Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17

Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S⁸⁸VS⁹⁰K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies.Members of the genus Acinetobacter are nonmotile Gram-negative bacteria, many of which cause severe, life-threatening infections and hospital outbreaks (1). Although Acinetobacter baumannii is regarded as an opportunistic pathogen with low virulence, this species infects the soft tissues, bone, bloodstream, and urinary tract and is an important cause of pneumonia and meningitis in immune-compromised patients (2). Crude mortalities because of nosocomial pneumonia and bloodstream infections caused by A. baumannii ranged from 30–75% and 25–54%, respectively (3–5). In intensive care units (ICU), outbreaks of infection caused by multidrug-resistant A. baumannii strains exhibit a crude mortality rate as high as 91.7% (4, 5). The poor outcome in patients with invasive multidrug-resistant A. baumannii infection highlights the urgent need for new therapeutic agents and vaccines to reduce the associated morbidity and mortality.The survival of A. baumannii is enhanced by its ability to acquire foreign genes, thus increasing the number of vulnerable hosts, producing biofilms, and displaying an open pan-genome (6, 7). These abilities enable A. baumannii to persist in nosocomial environments and to survive even under antibiotic treatment. Numerous studies have reported the emergence of A. baumannii clinical isolates that are resistant to multiple antimicrobials such as carbapenems, colistin, sulbactam, and tigecycline, thus reducing the number of effective therapeutic options (8, 9). In epidemiological studies, the incidence rate of carbapenem-resistant A. baumannii in countries such as Australia, Brazil, Singapore, Canada, South Korea, Taiwan, and Thailand is in the range of 47–80% (10). A study showed that 11% of nosocomial isolates of A. baumannii were carbapenem-resistant; resulting in a morbidity and mortality rate of 52% as compared with a rate of 19% of patients infected with carbapenem-sensitive isolates (4, 11–13). Among the many carbapenem derivatives, imipenem initially was highly effective in the treatment of patients with A. baumannii infections; however, imipenem resistance has been confirmed in 53.7% of Acinetobacter nosocomial infections since the early 1990s (4, 14, 15). The most common pathways leading to carbapenem resistance are associated with the loss of outer membrane porins, overexpression of efflux pumps, and overproduction of Ambler class B metallo-β-lactamases, class D oxacillinases, and AmpC cephalosporinase (16–18). In the case of Acinetobacter-derived cephalosporinase (ADC)¹, the key upstream insertion sequence (IS) element, ISAba1, provides promoter sequences that confer bacterial resistance to broad-spectrum cephalosporins (3, 19, 20). In a study of Pseudomonas aeruginosa, the overproduction of AmpC β-lactamase exhibited weak carbapenem-hydrolyzing activity and thus contributed to carbapenem resistance in porin-deficient isolates (21). Although the study suggested a link between AmpC β-lactamase and carbapenem resistance, the regulatory mechanisms remain unclear.Kinase-induced protein phosphorylation and phosphatase-induced protein dephosphorylation are crucial for signal transduction in both prokaryotic and eukaryotic species (22–26). Hence, bacterial phosphoproteomic analysis is a promising and accurate tool to study biological networks, including the mechanisms of antibiotic resistance. In a recent comparative phosphoproteomic study of A. baumannii ATCC17978 and the multidrug-resistant clinical isolate A. baumannii Abh12O-A2, the relationship between phosphoproteins and antibiotic resistance remained unclear because of the lack of biological confirmation (27). In this study, we used two clinical isolates of A. baumannii to establish comparative phosphoproteomic maps and to conduct biological validation to explore the mechanisms of imipenem resistance (28). Phosphoproteomic analysis of A. baumannii SK17 clinical strains was carried out using a shotgun strategy combined with phosphopeptides enrichment techniques and high-performance mass spectrometry, and thus the identified phosphosites were verified by site-directed mutagenesis (23, 29–31). Our findings clearly show that AmpC β-lactamase activity is regulated by phosphorylation and is involved in imipenem resistance.     

Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM.

Tumor therapy by the preferential activation of a prodrug at tumor cells targeted with an antibody-enzyme conjugate may allow improved treatment efficacy with reduced side effects. We examined antibody-mediated clearance of poly(ethylene glycol)-modified beta-glucuronidase (betaG-sPEG) as a method to reduce serum concentrations of enzyme and minimize systemic prodrug activation. Enzyme-linked immunosorbent assay and immunoblot analysis of two monoclonal antibodies generated by immunization of BALB/c mice with an antibody-betaG-sPEG conjugate showed that mAb 1E8 (IgG1) bound betaG and betaG-sPEG whereas mAb AGP3 (IgM) bound poly(ethylene glycol). Neither antibody affected the betaG activity. mAb 1E8 and AGP3 were modified with 36 and 208 galactose residues (1E8-36G and AGP3-208G) with retention of 72 and 48% antigen-binding activity, respectively, to target immune complexes to the asialoglycoprotein receptor on liver cells. mAb 1E8 and AGP3 cleared betaG-PEG from the circulation of mice as effectively as 1E8-36G and AGP3-208G, respectively. mAb AGP3, however, cleared betaG-sPEG more completely and rapidly than 1E8, reducing the serum concentration of betaG-sPEG by 38-fold in 8 h. AGP3 also reduced the concentration of an antibody-betaG-sPEG conjugate in blood by 280-fold in 2 h and 940-fold in 24 h. AGP3-mediated clearance did not produce obvious damage to liver, spleen, or kidney tissues. In addition, AGP3 clearance of betaG-sPEG before administration of BHAMG, a glucuronide prodrug of p-hydroxyaniline mustard, prevented toxicity associated with systemic activation of the prodrug based on mouse weight and blood cell numbers. AGP3 should be generally useful for accelerating the clearance of PEG-modified proteins as well as for improving the tumor/blood ratios of antibody-betaG-PEG conjugates for glucuronide prodrug therapy of cancer.     

bilbo, a non-LTR retrotransposon of Drosophila subobscura: a clue to the evolution of LINE-like elements in Drosophila

We used the repetitive character of transposable elements to isolate a non-LTR retrotransposon in Drosophila subobscura. bilbo, as we have called it, has homology to TRIM and LOA elements. Sequence analysis showed a 5' untranslated region (UTR), an open reading frame (ORF) with no RNA-binding domains, a downstream ORF that had structural homology to that of the I factor, and, finally, a 3' UTR which ended in several 5-nt repeats. The results of our phylogenetic and structural analyses shed light on the evolution of Drosophila non-LTR retrotransposons and support the hypothesis that an ancestor of these elements was structurally complex.      

Regulation of adenylyl cyclase in the central nervous system

Adenylyl cyclases (ACs) are a family of enzymes that synthesize one of the major second messengers, cAMP, upon stimulation. Since the report of the first adenylyl cyclase (AC) gene in 1989, tremendous efforts have been devoted to identifying and characterizing more AC isozymes. In the past decade, significant knowledge regarding the basic structure, tissue distribution, and regulation of AC isozymes has been accumulated. Because members of the AC superfamily are tightly controlled by various signals, one of the most important impacts of these AC isozymes is their contribution to the complexity and fine-tuning of cellular signalling, especially in the central nervous system (CNS) where multiple signals constantly occur. This review focuses on recent progress toward understanding the physiological roles of ACs in the CNS.     

Activation of protein kinase A and atypical protein kinase C by A(2A) adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells

We found in the present study that stimulation of A(2A) adenosine receptors (A(2A)-R) prevents apoptosis in PC12 cells. This A(2A)-protective effect was blocked by protein kinase A (PKA) inhibitors and was not observed in a PKA-deficient PC12 variant. Stimulation of PKA also prevented apoptosis, suggesting that PKA is required for the protective effect of A(2A)-R. A general PKC inhibitor, but not down-regulation of conventional and novel PKCs, readily blocked the protective effect of A(2A)-R stimulation and PKA activation, suggesting that atypical PKCs (aPKCs) serve a critical role downstream of PKA. Consistent with this hypothesis, stimulation of A(2A)-R or PKA enhanced nuclear aPKC activity. In addition, the A(2A)-protective effect was blocked by a specific inhibitor of one aPKC, PKCzeta, whereas overexpression of a dominant-positive PKCzeta enhanced survival. In contrast, inhibitors of MAP kinase and phosphatidylinositol 3-kinase did not modulate the A(2A)-protective effect. Dominant-negative Akt also did not alter the A(2A)-protective effect, whereas it significantly reduced the protective action of nerve growth factor. Collectively, these data suggest that aPKCs can function downstream of PKA to mediate the A(2A)-R-promoted survival of PC12 cells. Furthermore, the results indicate that different extracellular stimuli can employ distinct signaling pathways to protect against apoptosis induced by the same insult.     

The Influence of Pathological Mutations and Proline Substitutions in TDP-43 Glycine-Rich Peptides on Its Amyloid Properties and Cellular Toxicity

TAR DNA-binding protein (TDP-43) was identified as the major ubiquitinated component deposited in the inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) in 2006. Later on, numerous ALS-related mutations were found in either the glycine or glutamine/asparagine-rich region on the TDP-43 C-terminus, which hinted on the importance of mutations on the disease pathogenesis. However, how the structural conversion was influenced by the mutations and the biological significance of these peptides remains unclear. In this work, various peptides bearing pathogenic or de novo designed mutations were synthesized and displayed their ability to form twisted amyloid fibers, cause liposome leakage, and mediate cellular toxicity as confirmed by transmission electron microscopy (TEM), circular dichroism (CD), Thioflavin T (ThT) assay, Raman spectroscopy, calcein leakage assay, and cell viability assay. We have also shown that replacing glycines with prolines, known to obstruct β-sheet formation, at the different positions in these peptides may influence the amyloidogenesis process and neurotoxicity. In these cases, GGG308PPP mutant was not able to form beta-amyloid, cause liposome leakage, nor jeopardized cell survival, which hinted on the importance of the glycines (308–310) during amyloidogenesis.     

The effects of temperature and shading on mortality and development rates of Aedes aegypti (Diptera: Culicidae)

Urbanization has caused an increase in favorable habitats for Aedes aegypti (Diptera: Culicidae), given their ability to reproduce in small and often non‐degradable artificial water‐containers. While much work has been done on Ae. aegypti biology and ecology in urban landscapes, the role of shading on immature stages as an independent factor from temperature, and any possible interactions between these factors, remains unexamined. We assessed how temperature and shading affected egg hatch‐rate, larval/pupal mortality, and larval development to adult stage under different factorial temperature (28; 31; 34; 37; 40° C) and shade (0%, 3,100 lux; 40%, 1,860 lux; 75%, 775 lux; 100%, 0 lux) regimes. Hatch‐rate was significantly lower at 37° C (57 %), and no eggs hatched at 40° C. There was no significant effect caused by shading on hatchability. Larval and pupal mortality at 37° C was significantly higher (35%) compared to lower temperature groups, while the effects of shading were emergent at low temperatures. Developmental times from hatching to adult emergence were significantly reduced with increasing temperatures and with greater light exposures. The eco‐physiological response of Ae. aegypti larvae to temperature and light regimes suggest a photosensitivity previously unstudied in this species.     

Macroinvertebrates as bioindicators of water quality in the Mkondoa River,Tanzania, in an agricultural area

The suitability of using macroinvertebrates as bioindicators of stream water quality was tested in the Mkondoa River in an agricultural area at Kilosa, using the rapid bioassessment protocol. The family biotic index (FBI) showed marked variation in water quality along the stream from values ranging from 4.1 to 5.0 in the upstream reaches, indicating good water quality, 5.3 to 5.5 in the mid-reaches and 6.0 to 6.5 in the lower reaches. The water quality index (WQI) indicated that water quality was fair (77 ± 0.98) in the upstream reach of the Mkondoa, marginal (55 ± 0.86) in the midstream reach and poor (33 ± 0.45) in the downstream reach. There were significant relationships between biological oxygen demand and dissolved oxygen and the occurrence of specific taxa, mainly Chironomus and Caenis. Significant changes in macroinvertebrate abundance were mostly related to changes in water quality. As in other parts of the world, macroinvertebrate communities proved to be good biological indicators of water quality and they should be used as bioindicators in long-term monitoring of this river.