Autophagy suppression in DNA damaged cells occurs through a newly identified p53-proteasome-LC3 axis.

Macroautophagy is thought to have a critical role in shaping and refining cellular proteostasis in eukaryotic cells recovering from DNA damage. Here, we report a mechanism by which autophagy is suppressed in cells exposed to bacterial toxin-, chemical-, or radiation-mediated sources of genotoxicity. Autophagy suppression is directly linked to cellular responses to DNA damage, and specifically the stabilization of the tumor suppressor p53, which is both required and sufficient for regulating the ubiquitination and proteasome-dependent reduction in cellular pools of microtubule-associated protein 1 light chain 3 (LC3A/B), a key precursor of autophagosome biogenesis and maturation, in both epithelial cells and an ex vivo organoid model. Our data indicate that suppression of autophagy, through a newly identified p53-proteasome-LC3 axis, is a conserved cellular response to multiple sources of genotoxicity. Such a mechanism could potentially be important for realigning proteostasis in cells undergoing DNA damage repair.

Host cell sensing and restoration of mitochondrial function and metabolism within Helicobacter pylori VacA intoxicated cells.

IMPORTANCE: Persistent human gastric infection with Helicobacter pylori is the single most important risk factor for development of gastric malignancy, which is one of the leading causes of cancer-related deaths worldwide. An important virulence factor for Hp colonization and severity of gastric disease is the protein exotoxin VacA, which is secreted by the bacterium and modulates functional properties of gastric cells. VacA acts by damaging mitochondria, which impairs host cell metabolism through impairment of energy production. Here, we demonstrate that intoxicated cells have the capacity to detect VacA-mediated damage, and orchestrate the repair of mitochondrial function, thereby restoring cellular health and vitality. This study provides new insights into cellular recognition and responses to intracellular-acting toxin modulation of host cell function, which could be relevant for the growing list of pathogenic microbes and viruses identified that target mitochondria as part of their virulence strategies.

SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells.

Recovery from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanisms counteracting antibody-mediated immunity is therefore needed. Here, we report that ORF8 is a virally encoded SARS-CoV-2 factor that controls cellular Spike antigen levels. We show that ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. In conditions of limited Spike availability, we found ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions then leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Based on these findings, we propose that SARS-CoV-2 variants may adopt an ORF8-dependent strategy that facilitates immune evasion of infected cells for extended viral production.

SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein.

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response, independently of the Mitochondrial Antiviral Signaling Protein MAVS. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein.

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

Senescent cells promote tissue NAD+ decline during ageing via the activation of CD38+ macrophages.

Declining tissue nicotinamide adenine dinucleotide (NAD) levels are linked to ageing and its associated diseases. However, the mechanism for this decline is unclear. Here, we show that pro-inflammatory M1-like macrophages, but not naive or M2 macrophages, accumulate in metabolic tissues, including visceral white adipose tissue and liver, during ageing and acute responses to inflammation. These M1-like macrophages express high levels of the NAD-consuming enzyme CD38 and have enhanced CD38-dependent NADase activity, thereby reducing tissue NAD levels. We also find that senescent cells progressively accumulate in visceral white adipose tissue and liver during ageing and that inflammatory cytokines secreted by senescent cells (the senescence-associated secretory phenotype, SASP) induce macrophages to proliferate and express CD38. These results uncover a new causal link among resident tissue macrophages, cellular senescence and tissue NAD decline during ageing and offer novel therapeutic opportunities to maintain NAD levels during ageing.

Comparative study of new bone formation capability of zirconia bone graft material in rabbit calvarial.

PURPOSE: The purpose of this study was to compare the new bone formation capability of zirconia with those of other synthetic bone grafts. MATERIALS AND METHODS: Twelve rabbits were used and four 6-mm diameter transcortical defects were formed on each calvaria. Each defect was filled with Osteon II (Os), Tigran PTG (Ti), and zirconia (Zi) bone grafts. For the control group, the defects were left unfilled. The rabbits were sacrificed at 2, 4, and 8 weeks. Specimens were analyzed through micro computed tomography (CT) and histomorphometric analysis. RESULTS: The Ti and Zi groups showed significant differences in the amount of newly formed bone between 2 and 4 weeks and between 2 and 8 weeks (P<.05). The measurements of total bone using micro CT showed significant differences between the Os and Ti groups and between the Os and Zi groups at 2 and 8 weeks (P<.05). Comparing by week in each group, the Ti group showed a significant difference between 4 and 8 weeks. Histomorphometric analysis also showed significant differences in new bone formation between the control group and the experimental groups at 2, 4, and 8 weeks (P<.05). In the comparison of newly formed bone, significant differences were observed between 2 and 4 weeks and between 2 and 8 weeks (P<.05) in all groups. CONCLUSION: Zirconia bone graft material showed satisfactory results in new bone formation and zirconia could be used as a new synthetic bone graft material.

Helicobacter pylori Infection Modulates Host Cell Metabolism through VacA-Dependent Inhibition of mTORC1.

Helicobacter pylori (Hp) vacuolating cytotoxin (VacA) is a bacterial exotoxin that enters host cells and induces mitochondrial dysfunction. However, the extent to which VacA-dependent mitochondrial perturbations affect overall cellular metabolism is poorly understood. We report that VacA perturbations in mitochondria are linked to alterations in cellular amino acid homeostasis, which results in the inhibition of mammalian target of rapamycin complex 1 (mTORC1) and subsequent autophagy. mTORC1, which regulates cellular metabolism during nutrient stress, is inhibited during Hp infection by a VacA-dependent mechanism. This VacA-dependent inhibition of mTORC1 signaling is linked to the dissociation of mTORC1 from the lysosomal surface and results in activation of cellular autophagy through the Unc 51-like kinase 1 (Ulk1) complex. VacA intoxication results in reduced cellular amino acids, and bolstering amino acid pools prevents VacA-mediated mTORC1 inhibition. Overall, these studies support a model that Hp modulate host cell metabolism through the action of VacA at mitochondria.

Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo.

We describe a novel insulin-degrading enzyme, SidC, that contributes to the proliferation of the human bacterial pathogen Vibrio vulnificus in a mouse model. SidC is phylogenetically distinct from other known insulin-degrading enzymes and is expressed and secreted specifically during host infection. Purified SidC causes a significant decrease in serum insulin levels and an increase in blood glucose levels in mice. A comparison of mice infected with wild type V. vulnificus or an isogenic sidC-deletion strain showed that wild type bacteria proliferated to higher levels. Additionally, hyperglycemia leads to increased proliferation of V. vulnificus in diabetic mice. Consistent with these observations, the sid operon was up-regulated in response to low glucose levels through binding of the cAMP-receptor protein (CRP) complex to a region upstream of the operon. We conclude that glucose levels are important for the survival of V. vulnificus in the host, and that this pathogen uses SidC to actively manipulate host endocrine signals, making the host environment more favorable for bacterial survival and growth.

Remodeling the host environment: modulation of the gastric epithelium by the Helicobacter pylori vacuolating toxin (VacA).

Virulence mechanisms underlying Helicobacter pylori persistence and disease remain poorly understood, in part, because the factors underlying disease risk are multifactorial and complex. Among the bacterial factors that contribute to the cumulative pathophysiology associated with H. pylori infections, the vacuolating cytotoxin (VacA) is one of the most important. Analogous to a number of H. pylori genes, the vacA gene exhibits allelic mosaicism, and human epidemiological studies have revealed that several families of toxin alleles are predictive of more severe disease. Animal model studies suggest that VacA may contribute to pathogenesis in several ways. VacA functions as an intracellular-acting protein exotoxin. However, VacA does not fit the current prototype of AB intracellular-acting bacterial toxins, which elaborate modulatory effects through the action of an enzymatic domain translocated inside host cells. Rather, VacA may represent an alternative prototype for AB intracellular acting toxins that modulate cellular homeostasis by forming ion-conducting intracellular membrane channels. Although VacA seems to form channels in several different membranes, one of the most important target sites is the mitochondrial inner membrane. VacA apparently take advantage of an unusual intracellular trafficking pathway to mitochondria, where the toxin is imported and depolarizes the inner membrane to disrupt mitochondrial dynamics and cellular energy homeostasis as a mechanism for engaging the apoptotic machinery within host cells. VacA remodeling of the gastric environment appears to be fine-tuned through the action of the Type IV effector protein CagA which, in part, limits the cytotoxic effects of VacA in cells colonized by H. pylori.

Well-defined DNA-mimic brush polymers bearing adenine moieties: synthesis, layer-by-layer self-assembly, and biocompatibility.

Two new DNA-mimicking brush polymers were synthesized: poly[oxy(11-(3-(9-adeninyl)propionato)-undecanyl-1-thiomethyl)ethylene] (PECH-AP) and poly[oxy(11-(5-(9-adenylethyloxy)-4-oxopentanoato)undecanyl-1-thiomethyl)ethylene] (PECH-AS). These polymers were found to be thermally stable up to 220 °C and could be applied easily by conventional coating processes to produce good quality films. Interestingly, both brush polymers formed molecular multibilayer structures to provide an adenine-rich surface. Despite the structural similarities, PECH-AS surprisingly exhibited higher hydrophilicity and better water sorption properties than PECH-AP. These differences were attributed to the chemical structures in the bristles of the polymers. The adenine-rich surfaces of the polymer films demonstrated selective protein adsorption, suppressed bacterial adherence, facilitated HEp-2 cell adhesion, and exhibited good biocompatibility in mice. However, the high hydrophilicity and good water sorption characteristics of the PECH-AS film suggest that this brush polymer is better suited to applications requiring good biocompatibility and reduced chance of bacterial infection compared with the PECH-AP film.

The biocompatibility of self-assembled brush polymers bearing glycine derivatives.

We have synthesized brush polymers with various glycine derivatives as the end groups of their long alkyl bristles. The polymers are thermally stable up to 170-210 degrees C and form good quality films through conventional spin- or dip-coating and subsequent drying. Interestingly, the thin films of these brush polymers exhibit different molecular multi-layer structures that arise through the efficient self-assembly of the bristles with glycine derivative end groups. These brush polymer films have hydrophilic surfaces and exhibit some water sorption. The extent of the water sorption by these films depends upon the nature of the glycine derivatives in the bristle end. These films not only repel fibrinogen molecules and platelets from their surfaces, but also have high resistance to bacterial adherence. Moreover, the films were found to provide conducive surface environments for the successful anchoring and growth of HEp-2 cells, and to exhibit excellent biocompatibility in mice. These brush polymers have potential uses in biomedical applications including medical devices, especially blood contacting devices such as catheters, stents, blood vessels, and biosensors, due to their enhanced biocompatibility and the reduced possibility of post-operative infection.

The biocompatability of mesoporous inorganic-organic hybrid resin films with ionic and hydrophilic characteristics.

New mesoporous silicate-titania resin systems hybridized with 4,5-dihydroxy-m-benzenedisulfonic acid and poly(ethylene glycol)-dimethacrylate component were developed. These inorganic-organic hybrid resins were found to reveal highly controlled ionic and hydrophilic surface with excellent durability and adhesion onto various substrates. The resin films revealed high resistance to nonspecific adsorption of fibrinogen and to adherence by several bacterial pathogens such as Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. Furthermore, excellent biocompatibility of the developed resins was proved by both HEp-2 cell adhesion in vitro and subcutaneous implantation in mice. The inorganic-organic hybrid resins are strongly promising for biomedical applications including biomedical devices and biosensors.

Nonribosomal peptide synthase is responsible for the biosynthesis of siderophore in Vibrio vulnificus MO6-24/O.

Vibrio vulnificus produces siderophores, lowmolecular- weight iron-chelating compounds, to obtain iron under conditions of iron deprivation. To identify genes associated with the biosynthesis of siderophore in V. vulnificus MO6-24/ O, we screened clones with mini-Tn5 random insertions for those showing decreased production of siderophore. Among 6,000 clones screened, nine such clones were selected. These clones contain the transposon inserted in VV2_0830 (GenBank accession number) that is a homolog of a nonribosomal peptide synthetase (NRPS). There is an another NRPS module, VV2_0831, 49-bp upstream to VV2_0830. We named these two genes vvs (Vibrio vulnificus siderophore synthetase) A and B, respectively. Mutation of either vvsA or vvsB showed a decreased production of siderophore. The expression of an NRPS-lux fusion was negatively modulated by the presence of iron, and the regulation was dependent on Fur (ferric uptake regulator). However, the expression of the NRPS genes was still not fully derepressed in the iron-rich condition, even in fur-null mutant cells, suggesting that some other unknown factors are involved in the regulation of the genes. We also demonstrated that the NRPS genes are important for virulence of the pathogen in a mice model.

Synchrotron small-angle X-ray scattering studies of the structure of porcine pepsin under various pH conditions.

Structural characteristics of various conformational states of porcine pepsin in solution under different pH conditions were investigated in terms of size and shape by small-angle X-ray scattering (SAXS). Low-resolution structural models of porcine pepsin were reconstructed from SAXS data, which were made inside the search volume of maximum dimension (Dmax), calculated from the pair distance distribution function p(r). The reconstructed structural models were obtained without imposing any restrictions on the symmetry or anisometry of the pepsin molecule. Under conditions emulating those for physiological activity of the enzyme, the reconstructed structural models exhibited a more extended C-terminal domain compared to the crystal structure. The differences between the solution and crystal structures of pepsin can be explained by inherent conformations of the flexible subdomain in the C-terminal domain under the solution pH conditions. Under mild acidic conditions where the enzyme is inactive, the reconstructed structural models revealed a compact globular conformation similar in overall shape to the crystal structure. These results indicate that the changes in fluorescence and circular dichroism curves observed under acidic conditions could also arise from the inherent conformation of the flexible subdomain, which has a tendency to roll into a sphere in the overall structure, but without affecting the stability of internal structure. Furthermore, the conformational changes in the subdomain might explain the inactivity of pepsin under mildly acidic conditions. Finally, compared to neutral denaturing conditions, pepsin under alkaline denaturing conditions had a larger expanded vertical conformation in the reconstructed model, as a consequence of alkaline denaturation of the N-terminal domain and a fully extended conformation of the C-terminal domain. The structural evidence presented here may have important implications for understanding the relationship between the structure of porcine pepsin and enzymatic function.

Improvement of neutralizing activity of human scFv antibodies against hepatitis B virus binding using CDR3 V(H) mutant library.

CDR3 of the heavy-chain variable region of immunoglobulin is a region in which somatic mutation occurs heavily after secondary antibody response, resulting in an affinity maturation of antibodies in vivo. The aim of this study was to improve the affinity of a human single-chain variable fragment (scFv) specific for pre-S1 of hepatitis B virus (HBV) by introducing random mutagenesis in CDR3 variable region of heavy chain (V(H)) of the parental scFv clone 1E4. By using a BIAcore for panning and screening, we have selected three clones (A9, B2, and B9) with lower highest affinity (K(D)) than 1E4. Affinities of selected clones ranged from 1.7 x 10(7) mol/L to 6.3 x 10(8) mol/L, which were increased by factors of 1.4 to 4.0, respectively, compared to the parental clone. Binding inhibition assay using flow cytometry and polymerase chain reaction revealed that B2 (6.4 x 10(8) mol/L) had a higher neutralizing activity against pre-S1 or HBV virion binding to liver cell line. This anti-pre-S1 scFv can be considered as a potential therapeutic tool for a passive immunotherapy for HBV infection.

Hepatitis B virus-neutralizing anti-pre-S1 human antibody fragments from large naïve antibody phage library.

We report the construction of a large nonimmunized human phage antibody library in single-chain variable region fragment (scFv) format, which allowed the selection of antibodies that neutralize hepatitis B virus (HBV) in vitro. We generated 1.1 x 10(10) independent scFv clones using the cDNA of functional variable (V) gene segments of heavy and light chains purified from the peripheral blood mononuclear cells of 50 nonimmunized human donors. Using BIAcore, we selected two clones that recognized pre-S1 and neutralized pre-S1 and HBV binding to Chang liver cells. Clone G10 had the highest affinity (K(D)=1.69 x 10(-7)M), which was higher than that of clone 1E4 that was generated previously from a heavy chain-shuffled immune library. The off-rates of clones were within 10(-3)s(-1) as determined by BIAcore and were comparable to those of antibodies derived from a normal secondary immune response. In the inhibition assays of pre-S1 and virus binding to Chang liver cells using flow cytometry and the polymerase chain reaction, G10 had better neutralizing activity than 1E4. The new phage library may be a valuable source of antibodies with reasonable affinities to different targets, and the anti-pre-S1 G10 may be a good candidate for immunoprophylaxis against HBV infection.

Population structure of the Bacillus cereus group as determined by sequence analysis of six housekeeping genes and the plcR Gene.

The population structure of the Bacillus cereus group (52 strains of B. anthracis, B. cereus, and B. thuringiensis) was investigated by sequencing seven gene fragments (rpoB, gyrB, pycA, mdh, mbl, mutS, and plcR). Most of the strains were classifiable into two large subgroups in six housekeeping gene trees but not in the plcR tree. In addition, several consistent clusters were identified, which were unrelated to species distinction. Moreover, interrelationships among these clusters were incongruent in each gene tree. The incongruence length difference test and split decomposition analyses also showed incongruences between genes, suggesting horizontal gene transfer. The plcR gene was observed to have characteristics that differed from those of the other genes in terms of phylogenetic topology and pattern of sequence diversity. Thus, we suggest that the evolutionary history of the PlcR regulon differs from those of the other chromosomal genes and that recombination of the plcR gene may be frequent. The homogeneity of B. anthracis, which is depicted as an independent lineage in phylogenetic trees, is suggested to be of recent origin or to be due to the narrow taxonomic definition of species.

Identification of Bacillus anthracis by rpoB sequence analysis and multiplex PCR.

Comparative sequence analysis was performed upon Bacillus anthracis and its closest relatives, B. cereus and B. thuringiensis. Portions of rpoB DNA from 10 strains of B. anthracis, 16 of B. cereus, 10 of B. thuringiensis, 1 of B. mycoides, and 1 of B. megaterium were amplified and sequenced. The determined rpoB sequences (318 bp) of the 10 B. anthracis strains, including five Korean isolates, were identical to those of Ames, Florida, Kruger B, and Western NA strains. Strains of the "B. cereus group" were separated into two subgroups, in which the B. anthracis strains formed a separate clade in the phylogenetic tree. However, B. cereus and B. thuringiensis could not be differentiated. Sequence analysis confirmed the five Korean isolates as B. anthracis. Based on the rpoB sequences determined in the present study, multiplex PCR generating either B. anthracis-specific amplicons (359 and 208 bp) or cap DNA (291 bp) in a virulence plasmid could be used for the rapid differential detection and identification of virulent B. anthracis.