Metabolic shift underlies tumor progression and immune evasion in S-nitrosoglutathione reductase-deficient cancer.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR-low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR-low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR-Cas9-mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR-deficiency conferred higher tumorigenic and tumor-initiating capacities. Moreover, GSNOR-KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR-KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2-deoxyglucose (2DG), and a fragmented mitochondrial network. Real-time metabolic analysis revealed that GSNOR-KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient-derived xenografts and organoids from clinical GSNOR-low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Serotype-Specific Sugars Impact Structure but Not Functions of the Trimeric Autotransporter Adhesin EmaA of Aggregatibacter actinomycetemcomitans.

The human oral pathobiont Aggregatibacter actinomycetemcomitans expresses multiple virulence factors, including the trimeric, extracellular matrix protein adhesin A (EmaA). The posttranslational modification of EmaA is proposed to be dependent on the sugars and enzymes associated with O-polysaccharide (O-PS) synthesis of the lipopolysaccharide (LPS). This modification is important for the structure and function of this adhesin. To determine if the composition of the sugars alters structure and/or function, the prototypic 202-kDa protein was expressed in a non-serotype b, emaA mutant strain. The transformed strain displayed EmaA adhesins similar in appearance to the prototypic adhesin as observed by two-dimensional (2D) electron microscopy of whole-mount negatively stained bacterial preparations. Biochemical analysis indicated that the protein monomers were posttranslationally modified. 3D electron tomographic reconstruction and structure analyses of the functional domain revealed three well-defined subdomains (SI, SII, and SIII) with a linker region between SII and SIII. Structural changes were observed in all three subdomains and the linker region of the adhesins synthesized compared with the known structure. These changes, however, did not affect the ability of the strain to bind collagen or form biofilms. The data suggest that changes in the composition of the glycan moiety alter the 3D structure of the molecule without negatively affecting the function(s) associated with this adhesin. IMPORTANCE The human oral pathogen A. actinomycetemcomitans is a causative agent of periodontal and several systemic diseases. EmaA is a trimeric autotransporter protein adhesin important for colonization by this pathobiont in vivo. This adhesin is modified with sugars associated with the O-polysaccharide (O-PS), and the modification is mediated using the enzymes involved in lipopolysaccharide (LPS) biosynthesis. The interaction with collagen is not mediated by the specific binding between the glycans and collagen but is attributed to changes in the final quaternary structure necessary to maintain an active adhesin. In this study, we have determined that the composition of the sugars utilized in the posttranslational modification of this adhesin is exchangeable without compromising functional activities.

The serotype a-EmaA adhesin of Aggregatibacter actinomycetemcomitans does not require O-PS synthesis for collagen binding activity.

Aggregatibacter actinomycetemcomitans, a causative agent of periodontitis and non-oral diseases, synthesizes a trimeric extracellular matrix protein adhesin A (EmaA) that mediates collagen binding and biofilm formation. EmaA is found as two molecular forms, which correlate with the serotype of the bacterium. The canonical protein (b-EmaA), associated with serotypes b and c, has a monomeric molecular mass of 202 kDa. The collagen binding activity of b-EmaA is dependent on the presence of O-polysaccharide (O-PS), whereas biofilm activity is independent of O-PS synthesis. The EmaA associated with serotype a strains (a-EmaA) has a monomeric molecular mass of 173 kDa and differs in the amino acid sequence of the functional domain of the protein. In this study, a-emaA was confirmed to encode a protein that forms antenna-like appendages on the surface of the bacterium, which were found to be important for both collagen binding and biofilm formation. In an O-PS-deficient talose biosynthetic (tld) mutant strain, the electrophoretic mobility of the a-EmaA monomers was altered and the amount of membrane-associated EmaA was decreased when compared to the parent strain. The mass of biofilm formed remained unchanged. Interestingly, the collagen binding activity of the mutant strain was similar to the activity associated with the parent strain, which differs from that observed with the canonical b-EmaA isoform. These data suggest that the properties of the a-EmaA isoform are like those of b-EmaA, with the exception that collagen binding activity is independent of the presence or absence of the O-PS.

A DNA 3' phosphatase functions in active DNA demethylation in Arabidopsis.

DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3'- and 5'-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3' phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and colocalize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway.

Interactions between the Trimeric Autotransporter Adhesin EmaA and Collagen Revealed by Three-Dimensional Electron Tomography.

Bacterial adhesion to host tissues is considered the first and critical step of microbial infection. The extracellular matrix protein adhesin A (EmaA) is a collagen-binding adhesin of the periodontal pathogen Aggregatibacter actinomycetemcomitans Three 202-kDa EmaA monomers form antenna-like structures on the bacterial surface with the functional domain located at the apical end. The structure of the 30-nm functional domain has been determined by three-dimensional (3D) electron tomography and subvolume averaging. The region exhibits a complex architecture composed of three subdomains (SI to SIII) and a linker between subdomains SII and SIII. However, the molecular interaction between the adhesin receptor complexes has yet to be revealed. This study provides the first detailed 3D structure of reconstituted EmaA/collagen complexes obtained using 3D electron tomography and image processing techniques. The observed interactions of EmaA with collagen were not to whole, intact fibrils, but rather to individual collagen triple helices dissociated from the fibrils. The majority of the contacts with the EmaA functional domain encompassed subdomains SII and SIII and in some cases the tip of the apical domain, involving SI. These data suggest a multipronged mechanism for the interaction of Gram-negative bacteria with collagen.IMPORTANCE Bacterial adhesion is a crucial step for bacterial colonization and infection. In recent years, the number of antibiotic-resistant strains has dramatically increased; therefore, there is a need to search for novel antimicrobial agents. Thus, great efforts are being devoted to develop a clear understanding of the bacterial adhesion mechanism for preventing infections. In host/pathogen interactions, once repulsive forces are overcome, adhesins recognize and tightly bind to specific receptors on the host cell or tissue components. Here, we present the first 3D structure of the interaction between the collagen-binding adhesin EmaA and collagen, which is critical for the development of endocarditis in humans.

A Nonfimbrial Adhesin of Aggregatibacter actinomycetemcomitans Mediates Biofilm Biogenesis.

Periodontitis is an inflammatory disease caused by polymicrobial biofilms. The periodontal pathogen Aggregatibacter actinomycetemcomitans displays two proteinaceous surface structures, the fimbriae and the nonfimbrial extracellular matrix binding protein A (EmaA), as observed by electron microscopy. Fimbriae participate in biofilm biogenesis and the EmaA adhesins mediate collagen binding. However, in the absence of fimbriae, A. actinomycetemcomitans still retains the potential to form robust biofilms, suggesting that other surface macromolecules participate in biofilm development. Here, isogenic mutant strains lacking EmaA structures, but still expressing fimbriae, were observed to have reduced biofilm potential. In strains lacking both EmaA and fimbriae, biofilm mass was reduced by 80%. EmaA enhanced biofilm formation in different strains, independent of the fimbriation state or serotype. Confocal microscopy revealed differences in cell density within microcolonies between the EmaA positive and mutant strains. EmaA-mediated biofilm formation was found to be independent of the glycosylation state and the precise three-dimensional conformation of the protein, and thus this function is uncorrelated with collagen binding activity. The data suggest that EmaA is a multifunctional adhesin that utilizes different mechanisms to enhance bacterial binding to collagen and to enhance biofilm formation, both of which are important for A. actinomycetemcomitans colonization and subsequent infection.

Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila.

The indirect flight muscles (IFMs) of Drosophila and other insects with asynchronous flight muscles are characterized by a crystalline myofilament lattice structure. The high-order lattice regularity is considered an adaptation for enhanced power output, but supporting evidence for this claim is lacking. We show that IFMs from transgenic flies expressing flightin with a deletion of its poorly conserved N-terminal domain (flnΔN62 ) have reduced inter-thick filament spacing and a less regular lattice. This resulted in a decrease in flight ability by 33% and in skinned fibre oscillatory power output by 57%, but had no effect on wingbeat frequency or frequency of maximum power output, suggesting that the underlying actomyosin kinetics is not affected and that the flight impairment arises from deficits in force transmission. Moreover, we show that flnΔN62 males produced an abnormal courtship song characterized by a higher sine song frequency and a pulse song with longer pulses and longer inter-pulse intervals (IPIs), the latter implicated in male reproductive success. When presented with a choice, wild-type females chose control males over mutant males in 92% of the competition events. These results demonstrate that flightin N-terminal domain is required for optimal myofilament lattice regularity and IFM activity, enabling powered flight and courtship song production. As the courtship song is subject to female choice, we propose that the low amino acid sequence conservation of the N-terminal domain reflects its role in fine-tuning species-specific courtship songs.

Usefulness of multidetector computed tomography before and after pulmonary vein isolation. / Utilidad de la tomografía computarizada multidetector en la evaluación previa y el seguimiento de los pacientes sometidos a ablación de venas pulmonares.

OBJECTIVE: To analyze the usefulness of multidetector computed tomography (MDCT) in the preprocedural evaluation and follow-up of patients undergoing radiofrequency ablation of pulmonary veins and the impact of the MDCT findings on the approach to treatment. METHOD: We retrospectively analyzed 92 consecutive MDCT studies done in 80 patients between January 2011 and June 2013; 70 (76%) studies were done before a first ablation procedure and 22 (24%) were done in patients who had undergone an ablation procedure. RESULTS: Findings were useful in 34% of the patients who underwent MDCT before the first ablation procedure and in 68% of the studies done after a procedure. The incidence of stroke associated with the ablation procedure was 3%, similar to the incidence recorded in our center before we started to use MDCT to evaluate the anatomy of the left atrium. All symptomatic patients had some pulmonary vein stenosis, and 80% had significant stenosis. Furthermore, the stenoses progressed very rapidly; treatment with balloon angioplasty was associated with early restenosis. Stenting was an alternative in cases of failed angioplasty. CONCLUSION: In the preprocedural evaluation and postprocedural follow-up of patients undergoing pulmonary vein isolation, MDCT is useful for guiding treatment and detecting complications.

Inner-membrane protein MorC is involved in fimbriae production and biofilm formation in Aggregatibacter actinomycetemcomitans.

Fimbrial subunit synthesis, secretion and assembly on the surface of the periodontal pathogen Aggregatibacter actinomycetemcomitans are essential for biofilm formation. A recent quantitative proteomics study employing an afimbriated strain and a developed mutant isogenic for the inner-membrane protein morphogenesis protein C (MorC) revealed that the abundance of the proteins of the fimbrial secretion apparatus in the membrane is dependent on MorC. To investigate further the relationship between MorC and fimbriation, we identified and complemented the defect in fimbriae production in the afimbriated laboratory strain. The transformed strain expressing a plasmid containing genes encoding the WT fimbrial subunit and the prepilin peptidase displayed all of the hallmarks of a fimbriated bacterium including the distinct star-like colony morphology, robust biofilm formation, biofilm architecture composed of discrete microcolonies and the presence of fimbriae. When the identical plasmid was transformed into a morC mutant strain, the bacterium did not display any of the phenotypes of fimbriated strains. Extension of these studies to a naturally fimbriated clinical strain showed that the resulting morC mutant maintained the characteristic colony morphology of fimbriated strains. There was, however, a reduction in the secretion of fimbrial subunits, and fewer fimbriae were observed on the surface of the mutant strain. Furthermore, the morC mutant of the fimbriated strain displayed a significantly altered biofilm microcolony architecture, while maintaining a similar biofilm mass to the parent strain. These results suggest that MorC influences fimbrial secretion and microcolony formation in A. actinomycetemcomitans.

Profile of the Patient Who Refuses to Participate in the Expert Patients Program.

The Expert Patients Program (EPP), an educational intervention for chronic diseases aimed at nonprofessionals, has high nonparticipation and dropout rates. We used quantitative and qualitative methods to identify the prevalence of and reasons for nonparticipation in 100 patients with acute myocardial infarction and 69 with stroke. We confirmed a high prevalence of refusal, identifying three groups based on degree of participation: patients who refused to attend (51%), patients who attended and then dropped out (10%), and patients who attended (39%). Patients who refused to participate or dropped out were mainly women and former/current smokers with a better-than-average physical condition. In addition, they were invited to participate in the EPP long after their cardiovascular event. We found that the degree of participation depended upon the patient's individual and social context and the professional profile of the recruiter. Therefore, tailored strategic interventions should be designed for each type of participation.

The Trimeric Autotransporter Adhesin EmaA and Infective Endocarditis.

Infective endocarditis (IE), a disease of the endocardial surface of the heart, is usually of bacterial origin and disproportionally affects individuals with underlying structural heart disease. Although IE is typically associated with Gram-positive bacteria, a minority of cases are caused by a group of Gram-negative species referred to as the HACEK group. These species, classically associated with the oral cavity, consist of bacteria from the genera Haemophilus (excluding Haemophilus influenzae), Aggregatibacter, Cardiobacterium, Eikenella, and Kingella. Aggregatibacter actinomycetemcomitans, a bacterium of the Pasteurellaceae family, is classically associated with Aggressive Periodontitis and is also concomitant with the chronic form of the disease. Bacterial colonization of the oral cavity serves as a reservoir for infection at distal body sites via hematological spreading. A. actinomycetemcomitans adheres to and causes disease at multiple physiologic niches using a diverse array of bacterial cell surface structures, which include both fimbrial and nonfimbrial adhesins. The nonfimbrial adhesin EmaA (extracellular matrix binding protein adhesin A), which displays sequence heterogeneity dependent on the serotype of the bacterium, has been identified as a virulence determinant in the initiation of IE. In this chapter, we will discuss the known biochemical, molecular, and structural aspects of this protein, including its interactions with extracellular matrix components and how this multifunctional adhesin may contribute to the pathogenicity of A. actinomycetemcomitans.

Ultrastructural analysis of the rugose cell envelope of a member of the Pasteurellaceae family.

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wild-type and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

Gap2 promotes the formation of a stable protein complex required for mature Fap1 biogenesis.

Serine-rich repeat glycoproteins (SRRPs) are important bacterial adhesins conserved in streptococci and staphylococci. Fap1, a SRRP identified in Streptococcus parasanguinis, is the major constituent of bacterial fimbriae and is required for adhesion and biofilm formation. An 11-gene cluster is required for Fap1 glycosylation and secretion; however, the exact mechanism of Fap1 biogenesis remains a mystery. Two glycosylation-associated proteins within this cluster--Gap1 and Gap3--function together in Fap1 biogenesis. Here we report the role of the third glycosylation-associated protein, Gap2. A gap2 mutant exhibited the same phenotype as the gap1 and gap3 mutants in terms of Fap1 biogenesis, fimbrial assembly, and bacterial adhesion, suggesting that the three proteins interact. Indeed, all three proteins interacted with each other independently and together to form a stable protein complex. Mechanistically, Gap2 protected Gap3 from degradation by ClpP protease, and Gap2 required the presence of Gap1 for expression at the wild-type level. Gap2 augmented the function of Gap1 in stabilizing Gap3; this function was conserved in Gap homologs from Streptococcus agalactiae. Our studies demonstrate that the three Gap proteins work in concert in Fap1 biogenesis and reveal a new function of Gap2. This insight will help us elucidate the molecular mechanism of SRRP biogenesis in this bacterium and in pathogenic species.

Projection-based volume alignment.

When heterogeneous samples of macromolecular assemblies are being examined by 3D electron microscopy (3DEM), often multiple reconstructions are obtained. For example, subtomograms of individual particles can be acquired from tomography, or volumes of multiple 2D classes can be obtained by random conical tilt reconstruction. Of these, similar volumes can be averaged to achieve higher resolution. Volume alignment is an essential step before 3D classification and averaging. Here we present a projection-based volume alignment (PBVA) algorithm. We select a set of projections to represent the reference volume and align them to a second volume. Projection alignment is achieved by maximizing the cross-correlation function with respect to rotation and translation parameters. If data are missing, the cross-correlation functions are normalized accordingly. Accurate alignments are obtained by averaging and quadratic interpolation of the cross-correlation maximum. Comparisons of the computation time between PBVA and traditional 3D cross-correlation methods demonstrate that PBVA outperforms the traditional methods. Performance tests were carried out with different signal-to-noise ratios using modeled noise and with different percentages of missing data using a cryo-EM dataset. All tests show that the algorithm is robust and highly accurate. PBVA was applied to align the reconstructions of a subcomplex of the NADH: ubiquinone oxidoreductase (Complex I) from the yeast Yarrowia lipolytica, followed by classification and averaging.

Switching to an advanced hybrid closed-loop system in real-world practice improves hypoglycemia awareness and metabolic control in adults with type 1 diabetes, particularly in those with impaired perception of hypoglycemia symptoms.

AIMS: To evaluate the efficacy of an advance closed-loop (AHCL) system in restoring awareness of hypoglycemia in patients with type 1 diabetes (T1D). METHODS: We conducted a prospective study including 46 subjects with T1D flash glucose monitoring (FGM) or continuous glucose monitoring (CGM) switching to a Minimed 780G® system. Patients were classified in three groups according to the therapy used before switching to Minimed® 780G: multiple dose insulin (MDI) therapy + FGM (n = 6), continuous subcutaneous insulin infusion + FGM (n = 21), and sensor-augmented pump with predictive low-glucose suspend (n = 19). FGM/CGM data were analyzed at baseline, after 2 and 6 months on AHCL. Clarke's score of hypoglycemia awareness was compared at baseline and 6 months recordings. We also compared the efficacy of the AHCL system in improving A1c among patients with appropriate perception of symptoms of hypoglycemia compared to those presenting with impaired awareness of hypoglycemia (IAH). RESULTS: Participants had a mean age of 37 ± 15 and a diabetes duration of 20 ± 10 years. At baseline, 12 patients (27%) showed IAH as defined by a Clarke's score ≥ 3. Patients with IAH were older and had lower estimated glomerular filtration rate (eGFR) compared with those who did not have IAH; with no differences in baseline CGM metrics or A1c. An overall decrease in A1c was observed after 6 months on AHCL system (from 6.9 ± 0.5% to 6.7 ± 0.6%, P < 0.001), regardless of prior insulin therapy. The improvement in metabolic control was greater in patients with IAH, showing a reduction in A1c from 6.9 ± 0.5 to 6.4 ± 0.4% vs 6.9 ± 0.5 to 6.8 ± 0.6% (P = 0.003), showing a parallel increase in total daily boluses of insulin and automatic bolus correction administered by the AHCL system. In patients with IAH Clarke's score decreased from 3.6 ± 0.8 at baseline to 1.9 ± 1.6 after 6 months (P < 0.001). After 6 months on AHCL system, only 3 patients (7%) presented with a Clarke's score ≥ 3, resulting in an absolute risk reduction of 20% (95% confidence interval: 7-32) of having IAH. CONCLUSIONS: Switching from any type of insulin administration to AHCL system improves restoration of hypoglycemia awareness and metabolic control in patients with T1D, particularly in adults with impaired perception of hypoglycemia symptoms. TRIAL REGISTRATION: ClinicalTrial.gov ID NCT04900636.

CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

BACKGROUND: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated. RESULTS: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence. CONCLUSIONS: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans.

Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.

O-polysaccharide glycosylation is required for stability and function of the collagen adhesin EmaA of Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is hypothesized to colonize through the interaction with collagen and establish a reservoir for further dissemination. The trimeric adhesin EmaA of A. actinomycetemcomitans binds to collagen and is modified with sugars mediated by an O-antigen polysaccharide ligase (WaaL) that is associated with lipopolysaccharide (LPS) biosynthesis (G. Tang and K. Mintz, J. Bacteriol. 192:1395-1404, 2010). This investigation characterized the function and cellular localization of EmaA glycosylation. The interruption of LPS biogenesis by using genetic and pharmacological methods changed the amount and biophysical properties of EmaA molecules in the outer membrane. In rmlC and waaL mutant strains, the membrane-associated EmaA was reduced by 50% compared with the wild-type strain, without changes in mRNA levels. The membrane-associated EmaA protein levels were recovered by complementation with the corresponding O-polysaccharide (O-PS) biosynthetic genes. In contrast, another trimeric autotransporter, epithelial adhesin ApiA, was not affected in the same mutant background. The inhibition of undecaprenyl pyrophosphate recycling by bacitracin resulted in a similar decrease in the membrane-associated EmaA protein. This effect was reversed by removal of the compound. A significant decrease in collagen binding activity was observed in strains expressing the nonglycosylated form of EmaA. Furthermore, the electrophoretic mobility shifts of the EmaA monomers found in the O-PS mutant strains were associated only with the membrane-associated protein and not with the cytoplasmic pre-EmaA protein, suggesting that this modification does not occur in the cytoplasm. The glycan modification of EmaA appears to be required for collagen binding activity and protection of the protein against degradation by proteolytic enzymes.

Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence.

Cell senescence is a state of limited cell proliferation during a stress response or as part of a programmed process. When a senescent cell stops dividing, maintaining metabolic activity contributes to cellular homeostasis maintenance. In this process, the cell cycle is arrested at the G0/G1 phase. p16INK4A protein is a key regulator of this process via its cyclin­dependent kinase inhibitor (CDKI) function. CDKI 2A (CDKN2A)/p16 gene expression is regulated by DNA methylation and histone acetylation. Sirtuins (SIRTs) are nicotinamide dinucleotide (NAD+)­dependent deacetylases that have properties which prevent diseases and reverse certain aspects of aging (such as immune, metabolic and cardiovascular diseases). By performing quantitative PCR, Western blot, ChIP, and siRNAs assays, in this study it was demonstrated that CDKN2A/p16 gene transcriptional activation and repression were accompanied by selective deposition and elimination of histone acetylation during the senescence of MRC5 cells. Specifically, significant H3K9Ac and H3K18Ac enrichment in cells with a senescent phenotype concomitant with CDKN2A/p16 gene overexpression was demonstrated compared with the non­senescent phenotype. Furthermore, the presence of H3K18Ac in deacetyl­transferase SIRT7 knockdown MRC5 cells allowed CDKN2A/p16 promoter activation. These results suggested that SIRT7 served as a critical component of an epigenetic mechanism involved in senescence mediated by the CDKN2A/p16 gene.