Ceragenins were used to create biofouling resistant water-treatment membranes. Ceragenins are synthetically produced antimicrobial peptide mimics that display broad-spectrum bactericidal activity. While ceragenins have been used on bio-medical devices, use of ceragenins on water-treatment membranes is novel. Biofouling impacts membrane separation processes for many industrial applications such as desalination, waste-water treatment, oil and gas extraction, and power generation. Biofouling results in a loss of permeate flux and increase in energy use. Creation of biofouling resistant membranes will assist in creation of clean water with lower energy usage and energy with lower water usage. Five methods of attaching three different ceragenin molecules were conducted and tested. Biofouling reduction was observed in the majority of the tests, indicating the ceragenins are a viable solution to biofouling on water treatment membranes. Silane direct attachment appears to be the most promising attachment method if a high concentration of CSA-121a is used. Additional refinement of the attachment methods are needed in order to achieve our goal of several log-reduction in biofilm cell density without impacting the membrane flux. Concurrently, biofilm forming bacteria were isolated from source waters relevant for water treatment: wastewater, agricultural drainage, river water, seawater, and brackish groundwater. These isolates can be used for future testing of methods to control biofouling. Once isolated, the ability of the isolates to grow biofilms was tested with high-throughput multiwell methods. Based on these tests, the following species were selected for further testing in tube reactors and CDC reactors: Pseudomonas ssp. (wastewater, agricultural drainage, and Colorado River water), Nocardia coeliaca or Rhodococcus spp. (wastewater), Pseudomonas fluorescens and Hydrogenophaga palleronii (agricultural drainage), Sulfitobacter donghicola, Rhodococcus fascians, Rhodobacter katedanii, and Paracoccus marcusii (seawater), and Sphingopyxis spp. (groundwater). The testing demonstrated the ability of these isolates to be used for biofouling control testing under laboratory conditions. Biofilm forming bacteria were obtained from all the source water samples.
We have developed a novel modular automated processing system (MAPS) that enables reliable, high-throughput analysis as well as sample-customized processing. This system is comprised of a set of independent modules that carry out individual sample processing functions: cell lysis, protein concentration (based on hydrophobic, ion-exchange and affinity interactions), interferent depletion, buffer exchange, and enzymatic digestion of proteins of interest. Taking advantage of its unique capacity for enclosed processing of intact bioparticulates (viruses, spores) and complex serum samples, we have used MAPS for analysis of BSL1 and BSL2 samples to identify specific protein markers through integration with the portable microChemLab{trademark} and MALDI.
Cell membranes are dynamic substrates that achieve a diverse array of functions through multi-scale reconfigurations. We explore the morphological changes that occur upon protein interaction to model membrane systems that induce deformation of their planar structure to yield nanotube assemblies. In the two examples shown in this report we will describe the use of membrane adhesion and particle trajectory to form lipid nanotubes via mechanical stretching, and protein adsorption onto domains and the induction of membrane curvature through steric pressure. Through this work the relationship between membrane bending rigidity, protein affinity, and line tension of phase separated structures were examined and their relationship in biological membranes explored.
The innate immune system represents our first line of defense against microbial pathogens, and in many cases is activated by recognition of pathogen cellular components (dsRNA, flagella, LPS, etc.) by cell surface membrane proteins known as toll-like receptors (TLRs). As the initial trigger for innate immune response activation, TLRs also represent a means by which we can effectively control or modulate inflammatory responses. This proposal focused on TLR4, which is the cell-surface receptor primarily responsible for initiating the innate immune response to lipopolysaccharide (LPS), a major component of the outer membrane envelope of gram-negative bacteria. The goal was to better understand TLR4 activation and associated membrane proximal events, in order to enhance the design of small molecule therapeutics to modulate immune activation. Our approach was to reconstitute the receptor in biomimetic systems in-vitro to allow study of the structure and dynamics with biophysical methods. Structural studies were initiated in the first year but were halted after the crystal structure of the dimerized receptor was published early in the second year of the program. Methods were developed to determine the association constant for oligomerization of the soluble receptor. LPS-induced oligomerization was observed to be a strong function of buffer conditions. In 20 mM Tris pH 8.0 with 200 mM NaCl, the onset of receptor oligomerization occurred at 0.2 uM TLR4/MD2 with E coli LPS Ra mutant in excess. However, in the presence of 0.5 uM CD14 and 0.5 uM LBP, the onset of receptor oligomerization was observed to be less than 10 nM TLR4/MD2. Several methods were pursued to study LPS-induced oligomerization of the membrane-bound receptor, including CryoEM, FRET, colocalization and codiffusion followed by TIRF, and fluorescence correlation spectroscopy. However, there approaches met with only limited success.
Bioweapons and emerging infectious diseases pose formidable and growing threats to our national security. Rapid advances in biotechnology and the increasing efficiency of global transportation networks virtually guarantee that the United States will face potentially devastating infectious disease outbreaks caused by novel ('unknown') pathogens either intentionally or accidentally introduced into the population. Unfortunately, our nation's biodefense and public health infrastructure is primarily designed to handle previously characterized ('known') pathogens. While modern DNA assays can identify known pathogens quickly, identifying unknown pathogens currently depends upon slow, classical microbiological methods of isolation and culture that can take weeks to produce actionable information. In many scenarios that delay would be costly, in terms of casualties and economic damage; indeed, it can mean the difference between a manageable public health incident and a full-blown epidemic. To close this gap in our nation's biodefense capability, we will develop, validate, and optimize a system to extract nucleic acids from unknown pathogens present in clinical samples drawn from infected patients. This system will extract nucleic acids from a clinical sample, amplify pathogen and specific host response nucleic acid sequences. These sequences will then be suitable for ultra-high-throughput sequencing (UHTS) carried out by a third party. The data generated from UHTS will then be processed through a new data assimilation and Bioinformatic analysis pipeline that will allow us to characterize an unknown pathogen in hours to days instead of weeks to months. Our methods will require no a priori knowledge of the pathogen, and no isolation or culturing; therefore it will circumvent many of the major roadblocks confronting a clinical microbiologist or virologist when presented with an unknown or engineered pathogen.
This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.
This one year LDRD addresses problems of threat assessment and restoration of facilities following a bioterror incident like the incident that closed down mail facilities in late 2001. Facilities that are contaminated with pathogenic spores such as B. anthracis spores must be shut down while they are treated with a sporicidal agent and the effectiveness of the treatment is ascertained. This process involves measuring the viability of spore test strips, laid out in a grid throughout the facility; the CDC accepted methodologies require transporting the samples to a laboratory and carrying out a 48 hr outgrowth experiment. We proposed developing a technique that will ultimately lead to a fieldable microfluidic device that can rapidly assess (ideally less than 30 min) spore viability and effectiveness of sporicidal treatment, returning facilities to use in hours not days. The proposed method will determine viability of spores by detecting early protein synthesis after chemical germination. During this year, we established the feasibility of this approach and gathered preliminary results that should fuel a future more comprehensive effort. Such a proposal is currently under review with the NIH. Proteomic signatures of Bacillus spores and vegetative cells were assessed by both slab gel electrophoresis as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection. The conditions for germination using a number of chemical germinants were evaluated and optimized and the time course of protein synthesis was ascertained. Microseparations were carried out using both viable spores and spores inactivated by two different methods. A select number of the early synthesis proteins were digested into peptides for analysis by mass spectrometry.
Wild strains of Bacillus subtilis are capable of forming architecturally complex communities of cells known as biofilms. Critical to biofilm formation is the eps operon, which is believed to be responsible for the biosynthesis of an exopolysaccharide that binds chains of cells together in bundles. We report that transcription of eps is under the negative regulation of SinR, a repressor that was found to bind to multiple sites in the regulatory region of the operon. Mutations in sinR bypassed the requirement in biofilm formation of two genes of unknown function, ylbF and ymcA, and sinI, which is known to encode an antagonist of SinR. We propose that these genes are members of a pathway that is responsible for counteracting SinR-mediated repression. We further propose that SinR is a master regulator that governs the transition between a planktonic state in which the bacteria swim as single cells in liquid or swarm in small groups over surfaces, and a sessile state in which the bacteria adhere to each other to form bundled chains and assemble into multicellular communities.