Bioenergetic Heterogeneity in Mycobacterium tuberculosis Residing in Different Subcellular Niches.

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.

Evidence and speculation: the response of Salmonella confronted by autophagy in macrophages.

Bacteria of the Salmonella genus cause diseases ranging from self-limited gastroenteritis to typhoid fever. Macrophages are immune cells that engulf and restrict Salmonella. These cells will carry Salmonella into the circulatory system and provoke a systemic infection. Therefore, the interaction between macrophages and intracellular Salmonella is vital for its pathogenicity. As one of the immune responses of macrophages, autophagy, along with the fusion of autophagosomes with lysosomes, occupies an important position in eliminating Salmonella. However, Salmonella that can overcome cellular defensive responses and infect neighboring cells must derive strategies to escape autophagy. This review introduces novel findings on Salmonella and macrophage autophagy as a mechanism against infection and explores the strategies used by Salmonella to escape autophagy.

Invasion of Human Retinal Pigment Epithelial Cells by Porphyromonas gingivalis leading to Vacuolar/Cytosolic localization and Autophagy dysfunction In-Vitro.

Recent epidemiological  studies link Periodontal disease(PD) to age-related macular degeneration (AMD). We documented earlier that Porphyromonas gingivalis(Pg), keystone oral-pathobiont, causative of PD, efficiently invades human gingival epithelial and blood-dendritic cells. Here, we investigated the ability of dysbiotic Pg-strains to invade human-retinal pigment epithelial cells(ARPE-19), their survival, intracellular localization, and the pathological effects, as dysfunction of RPEs leads to AMD. We show that live, but not heat-killed Pg-strains adhere to and invade ARPEs. This involves early adhesion to ARPE cell membrane, internalization and localization of Pg within single-membrane vacuoles or cytosol, with some nuclear localization apparent. No degradation of Pg or localization inside double-membrane autophagosomes was evident, with dividing Pg suggesting a metabolically active state during invasion. We found significant downregulation of autophagy-related genes particularly, autophagosome complex. Antibiotic protection-based recovery assay further confirmed distinct processes of adhesion, invasion and amplification of Pg within ARPE cells. This is the first study to demonstrate invasion of human-RPEs, begin to characterize intracellular localization and survival of Pg within these cells. Collectively, invasion of RPE by Pg and its prolonged survival by autophagy evasion within these cells suggest a strong rationale for studying the link between oral infection and AMD pathogenesis in individuals with periodontitis.

Staphylococcus aureus Avoids Autophagy Clearance of Bovine Mammary Epithelial Cells by Impairing Lysosomal Function.

In dairy herds, mastitis caused by Staphylococcus aureus is difficult to completely cure on the account that S. aureus can invade bovine mammary epithelial cells (BMECs) and result in persistent infection in the mammary gland. Recent studies have demonstrated that autophagy can participate in cell homeostasis by eliminating intracellular microorganisms. The aim of the study was to investigate why S. aureus can evade autophagy clearance and survive in BMECs. The intracellular infection model was first constructed; then, the bacteria in autophagosome was detected by transmission electron microscopy. The autophagy flux induced by the S. aureus was also evaluated by immunoblot analysis and fluorescent labeling method for autophagy marker protein LC3. In addition, lysosomal alkalization and degradation ability were assessed using confocal microscopy. Results showed that, after infection, a double-layer membrane structure around the S. aureus was observed in BMECs, indicating that autophagy occurred. The change in autophagy marker protein and fluorescent labeling of autophagosome also confirmed autophagy. However, as time prolonged, the autophagy flux was markedly inhibited, leading to obvious autophagosome accumulation. At the same time, the lysosomal alkalization and degradation ability of BMECs were impaired. Collectively, these results indicated that S. aureus could escape autophagic degradation by inhibiting autophagy flux and damaging lysosomal function after invading BMECs.

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

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.

Cross-talk between CD38 and TTP Is Essential for Resolution of Inflammation during Microbial Sepsis.

The resolution phase of acute inflammation is essential for tissue homeostasis, yet the underlying mechanisms remain unclear. We demonstrate that resolution of inflammation involves interactions between CD38 and tristetraprolin (TTP). During the onset of acute inflammation, CD38 levels are increased, leading to the production of Ca2+-signaling messengers, nicotinic acid adenine dinucleotide phosphate (NAADP), ADP ribose (ADPR), and cyclic ADPR (cADPR) from NAD(P)+. To initiate the onset of resolution, TTP expression is increased by the second messengers, NAADP and cADPR, which downregulate CD38 expression. The activation of TTP by Sirt1-dependent deacetylation, in response to increased NAD+ levels, suppresses the acute inflammatory response and decreases Rheb expression, inhibits mTORC1, and induces autophagolysosomes for bacterial clearance. TTP may represent a mechanistic target of anti-inflammatory agents, such as carbon monoxide. TTP mediates crosstalk between acute inflammation and autophagic clearance of bacteria from damaged tissue in the resolution of inflammation during sepsis.

Autophagy Receptor Tollip Facilitates Bacterial Autophagy by Recruiting Galectin-7 in Response to Group A Streptococcus Infection.

Bacterial autophagy-a type of macroautophagy that is also termed xenophagy-selectively targets intracellular bacteria such as group A Streptococcus (GAS), a ubiquitous pathogen that causes numerous serious diseases, including pharyngitis, skin infections, and invasive life-threatening infections. Although bacterial autophagy is known to eliminate invading bacteria via the action of autophagy receptors, the underlying mechanism remains unclear. Herein, we elucidated that Tollip functions as a bacterial-autophagy receptor in addition to participating involved in the intracellular immunity mechanism that defends against bacterial infection. Tollip was recruited to GAS-containing endosomal vacuoles prior to the escape of GAS into the cytosol; additionally, Tollip knockout disrupted the recruitment of other autophagy receptors, such as NBR1, TAX1BP1, and NDP52, to GAS-containing autophagosomes and led to prolonged intracellular survival of GAS. Furthermore, Tollip was found to be required for the recruitment of galectin-1 and -7 to GAS-containing autophagosomes, and immunoprecipitation results indicated that Tollip interacts with galectin-7. Lastly, our data also revealed that galectin-1 and -7 are involved in the restriction of GAS replication in cells. These results demonstrated that Tollip modulates bacterial autophagy by recruiting other autophagy receptors and galectins.

The role of autophagy in the pathogenesis of periodontal disease.

Periodontal disease is a chronic inflammatory disease leading to destruction of periodontal tissues. As a local inflammation, periodontopathic bacterium, pro-inflammatory mediators, and local immune response play pivotal role in the progress of periodontal disease. Besides, cigarette smoke has long been associated with periodontal disease and tooth loss. Autophagy is an intracellular degradation process highly conserved from yeast to humans. As a lysosomal degradation pathway of self-digestion, it is critical for maintaining cells homeostasis and development. The role of autophagy has been investigated in oral diseases, such as oral cancer, periapical lesions, and oral candidiasis. Recently, increasing studies investigated the role of autophagy in periodontal disease. In this review, we try to illustrate the effect of autophagy on periodontal disease pathogenesis from 5 aspects: autophagy affects the intracellular infection and survival of bacteria; autophagy has an interaction with periodontal inflammation; autophagy is pivotal in periodontal cells biology and periodontal tissues destruction and reconstruction; autophagy can be induced by cigarette smoke; last but not least, autophagy may affect periodontal disease via endoplasmic reticulum stress.

Mycobacterium tuberculosis sensor kinase DosS modulates the autophagosome in a DosR-independent manner.

Dormancy is a key characteristic of the intracellular life-cycle of Mtb. The importance of sensor kinase DosS in mycobacteria are attributed in part to our current findings that DosS is required for both persistence and full virulence of Mtb. Here we show that DosS is also required for optimal replication in macrophages and involved in the suppression of TNF-α and autophagy pathways. Silencing of these pathways during the infection process restored full virulence in MtbΔdosS mutant. Notably, a mutant of the response regulator DosR did not exhibit the attenuation in macrophages, suggesting that DosS can function independently of DosR. We identified four DosS targets in Mtb genome; Rv0440, Rv2859c, Rv0994, and Rv0260c. These genes encode functions related to hypoxia adaptation, which are not directly controlled by DosR, e.g., protein recycling and chaperoning, biosynthesis of molybdenum cofactor and nitrogen metabolism. Our results strongly suggest a DosR-independent role for DosS in Mtb.

Diverse Cellular Roles of Autophagy.

Macroautophagy is an intracellular degradation system that delivers diverse cytoplasmic materials to lysosomes via autophagosomes. Recent advances have enabled identification of several selective autophagy substrates and receptors, greatly expanding our understanding of the cellular functions of autophagy. In this review, we describe the diverse cellular functions of macroautophagy, including its essential contribution to metabolic adaptation and cellular homeostasis. We also discuss emerging findings on the mechanisms and functions of various types of selective autophagy.

Celiac disease-associated Neisseria flavescens decreases mitochondrial respiration in CaCo-2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial-induced cellular imbalance.

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis.

Activation of the macroautophagy pathway by Yersinia enterocolitica promotes intracellular multiplication and egress of yersiniae from epithelial cells.

The virulence strategy of pathogenic Yersinia spp. involves cell-invasive as well as phagocytosis-preventing tactics to enable efficient colonisation of the host organism. Enteropathogenic yersiniae display an invasive phenotype in early infection stages, which facilitates penetration of the intestinal mucosa. Here we show that invasion of epithelial cells by Yersinia enterocolitica is followed by intracellular survival and multiplication of a subset of ingested bacteria. The replicating bacteria were enclosed in vacuoles with autophagy-related characteristics, showing phagophore formation, xenophagy, and recruitment of cytoplasmic autophagosomes to the bacteria-containing compartments. The subsequent fusion of these vacuoles with lysosomes and concomitant vesicle acidification were actively blocked by Yersinia. This resulted in increased intracellular proliferation and detectable egress of yersiniae from infected cells. Notably, deficiency of the core autophagy machinery component FIP200 impaired the development of autophagic features at Yersinia-containing vacuoles as well as intracellular replication and release of bacteria to the extracellular environment. These results suggest that Y. enterocolitica may take advantage of the macroautophagy pathway in epithelial cells to create an autophagosomal niche that supports intracellular bacterial survival, replication, and, eventually, spread of the bacteria from infected cells.

HIF1A and NFAT5 coordinate Na+-boosted antibacterial defense via enhanced autophagy and autolysosomal targeting.

Infection and inflammation are able to induce diet-independent Na+-accumulation without commensurate water retention in afflicted tissues, which favors the pro-inflammatory activation of mouse macrophages and augments their antibacterial and antiparasitic activity. While Na+-boosted host defense against the protozoan parasite Leishmania major is mediated by increased expression of the leishmanicidal NOS2 (nitric oxide synthase 2, inducible), the molecular mechanisms underpinning this enhanced antibacterial defense of mouse macrophages with high Na+ (HS) exposure are unknown. Here, we provide evidence that HS-increased antibacterial activity against E. coli was neither dependent on NOS2 nor on the phagocyte oxidase. In contrast, HS-augmented antibacterial defense hinged on HIF1A (hypoxia inducible factor 1, alpha subunit)-dependent increased autophagy, and NFAT5 (nuclear factor of activated T cells 5)-dependent targeting of intracellular E. coli to acidic autolysosomal compartments. Overall, these findings suggest that the autolysosomal compartment is a novel target of Na+-modulated cell autonomous innate immunity. Abbreviations: ACT: actins; AKT: AKT serine/threonine kinase 1; ATG2A: autophagy related 2A; ATG4C: autophagy related 4C, cysteine peptidase; ATG7: autophagy related 7; ATG12: autophagy related 12; BECN1: beclin 1; BMDM: bone marrow-derived macrophages; BNIP3: BCL2/adenovirus E1B interacting protein 3; CFU: colony forming units; CM-H2DCFDA: 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester; CTSB: cathepsin B; CYBB: cytochrome b-245 beta chain; DAPI: 4,6-diamidino-2-phenylindole; DMOG: dimethyloxallyl glycine; DPI: diphenyleneiodonium chloride; E. coli: Escherichia coli; FDR: false discovery rate; GFP: green fluorescent protein; GSEA: gene set enrichment analysis; GO: gene ontology; HIF1A: hypoxia inducible factor 1, alpha subunit; HUGO: human genome organization; HS: high salt (+ 40 mM of NaCl to standard cell culture conditions); HSP90: heat shock 90 kDa proteins; LDH: lactate dehydrogenase; LPS: lipopolysaccharide; Lyz2/LysM: lysozyme 2; NFAT5/TonEBP: nuclear factor of activated T cells 5; MΦ: macrophages; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MIC: minimum inhibitory concentration; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NaCl: sodium chloride; NES: normalized enrichment score; n.s.: not significant; NO: nitric oxide; NOS2/iNOS: nitric oxide synthase 2, inducible; NS: normal salt; PCR: polymerase chain reaction; PGK1: phosphoglycerate kinase 1; PHOX: phagocyte oxidase; RFP: red fluorescent protein; RNA: ribonucleic acid; ROS: reactive oxygen species; sCFP3A: super cyan fluorescent protein 3A; SBFI: sodium-binding benzofuran isophthalate; SLC2A1/GLUT1: solute carrier family 2 (facilitated glucose transporter), member 1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like kinase 1; v-ATPase: vacuolar-type H+-ATPase; WT: wild type.

Autophagy links antimicrobial activity with antigen presentation in Langerhans cells.

DC, through the uptake, processing, and presentation of antigen, are responsible for activation of T cell responses to defend the host against infection, yet it is not known if they can directly kill invading bacteria. Here, we studied in human leprosy, how Langerhans cells (LC), specialized DC, contribute to host defense against bacterial infection. IFN-γ treatment of LC isolated from human epidermis and infected with Mycobacterium leprae (M. leprae) activated an antimicrobial activity, which was dependent on the upregulation of the antimicrobial peptide cathelicidin and induction of autophagy. IFN-γ induction of autophagy promoted fusion of phagosomes containing M. leprae with lysosomes and the delivery of cathelicidin to the intracellular compartment containing the pathogen. Autophagy enhanced the ability of M. leprae-infected LC to present antigen to CD1a-restricted T cells. The frequency of IFN-γ labeling and LC containing both cathelicidin and autophagic vesicles was greater in the self-healing lesions vs. progressive lesions, thus correlating with the effectiveness of host defense against the pathogen. These data indicate that autophagy links the ability of DC to kill and degrade an invading pathogen, ensuring cell survival from the infection while facilitating presentation of microbial antigens to resident T cells.

Measuring Antibacterial Autophagy.

Bacteria that escape from membrane-enclosed vacuoles to the cytosol of cells are targeted by autophagy, which recognizes and captures bacteria into autophagosomes wherein their proliferation is restricted. Here we discuss two means by which antibacterial autophagy is assessed: (1) the visualization and enumeration of autophagy protein recruitment to the vicinity of cytosolic bacteria by means of immunofluorescence microscopy and (2) the measurement of autophagy-dependent restriction of bacterial proliferation by means of colony-forming unit assay.

Vitamin D3 activates the autolysosomal degradation function against Helicobacter pylori through the PDIA3 receptor in gastric epithelial cells.

Helicobacter pylori (H. pylori) is a common human pathogenic bacterium. Once infected, it is difficult for the host to clear this organism using the innate immune system. Increased antibiotic resistance further makes it challenging for effective eradication. However, the mechanisms of immune evasion still remain obscure, and novel strategies should be developed to efficiently eliminate H. pylori infection in stomachs. Here we uncovered desirable anti-H. pylori effect of vitamin D3 both in vitro and in vivo, even against antibiotic-resistant strains. We showed that H. pylori can invade into the gastric epithelium where they became sequestered and survived in autophagosomes with impaired lysosomal acidification. Vitamin D3 treatment caused a restored lysosomal degradation function by activating the PDIA3 receptor, thereby promoting the nuclear translocation of PDIA3-STAT3 protein complex and the subsequent upregulation of MCOLN3 channels, resulting in an enhanced Ca2+ release from lysosomes and normalized lysosomal acidification. The recovered lysosomal degradation function drives H. pylori to be eliminated through the autolysosomal pathway. These findings provide a novel pathogenic mechanism on how H. pylori can survive in the gastric epithelium, and a unique pathway for vitamin D3 to reactivate the autolysosomal degradation function, which is critical for the antibacterial action of vitamin D3 both in cells and in animals, and perhaps further in humans. Abbreviations: 1,25D3: 1α, 25-dihydroxyvitamin D3; ATG5: autophagy related 5; Baf A1: bafilomycin A1; BECN1: beclin 1; CagA: cytotoxin-associated gene A; CFU: colony-forming unit; ChIP-PCR: chromatin immunoprecipitation-polymerase chain reaction; Con A: concanamycin A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; CTSD: cathepsin D; GPN: Gly-Phe-ß-naphthylamide; H. pylori: Helicobacter pylori; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; MCOLN3: mucolipin 3; MCU: mitochondrial calcium uniporter; MOI: multiplicity of infection; NAGLU: N-acetyl-alpha-glucosaminidase; PDIA3: protein disulfide isomerase family A member 3; PMA: phorbol 12-myristate 13-acetate; PRKC: protein kinase C; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; SS1: Sydney Strain 1; TRP: transient receptor potential; VacA: vacuolating cytotoxin; VD3: vitamin D3; VDR: vitamin D receptor.

A Subpopulation of Intracellular Neisseria gonorrhoeae Escapes Autophagy-Mediated Killing Inside Epithelial Cells.

The bacterial pathogen Neisseria gonorrhoeae is able to transmigrate across the mucosal epithelia following the intracellular route and cause disseminated infections. It is currently unknown whether the autophagy pathway is able target intracellular N. gonorrhoeae for destruction in autolysosomes or whether this bacterium is able to escape autophagy-mediated killing. In this study, we demonstrate that during the early stage of epithelial cell invasion, N. gonorrhoeae is targeted by the autophagy pathway and sequestered into double-membrane autophagosomes that subsequently fuse with lysosomes for destruction. However, a subpopulation of the intracellular gonococci is able to escape early autophagy-mediated killing. N. gonorrhoeae is subsequently able to inhibit this pathway, allowing intracellular survival and exocytosis. During this stage, N. gonorrhoeae activates the autophagy repressor mammalian target of rapamycin complex 1 and inhibits autophagosome maturation and lysosome fusion. Thus, our results provide novel insight into the interactions between N. gonorrhoeae and the autophagy pathway during invasion and transcytosis of epithelial cells.

RAB30 regulates PI4KB (phosphatidylinositol 4-kinase beta)-dependent autophagy against group A Streptococcus.

Macroautophagy/autophagy plays an important role in the immune response to invasion by intracellular pathogens such as group A Streptococcus (GAS; Streptococcus pyogenes). We previously identified RAB30, a Golgi-resident GTPase, as a novel anti-bacterial autophagic regulator in the formation of GAS-containing autophagosome-like vacuoles (GcAVs); however, the precise mechanism underlying this process remains elusive. Here, we elucidate a novel property of RAB30: the ability to recruit PI4KB (phosphatidylinositol 4-kinase beta) to the Golgi apparatus and GcAVs. We found that trans-Golgi network (TGN) vesicles were incorporated into GcAVs via RAB30 to promote GcAV formation. Moreover, depletion of phosphatidylinositol-4-phosphate (PtdIns4P), a phosphatidylinositol enriched in the TGN, by wortmannin and phenylarsine oxide, followed by subsequent repletion with exogenous PtdIns4P revealed that PtdIns4P is crucial for GcAV formation. Furthermore, we identify an interaction between RAB30 and PI4KB, in which the knockdown of RAB30 decreased the localization of PI4KB to the TGN and GcAVs. Finally, PI4KB knockout suppressed autophagy by inhibiting GcAV formation, resulting in the increased survival of GAS. Our results demonstrate a novel autophagosomal formation mechanism involving coordinative functions of RAB30 and PI4KB distinct from those utilized in canonical autophagy. Abbreviations: GAS: group A Streptococcus; GcAVs: GAS-containing autophagosome-like vacuoles; PI4KB: phosphatidylinositol 4-kinase beta; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns5P: phosphatidylinositol-5-phosphate; SLO: streptolysin O; TGN: trans-Golgi network; TGOLN2: trans-golgi network protein 2; PH: plekstrin homology; OSBP: oxysterol binding protein.

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection.

CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.

Autophagy and Its Interaction With Intracellular Bacterial Pathogens.

Cellular responses to stress can be defined by the overwhelming number of changes that cells go through upon contact with and stressful conditions such as infection and modifications in nutritional status. One of the main cellular responses to stress is autophagy. Much progress has been made in the understanding of the mechanisms involved in the induction of autophagy during infection by intracellular bacteria. This review aims to discuss recent findings on the role of autophagy as a cellular response to intracellular bacterial pathogens such as, Streptococcus pyogenes, Mycobacterium tuberculosis, Shigella flexneri, Salmonella typhimurium, Listeria monocytogenes, and Legionella pneumophila, how the autophagic machinery senses these bacteria directly or indirectly (through the detection of bacteria-induced nutritional stress), and how some of these bacterial pathogens manage to escape from autophagy.