pathways. For years, the obligate intracellular nature of C. burnetii imposed significant experimental obstacles to the study of its pathogenic traits. With the development of an axenic culture medium in 2009, C. burnetii became genetically tractable, thus allowing the implementation of mutagenesis tools and screening approaches to identify its virulence determinants and investigate its complex interaction with host cells. Here, we review the key advances that have contributed to our knowledge of C. burnetii pathogenesis, leading to the rise of this once-neglected pathogen to an exceptional organism to study the intravacuolar lifestyle.

https://hal.archives-ouvertes.fr/hal-02993239/document

Melanie Burette

Papers

Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence.

Modulation of innate immune signaling by a Coxiella burnetii eukaryotic-like effector protein

The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5

From neglected to dissected: How technological advances are leading the way to the study of Coxiella burnetii pathogenesis