Comparison and analysis of Clostridium botulinum autolysins
Jessica Jackson, Jade Hardy, and Wei-Jen Lin
Department of Biological Sciences, California State Polytechnic University, Pomona
Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium. It produces botulinum neurotoxin (BoNT), a potent toxin responsible for botulism in humans and animals. BoNT poses severe health risks to humans, and is a major concern for public health. Previous studies in our lab have shown that the toxin is produced during cell growth and released as early as mid-log phase of bacterial growth. We believe autolysins, which hydrolyze the cell wall, are responsible for the cell leakage and early release of the toxin. Better understanding of the toxin and how it is released could lead to treatment and prevention of botulism. In this study we analyzed the autolysins of C. botulinum using microarray and genomic sequence analysis. Bioinformatics analysis was done comparing C. botulinumtype A strain ATCC3502 to Bacillus subtilis strain 168, a model strain with characterized autolysins. BLAST was performed on the protein sequences of B. subtilis compared to C. botulinum. Transglycosylase CBO 3012 was further analyzed for its expression pattern by microarray and for its phylogenetic comparison to other species using ClustalW. The BLAST results of the genomic sequence analysis showed over 100 C. botulinum genes with similarities and homologies to B. subtilisstr. 168 autolysins. Further analysis of CBO 3012, a transglycosylase, showed greater than 98% identity and homology to other C. botulinum type A strains. Different C. botulinum serotypes showed greater than 53% identity and 73% homology, and other Clostridium species showed between 52-96% identity and 70-99% homology to CBO 3012. There were less than 57 and 77% identity and homology, respectively, for other bacterial genera compared to CBO 3012. Phylogenetic analysis of CBO 3012 showed the evolutionary relationship of this autolysin in a variety of bacteria, which could lead to a better understanding of the function of this gene, as well as its evolutionary path. Microarray expression analysis of C. botulinum ATCC3502 reveals autolysin activity in early mid-log phase of bacterial growth. Microarray data confirms that several autolysins are active around the same time the toxin is being released, possibly correlating autolysins being responsible for early cell lysis and release of the toxin. Identities and homologies shown between C. botulinum ATCC3502 and B. subtilis str. 168 brings us one step closer to defining the precise functions of autolysins in C. botulinum.
The Effect of Nectar levels on Honey Bee (Apis mellifera) Visit Duration
and Pollen position in Watermelon (Citrullus lanatus)
Accurate estimates of pollinator efficiency of bee species are essential to optimizing commercial crop pollination. One common method of estimating pollinator efficiency is to quantify the number of pollen grains deposited from a single visit to a previously unvisited flower. The amount of pollen deposited by this first visit is presumed similar to the amount of pollen deposited per visit for all visits a flower receives from a given kind of pollinator. In watermelon, floral nectar levels are higher initially and then decrease throughout the day. If higher nectar levels cause longer visits and greater pollen deposition, then per visit pollen deposition is likely to vary temporally and cannot be approximated by pollen deposition from the first visit a flower receives. This study investigated the effect varying floral nectar levels have on visit duration and pollen deposition by honey bees. Nectar from virgin flowers was removed with microcapillary tubes (with full extraction, 1 μL extraction and no extraction control treatments) and offered to honey bees for a single visit. The nectar left in full extraction flowers was similar to levels in open pollinated flowers. Each visit duration was recorded. Pollen deposited was counted. Significant differences were identified using ad hoc Tukey-Kramer tests. Flowers from 1 μL extraction and control treatments received longer visits than full extraction flowers (p=.0121 and p=.0002, respectively). Control and 1 μL extraction flowers had more pollen deposited on than full extraction flowers in terms of stigmatic pollen (p=.0016, p=.0039) and total pollen (p=.0033, p=.0246). Mean total pollen deposition by honey bees was 2.7 times greater on control flowers than full extraction flowers. Our data indicate that honey bee visit duration and per visit pollen deposition is strongly influenced by nectar levels in watermelon, which has important consequences for understanding and measuring honey bee pollinator efficiency.
CSU-Research Competition 2011
Functional Consequences of Sulfhydryl Modification of the g-Aminobutyric Acid Transporter 1 (GAT1) at a Single Solvent-Exposed Cysteine Residue
Rachel Sanchez, Jaison Omoto, Ali Rahnama-Vaghef, Matthew Maestas Cynthia Anderson, Esther Choi, Gerardo Salto, and Sepehr Eskandari
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system (CNS). GABA molecules act on GABA receptors on the postsynaptic neuron to induce an inhibitory postsynaptic response, thus reducing excitatory transmission. GABA concentrations in the brain extracellular fluid must be maintained at low resting levels to ensure a proper balance of excitatory-inhibitory response in the CNS. GABA transporters located on the presynaptic neuron and surrounding glia regulate inhibitory neurotransmission by removing GABA molecules from synaptic and extra-synaptic regions. GABA transporters are electrogenic cotransporter that utilizes the Na+ electrochemical gradient to translocate GABA molecules across the plasma membrane and into the presynaptic neuron and glial cells. Each translocation cycle, transports 2 Na+ : 1 Cl−: 1 GABA into the cell.
The focus of this study was to label the cysteine residues of GABA transporter 1 (GAT1) facing the extracellular milieu in order to gain an understanding of the functional consequences on steady-state kinetics resulting from the labeled residues. Three of the fourteen cysteine residues are predicted to be facing the extracellular fluid. Residue C74 is positioned at the membrane/extracellular interface of transmembrane domain one and is thought to contain the only exposed free thiol group. Cysteine residues C163 and C174 are located in the extracellular loop, which links transmembrane domains three and four. Under most conditions, it is suspected that C163 and C174 interact to form a disulfide bridge. Human GAT1 was expressed in Xenopus laevis oocytes by injecting the cells with human GAT1 cRNA. Electrophysiological methods were performed using a two-microelectrode voltage clamp method in order to assay for transporter function before and after labeling. GAT1 labeling was accomplished by sulfhydryl modification using either [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) or N-ethylmaleimide (NEM). NEM is a membrane-permeant reagent, whereas MTSET is a membrane-impermeant reagent. In the presence of NaCl, transporter exposure to MTSET (1 mM for 5 min.) inhibited GAT1-mediated transport by 48%. Increasing the concentration of MTSET to 2.5 mM (5 min.) inhibited transport by 52%, and increasing the incubation time with MTSET to 20 min. (1 mM) inhibited transport by 66%. Loss of transporter function caused by MTSET modification was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas NEM inhibited GABA transport mediated by GAT1 C74A by 15%. Thus, C74 is the only functionally significant cysteine residue that is accessible from the extracellular fluid. Sulfhydryl modification of the mutant GAT1 C74 is an efficient labeling method that paves the way for future experiments that seek to quantify GAT1 transporters expressed at the cell membrane.
CSUPERB 2011 ABSTRACTS
Antifungal Effects of Combining Non-Drug Containing Liposomes with Amphotericin B Liposomes
Carla Noguera*, Davin Hahka, Jill Adler-Moore
Biological Sciences Department, Cal Poly Pomona
Introduction: Antifungal drugs, e.g. liposomal amphotericin B (AmBisome®), are used to kill fungi that cause life-threatening infections but the immune response of the body to the fungus is also needed to help resolve the infection. Aspergillus flavus and Aspergillus fumigatus infections often occur in immunocompromised patients and immunostimulating agents, such as non-drug containing liposomes (ndL), have been shown to help clear fungal infections in immunocompromised animals. It has not been determined if this effect of the ndLs is due to immunomodulation only, direct antifungal activity or if ndLs enhance the antifungal effects of AmBisome. We used microtiter broth assays to address the question of the antifungal activity of ndLs.
Methods: In vitro microtiter broth assays were used to determine the minimal amount of drug needed to inhibit (MIC) and kill (MFC) A. fumigatus and A. flavus. We tested AmBisome, ndLs and the combination of AmBisome and ndLs. We used 96 well microtiter plates with 1.7-3.8 x10^3 spores per well (A. flavus ) or 1.7-9.3x10^3 spores/well ( A. fumigatus )
Results: From several replicates (n= 15) of the microtiter broth assay, we determined that for A. flavus, the AmBisome MIC was from 0.78-3.1ug/ml. When AmBisome was combined with ndLs, the MIC was similar, ie. 1.56ug/ml, indicating that ndLs did not alter the antifungal activity of AmBisome for A. flavus. For A. fumigatus, the AmBisome MIC was from 0.58-2.34ug/ml. With the combination of AmBisome and ndLs, the MIC was also 0.58-2.34ug/ml. The AmBisome MFC for A. fumigatus was between 0.97-1.93ug/ml and for AmBisome plus, ndLs, it was slightly higher, i.e. 2.3-3.9ug/ml. Again, this showed that the ndLs had little to no effect on the antifungal activity of AmBisome for A. fumigatus. Importantly, the ndLs alone had no antifungal activity against either fungus.
Conclusions: The absence of any direct antifungal activity by the ndLs in the microtiter assay indicates that the beneficial effects of ndLs in the animals is probably due to their immunostimulating properties and that treating the animals with both AmBisome and ndLs does not interfere with AmBisome antifungal activity. For our future studies we will work with macrophages in vitro incubating them with fungal spores, AmBisome and ndLs to determine the antifungal and immunomodulating effects of these agents on yeast infections of macrophages. Research supported by NIH MBRS RISE (2 R25 GM061190-05A2).
Liposomes, Aspergillus, drugs
Pseudomonas aeruginosa and Staphylococcus epidermidis enhance Biofilm formation on Titanium Prosthetic Metals that Incorporate Boron
Alas, Steve — Pomona, Biological Sciences
Brelles-Marino, Graciela — Pomona, Biological Sciences
Marzano, Scott [U] — Pomona, Biological Sciences
Quijano, Jessamine [U] — Pomona, Biological Sciences
Prosthetics are the art and science of making artificial parts and implants for the human body. Human prosthetics are being utilized more frequently as the populations that require their use expand. These cohorts include an increasing elderly population, American soldiers returning with war injuries, and members of the general public who suffer serious injury from car collisions and sport related injuries. Human prosthetics commonly fail, however, due to chronic infection. Chronic infections that arise from biofilm forming bacteria are largely untreatable with antibiotics. Biofilm is a highly organized layer of microorganisms that firmly attach onto a surface using a extracellular polymer matrix (EPS). By testing new biometals comprised of titanium alloy with boron, we hoped to observe less biofilm propagation when compared to tranditional biometals currently used.
Using Pseudomonas aeruginosa and Staphylococcus epidermidis, two bacteria that commonly create biofilm on patient implants, we tested novel titanium alloys with regard to their susceptibility to biofilm propagation. The biometals examined were stainless steel (SS), commercially pure titanium (CPTi), Ti-6Al-4V (Ti64) and 3 novel titanium alloys that contain 0.05%, 0.4%, or 1.0% boron. Biofilm formation was analyzed using crystal violet staining and fluorescent microscopy. Ideal experimental conditions were obtained a biofilm reactor with TSB media, and incubation for 48 hours at 37˚C. Biofilm reactor experiments were performed with or without a constant infusion of growth media during biofilm formation.
The Ti64 metal alloy permits less biofilm formation than SS, CPTi and the titanium-boron alloys, by both Pseudomonas aeruginosa and Staphylococcus epidermidis, using both the flask method and biofilm reactor batch phase method. The same results were obtained with Pseudomonas aeruginosa in the method with constant flow rate in the biofilm reactor. These findings were corroborated using BacLight staining and fluorescent microscopy to verify the extent and viability of biofilm formed on the implant metals.
The Ti-6Al-4V alloy may be a better alternative to traditional metals, stainless steel and pure titanium, as a modern prosthetic biometal. Our findings indicate that the introduction of boron to the titaniu
Expression of Clostridial Autolysins and Their Role in Botulinum Neurotoxin Release
Dr. Wei-Jen Lin
Introduction: Clostridium botulinum is an anaerobic, Gram-postive bacteria that is responsible for the disease botulism, a deadly neurological and paralyzing disease. Botulism is usually derived by eating contaminated food. The ability to form spores gives them the advantage to survive in extreme conditions of drought, heat, or lack of nutrients and regrow when conditions are suitable. Clostridium botulinum produces the most dangerous toxin known to humans so far. It is one of the top biological risks that exists, which lead the organism to be strictly monitored and regulated by the government. The objective of this study is to clone and express autolysins from C. botulinum for in vitrocharacterization and study of their roles in the release of botulinum neurotoxin.
Methods: The first autolysin we chose to clone and characterize is the lysozyme CBO3274 which cleaves cell wall peptidoglycan. Gene specific primers were designed and the gene was amplified by the polymerase chain reaction and then cloned to a cloning vector. After verification of the gene sequence, the DNA was sub-cloned to an expression vector to create a poly-histidine-tagged fusion protein for expression and purification from an E. coli strain.
Results: PCR product show successful amplication of the target gene sequence. After introducing amplified PCR product into a cloning vector, DNA sequencing showed a 100% match by BLAST Analysis. Transformation of E. coli with pET30 expression vector and target insert was confirmed by gel electrophoresis. SDS-PAGE and Western blot analyses confirmed the presence and size of clostridial lysozyme in transformed E. coli.
Conclusions: We have successfully introduced clostridial genes in E.coli cells and induced expression of the proteins. For future studies, we want to characterize these clostridial proteins and investigate their function by introducing them in E. coli or other Clostridium species and observe cell leakage during early growth phases.
Comparison of the Efficacy of a Liposomal M2 Epitope Vaccine Prepared by Different Methods of Production
Natasha Ward1, Hana Kim1, Jill Adler-Moore1, and Suming Chiang2
1Department of Biological Sciences, California State Polytechnic University, Pomona, CA
2Molecular Express, Inc., Rancho Dominguez, CA
Introduction: The target for the present commercial influenza vaccines are the viral proteins, hemagglutinin (HA) and neuraminindase (NA), which are prone to yearly mutations (antigenic drift) or reassortment (antigenic shift) requiring new vaccines to be made every year. By incorporating the highly conserved region of the viral M2 protein (1-23aa) within a liposome, we can elicit significant protection against influenza strains, which have different HA and NA proteins. Our goal is to move this liposomal M2 epitope vaccine (L-M2e) into clinical trials and therefore, we needed to determine the optimum liposomal production method.
Methods: Liposomes were prepared as follows: 1) microfluidization using ethanol to solubilize the lipids (emicro), 2) sonication of a lipid film prepared with ethanol (efilm) or 3) sonication of a lipid film prepared with chloroform/methanol (c/mfilm). Female BALB/c mice (n=12/gp) were vaccinated with the liposome preparations (15ugM2e/dose) or liposome control (no M2e) (L-Con) on d0 s.c. and on d56 intranasally (i.n.). On d59, spleens were collected (n=5/gp). The splenocytes were used in an ELISPOT assay to determine cytokine production (IFN-γ for Th1 response vs IL4 for Th2 response). d62 blood (n=5/gp) was collected for ELISA antibody isotype titers. Remaining mice (n=7/gp) were challenged i.n. with 10LD50 H1N1 (A/PR/8/34) on d63 and monitored for morbidity.
Results: The mice vaccinated with efilm or c/mfilm L-M2e had 100% survival and mice given emicro L-M2e, had 71% survival; all but one mouse in the L-Con group survived (14%) (p<0.005 vs L-Con). Efilm L-M2e vaccinated mice lost the least weight and recovered faster compared to the other L-M2e groups although all the L-M2e groups lost significantly less weight (p<0.0001 vs L-Con) and recovered faster than the L-Con gp. In comparison, IL4 production was greater in the emicro L-M2e vaccinated mice (p<0.05 vs other groups) indicating upregulation of the Th2 response. All L-M2e formulations stimulated similar amounts of anti-M2e IgG1 and IgG2a.
Conclusions: The data support use of ethanol microfluidization as a scale-up method for making L-M2e vaccine needed for clinical trials. In addition, this technique allows the substitution of ethanol for the toxic organic solvents, methanol and chloroform, used currently in liposome production. Research supported by NIH MBRS RISE (2 R25 GM061190-05A2) and Cal Poly ARI.
Vaccine, Influenza virus, Liposomes