Signal peptide replacement of Ag43 enables an efficient bacterial cell surface display of receptor-binding domain of coronavirus.

To enable an efficient bacterial cell surface display with effective protein expression and cell surface loading ability via autotransporter for potential vaccine development applications, the inner membrane protein translocation efficiency was investigated via a trial-and-error strategy by replacing the original unusual long signal peptide of E. coli Ag43 with 11 different signal peptides. The receptor-binding domain (RBD) of coronavirus was used as a neutral display substrate to optimize the expression conditions, and the results showed that signal peptides from PelB, OmpC, OmpF, and PhoA protein enhance the bacterial cell surface display efficiency of RBD. In addition, the temperature has also a significant effect on the autodisplay efficiency of RBD. Our data provide further technical basis for the biotechnological application of Ag43 as a bacterial surface display carrier system and further potential application in vaccine development.

A minimum functional form of the Escherichia coli BAM complex constituted by BamADE assembles outer membrane proteins in vitro.

The biogenesis of outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM), which is a heteropentomeric complex composed of five proteins named BamA-E in Escherichia coli. Despite great progress in the BAM structural analysis, the molecular details of BAM-mediated processes as well as the exact function of each BAM component during OMP assembly are still not fully understood. To enable a distinguishment of the function of each BAM component, it is the aim of the present work to examine and identify the effective minimum form of the E. coli BAM complex by use of a well-defined reconstitution strategy based on a previously developed versatile assay. Our data demonstrate that BamADE is the core BAM component and constitutes a minimum functional form for OMP assembly in E. coli, which can be stimulated by BamB and BamC. While BamB and BamC have a redundant function based on the minimum form, both together seem to cooperate with each other to substitute for the function of the missing BamD or BamE. Moreover, the BamAE470K mutant also requires the function of BamD and BamE to assemble OMPs in vitro, which vice verse suggests that BamADE are the effective minimum functional form of the E. coli BAM complex.

Licochalcone A inhibits the assembly function of ß-barrel assembly machinery in Escherichia coli.

Antimicrobial resistance (AMR) crisis urges the development of new antibiotics. In the present work, we for the first time used bio-affinity ultrafiltration combined with HPLC-MS (UF-HPLC-MS) to examine the interaction between the outer membrane ß-barrel proteins and natural products. Our results showed that natural product licochalcone A from licorice interacts with BamA and BamD with the enrichment factor of 6.38 ± 1.46 and 4.80 ± 1.23, respectively. The interaction was further confirmed by use of biacore analysis, which demonstrated that the Kd value between BamA/D and licochalcone was 6.63/28.27 µM, suggesting a good affinity. To examine the effect of licochalcone A on BamA/D function, the developed versatile in vitro reconstitution assay was used and the results showed that 128 µg/mL licochalcone A could reduce the outer membrane protein A integration efficiency to 20%. Although licochalcone A alone can not inhibit the growth of E. coli, but it can affect the membrane permeability, suggesting that licochalcone A holds the potential to be used as a sensitizer to combat AMR.

What Approaches to Thwart Bacterial Efflux Pumps-Mediated Resistance?

An effective response that combines prevention and treatment is still the most anticipated solution to the increasing incidence of antimicrobial resistance (AMR). As the phenomenon continues to evolve, AMR is driving an escalation of hard-to-treat infections and mortality rates. Over the years, bacteria have devised a variety of survival tactics to outwit the antibiotic's effects, yet given their great adaptability, unexpected mechanisms are still to be discovered. Over-expression of efflux pumps (EPs) constitutes the leading strategy of bacterial resistance, and it is also a primary driver in the establishment of multidrug resistance (MDR). Extensive efforts are being made to develop antibiotic resistance breakers (ARBs) with the ultimate goal of re-sensitizing bacteria to medications to which they have become unresponsive. EP inhibitors (EPIs) appear to be the principal group of ARBs used to impair the efflux system machinery. Due to the high toxicity of synthetic EPIs, there is a growing interest in natural, safe, and innocuous ones, whereby plant extracts emerge to be excellent candidates. Besides EPIs, further alternatives are being explored including the development of nanoparticle carriers, biologics, and phage therapy, among others. What roles do EPs play in the occurrence of MDR? What weapons do we have to thwart EP-mediated resistance? What are the obstacles to their development? These are some of the core questions addressed in the present review.

Targeting Bacterial Membrane Proteins to Explore the Beneficial Effects of Natural Products: New Antibiotics against Drug Resistance.

Antibiotic resistance is currently a world health crisis that urges the development of new antibacterial substances. To this end, natural products, including flavonoids, alkaloids, terpenoids, steroids, peptides and organic acids play a vital role in the development of medicines and thus constitute a rich source in clinical practices, providing an important source of drugs directly or for the screen of lead compounds for new antibiotic development. Because membrane proteins, which comprise more than 60% of the current clinical drug targets, play crucial roles in signal transduction, transport, bacterial pathogenicity and drug resistance, as well as immunogenicity, it is our aim to summarize those natural products with different structures that target bacterial membrane proteins, such as efflux pumps and enzymes, to provide an overview for the development of new antibiotics to deal with antibiotic resistance.

Severe Acute Respiratory Syndrome Coronavirus 2 Viral RNA Load Status and Antibody Distribution Among Patients and Asymptomatic Carriers in Central China.

This study aimed to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral loads and specific serum-antibodies (immunoglobulin [Ig] G and M) among confirmed patients and asymptomatic carriers from returning healthy travelers. The throat swabs, sputum, and stool samples from 57 hospitalized coronavirus disease (COVID-19) patients and 8 asymptomatic carriers, among 170 returning healthy travelers were tested using reverse-transcription real-time polymerase chain reaction. SARS-CoV-2 IgM/IgG antibodies were detected via serum chemiluminescence assay. Sequential results showed higher viral RNA loads in the throat, sputum, and stool samples at 3-12 and 6-21 days after symptom onset among severely ill COVID-19 patients. Shorter viral habitation time (1-8 days) was observed in the oropharyngeal site and intestinal tract of asymptomatic carriers. The IgG and IgM response rates were 19/37 (51.4%) and 23/37 (62.6%) among the 29 confirmed patients and 8 asymptomatic carriers, respectively, within 66 days from symptom or detection onset. The median duration between symptom onset and positive IgG and IgM results was 30 (n=23; interquartile range [IQR]=20-66) and 23 (n=19; IQR=12-28) days, respectively. Of 170 returning healthy-travelers to China, 4.7% were asymptomatic carriers (8/170) within 2 weeks, and the IgG and IgM positivity rate was 12.8% (12/94). IgM/IgG-positivity confirmed 3 suspected SARS-CoV-2 cases, despite negative results for SARS-CoV-2 RNA. Compared with other respiratory viral infectious diseases, COVID-19 has fewer asymptomatic carriers, lower antibody response rates, and a longer antibody production duration in recovered patients and the contacted healthy population. This is an indication of the complexity of COVID-19 transmission.

Response surface methodology optimised solvothermal system enables an efficient extraction of echinacoside and oleuropein from Syringa pubescens Turcz.

INTRODUCTION: Syringa pubescens Turcz. was reported to be abundant in the Funiu Mountains of Henan Province and can be used to treat hepatitis and cirrhosis. In order to develop and utilise the resource, a fast and simple technique to extract bioactive compounds is needed. OBJECTIVES: Our aims were to provide an extraction technique of glycosides from S. pubescens and study the antioxidant activity of this material. METHODS: Box-Behnken design (BBD) was employed with three factors at three levels. The process parameters such as ethanol concentration (X1 ), temperature (X2 ), and solvent-solid ratio (X3 ) could significantly influence efficiency and yield of target compounds. High-performance liquid chromatography (HPLC) was used to determine the content of glycosides. DPPH (α,α-diphenyl-ß-picrylhydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and reducing power were used to evaluate the antioxidant activity of S. pubescens extract. RESULTS: The optimal parameters for the maximal extraction yield were obtained with ethanol concentration of 68%, temperature of 89°C, solvent-solid ratio of 46 mL/g, and time of 20 min. The S. pubescens extract showed strong antioxidant properties in vitro. CONCLUSION: The findings indicated the potential application of solvothermal extraction method to extract bioactive compounds from S. pubescens Turcz. Furthermore, the S. pubescens extract could be used as an important resource of antioxidant activity.

TtOmp85, a single Omp85 member protein functions as a ß-barrel protein insertase and an autotransporter translocase without any accessory proteins.

The biogenesis of outer membrane proteins requires the function of ß-barrel assembly machinery (BAM), whose function is highly conserved while its composition is variable. The Escherichia coli BAM is composed of five subunits, while Thermus thermophilus seems to contain a single BAM protein, named TtOmp85. To search for the primitive form of a functional BAM, we investigated and compared the function of TtOmp85 and E. coli BAM by use of a reconstitution assay that examines the integration of OmpA and BamA from E. coli and TtoA from T. thermophilus, as well as the translocation of the E. coli Ag43. Our results show that a single TtOmp85 protein can substitute for the collective function of the five subunits constituting E. coli BAM.

Development of a fourth-order compact finite difference scheme for simulation of simulated-moving-bed process.

A fourth-order compact finite difference scheme was developed to solve the model equation of simulated moving bed, which has a boundary condition that is updated along the calculation process and cannot be described as an explicit function of time. Two different methods, direct method and pseudo grid point method, were proposed to deal with the boundary condition. The high accuracy of the two methods was confirmed by a case study of solving an advection-diffusion equation with exact solution. The developed compact finite difference scheme was then used to simulate the SMB processes for glucose-fructose separation and enantioseparation of 1,1'-bi-2-naphtol. It was found that the simulated results fit well with the experimental data. Furthermore, the developed method was further combined with the continuous prediction method to shorten the computational time and the results showed that, the computational time can be saved about 45%.

An Integrated Approach for Combinatorial Readout of Dual Histone Modifications by Epigenetic Tandem Domains.

Histone post-translational modifications (HPTMs) serve as signal platforms for recruitment of binding proteins (readers) to regulate gene expression. Accumulated evidence suggests that the intensive distribution of HPTMs may result in crosstalk, which increases or inhibits the recruitment of reader proteins, further altering the functional outcome of HPTMs. Therefore, the comprehensive identification of multiple interactions between combinatorial HPTMs and reading domains is essential to understand the chromatin-templated processes. However, it is still a big challenge to profile these complicated interactions due to various limitations including rather weak, transient and multiple interactions between HPTMs and readers, the high dynamic property of HPTMs as well as the low abundance of reader proteins. Here we developed an integrated approach to profile the complicated interactions between combinatorial HPTMs and dual domains. Based on a combinatorial HPTM peptide library (trimethylation of histone H3 lysine 4 and its neighboring PTMs) and five affinity tag proteins containing tandem-domain probes, histone interactions can be profiled by pull-down assay combined with mass spectrometry analysis. The interactions were further verified by isothermal titration calorimetry and proximity ligation assay, as well as molecular docking. By use of combinatorial HPTMs, we demonstrated that this integrated approach can be successfully utilized for the characterization of multiple interactions between reading domains and combinatorial HPTMs including novel HPTMs with low stoichiometry. Thus, a novel chemical proteomics tool for profiling of multiple PTM-mediated protein-protein interactions was successfully developed and can be adapted for broad biomedical applications.

Risk Factors for Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections and Outcomes.

PURPOSE: The incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) bloodstream infections (BSIs) is increasing globally; however, little has been reported on the risk factors and outcomes of CRKP BSIs in central China. This study aimed to determine the clinical risk factors for CRKP BSIs and the outcomes of CRKP BSIs. PATIENTS AND METHODS: We performed a case-control study of 239 patients with K. pneumoniae BSIs who were treated at Henan Provincial People's Hospital between July 2017 and July 2018. The cases (n=98, 41%) had CRKP BSIs, and the controls (n=141, 59%) had non-carbapenem-resistant K. pneumoniae (non-CRKP) BSIs. Antimicrobial sensitivity was determined using automated broth microdilution and an agar disk diffusion method. Data were obtained from clinical and laboratory records. Multivariate logistic regression and Pearson chi-square tests were used to identify clinical factors and outcomes associated with carbapenem resistance. RESULTS: Risk factors for carbapenem resistance included recent carbapenem use (odds ratio [OR]: 9.98, 95% confidence interval [CI]: 5.2-17.1, P<0.001), invasive procedures (OR: 11.1, 95% CI: 3.3-37.7, P<0.001), and pre-existing diseases of the digestive system (OR: 8.22, 95% CI: 1.73-39.2, P=0.008). Treatment failure was more frequent in the cases (84.7%) than in the controls (32.6%). CONCLUSION: Exposure to antibiotics, especially carbapenems, and invasive procedures were the major risk factors for carbapenem resistance among patients with K. pneumoniae BSIs. Strict control measures should be implemented to prevent the emergence and spread of CRKP.

The first imported case of melioidosis in a patient in central China.

Here, we report the first imported case of melioidosis from Laos in central China. COMPACT VITEK2 identification system and PCR, as well as sequencing methods confirmed that the patient was infected by Burkholderia pseudomallei, a bacterial species closely related to an isolate detected in Thailand. These findings are highly valuable for an early diagnosis, treatment and to prevent the spread of this emerging infectious disease in central China.

Protein lysine de-2-hydroxyisobutyrylation by CobB in prokaryotes.

Lysine 2-hydroxyisobutyrylation (Khib) has recently been shown to be an evolutionarily conserved histone mark. Here, we report that CobB serves as a lysine de-2-hydroxyisobutyrylation enzyme that regulates glycolysis and cell growth in prokaryotes. We identified the specific binding of CobB to Khib using a novel self-assembled multivalent photocrosslinking peptide probe and demonstrated that CobB can catalyze lysine de-2-hydroxyisobutyrylation both in vivo and in vitro. R58 of CobB is a critical site for its de-2-hydroxyisobutyrylase activity. Using a quantitative proteomics approach, we identified 99 endogenous substrates that are targeted by CobB for de-2-hydroxyisobutyrylation. We further demonstrated that CobB can regulate the catalytic activities of enolase (ENO) by removing K343hib and K326ac of ENO simultaneously, which account for changes of bacterial growth. In brief, our study dissects a Khib-mediated molecular mechanism that is catalyzed by CobB for the regulation of the activity of metabolic enzymes as well as the cell growth of bacteria.

Reactive oxygen species inhibit biofilm formation of Listeria monocytogenes.

Although the level of reactive oxygen species (ROS) is altered upon the formation of bacterial biofilm, the relationship between ROS alteration and biofilm formation is still unclear. The aim of the present study is to use Listeria monocytogenes (L. monocytogenes) as a model organism to examine whether ROS have an effect on the biofilm formation. After eliminating ROS by treatment with NAD(P)H oxidase inhibitor Diphenyleneiodonium chloride (DPI) or scavenging reagents N-acetylcysteine (NAC), the biofilm formation of L. monocytogenes was examined. Our data demonstrate that DPI and NAC induced-reduction of ROS enhances the biofilm formation in L. monocytogenes without affecting bacterial growth and activity. These data provide the evidence that ROS produced by L. monocytogenes inhibit the biofilm formation.

Reactive oxygen species inhibits Listeria monocytogenes invasion into HepG2 epithelial cells.

Listeria monocytogenes (Lm) can colonize human gastrointestinal tract and subsequently cross the intestinal barrier. Reactive oxygen species (ROS) are produced by NADPH oxidase. However, the role of ROS in bacterial invasion remains to be less understood. Herein, we investigated the impact of ROS on Lm invasion to HepG2 using NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), as well as the ROS scavenger, N-acetyl cysteine (NAC). Our results showed that inhibiting ROS increased the invasive capability of Lm. Moreover, after Lm infection, inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL-1ß) in HepG2 were significantly upregulated. However, after inhibiting ROS, the expression levels of TNF-α and IL-1ß were downregulated, indicating a failure of host cells to activate the immune mechanism. Taken together, ROS in Lm might be as a signal for host cells to sense Lm invasion and then stimulate cells to activate the immune mechanism.

An Efficient Approach for Selective Enrichment of Histone Modification Readers Using Self-Assembled Multivalent Photoaffinity Peptide Probes.

Histone post-translational modifications (HPTMs) provide signaling platforms to recruit proteins or protein complexes (e.g., transcription factors, the so-called "readers" of the histone code), changing DNA accessibility in the regulation of gene expression. Thus, it is an essential task to identify HPTM readers for understanding of epigenetic regulation. Herein we designed and prepared a novel HPTM probe based on self-assembled multivalent photo-cross-linking technique for selective enrichment and identification of HPTM readers. By use of trimethylation of histone H3 lysine 4, we showcased that the functionalized HPTM probe was able to capture its reader with high enrichment efficiency and remarkable specificity even in a complex environment. Notably, this approach was readily applicable for exploring crosstalk among multiple HPTMs. Combining the probes with a mass spectrometry-based proteomic approach, our approach reached a fairly high coverage of known H3K4me3 readers. We further demonstrated that the HPTM probes can enrich a new type of HPTM readers and uncovered several novel putative binders of crotonylation of histone H3 lysine 9, expanding the repertoire of readers for this epigenetic mark. More broadly, our work provides a general strategy for rapid and robust interrogating HPTM readers and will be of great importance to elucidate epigenetic mechanism in regulating gene activity.

Systematic Identification of Lysine 2-hydroxyisobutyrylated Proteins in Proteus mirabilis.

Lysine 2-hydroxyisobutyrylation (Khib) is a novel post-translational modification (PTM), which was thought to play a role in active gene transcription and cellular proliferation. Here we report a comprehensive identification of Khib in Proteus mirabilis (P. mirabilis). By combining affinity enrichment with two-dimensional liquid chromatography and high-resolution mass spectrometry, 4735 2-hydroxyisobutyrylation sites were identified on 1051 proteins in P. mirabilis. These proteins bearing modifications were further characterized in abundance, distribution and functions. The interaction networks and domain architectures of these proteins with high confidence were revealed using bioinformatic tools. Our data demonstrate that many 2-hydroxyisobutyrylated proteins are involved in metabolic pathways, such as purine metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis. The extensive distribution of Khib also indicates that the modification may play important influence to bacterial metabolism. The speculation is further supported by the observation that carbon sources can influence the occurrence of Khib Furthermore, we demonstrate that 2-hydroxyisobutyrylation on K343 was a negative regulatory modification on Enolase (ENO) activity, and molecular docking results indicate the regulatory mechanism that Khib may change the binding formation of ENO and its substrate 2-phospho-d-glycerate (2PG) and cause the substrate far from the active sites of enzyme. We hope this first comprehensive analysis of nonhistone Khib in prokaryotes is valuable for further functional investigation of this modification.

Plant Phenolics Extraction from Flos Chrysanthemi: Response Surface Methodology Based Optimization and the Correlation Between Extracts and Free Radical Scavenging Activity.

Huaiju is one of the most famous and widely used Flos Chrysanthemi (FC) for medicinal purposes in China. Although various investigations aimed at phenolics extraction from other FC have been reported, a thorough optimization of the phenolics extraction conditions from Huaiju has not been achieved. This work applied the widely used response surface methodology (RSM) to investigate the effects of 3 independent variables including ethanol concentration (%), extraction time (min), and solvent-to-material ratio (mL/g) on the ultrasound-assisted extraction (UAE) of phenolics from FC. The data suggested the optimal UAE condition was an ethanol concentration of 75.3% and extraction time of 43.5 min, whereas the ratio of solvent to material has no significant effect. When the free radical scavenging ability was used as an indicator for a successful extraction, a similar optimal extraction was achieved with an ethanol concentration of 72.8%, extraction time of 44.3 min, and the ratio of solvent to material was 29.5 mL/g. Furthermore, a moderate correlation between the antioxidant activity of TP extract and the content of extracted phenolic compounds was observed. Moreover, a well consistent of the experimental values under optimal conditions with those predicted values suggests RSM successfully optimized the UAE conditions for phenolics extraction from FC. PRACTICAL APPLICATION: The work of the research investigated the plant phenolics in Flos Chrysanthemi and antioxidant capacities. These results of this study can support the development of antioxidant additive and relative food.

Maleic Anhydride Labeling-Based Approach for Quantitative Proteomics and Successive Derivatization of Peptides.

Chemical derivatization is a simple approach for stable-isotope covalent labeling of proteins in quantitative proteomics. Herein we describe the development of a novel maleyl-labeling-based approach for protein quantification. Under optimized conditions, maleic anhydride can serve as a highly efficient reagent to label the amino groups of tryptic peptides. Furthermore, "click chemistry" was successfully applied to obtain the second modification of maleylated peptides via thiol-Michael addition reaction. Accurate quantification was further achieved via the first or/and second step stable-isotope labeling in this study. Our data thus demonstrate that the maleyl-labeling-based method is simple, accurate, and reliable for quantitative proteomics. The developed method not only enables an enhanced sequence coverage of proteins by improving the identification of small and hydrophilic peptides, but also enables a controllable, successive, second derivatization of labeled peptides or proteins, and therefore holds a very promising potential for in-depth analysis of protein structures and dynamics.

DNA-Templated Aptamer Probe for Identification of Target Proteins.

Using aptamers as molecular probes for biomarker discovery has attracted a great deal of attention in recent years. However, it is still a big challenge to accurately identify those protein markers that are targeted by aptamers under physiological conditions due to weak and noncovalent aptamer-protein interactions. Herein, we developed an aptamer based dual-probe using DNA-templated chemistry and photo-cross-linking technique for the identification of target proteins that are recognized by aptamers. In this system, the aptamer was modified by a single strand DNA as binding probe (BP), and another complementary DNA with a photoactive group and reporter group was modified as capture probe (CP). BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the cross-linker close to the target via DNA self-assembly, and then a covalent bond between CP and its binding protein was achieved via photo-cross-linking reaction. The captured protein can be detected or affinity enrichment using the tag, finally identified by MS. By use of lysozyme as a model substrate, we demonstrated that this multiple functionalized probe can be utilized for a successful labeling and enrichment of target protein even under a complicated and real environment. Thus, a novel method to precisely identify the aptamer-targeted proteins has been developed and it has a potential application for discovery of aptamer-based biomarkers.