Matching Items (11)

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A Spatial Control for Correct Timing of Gene Expression during the Escherichia coli Cell Cycle

Description

Temporal transcriptions of genes are achieved by different mechanisms such as dynamic interaction of activator and repressor proteins with promoters, and accumulation and/or degradation of key regulators as a function

Temporal transcriptions of genes are achieved by different mechanisms such as dynamic interaction of activator and repressor proteins with promoters, and accumulation and/or degradation of key regulators as a function of cell cycle. We find that the TorR protein localizes to the old poles of the Escherichia coli cells, forming a functional focus. The TorR focus co-localizes with the nucleoid in a cell-cycle-dependent manner, and consequently regulates transcription of a number of genes. Formation of one TorR focus at the old poles of cells requires interaction with the MreB and DnaK proteins, and ATP, suggesting that TorR delivery requires cytoskeleton organization and ATP. Further, absence of the protein–protein interactions and ATP leads to loss in function of TorR as a transcription factor. We propose a mechanism for timing of cell-cycle-dependent gene transcription, where a transcription factor interacts with its target genes during a specific period of the cell cycle by limiting its own spatial distribution.

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Date Created
  • 2016-12-23

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The genes slyA, STM3120 and htrA are required for the anticancer ability of VNP20009

Description

VNP20009 is a very effective anti-cancer agent and can specifically target tumors and inhibit tumor growth. It was assumed that the tumor targeting ability of VNP20009 correlated to its anticancer

VNP20009 is a very effective anti-cancer agent and can specifically target tumors and inhibit tumor growth. It was assumed that the tumor targeting ability of VNP20009 correlated to its anticancer capacity. However, our observation contradicted to this assumption. Three VNP20009 mutant strains (ΔslyA, ΔSTM3120 and ΔhtrA) with reduced fitness in normal tissues and unchanged fitness in tumors partially or completely lost their anti-cancer capacities. The genes slyA, STM3120 and htrA were required for survival within macrophages and were indispensable for tumor microenvironment remodeling by VNP20009. The infiltration of immune cells occurred less in the tumors of mice infected with the mutant strains. In addition, the mRNA levels of TNF-α and IL-1β were significantly decreased in the tumors of mice treated with the mutant strains. Our results indicate that the immune responses elicited by bacteria rather than the bacterial titer in tumors play a “decisive” role in VNP20009-mediated bacterial cancer therapy, which provides a novel perspective for the underlying mechanism of bacterial cancer therapy.

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Date Created
  • 2016-11-08

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Codon Optimization of Human TRAIL Gene for Maximal Expression in a Self-Destructing Salmonella Strain for Efficient Colorectal Cancer Treatment

Description

Colorectal cancer is the third most common type of cancer that affects both men and women and the second leading cause of death in cancer related deaths[1, 2]. The most

Colorectal cancer is the third most common type of cancer that affects both men and women and the second leading cause of death in cancer related deaths[1, 2]. The most common form of treatment is chemotherapy followed by radiation, which is insufficient to cure stage four cancers[3]. Salmonella enteric has long been shown to have inherent tumor targeting properties and have been able to penetrate and exist in all aspects of the tumor environment, something that chemotherapy is unable to achieve. This lab has developed a genetically modified Salmonella typhimurium (GMS) which is able to deliver DNA vaccines or synthesized proteins directly to tumor sites. These GMS strains have been used to deliver human TNF-related apoptosis inducing ligand (TRAIL) protein directly to tumor sites, but expression level was limited. It is the hope of the experiment that codon optimization of TRAIL to S. typhimurium preferred codons will lead to increased TRAIL expression in the GMS. For preliminary studies, BALB/c mice were subcutaneously challenged with CT-26 murine colorectal cancer cells and treated with an intra-tumor injection with either PBS, strain GMS + PCMV FasL (P2), or strain GMS + Pmus FasL). APC/CDX2 mutant mice were also induced to develop human colon polyps and treated with either PBS, strain GMS + vector (P1), P2, or P3. The BALB/c mouse showed statistically significant levels of decreased tumor size in groups treated with P2 or P3. The APC/CDX2 mouse study showed statistically significant levels of decreased colon polyp numbers in groups treated with P3, as expected, but was not significantly significant for groups treated with P1 and P2. In addition, TRAIL was codon optimized for robust synthesis in Salmonella. The construct will be characterized and evaluated in vitro and in vivo. Hopefully, the therapeutic effect of codon optimized TRAIL will be maximal while almost completely minimizing any unintended side effects.

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Date Created
  • 2019-05

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Genomic Location and Intensity of Transcription of Non-coding RNA Regulation Candidates in Porphyromonas gingivalis

Description

Many pathogens are bacteria and antibiotic resistance is increasing. The development of novel treatments is hampered by a poor understanding of the mechanisms of their regulation. Specifically, non-coding RNAs play

Many pathogens are bacteria and antibiotic resistance is increasing. The development of novel treatments is hampered by a poor understanding of the mechanisms of their regulation. Specifically, non-coding RNAs play an important role in the internal regulation of bacteria. To further the investigation of non-coding RNA and pathogenicity, RNA sequencing data for PorX/PorY dependent regulation in P. gingivalis, a Gram negative oral pathogen was studied. The PorX/PorY two component regulatory system controls phenotypes for this bacteria's virulence including an important type IX secretion system for gingipain proteases, which degrades host cytokines, down regulating the host response by reducing inflammation. This study compared transcription of non-coding RNA in wild type and PorX knockout mutant strain, in the 33277 strain and the more virulent W83 strain in both liquid and solid cultures to identify and categorize loci of genomic sequence for further study of porX/porY regulation.

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Date Created
  • 2017-05

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The Environmental Contribution to Emerging Antibiotic Resistance

Description

Antibiotic resistance in the modern era has reached near-epidemic levels, resulting in much more difficult treatment of previously well-managed pathogens. Previous understandings of how antibiotic resistance emerges failed to account

Antibiotic resistance in the modern era has reached near-epidemic levels, resulting in much more difficult treatment of previously well-managed pathogens. Previous understandings of how antibiotic resistance emerges failed to account for the function of the environment. Over the past 15 years, new research has provided a link between the environmental and clinical spheres of antibiotic use. This data suggests that environmental bacteria, particularly those found in livestock farming ecosystems, may significantly contribute to the overall flow of antibiotic resistance genes into human populations. The main force behind this is the utilization of antibiotics as growth promoters in animal feed supplements, seeding individual animals and their surroundings with low doses of antibiotics. Notable increases in resistance have been observed within areas that utilize these supplements, as well as in connected but unrelated systems. Waste management strategies are poorly implemented, leading to the dispersal of contaminated runoff into groundwater and riverine environments. Furthermore, existing waste processing is limited in efficacy, often releasing large amounts of unprocessed antibiotics as well as a concentrated population of resistant bacteria. Within these resistant populations, horizontal gene transfer has emerged as a vehicle for the distribution of resistance genes into other populations of bacteria. Due to the prevalence of these transfer events, a new role for the environment as a reservoir and incubator of resistance genes is proposed. Current strategies for managing the spread of antibiotic resistance are woefully inadequate, and the continued emergence of new resistance mechanisms due to negligence highlights the need for global, multidisciplinary solutions. To corral the spread of antibiotic resistance, a system is proposed that utilizes metagenomic monitoring and the enforcement of core global policies to slow the advance of resistance while waiting for novel treatment strategies to bear fruit.

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Date Created
  • 2016-05

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Molecular Cloning of PGN_1740 and PGN_0012 Porphyromonas gingivalis Bacterium DNA Fragments into the pTCOW Plasmid

Description

Porphyromonas gingivalis (P. gingivalis) is an oral pathogen known for causing periodontal diseases like periodontitis and alveolar bone loss. In this study, we investigate the molecular mechanisms of P. gingivalis

Porphyromonas gingivalis (P. gingivalis) is an oral pathogen known for causing periodontal diseases like periodontitis and alveolar bone loss. In this study, we investigate the molecular mechanisms of P. gingivalis with focus of the molecular cloning of the two DNA strains of the bacteria PGN_1740 and PGN_0012 in the Ampr pTCow. PGN_1740 is an RNA polymerase ECF-type sigma factor used for transcription. PGN_0012 is a two-component system regulator gene that is important in signal transduction. We demonstrated the cloning mechanism through transformation and confirmed the results through gel electrophoresis and using a positive transformant as a control. The process of cloning the DNA inserts into the bacteria followed a polymerase chain reaction for the amplification of the DNA fragments, digestion of the plasmid and DNA fragments with the restriction endonucleases (BamHI and HindIII), ligation and finally heat shock transformation are presented in this thesis. The effectiveness of these procedures was observed through agarose gel electrophoresis and ethanol precipitation for the purification of the PCR products. In this investigation, we discuss molecular and biological characterization of the P. gingivalis bacteria in regard to cloning and ampicillin resistance.

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Date Created
  • 2019-05

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Identification of novel genetic mechanisms required for bacterial resistance to antimicrobial peptides

Description

The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat

The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.

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Date Created
  • 2013

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Genetics of functional AcrAB-TolC tripartite complex assembly

Description

Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of

Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of these three proteins (and many of their homologs) are known, the exact mechanisms of interaction are still poorly understood. By mutational analysis of the TolC turn 1 residues, a drug hypersensitive mutant has been identified which is defective in functional interactions with AcrA and AcrB. Antibiotic resistant revertants carry alterations in both TolC and AcrA act by stabilizing functional complex assembly and opening of the TolC aperture, as monitored by stability of a labile TolC mutant and sensitivity to vancomycin, respectively. Alterations in the AcrB periplasmic hairpin loops lead to a similar antibiotic hypersensitivity phenotype and destabilized complex assembly. Likewise, alterations in TolC which constitutively open the aperture suppress this antibiotic sensitivity. Suppressor alterations in AcrA and AcrB partially restore antibiotic resistance by mediating stability of the complex. The AcrA suppressor alterations isolated in these studies map to the three crystallized domains and it is concluded they alter the AcrA conformation such that it is permanently fixed in an active state, which wild type only transiently goes through when activated by AcrB. Through this genetic evidence, a direct interaction between TolC and AcrB which is stabilized by AcrA has been proposed. In addition to stabilizing the interactions between TolC and AcrB, AcrA is also responsible for triggering opening of the TolC aperture by mediating energy flow from AcrB to TolC. By permanently altering the conformation of AcrA, suppressor mutants allow defective TolC or AcrB mutants to regain functional interactions lost by the initial mutations. The data provide the genetic proof for direct interaction between AcrB and that AcrA mediated opening of TolC requires AcrB as a scaffold.

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Date Created
  • 2012

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Study of Edwardsiella ictaluri conserved genes towards the development of an attenuated recombinant vaccine for fish host

Description

Teleosts have the most primitive adaptive immune system. However, in terms of functionality the teleost immune system is similar to birds and mammals. On the other hand, enteric bacterial pathogens

Teleosts have the most primitive adaptive immune system. However, in terms of functionality the teleost immune system is similar to birds and mammals. On the other hand, enteric bacterial pathogens of mammals and birds present conserved regulatory mechanisms that control virulence factors. In this context, deletion of conserved genes that control virulence factors have been successfully used as measure to construct live attenuated bacterial vaccines for mammals and birds. Here, I hypothesize that evolutionary conserved genes, which control virulence factors or are essential for bacterial physiology in Enterobacteriaceae, could be used as universal tools to design live attenuated recombinant bacterial vaccines from fish to mammals. The evolutionary conserved genes that control virulence factors, crp and fur, and the essential gene for the synthesis of the cell wall, asd, were studied in Edwardsiella ictaluri to develop a live recombinant vaccine for fish host. The genus Edwardsiella is one of the most ancient represent of the Enterobacteriaceae family. E. ictaluri, a host restricted pathogen of catfish (Ictalurus punctatus), is the causative agent of the enteric septicemia and one of the most important pathogens of this fish aquaculture. Although, crp and fur control different virulence factors in Edwardsiella, in comparison to other enterics, individual deletion of these genes triggered protective immune response at the systemic and mucosal level of the fish. Deletion of asdA gene allowed the creation of a balanced-lethal system to syntheses heterologous antigens. I concluded that crp, fur and asd could be universally used to develop live attenuate recombinant Enterobacteriaceae base vaccines for different hosts.

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Date Created
  • 2012

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A study of protein-protein interactions in Salmonella typhimurium

Description

The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able

The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators.

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Date Created
  • 2018