Microbiology and Immunology

An avian live attenuated master backbone for potential use in epidemic and pandemic influenza vaccines


Authors: Danielle Hickman, Jaber Hossain, Haichen Song, Erin M. Sorrell, Yonas Araya, and Daniel R. Pérez
Department or Program: Virginia Regional College of Veterinary Medicine
Presented by: Danielle Hickman
Abstract: The unprecedented emergence in Asia of multiple avian influenza subtypes with broad host range poses a major challenge in the design of vaccines that are both effective and quickly available. The present study focuses on the protective effects of a genetically modified avian influenza virus as a source for the preparation of vaccines for epidemic and pandemic influenza. We have previously demonstrated that a live attenuated (att) avian influenza virus based on the internal backbone of influenza A/Guinea fowl/Hong Kong/WF10/99 (H9N2) was effective at protecting poultry species against low and highly pathogenic influenza strains. More importantly, our live attenuated virus provided effective protection when administered in ovo. In order to further characterize our att backbone for the use in epidemic and pandemic influenza vaccines, we evaluated its protective effects in mice and ferrets. Intranasal inoculation of the modified attenuated viruses in mice and ferrets provided adequate protective immunity against homologous lethal challenges with both the wild type influenza A/WSN/33 (H1N1) or A/Vietnam/1203/04 (H5N1) viruses. A difference in heterosubtypic immunity was also observed in mice and ferrets vaccinated with modified attenuated viruses carrying the H7N2 and H9N2 surface proteins. The results presented suggest that the internal genes of a genetically modified avian influenza virus confer protection the mouse and ferret models and thus could be used as a master donor strain for the generation of live attenuated vaccines for epidemic and pandemic influenza.

The Role of Processive Endoglucanases in the Degradation of Cellulose by Saccharophagus degradans 2-40


Authors: Brian J. Watson, Haitao Zhang, Young-Hwan Moon, Atkinson G. Longmire, and Steven W. Hutcheson
Department or Program: ENMA
Presented by: Brian Watson
Abstract: Bacteria and fungi are thought to degrade cellulose by either a complexed or noncomplexed cellulolytic system composed of endoglucanases, cellobiohydrolases and β-glucosidases. The marine bacterium Saccharophagus degradans 2-40 produces a multi-component cellulolytic system that is unusual in the abundance of GH5-containing endoglucanases. Secreted enzymes of this bacterium release high levels of cellobiose from cellulose, but there is a deficiency of cellobiohydrolases. With the exception of the proposed cellulobiohydrolase Cel6A, which is a classic endoglucanase, the predicted biochemical activities of the annotated GH5-containing endoglucanases were confirmed after cloning and purification. Through analysis of transcript levels, Cel5H was the highest expressed cellulase. Cel5H showed significantly greater activity on several types of cellulose and processively-released cellobiose during digestions independently of the activity of other endoglucanases. Phylogenetic analyses indicated that Cel5H is a member of a separate clade of GH5-containing enzymes that also included S. degradans Cel5G and Cel5J. These enzymes were also found to be processive endoglucanases. The processive endoglucanases were functionally equivalent to the endoglucanase/cellobiohydrolase combination of other cellulolytic systems. Thus the activity of a processive endoglucanase and a β-glucosidase are sufficient for this bacterium to convert cellulose to glucose.

Characterizing Pseudomonas Biofilm Virulence and Host Immune Responses in Drosophila Infection Models


Authors: Tuo Dong, Louisa Wu, Vincent Lee
Department or Program: CBMG
Presented by: Tuo Dong
Abstract: Many bacterial pathogens form surface-associated extracellular matrix to endure unfavorable external conditions. Bacteria that utilize this strategy reside in structured communities called biofilms. In clinical settings, Pseudomonas aeruginosa (PA) biofilms contribute to higher antibiotic resistance and chronic lung infections in cystic fibrosis patients. Synthesis of these exopolysaccharides requires the expression of pel genes, which can be induced by several mechanisms. First, the histidine kinases RetS and LadS act on the GacA/GacS system to modulate the translation of pel operon transcripts using small regulatory RNAs (srRNAs). The second pathway relies on high intracellular levels of c-di-GMP that also results in increase of pel transcripts. Using a Drosophila host model, I have characterized virulence of biofilm expression induced by the two different pathways. I hope to determine Drosophila host mechanisms that interact with the PA biofilm. I will examine susceptibility of Drosophila immune pathway mutants (i.e. IMD, Toll, and JNK) to biofilm infection, and analyze antimicrobial peptide expression during infection with and without biofilm expression. The finding of this research will determine how biofilms affect virulence and specific host responses that defend against biofilms. These studies may contribute to the development of strategies to help the host resist biofilm related diseases.

Evaluation of monoclonal antibodies for diagnostic identification of Streptococcus equi


Authors: Bronessa Fernandes, James T. Hoopes, Caren J. Stark, and Daniel C. Nelson
Department or Program: BIOL
Presented by: Bronessa Fernandes
Abstract: Streptococcus equi are important pathogens in equine respiratory disease. S.equi was originally regarded as a single species; however, recently they have been divided into two sub species, equi and zooepidimicus. The two species are very similar, and infect horses; however, they cause very different types of infections in horses. S.equi subspecies equi (S.equi) is the cause of the highly infectious equine disease known as Strangles, while S.equi subsp. zooepidemicus (S.zoo) is commonly found in the respiratory tracts of healthy horses and can cause secondary infections. S.equi is thus far more pathogenic than S. zoo and hence S.equi identification is quite serious and requires immediate isolation of horses and antibiotic treatment whereas S. zoo is considered non-pathogenic. Therefore, we need better diagnostics to distinguish between these two closely related species quickly and accurately. The current ways of distinguishing between the two species is very time consuming and hence there is a need to develop a quick and easy diagnostic assay to tell the two species apart. Research has shown that all streptococci, including those that infect humans, pigs, cows and horses have “M-proteins” on the surface. These M-proteins differ slightly between different species. We can thus use these M-proteins as a fingerprint to identify the exact streptococci species that they belong to by targeting them with monoclonal antibodies. Maine Biotechnology, Inc. will provide the antibodies. We will then test the binding specificity of these antibodies to the streptococcal surface using fluorescent microscopy. Our goal is to identify a monoclonal antibody that can distinguish S. equi from S. zooepidemicus for diagnosis of equine strangles.

Dynamin links the signal transduction and internalization pathways of the B cell receptor


Authors: Margaret K. Seeley, Olusegun O. Onabajo, Bruce K. Brown, Huan Wang, Hong Cao, Mark McNiven, and Wenxia Song
Department or Program: CBMG
Presented by: Margaret Seeley
Abstract: The B cell receptor (BCR) serves as both signal-transducer and antigen-transporter. Binding of antigens to the BCR induces signaling cascades and antigen internalization. BCR-initiated signaling is required for antigen-induced BCR internalization. Here we study the role of dynamin II in coupling BCR signaling with its internalization pathway, as well as dynamin’s ability to serve as a link between the cellular endocytosis machinery and the actin cytoskeleton. The binding of the BCR to antigen, which triggers BCR signaling, induces the recruitment of dynamin to the BCR at the B cell surface. This recruitment is abolished by the Src kinase inhibitor PP2, which blocks BCR signaling, but not by latrunculin that depolymerizes F-actin. These results suggest BCR signaling regulates the cellular location of dynamin. The PRD domain deletion, but not the GTPase (K44A) or phosphorylation mutants (Y231/597F) mutations, blocks the recruitment of dynamin to the cell surface. Over-expression of the dynamin PRD deletion, K44A, or Y231/597F mutants reduces BCR internalization to varying levels. Additionally, the small molecule inhibitor of dynamin, Dynasore, reduced BCR internalization. In B cells over-expressing the dynamin K44A mutant, the BCRs accumulated in plasma membrane-associated vesicles where the BCR colocalizes with dynamin. Furthermore, dynamin interacts with actin-binding protein 1 (Abp1/SH3P7/Hip55), and cells over-expressing the dynamin PRD domain mutant show reduced Abp1 redistribution to the plasma membrane upon activation. These results demonstrate that the BCR regulates the cellular location of dynamin through its PRD domain, and that dynamin is required for signaling stimulated BCR internalization.

Comparison of Inflammatory Response to Food Allergen in Two Rodent Models


Authors: Akshita Mehta and Dr. Uma Babu
Department or Program: CBMG
Presented by: Akshita Mehta
Abstract: Asthma affects 15 million adults and children in the United States and causes about 30,000 emergency room visits with 100 to 200 deaths a year in the United States. However, animal models exhibiting the characteristics of asthma and allergy have not been well studied with regard to differential expression of inflammatory genes and their receptors. The goal of the study was to compare the characteristic inflammatory gene activation profiles in spleens and lungs of Brown Norway and OASRA Dyspenic Rats (ODR) in response to Ovalbumin (OVA) sensitization using Microarray techniques. Our data showed differential up-regulation of several genes such as those from chemokine ligand family, interleukins 4, 5, 10, and 13, and cytokine receptors between Brown Norway and ODR rats. Overall, the ODR rats demonstrated a much greater up-regulation of inflammatory genes and their receptors compared to the Brown Norway rats, which correlated with the OVA induced asthmatic response. Our data suggests that ODR can be used as an animal model to study food allergy induced asthmatic responses.

Cyclic-di-GMP suppression of Pseudomonas aeruginosa swimming motility


Authors: Gregory P. Donaldson and Vincent T. Lee
Department or Program: CBMG
Presented by: Gregory Donaldson
Abstract: The opportunistic pathogen Pseudomonas aeruginosa is responsible for acute and chronic infections in immunocompromised individuals. In acute infection, the flagellated soil bacterium exists in a mobile, planktonic state. P. aeruginosa adopts an alternative lifestyle in chronic infections by forming biofilm communities. This lifestyle decision is due to broad reciprocal regulation of relevant genes which is in part coordinated by the secondary messenger cyclic-di-GMP (cdiGMP) in P. aeruginosa as well as Escherichia coli, Vibrio cholera, and Salmonella typhimirium. An unknown signaling pathway causes suppression of swimming motility in response to increases in the intracellular concentration of cdiGMP. This is correlated with stimulation of biofilm formation. I have developed several useful tools for elucidating details of the swimming response to this regulatory dinucleotide. Swim assays with an overexpressed diguanylate cyclase, which synthesizes cdiGMP, reveal that strains of the P. aeruginosa PAO1 strain can respond to artificially elevated levels of cdiGMP by either suppressing swimming or having unaltered flagella motility. The underlying genetic explanation for this marked difference between closely related strains could provide great insight into how the down-regulation of swimming occurs. I am setting up a system to use cosmid libraries and a phenotypic screen to identify the genetic difference between strains that respond to cdiGMP and those that do not. Determining the link between cdiGMP and flagellar motility suppression would be an important step forward in understanding the reciprocal lifestyle decision of flagellated bacteria to remain motile or settle into a biofilm community. This decision is relevant for the acute and chronic diseases caused by P. aeruginosa.

In Vitro Production and Purification of Hepatitis C Virus Particles for Cryo-Electron Microscopy


Authors: Alice Nieh, Zongyi Hu, T. Jake Liang
Department or Program: BIOL
Presented by: Alice Nieh
Abstract: Hepatitis C virus (HCV) infection is a major health threat in the world. Understanding HCV structure is important for the study of HCV infection and drug design. Structural information of HCV is so far unavailable due to the limitation of getting viral particles of adequate quantity and decent quality. Two in vitro systems to produce HCV-like particles (HCV-LP) or infectious HCV virions have recently been established. HCV-LP assembled in insect cells using baculovirus expression system has similar morphologic, biophysical, and antigenic properties as the HCV virions. HCVcc (JFH-1 clone) was propagated in Huh7 hepatoma cell line. The results of HCV-LP and HCV virions were compared after separating in Iodixanol gradient emphasized that both were found in the same density with similar sizes, but HCV virions was far less efficient in producing adequate quantity of particles. HCV-LP was subjected to antibody labeling of the structure proteins and 3D reconstruction of HCV-LP using electron cryomicroscopy. Results shown HCV-LP E1 and E2 proteins may form a tetramer that corresponds morphologically and functionally to the flavivirus E dimer. Also, E1-E2 tetramer of HCV-LP forms 3- and 5- fold symmetric axes.

Effect of HIV-1 Nucleocapsid on Template Extension and Strand Transfer


Authors: Katherine Fenstermacher, Deena Jacob, and Jeffrey DeStefano
Department or Program: CBMG
Presented by: Katherine Fenstermacher
Abstract: The HIV-1 nucleocapsid (NC) protein possesses nucleic acid chaperone properties: it unravels weak secondary structures in RNA, allowing the RNA to fold in a more thermodynamically stable conformation; aggregates nucleic acids, which helps package genomic RNA; and is necessary for recombination and genome replication. Secondary structures hinder full-length extension by causing the reverse transcriptase (RT) to pause while melting the structure, which increases the probability that RT will fall off the template before completing extension. If a second template is available, the RT might undergo strand transfer and complete extension on the new RNA, resulting in a chimeric genome. We developed an assay to detect both extension and recombination under varying concentrations of NC, as well as Mg2+ (a secondary structure stabilizer that binds dNTPs and is necessary for RT function). We determined that the presence of Mg2+ hinders extension and increases strand transfer, probably due to its stabilization of secondary structures. Addition of NC reduces these structures and enhances extension. NC also helps spread the probability of recombination along an entire template. Genetic diversity in HIV is driven by recombination and determination of its mechanisms might one day be used to help design vaccines.

Glutamine metabolism in activated T cells


Authors: Alina Kelman and Kenneth Frauwirth
Department or Program: CBMG
Presented by: Alina Kelman
Abstract: When a T cell becomes activated, it must proliferate rapidly in order to mount an immune response. This proliferation requires specific sugars and amino acids. Although previous research has shown that T cells exhibit a high rate of glycolysis, most of the resulting pyruvate is converted to lactate and does not enter the Krebs cycle. Glutamine may represent an important additional energy source for activated T cells through its role in the Krebs cycle. We report that the expression of glutaminase (GLS), the enzyme which converts glutamine to glutamate, is induced during T cell stimulation. Three other enzymes involved in glutamine metabolism - glutamate dehydrogenase (GDH), glutamic pyruvic transaminase (GPT), and glutamic oxaloacetic transaminase (GOT) – are also induced although their expression peaks later in stimulation than that of GLS. GDH, GOT, and GPT can each convert glutamate to alpha-ketoglutarate, the main entry point for glutamine into the Krebs cycle. GDH activity in stimulated T cells is about double that of resting cells while GOT and GPT activity is 2.5 to 3 times higher after stimulation. These findings suggest that the upregulation of these enzymes enhances the ability of glutamine to enter the Krebs cycle during T cell activation. Next, we explored the role of extracellular signal-regulated kinase (ERK), which is known to be important in T cell activation, in the regulation of glutamine metabolism. The addition of an ERK inhibitor to stimulation media lowers the activity of GDH, GPT, and GOT to levels that are not significantly different from resting T cell activity. This result suggests a link between a signal transduction pathway in T cells and the control of metabolism indicating that metabolism may be directed in stimulated T cells rather than influenced exclusively by prevailing conditions.

Scientists and Engineers for America at the University of Maryland: Informing and empowering the leaders of tomorrow


Authors: Katherine Pankow and Kevin Roelofs
Department or Program: CBMG
Presented by: Kevin Roelofs
Abstract: Misinterpretation, lack of understanding, or even willful ignorance on the part of special interests have become increasingly damaging to science as a respected source of objective information. In order to combat this trend, the Scientists and Engineers of America: UMCP chapter was started. SEA: UMCP’s mission is to train members to communicate ideas more accurately, empower student leaders to pursue careers within the scientific and political arenas, and educate and inspire the general public. Through our 1.5 semesters of development and outreach, we have completed a number of successful outreach events, participated in the scientific and political communities, and continue to develop new ideas and projects. In the future, we will further our goal of communicating science accurately to the public by perpetuating the programs we have already started, as well as seeking out new opportunities to advance our goal of communicating science accurately to the public.

Engineering Nanoparticle Specificity for Thermal Ablation Therapy


Authors: Haoming Pang, James T. Hoopes, Caren J. Stark, and Daniel C. Nelson
Department or Program: BIOL
Presented by: Haoming Pang
Abstract: Infectious diseases are the second leading cause of death worldwide. As development of new antibiotics continues to decrease, antibiotic resistance is increasing at an alarming rate. If we are to survive this evolutionary war against bacteria, we need an alternative to antibiotics. One potential antimicrobial approach to fight antibiotic resistant bacteria includes identification of bacterial cell wall binding proteins with nanomolar affinity that could be exploited for the delivery of an antimicrobial payload. Lysins are a class of proteins that have a modular design consisting of a catalytic domain and a cell wall binding domain (CBD). The CBDs can distinguish discrete epitopes present within the cell wall, giving rise to the strain-specific activity of a particular lysin. One CBD in particular, PlyCB, is of interest because it acts as a scaffold protein for development into a bionanotechnology platform3. The “top” surface contains the flexible N-terminus that allows covalent conjugation with a nanoparticle. The C-terminus on the “bottom” surface has C-terminal fusions to other lysin CBDs which could change the specificity of PlyCB for other targets. By linking PlyCB and choline CBD DNA derived from a pneumococcal lysin4, we were able to encode for chimeric protein containing both subunits. The CBD acts as the variable subunit that can be changed to be strain-specific while PlyCB constant subunit that binds Nanoparticles. In other words, the chimeric protein anchors gold-nanoparticles onto bacteria. Near infrared (IR) radiation, gold is known to heat to temperatures (>70°C) which, in principle, could thermally ablate bound bacterial cells.

BCR-induced actin rearrangement provides the driving force for the formation of surface BCR signalosomes


Authors: Chaohong Liu, Heather Miller, Gregory Orlowsky, Shruti Sharma, Hui-King Lam, Brian Grooman, Arpita Upadhyaya, and Wenxia Song
Department or Program: CBMG
Presented by: Chaohong Liu
Abstract: BCR-induced actin rearrangement provides the driving force for the formation of surface BCR signalosomes Chaohong Liu*, Heather Miller*, Gregory Orlowsky*, Shruti Sharma*, Hui-King Lam#, Brian Grooman#, Arpita Upadhyaya#, and Wenxia Song* * Department of Cell Biology and Molecular Genetics and #Department of Physics, University of Maryland, College Park, MD 20742. The binding of the BCR to antigens induces the clustering of the surface BCRs and the formation of surface BCR signalosomes, where the BCR orchestrates signaling cascades and antigen internalization. Here, we examine the role of the actin cytoskeleton in the formation of BCR surface signalosomes using live cell and 3-D cell imaging by confocal and total internal reflection fluorescence (TIRF) microscopes. Both multi-valent soluble antigens and membrane-associated antigens induce BCR clustering at the B cell surface, and these BCR micro-clusters are positive for phosphorylated tyrosine and Erk. Concurrent with BCR clustering, actin is actively polymerized at BCR micro-clusters during the first few minutes and surrounding BCR central clusters at later times of BCR activation. Actin regulators, including Wiskott-Aldrich syndrome protein (WASP), an actin polymerization promoting factor, cofilin, an actin depolymerization promoting factor, and gelsolin, an F-actin severing protein, are recruited to BCR clusters. The treatment of latrunculin or jasplakinolide, which block BCR-induced actin reorganization by depolymerizing and stabilizing F-actin, inhibits antigen-triggered BCR micro-clustering and B cell spreading. In the absence of antigenic stimulation, latrunculin treatment alone induces BCR micro-clustering and tyrosine phosphorylation in the BCR clusters, however, in a much delayed fashion. The deficiency of Btk that controls BCR-induced actin reorganization through WASP not only inhibits antigen-induced B cell spread and contraction, but also dramatically reduces the dynamics of BCR clustering. These results indicate that Btk-mediated actin reorganization, including both actin polymerization and depolymerization events, facilitates the formation of surface BCR signalosomes, and BCR-induced actin reorganization provides a positive feedback for BCR signaling by increasing the speed and the extend of BCR clustering.

In vitro activity of the aminoglycoside antibiotic arbekacin against Acinetobacter baumannii-calcoaceticus isolated from war wounded at Walter Reed Army Medical Center


Authors: Michael J. Zapor, MD, PhD; Melissa Barber, AS, MLT (AMT); Amy Summers, BS; George Miller, PhD; Lee Feeney, BS; Lynn E. Eber
Department or Program: BIOL
Presented by: Melissa Barber
Abstract: Using the broth microdilution method, we determined the minimum inhibitory concentration of arbekacin, an aminoglycoside licensed in Japan, against two hundred Acinetobacter isolated during 2007-2008 from hospitalized war wounded patients. We then assayed for synergy between arbekacin and twenty eight other antibiotics using the disk diffusion and checkerboard methods. Finally, we performed pulse field gel electrophoresis on each isolate to determine if any association exists between clonality and in vitro inhibition of Acinetobacter by arbekacin.

ESTROGEN AND PROGESTERONE INCREASE NEISSERIA GONORRHOEAE TRANSMIGRATION ACROSS POLARIZED EPITHELIAL CELLS


Authors: Edwards, V. L., Stein, D. C. and Song, W.
Department or Program: CBMG
Presented by: Vonetta Edwards
Abstract: Gonorrhea, caused by the obligate human bacterial pathogen Neisseria gonorrhoeae, is one of the most common sexually transmitted diseases. N. gonorrhoeae (GC) infection is generally initiated at the genital epithelium and in women can lead to significant morbidity. In order to enter underlying host tissues, GC must first transmigrate across an epithelial barrier. This could occur via transcytosis through or transmigration between epithelial cells. In this study, we examined the relationship of GC with host cell junctional complexes in polarized cells of the human endometrial epithelial cell line, HEC-1-B, and the effect of sex hormones on these interactions and gonococcal transmigration. Studies using immunofluorescence microscopy showed that live GC preferentially localize at the epithelial apical junction, marked by ZO-1 and E-cadherin and induce redistribution of the apical junctional proteins, ZO-1 and E-cadherin. Treatment with estrogen and progesterone, two major hormones of the female reproductive system, enhanced GC-induced cellular redistribution of ZO-1. Under steroid hormone-free condition, GC transmigrated across polarized HEC-1-B cells at a low rate in a time dependent manner. Treatment with progesterone or estrogen increased GC transmigration at least five fold. Disruption of the apical junction by treatment of cells with the calcium chelator EGTA, increased GC transmigration across the polarized epithelial monolayer. These results suggest that GC target the apical junctional complex of polarized epithelial cells and work in synergy with estrogen and progesterone to disrupt the apical junction, thereby facilitating GC transmigration across the host epithelium.