Single-step protein purification by back flush in ion exchange chromatography

Ion exchange chromatography, one of the major procedures for protein purification, seldom provides single-step purification due to a lack of specific affinity. In this work, a novel and simple method called “back flush” (i.e., reversing the flow direction of elution relative to that of sample loading) was developed to achieve single-step purification on an ion exchanger. Tips for the conditions and operation by back flush are presented. Our study demonstrates, for the first time, the feasibility and dramatic improvement for protein purification by the back-flush method.     

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.     

Suppression of bacterial infection in rice by treatment with a sulfated peptide

The rice XA21 receptor kinase confers robust resistance to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). A tyrosine‐sulfated peptide from Xoo, called RaxX, triggers XA21‐mediated immune responses, including the production of ethylene and reactive oxygen species and the induction of defence gene expression. It has not been tested previously whether these responses confer effective resistance to Xoo. Here, we describe a newly established post‐inoculation treatment assay that facilitates investigations into the effect of the sulfated RaxX peptide in planta. In this assay, rice plants were inoculated with a virulent strain of Xoo and then treated with the RaxX peptide 2 days after inoculation. We found that post‐inoculation treatment of XA21 plants with the sulfated RaxX peptide suppresses the development of Xoo infection in XA21 rice plants. The treated plants display restricted lesion development and reduced bacterial growth. Our findings demonstrate that exogenous application of sulfated RaxX activates XA21‐mediated immunity in planta, and provides a potential strategy for the control of bacterial disease in the field.     

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.     

Significance of Pyruvate Carboxylase in Sugar Metabolism of Agrobacterium tumefaciens

Mutants, which fail to grow on glucose medium but can grow on succinate medium, were isolated by treatment with N-methyl-N′-nitro-N-nitrosoganidine from the wild-type strain of Agrobacterium tumefaciens, and were found to lose growth on several hexoses and three-carbon intermediates. The revertant mutants, which recovered the ability to grow on glucose medium, simultaneously regained the ability to grow on hexoses and three-carbon intermediates. By comparison of biochemical properties of the wild-type, the mutants and the revertant mutants, two mutant strains were characterized to be pyruvate carboxylase-deficient. Then, we concluded that these mutants might be induced by a single mutation at a genetic locus of pyruvate carboxylase and that the deficiency in the enzyme gave a pleiotropic effect on the ability to grow on hexoses and three-carbon intermediates. Some properties of pyruvate carboxylase of this bacterium were also presented.     

Evaluation of Propionibacterium acnes Isolates Using Contour-clamped Homogeneous Electric Field Gel Electrophoresis

Contour-clamped homogeneous electric field electrophoresis was performed to compare strains ofPropionibacterium acnes isolated from patients with chronic postoperative endophthalmitis. Propionibacterium acnes isolates were obtained from the vitreous humor of nine patients with chronic postoperative endophthalmitis following cataract surgery. In two of the patients, P. acnes isolates were also obtained from the aqueous humor as well as from the vitreous humor. Bacterial DNA was digested using Not I and Spe I restriction endonucleases. The DNA fragments were then subjected to contour-clamped homogeneous electric field electrophoresis and the DNA banding patterns were analysed. Eight nonidentical banding patterns were identified among the nine vitreous isolates of P. acnes. In each of the two cases from which aqueous and vitreous isolates were recovered from the same eye, the banding patterns were identical. Contour-clamped homogeneous electric field electrophoresis is a powerful method to distinguish P. acnes isolates based on DNA banding patterns and could be used in the epidemiological study of clinical processes caused by this organism.