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Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large‐scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac‐T3SS)‐mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self‐limiting growth. Compared to the method of transfection, the Bac‐T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard‐to‐transfect human cell lines. Furthermore, through the delivery of β‐lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac‐T3SS. This study provided an efficient and low‐cost proteofection strategy for laboratory use as well as for application in large‐scale cell manipulations. [ABSTRACT FROM AUTHOR]

Summary: The biofilm formation by Pseudomonas aeruginosa highly increases the bacterial resistance to antimicrobial agents and host immune clearance. The biofilm formation is positively regulated by two small RNAs, RsmY and RsmZ. Previously, we reported that mutation in the polynucleotide phosphorylase (PNPase) coding gene pnp increases the levels of RsmY/Z. However, in this study, we found that the biofilm formation is decreased in the pnp mutant, which is due to a defect in rhamnolipids production. The rhamnolipids production is regulated by the RhlI‐RhlR quorum sensing system. We found that PNPase influences the translation of RhlI through its 5′‐untranslated region (UTR) and identified that the sRNA P27 is responsible for the translational repression. In vitro translation experiments demonstrated that P27 directly represses the translation of the rhlI mRNA through its 5′UTR in an Hfq‐dependent manner. Point mutations in the rhlI 5′UTR or P27, which abolish the pairing between the two RNAs restore the rhlI expression and rhamnolipids production as well as the biofilm formation in the pnp mutant. Overall, our results reveal a novel layer of regulation of the Rhl quorum sensing system by the sRNA P27. [ABSTRACT FROM AUTHOR]

Bacterial biofilms are surface‐attached communities of non‐replicating bacteria innately tolerant to antibiotics. Biofilms display differential gene expression profiles and physiologies as compared to their planktonic counterparts; however, their biology remains largely unknown. In this study, we used a halogenated phenazine (HP) biofilm eradicator in transcript profiling experiments (RNA‐seq) to define cellular targets and pathways critical to biofilm viability. WoPPER analysis with time–course validation (RT‐qPCR) revealed that HP‐14 induces rapid iron starvation in MRSA biofilms, as evident by the activation of iron‐acquisition gene clusters in 1 hour. Serine proteases and oligopeptide transporters were also found to be up‐regulated, whereas glycolysis, arginine deiminase, and urease gene clusters were down‐regulated. KEGG analysis revealed that HP‐14 impacts metabolic and ABC transporter functional pathways. These findings suggest that MRSA biofilm viability relies on iron homeostasis. Exploring ways to end a film: A halogenated phenazine probe molecule was used in transcript‐profiling investigations with RNA‐seq technology to define cellular targets and pathways critical to biofilm survival (see picture; MRSA, methicillin‐resistant Staphylococcus aureus). The results suggest that MRSA biofilm viability relies on iron homeostasis and establish a framework for the development of next‐generation therapeutics to eradicate biofilms. [ABSTRACT FROM AUTHOR]

Agents capable of eradicating bacterial biofilms are of great importance to human health as biofilm-associated infections are tolerant to our current antibiotic therapies. We have recently discovered that halogenated quinoline (HQ) small molecules are: 1) capable of eradicating methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, and 2) synthetic tuning of the 2-position of the HQ scaffold has a significant impact on antibacterial and antibiofilm activities. Here, we report the chemical synthesis and biological evaluation of 39 HQ analogues that have a high degree of structural diversity at the 2-position. We identified diverse analogues that are alkylated and aminated at the 2-position of the HQ scaffold and demonstrate potent antibacterial (MIC≤0.39 μ m) and biofilm eradication (MBEC 1.0-93.8 μ m) activities against drug-resistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium strains while demonstrating <5 % haemolysis activity against human red blood cells (RBCs) at 200 μ m. In addition, these HQs demonstrated low cytotoxicity against HeLa cells. Halogenated quinolines are a promising class of antibiofilm agents against Gram-positive pathogens that could lead to useful treatments against persistent bacterial infections. [ABSTRACT FROM AUTHOR]

ABSTRACT Cell-free protein synthesis (CFPS), which entails synthesizing proteins outside of intact cells, is conducted in several formats with the continuous-exchange cell-free (CECF) format generally having the greatest protein expression yields. With this format, continuous chemical exchange occurs through a dialysis membrane separating a reaction solution from a feeding solution containing supplemental nutrient/energy molecules. Here, we describe the optimization of the miniaturized fluid array device (µFAD) by studying the effects of structural and experimental parameters responsible for the heightened chemical exchange across the dialysis membranes and enhanced protein expression capabilities of the high-throughput device. The interface area and number between the reaction and feeding solutions have a direct impact on protein expression, with a 1.6% enhancement in protein expression yield with each square millimeter increase in area and a 20% decrease with each additional interface. For nutrient/energy availability, an increasing solution volume ratio and height difference increase protein expression yield until the expression yield plateaus at a volume ratio of 20 to 1 (feeding to reaction solution) and a solution height difference of 2 mm. This yield can be further increased by 7% every 30 min with feeding solution replacement. Of the studied experimental factors (feeding solution stirring, device shaking, and temperature increase), feeding solution stirring has a significant effect on protein expression in this device. In the optimized system, green fluorescent protein (GFP), ß-glucuronidase (GUS), ß-galactosidase (LacZ), luciferase, and tissue plasminogen activator (tPA) expression increased 77.8-, 212-, 3.66-, 463-, and 5.43-fold, respectively, compared to the conventional batch format in a standard microplate. These results highlight the significance of structural/experimental conditions on the productive expression of proteins in the CECF format. Biotechnol. Bioeng. 2015;112: 2459-2467. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Conventional antibiotics are ineffective against nonreplicating bacteria (for example, bacteria within biofilms). We report a series of halogenated phenazines (HP), inspired by marine antibiotic 1, that targets persistent bacteria. HP 14 demonstrated the most potent biofilm eradication activities to date against MRSA, MRSE, and VRE biofilms (MBEC=0.2- 12.5 μM), as well as the effective killing of MRSA persister cells in non-biofilm cultures. Frontline MRSA treatments, vancomycin and daptomycin, were unable to eradicate MRSA biofilms or non-biofilm persisters alongside 14. HP 13 displayed potent antibacterial activity against slow-growing M. tuberculosis (MIC=3.13 μM), the leading cause of death by bacterial infection around the world. HP analogues effectively target persistent bacteria through a mechanism that is non-toxic to mammalian cells and could have a significant impact on treatments for chronic bacterial infections. [ABSTRACT FROM AUTHOR]

Translation elongation is modulated by various ribosome-binding proteins. Environmental stresses, such as starvation and antibiotics, can cause stalling of bacterial ribosomes, which may alter gene expression through a transcription or translation attenuation mechanism. In P seudomonas aeruginosa, the expression of MexXY multidrug efflux system, which plays a significant role in resistance against aminoglycoside antibiotics, is controlled by a translation surveillance mechanism. Stalling of ribosome at the PA5471 leader peptide ( PA5471.1) m RNA leads to transcription of PA5471, which subsequently up-regulates the expression of MexXY. In this study, we found that mutation in a suh B gene leads to decreased susceptibility to aminoglycosides. Transcriptomic analysis revealed an up-regulation of MexXY and PA5471, which were demonstrated to be responsible for the decreased susceptibility of the suh B mutant. We further demonstrated that PA5471.1 is essential for the up-regulation of PA5471 in the suh B mutant. Co-immunoprecipitation assay revealed an interaction between SuhB and ribosome, suggesting a role of SuhB in translation. Indeed, higher amount of PA5471.1 m RNA was found to associate with ribosome isolated from the suh B mutant, indicating increased ribosome stalling. Therefore, this study identified SuhB as a novel ribosome associated protein that is involved in modulating ribosome activity. [ABSTRACT FROM AUTHOR]

Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a Δ lasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms. [ABSTRACT FROM AUTHOR]

The 2 ‐ position of the halogenated quinoline (HQ) scaffold can be dramatically tuned through diverse and practical synthetic pathways, including reductive amination and alkylation reactions. New HQs discovered during these investigations potently eradicate methicillin ‐ resistant Staphylococcus aureus (MRSA), methicillin ‐ resistant Staphylococcus epidermidis (MRSE) and vancomycin ‐ resistant Enterococcus faecium (VRE) biofilms, which display high levels of tolerance towards conventional antibiotic therapies. The background image is reproduced with permission and copyright of the British Editorial Society of Bone and Joint Surgery (Bone Joint J. 2013, 95B, 678 – 682; Figure 2 b). More information can be found in the Full Paper by R. W. Huigens III et al. on page 9181 ff. [ABSTRACT FROM AUTHOR]

We describe a miniaturized fluid array device for high-throughput cell-free protein synthesis (CFPS), aiming to match the throughput and scale of gene discovery. Current practice of using E. coli cells for production of recombinant proteins is difficult and cost-prohibitive to implement in a high-throughput format. As more and more new genes are being identified, there is a considerable need to have high-throughput methods to produce a large number of proteins for studying structures and functions of the corresponding genes. The device consists of 96 units and each unit is for expression of one protein; thus up to 96 proteins can be produced simultaneously. The function of the fluid array was demonstrated by expression of a variety of proteins, with more than two orders of magnitude reduction in reagent consumption compared with a commercially available CFPS instrument. The protein expression yield in the device was up to 87 times higher for β-glucoronidase than that in a conventional microplate. The concentration of β-galactosidase expressed in the device was determined at 5.5 μg/μL. The feasibility of using the device for drug screening was demonstrated by measuring the inhibitory effects of mock drug compounds on synthesized β-lactamase without the need for harvesting proteins, which enabled us to reduce the analysis time from days to hours., (Copyright © 2010 American Institute of Chemical Engineers (AIChE).)

Choline acetyltransferase (ChAT) catalyzes the biosynthesis of the neurotransmitter acetylcholine from acetyl-CoA and choline in cholinergic neurons. Rat ChAT (rChAT) was overexpressed in Escherichia coli, purified by affinity chromatography and crystallized. Diffraction data were collected from a single crystal under cryoconditions at the F1 beamline at the Cornell High Energy Synchrotron Source, with a maximal useful diffraction pattern to 1.55 A resolution. The crystals were shown to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 138.97, b = 77.67, c = 59.67 A and a scaling R(sym) of 0.054 for 72 446 unique reflections. Packing considerations indicate there to be one molecule per asymmetric unit. It is expected that in the near future the structure of rChAT will be obtained using molecular-replacement methods. Elucidation of the structure of rChAT will aid in the development of therapeutic agents for Alzheimer's disease.