Nancy Connell - Academia.edu (original) (raw)

Papers by Nancy Connell

Journal of Chemical Health and Safety, 2013

Local Planning for Terror and Disaster presents a truly integrated approach to emergency response... more Local Planning for Terror and Disaster presents a truly integrated approach to emergency response. It offers real–world strategies for handling all types of emergencies, covering such areas as bioterrorism, natural disasters, infectious disease outbreaks, public health policy, and emergency medicine. Written by leading experts on the cutting edge of terror medicine and other disciplines involved in local preparedness, the book clearly shows how proven techniques for dealing with terrorist attacks can be applied to accidental and natural disasters as well.

mBio, 2018

Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We... more Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA—a key component for biosynthesis of the mycolic acid layer of the bacterium’s cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.

Journal of Bacteriology, 1997

Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carb... more Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.

Journal of Bacteriology, 1991

Many of the changes in gene expression observed when Escherichia coli cells enter stationary phas... more Many of the changes in gene expression observed when Escherichia coli cells enter stationary phase are regulated at the level of transcription initiation. A group of stationary-phase-inducible promoters, known as "gearbox" promoter, display a characteristic sequence in the -10 region which differs greatly from the consensus sequence for sigma 70-dependent promoters. Here we describe our studies on the gearbox promoters bolAp1 and mcbAp, responsible for the temporally regulated transcription of bolA and the genes involved in the synthesis of the peptide antibiotic microcin B17, respectively. Deletion analysis of mcbAp demonstrated that the stationary-phase-inducible properties of this promoter are found in a DNA fragment extending from -54 to +11 bp, surrounding the transcriptional start site, and are separable from DNA sequences responsible for the OmpR-dependent stimulation of transcription of mcbAp. In vitro transcription studies indicate that the RNA polymerase holoenzy...

Journal of clinical microbiology, Oct 26, 2017

BACKGROUND.Bacillus anthracis is a tier1 select agent with the potential to quickly cause severe ... more BACKGROUND.Bacillus anthracis is a tier1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point of care testing.METHODS. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. Assay limit of detection (LOD), and dynamic range were determined by spiking B. anthracis DNA into individual PCR reactions, and B. anthracis colony forming units (CFU) into human blood. One-ml blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates, or by testing blood samples drawn from p...

Scientific reports, Mar 6, 2017

Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are Gram-negative proteobacteria that a... more Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are Gram-negative proteobacteria that are obligate predators of other Gram-negative bacteria and are considered potential alternatives to antibiotics. Most studies focusing on predatory bacteria have been performed in vitro, thus the effect of predatory bacteria on a live host, including the impact on the ecology of the native microbiota, has yet to be fully examined. In this study, intrarectal inoculations of Sprague-Dawley rats with predatory bacteria were performed. Additionally, feces were collected for seven days post-inoculation to determine the effect on gut bacterial diversity. Rat colonic tissue exhibited no abnormal histopathological effects due to predatory bacteria. A modest increase in pro-inflammatory cytokines was measured in the colons of rats inoculated with predatory bacteria by 24 and 48 hours, with all but IL-13 returning to baseline by seven days. V4 16S rRNA gene sequencing of fecal DNA demonstrated mini...

mBio, Feb 14, 2017

Active tuberculosis (TB) and latent Mycobacterium tuberculosis infection both require lengthy tre... more Active tuberculosis (TB) and latent Mycobacterium tuberculosis infection both require lengthy treatments to achieve durable cures. This problem has partly been attributable to the existence of nonreplicating M. tuberculosis "persisters" that are difficult to kill using conventional anti-TB treatments. Compounds that target the respiratory pathway have the potential to kill both replicating and persistent M. tuberculosis and shorten TB treatment, as this pathway is essential in both metabolic states. We developed a novel respiratory pathway-specific whole-cell screen to identify new respiration inhibitors. This screen identified the biphenyl amide GSK1733953A (DG70) as a likely respiration inhibitor. DG70 inhibited both clinical drug-susceptible and drug-resistant M. tuberculosis strains. Whole-genome sequencing of DG70-resistant colonies identified mutations in menG (rv0558), which is responsible for the final step in menaquinone biosynthesis and required for respiration. ...

One Sentence SummaryStructures ofMycobacterium tuberculosisRNA polymerase reveal taxon-specific p... more One Sentence SummaryStructures ofMycobacterium tuberculosisRNA polymerase reveal taxon-specific properties and binding sites of known and new antituberculosis agentsMycobacterium tuberculosis(Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually.MtbRNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures ofMtbRNAP, alone and in complex with Rif. The results identify anMtb-specific structural module ofMtbRNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report novel non-Rif-related compounds–Nα-aroyl-N-aryl-phenylalaninamides (AAPs)–that potently and selectively inhibitMtbRNAP andMtbgrowth, and we report crystal structures ofMtbRNAP in complex with AAPs. AAPs bind to a different site onMtbRNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-admini...

Journal of clinical microbiology, 2017

Francisella tularensis is a potential bioterrorism agent that is highly infectious at very low do... more Francisella tularensis is a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitive F. tularensis assay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system. F. tularensis DNA in buffer or CFU of F. tularensis was spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected with F. tularensis Schu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/ml F. tularensis in both human and macaque blood. In infected macaques, th...

A workshop was held in August 2014 under the auspices of the IAP Biosecurity Working Group to dis... more A workshop was held in August 2014 under the auspices of the IAP Biosecurity Working Group to discuss understanding and modulating pathogen virulence mechanisms and host immune responses. The workshop focused on two complementary strategies for combating infectious diseases: targeting pathogen virulence factors and modifying a host's immune responses. These issues were directly relevant to the 2014 intersessional focus of the Biological Weapons Convention (BWC). An understanding of pathogenicity and immunology has the potential to be misapplied to create pathogens with increased virulence or to decrease the effectiveness of responses to infection. Alternatively, advances in this understanding offer promising new strategies in disease treatment. The workshop brought together approximately 35 scientists from academia and industry, scientific and technical experts from BWC delegations, and members of stakeholder communities interested in BWC issues. The workshop did not attempt to arrive at consensus conclusions, although several points were made by multiple participants, including the caution that novel approaches to alter host and pathogen responses are possible but enormously complex. The methods discussed present interesting opportunities, and would likely be used as additional lines of defense in concert with traditional therapeutics. The presentations at the workshop also raised the point that lines of research may have unexpected positive, as well as potential negative results for other fields of study. As a result, many participants highlighted the need for continuing communication between scientists and policy-makers and for members of the scientific community to be aware of how they present the findings and implications of their work. An open question of significant interest remains the issue of how to evaluate the risks and benefits of certain areas of research and the control of resulting information: who should determine whether the research is conducted, how the results are distributed, and based on what criteria?

Microorganisms, 2015

Select Agents are microorganisms and toxins considered to be exploitable as biological weapons. A... more Select Agents are microorganisms and toxins considered to be exploitable as biological weapons. Although infections by many Select Agents can be treated by conventional antibiotics, the risk of an emerging or engineered drug resistant strain is of great concern. One group of microorganisms that is showing potential to control drug resistant Gram-negative bacteria are the predatory bacteria from the genera Bdellovibrio spp. and Micavibrio spp. In this study, we have examined the ability of Bdellovibrio bacteriovorus (B. bacteriovorus) strain 109J, HD100 and Micavibrio aeruginosavorus (M. aeruginosavorus) ARL-13 to prey on a variety of Select Agents. Our findings demonstrate that B. bacteriovorus and M. aeruginosavorus are able to prey efficiently on Yersinia pestis and Burkholderia mallei. Modest predation was also measured in co-cultures of B. bacteriovorus and Francisella tularensis. However, neither of the predators showed predation when Burkholderia pseudomallei and Brucella melitensis were used as prey.

Scientific reports, Jan 7, 2015

Bdellovibrio spp. and Micavibrio spp. are Gram-negative predators that feed on other Gram-negativ... more Bdellovibrio spp. and Micavibrio spp. are Gram-negative predators that feed on other Gram-negative bacteria, making predatory bacteria potential alternatives to antibiotics for treating multi-drug resistant infections. While the ability of predatory bacteria to control bacterial infections in vitro is well documented, the in vivo effect of predators on a living host has yet to be extensively examined. In this study, respiratory and intravenous inoculations were used to determine the effects of predatory bacteria in mice. We found no reduction in mouse viability after intranasal or intravenous inoculation of B. bacteriovorus 109J, HD100 or M. aeruginosavorus. Introducing predators into the respiratory tract of mice provoked a modest inflammatory response at 1 hour post-exposure, but was not sustained at 24 hours, as measured by RT-qPCR and ELISA. Intravenous injection caused an increase of IL-6 in the kidney and spleen, TNF in the liver and CXCL-1/KC in the blood at 3 hours post-expo...

PLOS ONE, 2015

Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pa... more Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

eLife, Jan 30, 2014

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipept... more We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center 'bridge-helix cap' comprising the 'bridge-helix N-terminal hinge', 'F-loop', and…

Proceedings of the National Academy of Sciences, 1997

One-third of humans are infected with Mycobacterium tuberculosis , the causative agent of tubercu... more One-third of humans are infected with Mycobacterium tuberculosis , the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis , the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS 6110 subtypes of t...

Molecular Cell, 2005

Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 Å resolu... more Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 Å resolution structure of the Thermus thermophilus RNAP-Stl complex showed that, in full agreement with the available genetic data, the inhibitor binding site is located 20 Å away from the RNAP active site and encompasses the bridge helix and the trigger loop, two elements that are considered to be crucial for RNAP catalytic center function. Structure-based biochemical experiments revealed additional determinants of Stl binding and demonstrated that Stl does not affect NTP substrate binding, DNA translocation, and phosphodiester bond formation. The RNAP-Stl complex structure, its comparison with the closely related substrate bound eukaryotic transcription elongation complexes, and biochemical analysis suggest an inhibitory mechanism in which Stl stabilizes catalytically inactive (preinsertion) substrate bound transcription intermediate, thereby blocking structural isomerization of RNAP to an active configuration. The results provide a basis for a design of new antibiotics utilizing the Stl-like mechanism.

Journal of Microbiological Methods, 2007

Bacterial 16S ribosomal RNA (rRNA) genes contain nine "hypervariable regions" (V1-V9) that demons... more Bacterial 16S ribosomal RNA (rRNA) genes contain nine "hypervariable regions" (V1-V9) that demonstrate considerable sequence diversity among different bacteria. Species-specific sequences within a given hypervariable region constitute useful targets for diagnostic assays and other scientific investigations. No single region can differentiate among all bacteria; therefore, systematic studies that compare the relative advantage of each region for specific diagnostic goals are needed. We characterized V1-V8 in 110 different bacterial species including common blood borne pathogens, CDC-defined select agents and environmental microflora. Sequence similarity dendrograms were created for hypervariable regions V1-V8, and for selected combinations of regions or short segments within individual hypervariable regions that might be appropriate for DNA probing and real-time PCR. We determined that V1 best differentiated among Staphylococcus aureus and coagulase negative Staphylococcus sp. V2 and V3 were most suitable for distinguishing all bacterial species to the genus level except for closely related enterobacteriaceae. V2 best distinguished among Mycobacterial species and V3 among Haemophilus species. The 58 nucleotides-long V6 could distinguish among most bacterial species except enterobacteriaceae. V6 was also noteworthy for being able to differentiate among all CDC-defined select agents including Bacillus anthracis, which differed from B. cereus by a single polymorphism. V4, V5, V7 and V8 were less useful targets for genus or species-specific probes. The hypervariable sequence-specific dendrograms and the "MEGALIGN" files provided online will be highly useful tools for designing specific probes and primers for molecular assays to detect pathogenic bacteria, including select agents.

Journal of Cellular and Molecular Medicine, 2008

Bacillus anthracis (B. anthracis) is a virulent spore-producing bacterium. The need to protect th... more Bacillus anthracis (B. anthracis) is a virulent spore-producing bacterium. The need to protect the civilian population was heightened by the anthrax mail attacks of 2001. The threat of bioterrorism and the potential use of biological weapons against both military and civilian populations have become a reality. Although immediate treatment could clear early anthrax infection, the subtle effects of an infection on the emerging immune system has not been examined [1]. The use of B. anthracis as a lethal biological weapon has provoked renewed research interest. Although there is a rapid movement to develop effective vaccines against B. anthracis, there is no effective vaccine [2]. The promise of an effective vaccine and host response rely on a functional emerging immune system, which encompass the early period of immune cell development up to the generation of mature immune cells. The complexity of anthrax toxins and the presence of its receptors in different cells provide challenges for vaccine development [3]. This study addresses one area of the immune system, the early developmental process, namely haematopoietic system. B. anthracis releases three monomeric proteins, lethal factor (LF), edema factor (EF) and protective antigen (PA) [4, 5]. PA facilitates the biological activities of LF and EF by forming lethal toxin (LT) and edema toxin (ET), respectively [5-8]. Once intracellular, PA is disassociated to release LT and ET where they mediate intracellular responses with pathophysiological effects, including septicaemia [9, 10]. The anthrax vaccine immunization program, which was initially developed in the 1970s, uses a PA-based cell-free subunit, also known as 'anthrax vaccine absorbed' (AVA) [11]. The AVA method requires multiple subcutaneous injections, mostly every 2 months up to 18 months, followed by annual boosts [12, 13]. The vaccine

Journal of Bacteriology, 2000

Genes encoding l -arginine biosynthetic and transport proteins have been shown in a number of pat... more Genes encoding l -arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter from Mycobacterium bovis BCG and the effects of its deletion on l -arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to the rocE gene of Bacillus subtilis , a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using 14 C-labeled substrates. Greatly reduced uptake of l -arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook ...

Journal of Bacteriology, 2006

ABSTRACTGlutathione is a tripeptide and antioxidant, synthesized at high levels by cells during t... more ABSTRACTGlutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form ofS-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione andS-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione andS-nitrosoglutathione is brought about by the action of the enzyme γ-glutamyl transpeptidase. The function of γ-glutamyl transpeptidase is to cleave glutathione andS-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain ofMycobacterium ...