VTechWorks Repository :: Browsing by Author "Scharf, Birgit E."

Browsing by Author "Scharf, Birgit E."

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Bacteria-Enabled Autonomous Drug Delivery Systems: Design, Modeling, and Characterization of Transport and Sensing
Traore, Mahama Aziz (Virginia Tech, 2014-06-25)
The lack of efficacy of existing chemotherapeutic treatments of solid tumors is partially attributed to the limited diffusion distance of therapeutics and the low selectivity of anti-cancer drugs with respect to cancerous tissue, which also leads to high levels of systemic toxicity in patients. Thus, chemotherapy can be enhanced through improving anti-cancer drug carrier selectivity and transport properties. Several strains of gram positive (e.g. Clostridium and Bifidobacterium) and gram-negative (e.g. Salmonella Typhimurium and Escherichia coli) bacteria have been shown to possess the innate ability to preferentially colonize tumor tissues. The overall goal of this dissertation is to characterize the transport and sensing of Bacteria-Enabled Drug Delivery Systems (BEADS) in select relevant environments and to investigate the associated underlying principles. BEADS consist of an engineered abiotic load (i.e. drug-laden micro or nano-particles) and a living component (i.e. bacteria) for sensing and actuation purposes. Findings of this dissertation work are culminated in experimental demonstration of deeper penetration of the NanoBEADS within tumor tissue when compared to passively diffusing chemotherapeutic nanoparticles. Lastly, the transport mechanisms that Salmonella Typhimurium VNP20009 utilize to preferentially colonize solid tumors are also examined with the ultimate goal of engineering intelligent and more efficacious drug delivery vehicles for cancer therapy.
Bacteria-Enabled Autonomous Drug Delivery Systems: Development, Characterization of Intratumoral Transport and Modeling
Suh, SeungBeum (Virginia Tech, 2017-08-17)
Systemic chemotherapy is a major therapeutic approach for nearly all types and stages of cancer. Success of this treatment depends not only on the efficacy of the therapeutics but also on the transport of the drug to all tumor cells in sufficient concentrations. Intratumoral drug transport is limited by characteristics of the tumor microenvironment such as elevated interstitial pressure and sparse, irregular vascularization. Moreover, poor tumor selectivity, leads to systemic toxicity. Bacteria possess a host of characteristics that address the aforementioned challenges in conventional drug delivery approaches including tumor selectivity, preferential tumor colonization, effective tumor penetration, which can be augmented via genetic engineering. However, in clinical trials conducted to date, bacteria have rarely been able to inhibit tumor growth solely by their presence in the tumor. The overall goal of this doctoral dissertation is to develop a novel tumor treatment system based on Salmonella Typhimurium VNP20009 (genetically modified for preferential tumor colonization and attenuation) coupled with biodegradable copolymer, poly(lactic-co-glycolic acid) nanoparticles, hereafter referred to as NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery System). To this end, a NanoBEADS fabrication procedure that is robust and repeatable was established and a microfluidic chemotaxis-based sorting platform for the separation NanoBEADS from unattached nanoparticles was developed. The transport efficacy of NanoBEADS compared to the commonly used passively-diffusing nanoparticle was investigated in vitro and in vivo and the intratumoral penetration of the therapeutic vectors was quantified using a custom image processing algorithm. The mechanism of intratumoral penetration was elucidated through 2D and 3D invasion assays. Lastly, we developed a biophysical model of intratumoral transport of NanoBEADS based on the intratumoral penetration experimental results towards the theoretical evaluation of the drug transport profile following the administration of NanoBEADS.
Cell-type specificity and herbivore-induced responses of primary and terpene secondary metabolism in Arabidopsis roots
Zhang, Jingyu (Virginia Tech, 2013-09-02)
Plants employ diverse defense mechanisms to combat attack by harmful organisms. For instance, plants produce constitutive physical barriers or use chemical compounds such as specialized secondary metabolites to resist herbivore or pathogen invasion. Considering the cost-efficiency and energy balance between defense, growth and reproduction, defense reactions in plants have to be regulated temporally and spatially. As more cost-efficient strategies, plants may induce their defense response only in the presence of the attacker or restrict constitutive defenses to specific tissues or cells. In this study, we investigated aspects of the spatial regulation and induced changes of primary and secondary metabolism in roots of Arabidopsis thaliana. Roots represent important organs for anchoring plants in the soil and taking up water and nutrients. Hence, it is assumed that roots are as well protected as aerial tissues by different defense mechanisms. The first part of this work is focused on the cell-type-specific biosynthesis of volatile terpenes in Arabidopsis roots. Terpenes are the most abundant specialized metabolites in plants and play an important role in plant defense against pathogens or herbivores. Terpene biosynthetic enzyme activities are often coordinated in specific tissues and cellular compartments. Fine-scale transcriptome maps of Arabidopsis roots have shown that terpene biosynthesis is restricted to particular cell types. However, the reasons and significance of this cell-type specificity are not well understood. We hypothesized that the formation of terpene metabolites is not restricted to specific cells but can be supported by different cell types. We, therefore, probed the plasticity of the cell-specific formation of terpenes by swapping the expression of the terpene synthase (TPS) genes, TPS08, TPS13 and TPS25, between different root cell types in the respective mutant background. To investigate the ectopic expression of TPSs at different levels, quantitative real-time PCR (qRT-PCR), confocal microscopy, and gas chromatography-mass spectrometry (GC-MS) were performed. We found that terpene synthase TPS08, which produces the diterpene rhizathalene and is normally expressed in the root vascular tissue, is functionally active when expressed in the epidermis or cortex, although at substantially lower levels compared to the wild type. We did not find an obvious correlation between the volatile emission level and gene transcript level of TPS08, which may be attributed to a reduced activity of the expressed TPS08-yellow fluorescent protein (YFP) fusion protein. When expression of TPS13 (producing the sesquiterpene (Z)-"-bisabolene) was directed from the cortex to the epidermis or stele, TPS13 gene expression and (Z)-"-bisabolene formation was supported by these cell types although to varying levels in comparison to wild type. TPS13-YFP fluorescent signal driven by the epidermal WER and GL3 promoters was primarily detected at the root tip. Terpene production was also observed for the (E)-"-farnesene sesquiterpene synthase TPS25 when its expression was switched from the endodermis and non-hair producing epidermal cells to hair producing epidermal cells although only a weak fluorescent signal was detected from the expressed TPS25-mGFP protein. Together, the results provide preliminary evidence for a relaxed cell specificity of terpene biosynthesis in Arabidopsis roots and suggest that tissue-specific terpene metabolite patterns could change depending on different selective pressures in rhizosphere. In the second part of this study, we performed global gene transcript profiling and primary metabolite analysis of Arabidopsis roots upon feeding by the generalist root herbivore, Bradysia (fungus gnat). In a microarray analysis, we identified 451 of 22,810 genes that were up-regulated more than 2-fold. Gene ontology (GO) analysis showed that 26% of those genes with predicted or known functions play a role in primary or secondary metabolism, while 24% are involved in cell signaling or in responses to stimulating factors, such as jasmonic acid (JA), ethylene, wounding, and oxidative stress. At the metabolite level, we observed only marginal changes of amino acid, sugar and carboxylic acid relative levels over a time course of 4 days of Bradysia feeding. There was a trend for increased levels of amino acids and the relative levels of sucrose were increased significantly ("=0.05) at the fourth day of feeding. In conclusion, the study provided evidence for the induction of genes related to primary and secondary metabolism and stress responses in Arabidopsis roots, but showed only marginal changes at the primary metabolite level. In addition, the work indicated that the formation of terpene-specialized metabolites in Arabidopsis roots is not restricted to specific cells, but can be supported by different cell types.
Characterization of Bacillus Spore Membrane Proteomes and Investigation of Their Roles in the Spore Germination Process
Chen, Yan (Virginia Tech, 2014-09-23)
Components of the bacterial spore germination apparatus are crucial for survival and for initiation of infection by some pathogens. While some components of the germination apparatus are well conserved in spore-forming species, such as the spoVA operon, each species may possess a different and possibly unique germinant recognition mechanism. The significance of several individual proteins in the germination process has been characterized. However, the mechanisms of how these proteins perform their functions and the network connecting these proteins in the complete germination process are still a mystery. In this study, we characterized a Bacillus subtilis superdormant spore population and investigated the abundance of 11 germination-related proteins. The relative quantities of these proteins in dormant, germinating and superdormant spores suggested that variation in the levels of proteins, other than germinant receptor proteins may result in superdormancy. Specifically, variation in the abundance of the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Spore membrane proteomes of Bacillus anthracis and B. subtilis were characterized to generate a candidate protein list that can be further investigated. Proteins that were not previously known to be spore-associated were identified, and many of these proteins shared great similarity in both Bacillus species. A significant number of these proteins are implicated in functions that play major roles in spore formation and germination, such as amino acid or inorganic ion transport and protein fate determination. By analyzing the in vivo and in vitro activity of HtrC, we proved that the protease is responsible for YpeB proteolytic processing at specific sites during germination. However, without HtrC present in the spore, other proteases appear to degrade YpeB at a reduced rate. The activity of purified HtrC in vitro was stimulated by a relatively high concentration of Mn²⁺ or Ca²⁺ ions, but the mechanism behind the stimulation is not clear. We also demonstrated that YpeB and SleB, in the absence of their partner protein, were degraded by unknown proteases other than HtrC during spore formation. Identification and characterization of these unknown proteases would be a future direction for revealing the roles of proteases in spore germination.
Characterization of proteins involved in Bacillus subtilis spore formation and germination
Barat, Bidisha (Virginia Tech, 2020-05-22)
Members of the Bacillus genus, when faced with unfavorable environmental conditions such as depletion of nutrients, undergo an asymmetric division process ultimately leading to the formation of an endospore. In some instances, the spore serves as the infectious agent of an associated disease; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of unfavorable environmental conditions including traditional decontamination techniques. Spore resistance is due to the formation of specialized structures that contribute to spore dormancy through several mechanisms, including maintenance of the dehydrated state of the spore core. Spore germination is a rapid process resulting in the irrevocable transformation of the non-metabolizing dehydrated spore into a vegetative outgrowing bacterium. The exact mechanism by which individual proteins function in the germination pathway remains unknown. In this study, we have focused on the roles of putative ion transporters and other germination-active proteins in affecting spore formation and germination. Metal ions can activate enzymes during the sporulation process and/or be factors in spore resistance properties. In B. subtilis, six proteins within the spore membrane proteome (ChaA, YcnL,YflS, YloB, YugS, ZnuA) are similar to components of known cation transport systems. These proteins may play roles in the accumulation of ions during sporulation and/or the release of ions during germination. Multiple mutants altered in the putative ion transporter genes were generated, and the effects of these mutations were analyzed. All strains containing a yloB deletion showed a decrease in heat resistant cfu/ml, and >40% of the spores appeared phase dark during microscopy, indicating the formation of unstable spores. Studies were conducted to quantify the amounts of individual ions in phase-bright spores using atomic emission spectroscopy and to analyze the rate at which ions are released from germinating spores. The transport of Ca2+ from mother cell to forespore during sporulation seems to be affected in the yloB deletion mutant. This Ca2+ deficit apparently renders the spores unstable, heat sensitive, and partially germination defective, suggesting that a high-affinity transporter for Ca2+ is nonfunctional. To better understand the underlying mechanisms of germination, a high-throughput genetic screening method called transposon sequencing was used. This analysis identified genes that had not been previously implicated in germination. To investigate their functions, a number of functional assays of all the Ger mutant strains were performed that indicated a delay in stage I of germination. The mutant strains showed significant reduction in germination efficiency with L-valine: about 50% of the population failed to initiate germination suggesting a defect in the GerA-mediated response. The expression of gerA was studied using a lacZ transcriptional fusion followed by quantitative western blot analyses to determine abundance of GerA in mutant strains. The mutants were classified based upon normal or decreased gerA transcription and normal or reduced GerA protein. Further work involves understanding the functions of the identified genes and their correlation to the GerA receptor. Insight into ion transporters of spore-forming bacteria and understanding the germination apparatus may lead to promising new applications, detection methods, or therapeutics for spores, and may allow the development of better spore decontamination procedures.
Characterization of symbiotically important processes in Sinorhizobium meliloti
Zatakia, Hardik M. (Virginia Tech, 2015-09-15)
Bacteria perform biological nitrogen fixation (BNF) which leads to conversion of N2 to ammonia. One of the best studied models of BNF is the symbiotic association of Sinorhizobium meliloti - Medicago sativa (alfalfa). Since alfalfa is a major source of animal feed and the fourth largest crop grown in the USA, enhanced understanding of this symbiosis can have implications for increasing crop yields, reducing environmental contamination and food costs. Studies discussed here focus on two symbiotically important bacterial traits, type IVb pili and chemotaxis. Chapter 2 characterizes S. meliloti type IVb pili encoded by flp-1 and establishes their role in nodulation. Bundle-forming pili were visualized in wild-type cells, while cells lacking pilA1, the pilin-encoding gene, showed an absence of pili. Competitive nodulation assays with alfalfa concluded that cells lacking pili had a significant nodulation defect. Regulation of pilA1 expression via a quorum sensing regulator, ExpR, was confirmed. Chapter 3 describes the role of the flp-2 cluster in establishing symbiosis. PilA2 is a pilin subunit encoded from flp-2. The pilA2 deletion strain was defective in nodulation by 31% as compared to the wild type. A non-significant change in nodulation was seen in pilA1pilA2 strain. Thus, both flp-1 and flp-2 have a significant role in establishing symbiosis. Chapter 4 focuses on the deviations of S. meliloti chemotaxis from the enterobacterial paradigm. Transcriptional fusions showed that S. meliloti chemoreceptors (MCPs) are class III genes and regulated by FlbT. Quantitative immunoblots determined the cellular amounts of chemoreceptors. Chemoreceptors were grouped in three classes; high, low, and extremely-low abundance, similar to the high and low abundance chemoreceptors of Escherichia coli. Importantly, the MCP:CheA ratio in an S. meliloti cell was observed to be 37:1, similar to that in Bacillus subtilis of 24:1, but quite different from that in E. coli of 3.4:1. In conclusion, our data indicates that soil bacteria may have optimized their chemotaxis system based on their milieu, which is different from enteric bacteria. These studies have enhanced our understanding of two symbiotically important processes in S. meliloti, and pave the way for future manipulations of the system to increase symbiosis and reduce our dependence on synthetic fertilizers.
Characterization of the Sinorhizobum Meliloti Chotaxis System
Castaneda Saldana, Rafael (Virginia Tech, 2019-12-19)
Increasing awareness to global climate change has drastically focused attention on finding solutions to reduce environmental impacts while still providing sufficient food for the increasing world population. Beneficial Nitrogen Fixing (BNF) microbes provide a possible solution by delivering biological nitrogen to plants resulting in reduced environmental impacts due to fertilizer runoff and eutrophication. One well studied model is that of Sinorhizobium meliloti and its legume host Medicago sativa (alfalfa), the fourth largest USA crop used for animal feed. Advancing research for this symbiosis model can provide solutions to enhance yield while minimizing environmental impacts. Chapter 2 focuses on the deviation of the S. meliloti chemotaxis system from the enteric paradigm. Quantitative immunoblots determined the cellular amounts of chemotaxis proteins. Overall, chemotaxis protein levels were approximately 10-fold lower in S. meliloti compared to B. subtilis and E. coli. Focusing on cellular stoichiometric ratios, S. meliloti generally exhibits drastically higher values for CheB, CheR, and CheY to the histidine kinase CheA monomer compared to E. coli and B. subtilis. Chapter 3 characterizes the role of McpX to quaternary ammonium compound (QAC) sensing. QACs are exuded by germinating alfalfa seeds. In vitro binding assays were performed to determine ligand binding characteristics. S. meliloti chemotaxis behavior to QACs was analyzed in in vivo capillary assays under real-time imaging. These studies strengthen our knowledge of the chemotaxis system in the symbiosis model of S. meliloti and alfalfa. The data can further be used to create a mathematical model of the dynamics of bacteria-host interaction. The results can be used to optimize chemotaxis to host plants to improve crop yield and protect watersheds.
Characterization of the thioredoxin system in Methanosarcina mazei
Loganathan, Usha R. (Virginia Tech, 2014-12-18)
Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation.
Characterization of the VtlR regulons in Brucella abortus and Agrobacterium tumefaciens
Budnick, James Andrew (Virginia Tech, 2019-04-25)
Brucella abortus and Agrobacterium tumefaciens are pathogenic bacteria that infect animals and plants, respectively. These bacteria are genetically similar and are found within the same Class, Alphaproteobacteria, and Order, Rhizobiales, of the domain Eubacteria; however, they survive and replicate in vastly different environmental niches. In Order to adapt to different environments, bacteria utilize several mechanisms of gene regulation to tightly control gene expression. Two of these mechanisms include transcriptional regulators and small regulatory RNAs (sRNAs), which can activate and repress gene expression through various interactions with DNA, mRNA, and proteins. A well-conserved transcriptional regulator among the Rhizobiales is VtlR, a virulence-associated transcriptional LysR regulator. The objectives of this dissertation were three fold: 1) characterize the known regulon of VtlR in B. abortus with regards to gene regulatory function and virulence, 2) determine the regulon of VtlR in A. tumefaciens and define the mechanism by which this regulation occurs, and 3) define the role of an ABC-type transport system indirectly regulated by VtlR in B. abortus that putatively imports the non-proteinogenic amino acid gamma-aminobutyric acid (GABA). VtlR was characterized in B. abortus as a virulence-associated transcriptional regulator that directly activates four genes: the sRNA AbcR2, and the three small hypothetical proteins BAB1_0914, BAB2_0512, and BAB2_0574; and deletion of vtlR led to a significant defect in the ability of B. abortus to cause infection in vitro and in vivo. Since dysregulation of abcR2 alone could not account for the defect in virulence, it was hypothesized that one or all three hypothetical proteins could be responsible for a virulence phenotype observed in ΔvtlR. This turned out to not be the case, as a deletion of the entire VtlR regulon displayed no difference in virulence compared to the parental strain. Further characterization of the small hypothetical proteins is outlined in Chapter 2 and the data revealed bona fide translation of each small protein, and the deletion strain of the VtlR regulon displayed a growth defect when grown in the presence of the sugar fucose. This phenotype was subsequently observed in ΔvtlR as well. This led to the identification of a putative fucose transport and metabolism locus in B. abortus that has yet to be studied. In A. tumefaciens, VtlR is necessary for proper attachment to plant cells and biofilm formation and regulates over 200 genes, significantly more than the four genes VtlR regulates in B. abortus. The mechanism by which this occurs was unknown, and the relationship between VtlR and AbcR1 or AbcR2 was uncharacterized. The data in Chapter 3 outline the VtlR network by showing that VtlR regulation of myriad genes in A. tumefaciens is primarily indirect via the direct regulation of a few sRNAs. This direct interaction was shown experimentally and a VtlR binding box was identified in the A. tumefaciens genome. This project outlines the divergence of a regulatory element between phylogenetically related organisms that occupy different environmental niches. The AbcR sRNAs are conserved throughout the Rhizobiales and regulate numerous ABC-type transport systems within these bacteria. In A. tumefaciens, one of these transport systems specifically transports the amino acds proline and GABA. B. abortus contains homologs of this system, which led to the hypothesis that the brucellae may also transport GABA but for a yet unknown purpose. The data in Chapter 4 revealed that B. abortus also transports GABA in vitro and this transport is under the regulation of AbcR1 and AbcR2. This transport was increased under extreme nutrient limitations and was uninhibited by the presence of other amino acids. Metabolic studies showed GABA is not utilized by B. abortus under aerobic conditions, and transcriptomic data revealed increased expression of several loci in the presence of GABA. Altogether, this study uncovers a putative signaling role for the amino acid GABA that has been understudied in bacterial pathogens that infect animal hosts. Overall, the work presented in this dissertation is focused on further elucidating the biological role of downstream regulatory targets of both VtlR and the sRNAs AbcR1 and AbcR2 in the related organisms Brucella abortus and Agrobacterium tumefaciens. Findings show that while there are similarities between the two systems, there are also many differences that may be attributed to the vastly different lifestyles of each organism.
Characterizing the AbcR/VtlR system in the Rhizobiales
Sheehan, Lauren Marie (Virginia Tech, 2018-07-30)
Rhizobiales encompass a diverse group of microbes, ranging from free-living, soil-dwelling bacteria to disease-causing, intracellular pathogens. Although the lifestyle of these organisms vary, many genetic systems are well conserved. One system, named the AbcR/VtlR system, is found throughout rhizobiales, and even extends to bacteria in other orders within the Alphaproteobacteria. The AbcR sRNAs are an example of sibling sRNAs, where two copies of the abcR gene are typically present in the genome. The AbcRs are involved in the negative regulation of ABC-type transport systems, which are important components for nutrient acquisition. Although the AbcRs share several features amongst organisms, major differences can be found in their functional and regulatory redundancy, the targets they regulate and how they regulate them. Specifically, one major difference in the AbcRs lies in the nucleotide sequences utilized by the sRNAs to bind mRNA targets. In the present studies, the regulatory mechanisms of the AbcR sRNAs were further characterized in the mammalian pathogen Brucella abortus, and the full regulatory profiles of the AbcRs were defined in the plant pathogen Agrobacterium tumefaciens. As mentioned above, the AbcR sRNAs are important for the proper regulation of nutrient-acquiring transport systems in the Rhizobiales. Since these sRNAs are critical to the lifestyle of a bacterium, proper regulation of this system is key to survival. A LysR-type transcriptional regulator, named VtlR, was found to be the bonefide transcriptional activator of abcR1 in B. abortus. Furthermore, VtlR has been shown to be a key component in host interactions in several rhizobiales. The preset work has shed light on the evolutionary divergence of this regulator in bacteria, and further defined the regulatory capacity of VtlR in Agrobacterium. Overall, the studies described here have made significant advances in our knowledge of the AbcR/VtlR-regulatory systems in the Rhizobiales, and have further defined this system as being a vital part of host-microbe interactions.
Complete Genome Sequence of Curtobacterium sp. Isolated from Surface-Sterilized Germinating Alfalfa Seeds
Compton, K. Karl; Jensen, Roderick, V; Lahmers, Kevin K.; Scharf, Birgit E. (American Society for Microbiology, 2022-02)
We reported the complete genome sequence of a member of the pathogenic Curtobacterium genus. The sample includes a circular 3955-kb chromosome, a 164-kb megaplasmid and a 42-kb plasmid. This strain was isolated from surface-sterilized alfalfa seeds.
CRISPR3 Regulates Exopolysaccharide Production in Myxococcus xanthus
Wallace, Regina A. (Virginia Tech, 2013-10-10)
Myxococcus xanthus, a model organism for studying development and Type IV pili (T4P), harbors three Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) on its chromosome. CRISPR systems, which function as an adaptive immune system in prokaryotes, are classified into three types based on CRISPR-associated genes. Evidence suggests that these three types mediate immunity slightly differently. M. xanthus CRISPR1 and CRISPR2 are Type I systems while CRISPR3 is a Type III-B system. In a genetic screen, a mariner transposon insertion in the 13th spacer of CRISPR3 (3SP13) was found to restore exopolysaccharide (EPS) production to a pilA mutant. Since the deletion of CRISPR3 failed to suppress a pilA mutation and expression of CRISPR3 from a heterologous promoter led to pilA suppression, it was concluded that the 3SP13 transposon insertion is a gain-of-function mutation. Deletion of the adjacent Repeat Associated Mysterious Proteins (RAMP) genes indicated that they are essential for the 3SP13 transposon insertion to suppress pilA, providing evidence that Type III-B systems may be involved in the regulation of chromosomal genes. We suggest that one of the spacers, once expressed and processed, may inhibit the expression of a negative regulator of EPS production in M. xanthus.
Development of Bacteria-Based Bio-Hybrid Delivery Systems: Fabrication, and Characterization of Chemotaxis and Quorum Sensing
Sahari, Ali Akbar (Virginia Tech, 2014-10-09)
Bio-hybrid approaches have recently provided a possible solution to address the challenge of on-board actuation, control and communication modules for micro/nanoscale cargo-carrying vehicles by integrating live prokaryotic or eukaryotic cells with synthetic objects. More specifically, because micro/nanoparticles are able to transport cargos efficiently and bacteria can play the role of targeted and selective delivery agents, a hybrid of these two can advance the current strategies for environmental monitoring, drug delivery and medical imaging. The main goal of this dissertation was to fabricate, assemble, and characterize different components of a mobile network of bacteria-based bio-hybrid systems for long-term applications in drug delivery and biosensing. First, a new library of bacteria-enabled delivery systems was developed by coupling live engineered bacteria with non-spherical particles and the transport of these bacteria-based systems was investigated in the absence and presence of chemical cues using microfluidic platforms. Next, a quorum-sensing (QS) based bacterial cell-cell communication network was characterized in a high-throughput manner in order to understand the coordinated behavior of the bacterial species ferrying the cargoes. Lastly, the QS behavior of a chemotactic population of the bacterial species in response to the endogenously produced signaling molecules was studied. The work presented in this dissertation lays the foundation for a well-characterized generation of bacteria-assisted cargo delivery devices with enhanced transport properties and capable of executing pre-programmed multi-agent coordinated tasks upon their arrival at the target site.
Differential Expression Analysis of Type II Toxin-Antitoxin Genes of Pseudomonas aeruginosa PAO1 under Different Environmental Conditions
Haque, Anamul (Virginia Tech, 2018-07-02)
Bacterial persistence is considered as one of the primary reason for antibiotic tolerance besides genetically acquired antibiotic resistance. Persisters are the subpopulation of a clonal bacterial population, which can survive environmental extremes and become invulnerable to stresses due to limited metabolic activities and physiological functions. Cognate toxin and antitoxin (TA) pairs, which are transcribed simultaneously from the same or different operons within the bacterial chromosomes or plasmids, play an important role for bacterial survival during stressful growth environments. Pseudomonas aeruginosa PAO1 is one of the most versatile microorganisms in the environment. Despite its ubiquitous presence, no studies have shown the differential expression pattern of its toxin-antitoxins, and persistence related genes. The purpose of the following study is to analyze differential expression of P. aeruginosa PAO1 type II toxin-antitoxins and persistence related genes under different growth conditions and to show how their stoichiometric ratio changes during different growth conditions. Differential expression analysis indicated that the toxins and antitoxin pairs behave differently under different growth conditions. In addition, the genes related to persistence presented relatively consistent differential expression pattern under different growth environment.
Effect of Salmonella enterica serovar Typhimurium VNP20009 and VNP20009 with restored chemotaxis on 4T1 mouse mammary carcinoma progression
Coutermarsh-Ott, Sheryl; Broadway, Katherine M.; Scharf, Birgit E.; Allen, Irving C. (Impact Journals, 2017-05-16)
A variety of bacterial strains have been evaluated as bio-therapeutic and immunomodulatory agents to treat cancer. One such strain, Salmonella enterica serovar Typhimurium VNP20009, which is attenuated by a purine auxotrophic mutation and modified lipid A, is characterized in previous models as a safely administered, tumor colonizing agent. However, earlier work tended to use less aggressive cancer cell lines and immunocompromised animal models. Here, we investigated the safety and efficacy of VNP20009 in a highly malignant murine model of human breast cancer. Additionally, as VNP20009 has recently been found to have a defective chemotaxis system, we tested whether restoring chemotaxis would improve anti-cancer properties in this model system. Exposure to VNP20009 had no significant effect on primary mammary tumor size or pulmonary metastasis, and the tumor colonizing process appeared chemotaxis independent. Moreover, tumor-bearing mice exposed to Salmonella exhibited increased morbidity that was associated with significant liver disease. Our results suggest that VNP20009 may not be safe or efficacious when used in aggressive, metastatic breast cancer models utilizing immunocompetent animals.
Elucidating three novel mechanisms of Pseudomonas syringae pathogenicity
Clarke, Christopher R. (Virginia Tech, 2012-02-10)
Pseudomonas syringae is an important bacterial plant pathogen that, as a species, is known to cause disease on hundreds of different plant species. However, any individual pathovar of P. syringae typically only causes disease on one or a few plant species, which constitute the host range of the pathovar. Plants are generally resistant to most pathogens primarily because the plant innate immune system is capable of recognizing conserved microbial-associated molecular patterns (MAMPs). Adapted pathovars of P. syringae secrete effector proteins through a Type Three Secretion System (T3SS) to suppress the immune response elicited by their MAMPs. However, secretion of effectors can also trigger a strong plant immune response if the plant harbors resistance proteins capable of recognizing the secreted effectors. Successful pathovars, therefore, must secrete a combination of effectors capable of suppressing MAMP/Pattern-Triggered Immunity (PTI) without eliciting Effector-Triggered Immunity. Here we identify several novel strategies employed by P. syringae to overcome the plant immune system and cause disease. First, we demonstrate that, in place of the canonical T3SS used by all known pathogens of P. syringae, several apparently nonpathogenic isolates of P. syringae employ a novel T3SS that is functional but not necessary for colonization of plants. Despite being closely related to pathogenic isolates of P. syringae, the isolates employing the noncanonical T3SS do not cause disease on any tested plants and instead appear to act more as commensal organisms. Second, we advance the understanding of PTI by identifying a second region of bacterial flagellin that triggers PTI in addition to the archetypical MAMP flg22, which is recognized by the archetypical plant receptor FLS2. This new elicitor, termed flgII-28, is also detected by FLS2 and appears to be under selection in very closely related lineages of P. syringae. Alleles of flagellin present in one recently expanded and agriculturally problematic lineage of P. syringae appear to trigger less PTI on their host plant, tomato, than the ancestral allele suggesting that avoidance of PTI through allelic diversity in MAMPs is an effective alternative strategy to suppression of PTI through delivery of effectors. Finally, we start to elucidate a role for chemotaxis (chemical-directed movement) in P. syringae pathogenicity. Not only is chemotaxis required for pathogenicity of P. syringae on plants, but it also appears to contribute to delimiting the host range of several P. syringae pathovars. These results highlight that additional aspects of P. syringae pathogenicity, such as chemotaxis, can directly contribute to defining the host range of individual P. syringae pathovars. The current paradigm of P. syringae pathogenicity posits that MAMPS and the repertoire of effector proteins are the primary determinant of the host range of any P. syringae pathovar; in contrast these results inspire a more nuanced view of pathogenicity that considers multiple aspects of the infection process.
Elucidation of the Specificity of S. meliloti Chemoreceptors for Host Derived Attractants
Webb, Benjamin A. (Virginia Tech, 2016-08-24)
The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields. Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor. Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates. Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline. In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar. These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields.
An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano
Sonani, Ravi R.; Palmer, Lee K.; Esteves, Nathaniel C.; Horton, Abigail A.; Sebastian, Amanda L.; Kelly, Rebecca J.; Wang, Fengbin; Kreutzberger, Mark A. B.; Russell, William K.; Leiman, Petr G.; Scharf, Birgit E.; Egelman, Edward H. (Springer, 2024-01-26)
A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.
Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments
Kreutzberger, Mark A. B.; Sobe, Richard C.; Sauder, Amber B.; Chatterjee, Sharanya; Pena, Alejandro; Wang, Fengbin; Giron, Jorge A.; Kiessling, Volker; Costa, Tiago RD D.; Conticello, Vincent P.; Frankel, Gad; Kendall, Melissa M.; Scharf, Birgit E.; Egelman, Edward H. (Nature Portfolio, 2022-03-17)
Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.
Flagellotropic Bacteriophages: Opportunities and Challenges for Antimicrobial Applications
Esteves, Nathaniel C.; Scharf, Birgit E. (MDPI, 2022-06-25)
Bacteriophages (phages) are the most abundant biological entities in the biosphere. As viruses that solely infect bacteria, phages have myriad healthcare and agricultural applications including phage therapy and antibacterial treatments in the foodservice industry. Phage therapy has been explored since the turn of the twentieth century but was no longer prioritized following the invention of antibiotics. As we approach a post-antibiotic society, phage therapy research has experienced a significant resurgence for the use of phages against antibiotic-resistant bacteria, a growing concern in modern medicine. Phages are extraordinarily diverse, as are their host receptor targets. Flagellotropic (flagellum-dependent) phages begin their infection cycle by attaching to the flagellum of their motile host, although the later stages of the infection process of most of these phages remain elusive. Flagella are helical appendages required for swimming and swarming motility and are also of great importance for virulence in many pathogenic bacteria of clinical relevance. Not only is bacterial motility itself frequently important for virulence, as it allows pathogenic bacteria to move toward their host and find nutrients more effectively, but flagella can also serve additional functions including mediating bacterial adhesion to surfaces. Flagella are also a potent antigen recognized by the human immune system. Phages utilizing the flagellum for infections are of particular interest due to the unique evolutionary tradeoff they force upon their hosts: by downregulating or abolishing motility to escape infection by a flagellotropic phage, a pathogenic bacterium would also likely attenuate its virulence. This factor may lead to flagellotropic phages becoming especially potent antibacterial agents. This review outlines past, present, and future research of flagellotropic phages, including their molecular mechanisms of infection and potential future applications.

Browsing by Author "Scharf, Birgit E."

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