Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

Apolipoprotein A-I (apoA-I) is the major component of HDL and central to the ability of HDL to stimulate ATP-binding cassette transporter A1 (ABCA1)-dependent, antiatherogenic export of cholesterol from macrophage foam cells, a key player in the pathology of atherosclerosis. Cell-mediated modifications of apoA-I, such as chlorination, nitration, oxidation, and proteolysis, can impair its antiatherogenic function, although it is unknown whether macrophages themselves contribute to such modifications. To investigate this, human monocyte-derived macrophages (HMDMs) were incubated with human apoA-I under conditions used to induce cholesterol export. Two-dimensional gel electrophoresis and Western blot analysis identified that apoA-I is cleaved (∼20-80%) by HMDMs in a time-dependent manner, generating apoA-I of lower MW and isoelectric point. Mass spectrometry analysis identified a novel C-terminal cleavage site of apoA-I between Ser228-Phe229 Recombinant apoA-I truncated at Ser228 demonstrated profound loss of capacity to solubilize lipid and to promote ABCA1-dependent cholesterol efflux. Protease inhibitors, small interfering RNA knockdown in HMDMs, mass spectrometry analysis, and cathepsin B activity assays identified secreted cathepsin B as responsible for apoA-I cleavage at Ser228 Importantly, C-terminal cleavage of apoA-I was also detected in human carotid plaque. Cleavage at Ser228 is a novel, functionally important post-translational modification of apoA-I mediated by HMDMs that limits the antiatherogenic properties of apoA-I.-Dinnes, D. L. M., White, M. Y., Kockx, M., Traini, M., Hsieh, V., Kim, M.-J., Hou, L., Jessup, W., Rye, K.-A., Thaysen-Andersen, M., Cordwell, S. J., Kritharides, L. Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

HDL particle size is a critical determinant of ABCA1-mediated macrophage cellular cholesterol export.

RATIONALE: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied. OBJECTIVE: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. METHODS AND RESULTS: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. CONCLUSIONS: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo.

Cellular cholesterol regulates ubiquitination and degradation of the cholesterol export proteins ABCA1 and ABCG1.

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes.

Stimulation of cholesterol efflux by LXR agonists in cholesterol-loaded human macrophages is ABCA1-dependent but ABCG1-independent.

OBJECTIVE: Maintenance of cholesterol homeostasis in human macrophages is essential to prevent foam cell formation. We evaluated the relative contribution of the ABCA1 and ABCG1 transporters to cholesterol efflux from human macrophages, and of the capacity of LXR agonists to reduce foam cell formation by stimulating export of cellular cholesterol. METHODS AND RESULTS: ABCG1 mRNA levels were strongly increased in acLDL-loaded THP-1 macrophages and in HMDM on stimulation with LXR agonists. However, silencing of ABCG1 expression using ABCG1-specific siRNA indicated that ABCG1 was not essential for cholesterol efflux to HDL in cholesterol-loaded human macrophages stimulated with LXR agonists. Indeed, ABCA1 was solely responsible for the stimulation of cholesterol efflux to HDL on LXR activation, as this effect was abolished in HMDM from Tangier patients. Furthermore, depletion of cellular ATP indicated that the LXR-induced export of cholesterol was an ATP-dependent transport mechanism in human macrophages. Finally, use of an anti-Cla-1 blocking antibody identified the Cla-1 receptor as a key component in cholesterol efflux to HDL from cholesterol-loaded human macrophages. CONCLUSIONS: Our data indicate that stimulation of cholesterol efflux to HDL by LXR agonists in human foam cells involves an ATP-dependent transport mechanism mediated by ABCA1 that it appears to be independent of ABCG1 expression.

Antibiotic resistance determinants in nosocomial strains of multidrug-resistant Acinetobacter baumannii.

OBJECTIVES: To investigate the presence of resistance genes in nosocomial multidrug-resistant (MDR) Acinetobacter baumannii isolated from outbreak and sporadic settings. METHODS: Thirty-two A. baumannii isolates were collected, 13 of which were involved in two outbreaks from different hospitals, which resulted in four deaths. The remaining 19 isolates were collected sporadically over 5 years from two other hospitals. The MICs of 25 antibiotics were determined for each isolate. PCR screening was carried out to identify possible genes that contributed to each resistance phenotype. Repetitive extragenic palindromic-PCR (REP-PCR) was performed to assess isolate clonality in conjunction with genotype data. RESULTS: Between eight and 12 resistance determinants were detected in the 32 MDR A. baumannii isolates examined. These resistance determinants included the genes blaOXA-23 and ampC, with the upstream element ISAba1 promoting increased gene expression and subsequent resistance to carbapenems and cephalosporins, respectively. In all isolates, resistance to quinolones and fluoroquinolones was conferred by an S83L mutation in GyrA. Twenty-eight of the 32 isolates were also positive for tet(B), a tetracycline resistance determinant, blaTEM-1, which contributed to beta-lactam resistance, and strB, which contributed to aminoglycoside resistance. Class 1 integrons that harboured aacC1, aadA1, qacEDelta1 and sul1 were identified in 10 of the 32 isolates (31%) together with the kanamycin resistance gene, aphA1. A putative trimethoprim resistance gene, folA, was also identified in all isolates. REP-PCR together with genotyping identified three main clonal types. CONCLUSIONS: Isolates of A. baumannii from both outbreak and sporadic cases possess at least eight resistance gene determinants that give rise to the MDR phenotype.

Norovirus excretion in an aged-care setting.

Norovirus genogroup II excretion during an outbreak of gastroenteritis was investigated in an aged-care facility. Viral shedding peaked in the acute stage of illness and continued for an average of 28.7 days. The viral decay rate was 0.76 per day, which corresponds to a viral half-life of 2.5 days.

The aadB gene cassette is associated with blaSHV genes in Klebsiella species producing extended-spectrum beta-lactamases.

Integrons were detected in 37 (72.5%) of 51 Klebsiella spp. producing extended-spectrum beta-lactamases by PCR with primers that targeted integrase genes and cassette regions. PCR and amplicon sequencing of the cassette regions revealed aadB and aadA2 gene cassettes that confer resistance to a range of aminoglycosides. aadB was associated with a class 1 integron on a 28-kb plasmid, pES1, that also contained bla(SHV-12) and IS26.

Integron-encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59-be site.

Integrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron-encoded IntI integrases. IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved. Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays. IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette-associated 59-be site and the cognate attI1 or attI3 site. Integrative recombination events involving a 59-be and a non-cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely. In cassette excision assays, the non-cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3. The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site.

Characterization of the class 3 integron and the site-specific recombination system it determines.

Integrons capture gene cassettes by using a site-specific recombination mechanism. As only one class of integron and integron-determined site-specific recombination system has been studied in detail, the properties of a second class, the only known class 3 integron, were examined. The configuration of the three potentially definitive features of integrons, the intI3 gene, the adjacent attI3 recombination site, and the P(c) promoter that directs transcription of the cassettes, was similar to that found in the corresponding region (5' conserved segment) of class 1 integrons. The integron features are flanked by a copy of the terminal inverted repeat, IRi, from class 1 integrons on one side and a resolvase-encoding tniR gene on the other, suggesting that they are part of a transposable element related to Tn402 but with the integron module in the opposite orientation. The IntI3 integrase was active and able to recognize and recombine both known types of IntI-specific recombination sites, the attI3 site in the integron, and different cassette-associated 59-be (59-base element) sites. Both integration of circularized cassettes into the attI3 site and excision of integrated cassettes were also catalyzed by IntI3. The attI3 site was localized to a short region adjacent to the intI3 gene. Recombination between a 59-be and secondary sites was also catalyzed by IntI3, but at frequencies significantly lower than observed with IntI1, the class 1 integron integrase.