Staphylococcus aureus phagocytosis is affected by senescence.

Senescent cells accumulate in multicellular animals with aging, resulting in organ or tissue dysfunction. These alterations increase the incidence of a variety of illnesses, including infectious diseases, and, in certain instances, its severity. In search of a rationale for this phenomenon, we focused on the endophagocytic pathway in senescent cells. We first described the endocytic vesicle populations at different stages of maturation using confocal microscopy. There was an increase in the number of vacuoles per cell, which was partially explained by an increase in cell size. No changes in vesicle maturation or degradation capacities were determined by microscopy or Western blot assays. Also, we studied the internalization of various endophagocytic cargoes in senescent cells and observed only a decrease in the intracellular recovery of bacteria such as Staphylococcus aureus. Afterwards, we studied the intracellular traffic of S. aureus, and observed no differences in the infection between control and senescent cells. In addition we quantified the recovery of bacteria from control and senescent cells infected in the presence of several inhibitors of endophagosomal maturation, and no changes were observed. These results suggest that bacterial internalization is affected in senescent cells. Indeed, we confirmed this hypothesis by determining minor bacterial adherence and internalization by confocal microscopy. Furthermore, it is important to highlight that we found very similar results with cells from aged animals, specifically BMDMs. This alteration in senescent cells enlightens the diminished bacterial clearance and may be a factor that increases the propensity to suffer severe infectious conditions in the elderly.

Comparative analysis of humoral immune response upon the three first vaccines applied in Argentina: IgG production and neutralizing capacity against SARS-CoV-2.

The population that has not received a SARS-CoV-2 vaccine is at high risk for infection whereas vaccination prevents COVID-19 severe disease, hospitalization, and death. In Argentina, to date, more than 50 million doses of vaccines against SARS-CoV-2 have been administered. The three main vaccines applied are Sputnik V, Oxford-AstraZeneca, and Sinopharm. In this study, we have compared the antibody response of voluntary individuals at day 0 (first dose vaccination day) and at 21-25 days post first and second dose. Our results indicate that at 21-25 days after the administration of the first doses of Sputnik V the large majority of the people vaccinated 80% (n = 15) presented high humoral responses as determined by the measurement of IgG against the Spike protein and the Receptor Binding Domain (RBD). In the case of those vaccinated with AstraZeneca, the percentage was 80% (n = 15) whereas this value was reduced to only 25% (n = 16) in persons that received Sinopharm. However, after the second doses, most of the recipients had significant levels of antibodies. The virus neutralizing capacity of the antibodies generated was evaluated using a pseudotyped VSV-SARS-CoV2 Spike expressing eGFP and the data was analyzed by fluorescence microscopy and flow cytometry. The results indicate that a good correlation exists between the levels of IgG and the neutralizing capacity of the antibodies against the recombinant virus. Our results stand out the importance of applying the second dose of Sinopharm. Thus, the present report provides data that will contribute to decisions making about the vaccine implementation plans of action for, not only our region but our country to support the fight against the COVID-19 global pandemic.

Receptor-binding domain-based SARS-CoV-2 vaccine adjuvanted with cyclic di-adenosine monophosphate enhances humoral and cellular immunity in mice.

Novel adjuvants are highly desired to improve immune responses of SARS-CoV-2 vaccines. This work reports the potential of the stimulator of interferon genes (STING) agonist adjuvant, the cyclic di-adenosine monophosphate (c-di-AMP), in a SARS-CoV-2 vaccine based on the receptor binding domain (RBD). Here, mice immunized with two doses of monomeric RBD adjuvanted with c-di-AMP intramuscularly were found to exhibit stronger immune responses compared to mice vaccinated with RBD adjuvanted with aluminum hydroxide (Al(OH)3 ) or without adjuvant. After two immunizations, consistent enhancements in the magnitude of RBD-specific immunoglobulin G (IgG) antibody response were observed by RBD + c-di-AMP (mean: 15360) compared to RBD + Al(OH)3 (mean: 3280) and RBD alone (n.d.). Analysis of IgG subtypes indicated a predominantly Th1-biased immune response (IgG2c, mean: 14480; IgG2b, mean: 1040, IgG1, mean: 470) in mice vaccinated with RBD + c-di-AMP compared to a Th2-biased response in those vaccinated with RBD + Al(OH)3 (IgG2c, mean: 60; IgG2b: n.d.; IgG1, mean: 16660). In addition, the RBD + c-di-AMP group showed better neutralizing antibody responses as determined by pseudovirus neutralization assay and by plaque reduction neutralization assay with SARS-CoV-2 wild type. Moreover, the RBD + c-di-AMP vaccine promoted interferon-γ secretion of spleen cell cultures after RBD stimulation. Furthermore, evaluation of IgG-antibody titers in aged mice showed that di-AMP was able to improve RBD-immunogenicity at old age after 3 doses (mean: 4000). These data suggest that c-di-AMP improves immune responses of a SARS-CoV-2 vaccine based on RBD, and would be considered a promising option for future COVID-19 vaccines.

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway.

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex. SUMMARY: In this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

PGE2 displays immunosuppressive effects during human active tuberculosis.

Prostaglandin E2 (PGE2), an active lipid compound derived from arachidonic acid, regulates different stages of the immune response of the host during several pathologies such as chronic infections or cancer. In fact, manipulation of PGE2 levels was proposed as an approach for countering the Type I IFN signature of tuberculosis (TB). However, very limited information regarding the PGE2 pathway in patients with active TB is currently available. In the present work, we demonstrated that PGE2 exerts a potent immunosuppressive action during the immune response of the human host against Mycobacterium tuberculosis (Mtb) infection. Actually, we showed that PGE2 significantly reduced the surface expression of several immunological receptors, the lymphoproliferation and the production of proinflammatory cytokines. In addition, PGE2 promoted autophagy in monocytes and neutrophils cultured with Mtb antigens. These results suggest that PGE2 might be attenuating the excessive inflammatory immune response caused by Mtb, emerging as an attractive therapeutic target. Taken together, our findings contribute to the knowledge of the role of PGE2 in the human host resistance to Mtb and highlight the potential of this lipid mediator as a tool to improve anti-TB treatment.

PKCα Is Recruited to Staphylococcus aureus-Containing Phagosomes and Impairs Bacterial Replication by Inhibition of Autophagy.

Hijacking the autophagic machinery is a key mechanism through which invasive pathogens such as Staphylococcus aureus replicate in their host cells. We have previously demonstrated that the bacteria replicate in phagosomes labeled with the autophagic protein LC3, before escaping to the cytoplasm. Here, we show that the Ca2+-dependent PKCα binds to S. aureus-containing phagosomes and that α-hemolysin, secreted by S. aureus, promotes this recruitment of PKCα to phagosomal membranes. Interestingly, the presence of PKCα prevents the association of the autophagic protein LC3. Live cell imaging experiments using the PKC activity reporter CKAR reveal that treatment of cells with S. aureus culture supernatants containing staphylococcal secreted factors transiently activates PKC. Functional studies reveal that overexpression of PKCα causes a marked inhibition of bacterial replication. Taken together, our data identify enhancing PKCα activity as a potential approach to inhibit S. aureus replication in mammalian cells.

Phosphatidylinositol 3-Phosphate Mediates the Establishment of Infectious Bursal Disease Virus Replication Complexes in Association with Early Endosomes.

Infectious bursal disease virus (IBDV) is the archetypal member of the family Birnaviridae and the etiological agent of Gumboro disease, a highly contagious immunosuppressive infection of concern to the global poultry sector for its adverse health effects in chicks. Unlike most double-stranded RNA (dsRNA) viruses, which enclose their genomes within specialized cores throughout their viral replication cycle, birnaviruses organize their bisegmented dsRNA genome in ribonucleoprotein (RNP) structures. Recently, we demonstrated that IBDV exploits endosomal membranes for replication. The establishment of IBDV replication machinery on the cytosolic leaflet of endosomal compartments is mediated by the viral protein VP3 and its intrinsic ability to target endosomes. In this study, we identified the early endosomal phosphatidylinositol 3-phosphate [PtdIns(3)P] as a key host factor of VP3 association with endosomal membranes and consequent establishment of IBDV replication complexes in early endosomes. Indeed, our data reveal a crucial role for PtdIns(3)P in IBDV replication. Overall, our findings provide new insights into the replicative strategy of birnaviruses and strongly suggest that it resembles those of positive-strand RNA (+ssRNA) viruses, which replicate in association with host membranes. Furthermore, our findings support the role of birnaviruses as evolutionary intermediaries between +ssRNA and dsRNA viruses and, importantly, demonstrate a novel role for PtdIns(3)P in the replication of a dsRNA virus.IMPORTANCEInfectious bursal disease virus (IBDV) infects chicks and is the causative agent of Gumboro disease. During IBDV outbreaks in recent decades, the emergence of very virulent variants and the lack of effective prevention/treatment strategies to fight this disease have had devastating consequences for the poultry industry. IBDV belongs to the peculiar family Birnaviridae Unlike most dsRNA viruses, birnaviruses organize their genomes in ribonucleoprotein complexes and replicate in a core-independent manner. We recently demonstrated that IBDV exploits host cell endosomes as platforms for viral replication, a process that depends on the VP3 viral protein. In this study, we delved deeper into the molecular characterization of IBDV-endosome association and investigated the role of host cell phosphatidylinositide lipids in VP3 protein localization and IBDV infection. Together, our findings demonstrate that PtdIns(3)P serves as a scaffold for the association of VP3 to endosomes and reveal its essential role for IBDV replication.

Neutrophil autophagy during human active tuberculosis is modulated by SLAMF1.

Neutrophils infected with Mycobacterium tuberculosis (Mtb) predominate in tuberculosis patients' lungs. Neutrophils phagocytose the pathogen, but the mechanism of pathogen elimination is controversial. Macroautophagy/autophagy, a crucial mechanism for several neutrophil functions, can be modulated by immunological mediators. The costimulatory molecule SLAMF1 can act as a microbial sensor in macrophages being also able to interact with autophagy-related proteins. Here, we demonstrate for the first time that human neutrophils express SLAMF1 upon Mtb-stimulation. Furthermore, SLAMF1 was found colocalizing with LC3B+ vesicles, and activation of SLAMF1 increased neutrophil autophagy induced by Mtb. Finally, tuberculosis patients' neutrophils displayed reduced levels of SLAMF1 and lower levels of autophagy against Mtb as compared to healthy controls. Altogether, these results indicate that SLAMF1 participates in neutrophil autophagy during active tuberculosis.Abbreviations: AFB: acid-fast bacilli; BafA1: bafilomycin A1; CLL: chronic lymphocytic leukemia; DPI: diphenyleneiodonium; EVs: extracellular vesicles; FBS: fetal bovine serum; HD: healthy donors; HR: high responder (tuberculosis patient); IFNG: interferon gamma; IL1B: interleukin 1 beta; IL17A: interleukin 17A; IL8: interleukin 8; LR: low responder (tuberculosis patient); mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK14/p38: mitogen-activated protein kinase 14; Mtb: Mycobacterium tuberculosis; Mtb-Ag: Mycobacterium tuberculosis, Strain H37Rv, whole cell lysate; NETs: neutrophils extracellular traps; PPD: purified protein derivative; ROS: reactive oxygen species; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; SLAMF1: signaling lymphocytic activation molecule family member 1; TB: tuberculosis; TLR: toll like receptor.

Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth.

VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth.

Autophagy plays a protective role against Trypanosoma cruzi infection in mice.

Autophagy is a catabolic pathway required for cellular and organism homeostasis. Autophagy participates in the innate and adaptive immune responses at different levels. Xenophagy is a class of selective autophagy that involves the elimination of intracellular pathogens. Trypanosoma cruzi is the causative agent of Chagas, a disease that affects 8 million individuals worldwide. Previously, our group has demonstrated that autophagy participates in the invasion of T. cruzi in non-phagocytic cells. In this work we have studied the involvement of autophagy in the development of T. cruzi infection in mice. Beclin-1 is a protein essential for autophagy, required for autophagosome biogenesis and maturation. We have performed an acute model of infection on the autophagic deficient Beclin-1 heterozygous knock-out mice (Bcln±) and compared to control Bcln+/+ animals. In addition, we have analyzed the infection process in both peritoneal cells and RAW macrophages. Our results have shown that the infection was more aggressive in the autophagy-deficient mice, which displayed higher numbers of parasitemia, heart´s parasitic nests and mortality rates. We have also found that peritoneal cells derived from Bcln± animals and RAW macrophages treated with autophagy inhibitors displayed higher levels of infection compared to controls. Interestingly, free cytosolic parasites recruited LC3 protein and other markers of xenophagy in control compared to autophagy-deficient cells. Taken together, these data suggest that autophagy plays a protective role against T. cruzi infection in mice, xenophagy being one of the processes activated as part of the repertoire of immune responses generated by the host.

The cAMP effectors, Rap2b and EPAC, are involved in the regulation of the development of the Coxiella burnetii containing vacuole by altering the fusogenic capacity of the vacuole.

Cyclic Adenosine 3',5'-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2. Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.

Hemin induces autophagy in a leukemic erythroblast cell line through the LRP1 receptor.

Hemin is an erythropoietic inductor capable of inducing autophagy in erythroid-like cell lines. Low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor involved in a wide range of cellular processes, such as proliferation, differentiation, and metabolism. Our aim was to evaluate whether LRP1 is responsible for hemin activity in K562 cells, with the results demonstrating a three-fold increase in LRP1 gene expression levels (P-values <0.001) when assessed by quantitative real-time RT-PCR (qRT-PCR). Moreover, a 70% higher protein amount was observed compared with control condition (P-values <0.01) by Western blot (WB). Time kinetic assays demonstrated a peak in light chain 3 (LC3) II (LC3II) levels after 8 h of hemin stimulation and the localization of LRP1 in the autophagosome structures. Silencing LRP1 by siRNA decreased drastically the hemin-induced autophagy activity by almost 80% compared with control cells (P-values <0.01). Confocal localization and biochemical analysis indicated a significant redistribution of LRP1 from early endosomes and recycling compartments to late endosomes and autophagolysosomes, where the receptor is degraded. We conclude that LRP1 is responsible for hemin-induced autophagy activity in the erythroblastic cell line and that hemin-LRP1 complex activation promotes a self-regulation of the receptor. Our results suggest that hemin, via the LRP1 receptor, favors erythroid maturation by inducing an autophagic response, making it a possible therapeutic candidate to help in the treatment of hematological disorders.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.