Antimicrobial Autophagy Research Articles

Atg8ylation as a host-protective mechanism against Mycobacterium tuberculosis.

Nearly two decades have passed since the first report on autophagy acting as a cell-autonomous defense against Mycobacterium tuberculosis. This helped usher a new area of research within the field of host-pathogen interactions and led to the recognition of autophagy as an immunological mechanism. Interest grew in the fundamental mechanisms of antimicrobial autophagy and in the prophylactic and therapeutic potential for tuberculosis. However, puzzling in vivo data have begun to emerge in murine models of M. tuberculosis infection. The control of infection in mice affirmed the effects of certain autophagy genes, specifically ATG5, but not of other ATGs. Recent studies with a more complete inactivation of ATG genes now show that multiple ATG genes are indeed necessary for protection against M. tuberculosis. These particular ATG genes are involved in the process of membrane atg8ylation. Atg8ylation in mammalian cells is a broad response to membrane stress, damage and remodeling of which canonical autophagy is one of the multiple downstream outputs. The current developments clarify the controversies and open new avenues for both fundamental and translational studies.

Porphyromonas gingivalis Evades Immune Clearance by Regulating Lysosome Efflux

Porphyromonas gingivalis, a major periodontal pathogen, invades autophagosomes of cells, including gingival epithelial cells, endothelial cells, gingival fibroblasts, macrophages, and dendritic cells, to escape antimicrobial autophagy and lysosome fusion. However, it is not known how P. gingivalis resists autophagic immunity, survives within cells, and induces inflammation. Thus, we investigated whether P. gingivalis could escape antimicrobial autophagy by promoting lysosome efflux to block autophagic maturation, leading to intracellular survival, and whether the growth of P. gingivalis within cells results in cellular oxidative stress, causing mitochondrial damage and inflammatory responses. P. gingivalis invaded human immortalized oral epithelial cells in vitro and mouse oral epithelial cells of gingival tissues in vivo. The production of reactive oxygen species (ROS) increased upon bacterial invasion, as well as mitochondrial dysfunction-related parameters with downregulated mitochondrial membrane potential and intracellular adenosine triphosphate (ATP), upregulated mitochondrial membrane permeability, intracellular Ca2+ influx, mitochondrial DNA expression, and extracellular ATP. Lysosome excretion was elevated, the number of intracellular lysosomes was diminished, and lysosomal-associated membrane protein 2 was downregulated. Expression of autophagy-related proteins, microtubule-associated protein light chain 3, sequestosome-1, the NLRP3 inflammasome, and interleukin-1β increased with P. gingivalis infection. P. gingivalis may survive in vivo by promoting lysosome efflux, blocking autophagosome-lysosome fusion, and destroying autophagic flux. As a result, ROS and damaged mitochondria accumulated and activated the NLRP3 inflammasome, which recruited the adaptor protein ASC and caspase 1, leading to the production of proinflammatory factor interleukin-1β and inflammation.

Bag it, tag it: ubiquitin ligases and host resistance to Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), remains a significant global epidemic. Host resistance to Mtb depends on both adaptive and innate immunity mechanisms, including development of antigen-specific CD4 and CD8 T cells, production of inflammatory cytokines, bacterial phagocytosis and destruction within phagolysosomes, host cell apoptosis, and autophagy. A key regulatory mechanism in innate immunity is the attachment of the small protein ubiquitin to protein and lipid targets by the enzymatic activity of ubiquitin ligases. Here, we summarize the latest advances on the role of ubiquitination and ubiquitin ligases in host immunity against Mtb, with a focus on innate immunity signaling, inflammation, and antimicrobial autophagy. Understanding how ubiquitin ligases mediate immunity to Mtb, and the specific substrates of distinct ubiquitin ligases in the context of Mtb infection, could facilitate development of new host-directed antimicrobials.

A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component

Beyond its role in cellular homeostasis, autophagy plays anti‐ and promicrobial roles in host–microbe interactions, both in animals and plants. One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well‐described in animals, the extent to which xenophagy contributes to plant–bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type‐III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense‐related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense‐related autophagy in plant–bacteria interactions.

Relish regulates innate immunity via mediating ATG5 activity in Antheraea pernyi

In innate immunity, autophagy is an important molecular mechanism that plays a critical role in the animal defense system. Given the importance of anti-microbial autophagy in the innate immune processes, the relationship between anti-microbial autophagy and LPS-induced innate immunity in A. pernyi was investigated. Quantitative RT-PCR analysis revealed that autophagy-related genes (ATG6, ATG5, and ATG12) were induced following LPS injection. LPS treatment in the Relish knockdown larvae reduced the expression of autophagy-related genes, especially ATG5. Furthermore, ATG5 depletion decreased the innate immune effect, while its over-expression with ATG12 was induced after the LPS challenge. The dual-luciferase assay revealed that Relish could regulate ATG5 expression by binding directly to the promoter of the ATG5 gene. Overall, our findings show that Relish regulates the ATG5 transcription to eliminate Gram-negative bacteria by anti-microbial autophagy, implying a strong connection between autophagy and innate immunity in immunologic homeostasis.

An oomycete effector subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface.

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phytophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway that antagonizes antimicrobial autophagy at the pathogen interface. Here, we show that PexRD54 induces autophagosome formation by bridging vesicles decorated by the small GTPase Rab8a with autophagic compartments labeled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing specific trafficking pathways underpin selective autophagy. By subverting Rab8a-mediated vesicle trafficking, PexRD54 utilizes lipid droplets to facilitate biogenesis of autophagosomes diverted to pathogen feeding sites. Altogether, we show that PexRD54 mimics starvation-induced autophagy to subvert endomembrane trafficking at the host-pathogen interface, revealing how effectors bridge distinct host compartments to expedite colonization.

Lysine methylation shields an intracellular pathogen from ubiquitylation and autophagy.

Many intracellular pathogens avoid detection by their host cells. However, it remains unknown how they avoid being tagged by ubiquitin, an initial step leading to antimicrobial autophagy. Here, we show that the intracellular bacterial pathogen Rickettsia parkeri uses two protein-lysine methyltransferases (PKMTs) to modify outer membrane proteins (OMPs) and prevent their ubiquitylation. Mutants deficient in the PKMTs were avirulent in mice and failed to grow in macrophages because of ubiquitylation and autophagic targeting. Lysine methylation protected the abundant surface protein OmpB from ubiquitin-dependent depletion from the bacterial surface. Analysis of the lysine-methylome revealed that PKMTs modify a subset of OMPs, including OmpB, by methylation at the same sites that are modified by host ubiquitin. These findings show that lysine methylation is an essential determinant of rickettsial pathogenesis that shields bacterial proteins from ubiquitylation to evade autophagic targeting.

Vitamin D and COVID-19: A Physiological Perspective

Already known to be capable of enhancing the performance of the immune system, preclude microbial infections, and reduce susceptibility to influenza, Vitamin D, or 1,25-dihydroxyvitamin D (1,25(OH)2D) as an active form, it has maintained a positive reputation in discourses regarding COVID-19 for a lengthy amount of time. Its deficiency is statistically correlated with infection of the disease, the severity thereof, and fatalities therefrom. There have also existed suggestions that Vitamin D supplements could either prevent contracting SARS-CoV-2, the Coronavirus that causes the disease, or alleviate the symptoms it is capable of begetting, since data has established a relationship between Vitamin D supplementation and the severity of respiratory illnesses and certain assays indicated optimistic results in COVID-19 patients supplemented with the vitamin. Accordingly, a great deal of research and efforts have been put into investigating the physiological mechanisms in the human body attributable to the vitamin's reputation against the Coronavirus and combined with what was already discovered before the advent of SARS-CoV-2, a great amount of knowledge has consequently been unveiled. Via regulation of various pathways, 1,25(OH)2D promotes the production of antimicrobial peptides, autophagy, the integrity and impermeability of cellular junctions against pathogens, as well as mitigate the consequences of SARS-CoV-2 infection such as cytokine storm through immunomodulation of the T cell differentiation pathways and lung injury through stimulation of the Angiotensin-Converting Enzyme 2 (ACE2) and manipulation of the Renin-Angiotensin System (RAS). Nevertheless, the unfortunate fact is that Vitamin D deficiency still plagues the global population across all age categories, which could have translated into humanity's heightened exposure to the Coronavirus. With the COVID-19 pandemic inexorably raging on with no prospect of termination observable in the foreseeable future, this review article provides a concise yet thorough insight into that vast knowledge, which could illuminate the significance of Vitamin D amidst the current predicament experienced by mankind, as well as instigate more curiosity as to any current unclarity related to the issue and ignite further discussions and studies that would deliver more understanding to the vitamin's role as a safeguard against SARS-CoV-2

Immunomodulatory Agents Combat Multidrug-Resistant Tuberculosis by Improving Antimicrobial Immunity.

BackgroundMultidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis.MethodsA clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (n = 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, β-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression).ResultsVitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and LC3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA–treated macrophages. The combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a >125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid.ConclusionsImmunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis.

Role of vitamin D in COVID‐19 infections and deaths

Role of vitamin D in COVID‐19 infections and deaths

Hypusination Orchestrates the Antimicrobial Response of Macrophages

SUMMARYInnate responses of myeloid cells defend against pathogenic bacteria via inducible effectors. Deoxyhypusine synthase (DHPS) catalyzes the transfer of the N-moiety of spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form the amino acid hypusine. Hypusinated EIF5A (EIF5AHyp) transports specific mRNAs to ribosomes for translation. We show that DHPS is induced in macrophages by two gastrointestinal pathogens, Helicobacter pylori and Citrobacter rodentium, resulting in enhanced hypusination of EIF5A. EIF5AHyp was also increased in gastric macrophages from patients with H. pylori gastritis. Furthermore, we identify the bacteria-induced immune effectors regulated by hypusination. This set of proteins includes essential constituents of antimicrobial response and autophagy. Mice with myeloid cell-specific deletion of Dhps exhibit reduced EIF5AHyp in macrophages and increased bacterial burden and inflammation. Thus, regulation of translation through hypusination is a critical hallmark of the defense of eukaryotic hosts against pathogenic bacteria.

Functions of Bombyx mori cathepsin L-like in innate immune response and anti-microbial autophagy

Cathepsins belongs to the cysteine protease family, which are activated by an acidic environment. They play essential biological roles in the innate immunity and development of animals. Here, we identified a 62 kDa cathepsin L-like protease from the silkworm Bombyx mori. It contained putative conserved domains, including an I29 inhibitor domain and a peptidase C1A domain. The expression analysis revealed that cathepsin L-like was highly produced in the fat body, and 20-hydroxyecdysone (20 E) induced its expression. After challenge with three different types of heat-killed pathogens (Escherichia coli, Beauveria bassiana, and Bacillus cereus), the mRNA levels of cathepsin L-like significantly increased and displayed variable expression patterns in the immune tissues, suggesting its potential role in the innate immune response. The suppression of cathepsin L-like altered the expression of immune-related genes associated with the Toll and IMD pathway. Besides, autophagy-related genes such as Atg6, Atg8, VAMP2, Vps4, and syntaxin expression were also altered, indicating that cathepsin L-like regulates innate immunity and autophagy. Fluorescence microscopic analysis exhibited that cathepsin L-like was localized in the cytoplasm, and it was activated and dispersed throughout the cytoplasm and nucleus following the induction of anti-microbial autophagy. Altogether, our data suggest that cathepsin L-like may regulate the innate immune response and anti-microbial autophagy in the silkworm, B. mori.

Vitamin D for COVID-19: a case to answer?

Vitamin D for COVID-19: a case to answer?

Outside in: Roles of complement in autophagy.

The complement system is a well-characterized cascade of extracellular serum proteins that is activated by pathogens and unwanted waste material. Products of activated complement signal to the host cells via cell surface receptors, eliciting responses such as removal of the stimulus by phagocytosis. The complement system therefore functions as a warning system, resulting in removal of unwanted material. This review describes how extracellular activation of the complement system can also trigger autophagic responses within cells, up-regulating protective homeostatic autophagy in response to perceived stress, but also initiating targeted anti-microbial autophagy in order to kill intracellular cytoinvasive pathogens. In particular, we will focus on recent discoveries that indicate that complement may also have roles in detection and autophagy-mediated disposal of unwanted materials within the intracellular environment. We therefore summarize the current evidence for complement involvement in autophagy, both by transducing signals across the cell membrane, as well as roles within the cellular environment. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

Regulation of anti-microbial autophagy by factors of the complement system.

The complement system is a major component of innate immunity that participates in the defense of the host against a myriad of pathogenic microorganisms. Activation of complement allows for both local inflammatory response and physical elimination of microbes through phagocytosis or lysis. The system is highly efficient and is therefore finely regulated. In addition to these well-established properties, recent works have revealed that components of the complement system can be involved in a variety of other functions including in autophagy, the conserved mechanism that allows for the targeting and degradation of cytosolic materials by the lysosomal pathway after confining them into specialized organelles called autophagosomes. Besides impacting cell death, development or metabolism, the complement factors-autophagy connection can greatly modulate the cell autonomous, anti-microbial activity of autophagy: xenophagy. Both surface receptor-ligand interactions and intracellular interactions are involved in the modulation of the autophagic response to intracellular microbes by complement factors. Here, we review works that relate to the recently discovered connections between factors of the complement system and the functioning of autophagy in the context of host-pathogen relationship.

Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence.

SUMMARYRickettsia are obligate intracellular bacteria that evade antimicrobial autophagy in the host cell cytosol by unknown mechanisms. Other cytosolic pathogens block different steps of autophagy targeting, including the initial step of polyubiquitin coat formation. One mechanism of evasion is to mobilize actin to the bacterial surface. Here, we show that actin mobilization is insufficient to block autophagy recognition of the pathogen Rickettsia parkeri. Instead, R. parkeri employs outer membrane protein B (OmpB) to block ubiquitylation of bacterial surface proteins, including OmpA, and subsequent recognition by autophagy receptors. OmpB is also required for the formation of a capsule-like layer. Although OmpB is dispensable for bacterial growth in endothelial cells, it is essential for R. parkeri to block autophagy in macrophages and to colonize mice because of its ability to promote autophagy evasion in immune cells. Our results indicate that OmpB acts as a protective shield to obstruct autophagy recognition, revealing a distinctive bacterial mechanism to evade antimicrobial autophagy.

The influence of vitamin D supplementation on local and systemic inflammatory markers in periodontitis patients: A pilot study.

Vitamin D deficiency/insufficiency is a worldwide public health issue that has been linked to numerous inflammatory disorders, including periodontitis. There is increasing support for a role for adequate vitamin D levels in overall health. Populations with darker skin color have a higher prevalence of vitamin D deficiency/insufficiency and periodontitis. The purpose of this small pilot study was to investigate the influence of 12weeks of 25(OH)D vitamin D supplementation (VDS) on mediators of systemic inflammation in dark-skinned, periodontitis patients. A total of 23 patients with moderate to severe periodontitis were randomly assigned to the vitamin D group or placebo group and received intensive single visit scaling and root planning to elicit a systemic inflammatory response. Vitamin D supplementation increased serum 25(OH)D levels approximately 2-fold over baseline levels; moreover, VDS group had reduced peripheral blood CD3 and CD3+CD8+ cytotoxic T lymphocyte (CTLs) counts and reduced pro-inflammatory salivary cytokines. In contrast, VDS group had higher levels of the autophagy-related proteins and other proteins crucial for anti-microbial autophagy in whole blood PBMCs. In conclusion, VDS has multiple benefits for reducing systemic inflammation and promoting induction of autophagy-related proteins related to anti-microbial functions.

The Rickettsia Surface Protein OmpB is Critical for Virulence and Evasion of Autophagy

Rickettsia are obligate intracellular bacteria that evade antimicrobial autophagy in the host cell cytosol by unknown mechanisms. Other cytosolic pathogens block different steps of autophagy targeting, including the initial step of polyubiquitin coat formation. One mechanism of evasion is to mobilize actin to the bacterial surface. Here, we show that actin mobilization is insufficient to block autophagy recognition of the pathogen Rickettsia parkeri. Instead, R. parkeri employs outer membrane protein B (OmpB) to block ubiquitylation of bacterial surface proteins, including OmpA, and subsequent recognition by autophagy receptors. Although OmpB is dispensable for bacterial growth in endothelial cells, it is essential for R. parkeri to block autophagy in macrophages and to colonize mice. Our results indicate that OmpB acts as a protective shield to obstruct autophagy recognition, revealing a distinctive bacterial mechanism to evade antimicrobial autophagy.

Neutrophils: Interplay between host defense, cellular metabolism and intracellular infection

Neutrophils are short-living innate immune cells present in abundance in the circulation and they provide the first line of defense against infection. Microbicidal effector functions of the immune cells are tightly linked with their metabolic state. Overall knowledge of the association of neutrophil defense with cellular metabolism is still elusive. Recent studies have reported that neutrophil metabolism during granulopoiesis is impacted by the homeostatic process of autophagy. Autophagy is a complex process of which the subtype xenophagy, an antimicrobial autophagic process, clears invading pathogens. Neutrophils, being sentinels of innate immunity, encounter microbes and digest them. Studies on neutrophil antimicrobial autophagy process is still in its primeval state and has not been much explored as in other professional phagocytes. However, several obligate-intracellular pathogens are able to subvert the toxic antimicrobial machineries and utilize neutrophils as their final abodes or “Trojan Horses” for further propagation. In this review, we would provide an insight into the relationship between neutrophil microbicidal effector functions emphasizing on selective antimicrobial autophagy and metabolism, highlighting on some successful pathogens which have evolved ways to subvert or exploit this defense.

Inflammation-Induced, STING-Dependent Autophagy Restricts Zika Virus Infection in the Drosophila Brain

The emerging arthropod-borne flavivirus Zika virus (ZIKV) is associated with neurological complications. Innate immunity is essential for the control of virus infection, but the innate immune mechanisms that impact viral infection of neurons remain poorly defined. Using the genetically tractable Drosophila system, we show that ZIKV infection of the adult flybrain leads to NF-kB-dependent inflammatory signaling, which serves to limit infection. ZIKV-dependent NF-kB activation induces the expression of Drosophila stimulator of interferon genes (dSTING) in the brain. dSTING protects against ZIKV by inducing autophagy in the brain. Loss of autophagy leads to increased ZIKV infection of the brain and death of the infected fly, while pharmacological activation of autophagy is protective. These data suggest an essential role for an inflammation-dependent STING pathway in the control of neuronal infection and a conserved role for STING in antimicrobial autophagy, which may represent an ancestral function for this essential innate immune sensor.