Publications by authors named "Kimberly R Marshall-Batty"

12 Publications

  • Page 1 of 1

Antisense Inhibitors Retain Activity in Pulmonary Models of Burkholderia Infection.

ACS Infect Dis 2018 05 1;4(5):806-814. Epub 2018 Mar 1.

Department of Internal Medicine , UT Southwestern , 5323 Harry Hines Blvd , Dallas , Texas 75390 , United States.

The Burkholderia cepacia complex is a group of Gram-negative bacteria that are opportunistic pathogens in immunocompromised individuals, such as those with cystic fibrosis (CF) or chronic granulomatous disease (CGD). Burkholderia are intrinsically resistant to many antibiotics and the lack of antibiotic development necessitates novel therapeutics. Peptide-conjugated phosphorodiamidate morpholino oligomers are antisense molecules that inhibit bacterial mRNA translation. Targeting of PPMOs to the gene acpP, which is essential for membrane synthesis, lead to defects in the membrane and ultimately bactericidal activity. Exploration of additional PPMO sequences identified the ATG and Shine-Dalgarno sites as the most efficacious for targeting acpP. The CF lung is a complex microenvironment, but PPMO inhibition was still efficacious in an artificial model of CF sputum. PPMOs had low toxicity in human CF cells at doses that were antibacterial. PPMOs also reduced the bacterial burden in the lungs of immunocompromised CyBB mice, a model of CGD. Finally, the use of multiple PPMOs was efficacious in inhibiting the growth of both Burkholderia and Pseudomonas in an in vitro model of coinfection. Due to the intrinsic resistance of Burkholderia to traditional antibiotics, PPMOs represent a novel and viable approach to the treatment of Burkholderia infections.
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http://dx.doi.org/10.1021/acsinfecdis.7b00235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669075PMC
May 2018

Antimicrobial Activity of Ibuprofen against Cystic Fibrosis-Associated Gram-Negative Pathogens.

Antimicrob Agents Chemother 2018 03 23;62(3). Epub 2018 Feb 23.

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USA

Clinical trials have demonstrated the benefits of ibuprofen therapy in cystic fibrosis (CF) patients, an effect that is currently attributed to ibuprofen's anti-inflammatory properties. Yet, a few previous reports demonstrated an antimicrobial activity of ibuprofen as well, although none investigated its direct effects on the pathogens found in the CF lung, which is the focus of this work. Determination of ibuprofen's antimicrobial activity against and species strains through measurements of the endpoint number of CFU and growth kinetics showed that ibuprofen reduced the growth rate and bacterial burden of the tested strains in a dose-dependent fashion. In an biofilm model, a reduction in the rate of biomass accumulation over 8 h of growth with ibuprofen treatment was observed. Next, an acute pneumonia model was used to test this antimicrobial activity after the oral delivery of ibuprofen. Following intranasal inoculation, ibuprofen-treated mice exhibited lower CFU counts and improved survival compared with the control animals. Preliminary biodistribution studies performed after the delivery of ibuprofen to mice by aerosol demonstrated a rapid accumulation of ibuprofen in serum and minimum retention in lung tissue and bronchoalveolar lavage fluid. Therefore, ibuprofen-encapsulated polymeric nanoparticles (Ibu-NPs) were formulated to improve the pharmacokinetic profile. Ibu-NPs formulated for aerosol delivery inhibited the growth of and may provide a convenient dosing method. These results provide an additional explanation for the previously observed therapeutic effects of ibuprofen in CF patients and further strengthen the argument for its use by these patients.
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http://dx.doi.org/10.1128/AAC.01574-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826130PMC
March 2018

Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers.

Antimicrob Agents Chemother 2017 04 24;61(4). Epub 2017 Mar 24.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

is a highly virulent, multidrug-resistant pathogen that causes significant morbidity and mortality in hospitalized patients and is particularly devastating in patients with cystic fibrosis. Increasing antibiotic resistance coupled with decreasing numbers of antibiotics in the developmental pipeline demands novel antibacterial approaches. Here, we tested peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which inhibit translation of complementary mRNA from specific, essential genes in PPMOs targeted to , , and , inhibited growth in many clinical strains and activity of PPMOs could be enhanced 2- to 8-fold by the addition of polymyxin B nonapeptide at subinhibitory concentrations. The PPMO targeting was also effective at preventing PAO1 biofilm formation and at reducing existing biofilms. Importantly, treatment with various combinations of a PPMO and a traditional antibiotic demonstrated synergistic growth inhibition, the most effective of which was the PPMO targeting with tobramycin. Furthermore, treatment of PA103-infected mice with PPMOs targeting , , or significantly reduced the bacterial burden in the lungs at 24 h by almost 3 logs. Altogether, this study demonstrates that PPMOs targeting the essential genes , , or in are highly effective at inhibiting growth and These data suggest that PPMOs alone or in combination with antibiotics represent a novel approach to addressing the problems associated with rapidly increasing antibiotic resistance in .
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http://dx.doi.org/10.1128/AAC.01938-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365667PMC
April 2017

Simultaneous Host-Pathogen Transcriptome Analysis during Granulibacter bethesdensis Infection of Neutrophils from Healthy Subjects and Patients with Chronic Granulomatous Disease.

Infect Immun 2015 Nov 17;83(11):4277-92. Epub 2015 Aug 17.

Laboratory of Host Defenses, NIAID, NIH, Bethesda, Maryland, USA

Polymorphonuclear leukocytes (PMN) from patients with chronic granulomatous disease (CGD) fail to produce microbicidal concentrations of reactive oxygen species (ROS) due to mutations in NOX2. Patients with CGD suffer from severe, life-threatening infections and inflammatory complications. Granulibacter bethesdensis is an emerging Gram-negative pathogen in CGD that resists killing by PMN of CGD patients (CGD PMN) and inhibits PMN apoptosis through unknown mechanisms. Microarray analysis was used to study mRNA expression in PMN from healthy subjects (normal PMN) and CGD PMN during incubation with G. bethesdensis and, simultaneously, in G. bethesdensis with normal and CGD PMN. We detected upregulation of antiapoptotic genes (e.g., XIAP and GADD45B) and downregulation of proapoptotic genes (e.g., CASP8 and APAF1) in infected PMN. Transcript and protein levels of inflammation- and immunity-related genes were also altered. Upon interaction with PMN, G. bethesdensis altered the expression of ROS resistance genes in the presence of normal but not CGD PMN. Levels of bacterial stress response genes, including the ClpB gene, increased during phagocytosis by both normal and CGD PMN demonstrating responses to oxygen-independent PMN antimicrobial systems. Antisense knockdown demonstrated that ClpB is dispensable for extracellular growth but is essential for bacterial resistance to both normal and CGD PMN. Metabolic adaptation of Granulibacter growth in PMN included the upregulation of pyruvate dehydrogenase. Pharmacological inhibition of pyruvate dehydrogenase by triphenylbismuthdichloride was lethal to Granulibacter. This study expands knowledge of microbial pathogenesis of Granulibacter in cells from permissive (CGD) and nonpermissive (normal) hosts and identifies potentially druggable microbial factors, such as pyruvate dehydrogenase and ClpB, to help combat this antibiotic-resistant pathogen.
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http://dx.doi.org/10.1128/IAI.00778-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598409PMC
November 2015

Serologic reactivity to the emerging pathogen Granulibacter bethesdensis.

J Infect Dis 2012 Sep 10;206(6):943-51. Epub 2012 Jul 10.

Laboratory of Clinical Infectious Diseases, Bethesda, MD, USA.

Background: Granulibacter bethesdensis is a recently described member of the Acetobacteraceae family that has been isolated from patients with chronic granulomatous disease (CGD). Its pathogenesis, environmental reservoir(s), and incidence of infection among CGD patients and the general population are unknown.

Methods: Detected antigens were identified by mass spectroscopy after 2-dimensional electrophoresis and immunoaffinity chromatography. The prevalence of Granulibacter immunoreactivity was assessed through immunoblotting and enzyme-linked immunosorbent assay (ELISA).

Results: Methanol dehydrogenase (MDH) and formaldehyde-activating enzyme were recognized during analysis of sera from infected patients. Unique patterns of immunoreactive bands were identified in Granulibacter extracts, compared with extracts of other Acetobacteraceae species. By use of criteria based on these specific bands, specimens from 79 of 175 CGD patients (45.1%) and 23 of 93 healthy donors (24.7%) reacted to all 11 bands. An ELISA that used native MDH to capture and detect immunoglobulin G was developed and revealed high-titer MDH seroreactivity in culture-confirmed cases and 5 additional CGD patients. Testing of samples collected prior to culture-confirmed infection demonstrated instances of recent seroconversion, as well as sustained seropositivity. Infection of CGD mice with G. bethesdensis confirmed acquisition of high-titer antibody-recognizing MDH.

Conclusions: These serologic tests suggest that Granulibacter immunoreactivity is more common among CGD patients and, perhaps, among healthy donors than was previously suspected. This finding raises the possibility that clinical presentations of Granulibacter infection may be underappreciated.
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http://dx.doi.org/10.1093/infdis/jis431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501152PMC
September 2012

Innate immunity against Granulibacter bethesdensis, an emerging gram-negative bacterial pathogen.

Infect Immun 2012 Mar 19;80(3):975-81. Epub 2011 Dec 19.

Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H(2)O(2) but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O(2)-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.
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http://dx.doi.org/10.1128/IAI.05557-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294668PMC
March 2012

Recurrent Granulibacter bethesdensis infections and chronic granulomatous disease.

Emerg Infect Dis 2010 Sep;16(9):1341-8

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1684, USA.

Chronic granulomatous disease (CGD) is characterized by frequent infections, most of which are curable. Granulibacter bethesdensis is an emerging pathogen in patients with CGD that causes fever and necrotizing lymphadenitis. However, unlike typical CGD organisms, this organism can cause relapse after clinical quiescence. To better define whether infections were newly acquired or recrudesced, we use comparative bacterial genomic hybridization to characterize 11 isolates obtained from 5 patients with CGD from North and Central America. Genomic typing showed that 3 patients had recurrent infection months to years after apparent clinical cure. Two patients were infected with the same strain as previously isolated, and 1 was infected with a genetically distinct strain. This organism is multidrug resistant, and therapy required surgery and combination antimicrobial drugs, including long-term ceftriaxone. G. bethesdensis causes necrotizing lymphadenitis in CGD, which may recur or relapse.
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http://dx.doi.org/10.3201/eid1609.091800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294967PMC
September 2010

Antisense phosphorodiamidate morpholino oligomers targeted to an essential gene inhibit Burkholderia cepacia complex.

J Infect Dis 2010 Jun;201(12):1822-30

Immunopathogenesis Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, Office of Research Services, National Institutes of Health, Bethesda, Maryland, USA.

Background: Members of the Burkholderia cepacia complex (Bcc) cause considerable morbidity and mortality in patients with chronic granulomatous disease and cystic fibrosis. Many Bcc strains are antibiotic resistant, which requires the exploration of novel antimicrobial approaches, including antisense technologies such as phosphorodiamidate morpholino oligomers (PMOs).

Methods: Peptide-conjugated PMOs (PPMOs) were developed to target acpP, which encodes an acyl carrier protein (AcpP) that is thought to be essential for growth. Their antimicrobial activities were tested against different strains of Bcc in vitro and in infection models.

Results: PPMOs targeting acpP were bactericidal against clinical isolates of Bcc (>4 log reduction), whereas a PPMO with a scrambled base sequence (scrambled PPMO) had no effect on growth. Human neutrophils were infected with Burkholderia multivorans and treated with AcpP PPMO. AcpP PPMO augmented killing, compared with neutrophils alone and compared with neutrophils alone plus scrambled PPMO. Mice with chronic granulomatous disease that were infected with B. multivorans were treated with AcpP PPMO, scrambled PPMO, or water at 0, 3, and 6 h after infection. Compared with water-treated control mice, the AcpP PPMO-treated mice showed an approximately 80% reduction in the risk of dying by day 30 of the experiment and relatively little pathology.

Conclusion: AcpP PPMO is active against Bcc infections in vitro and in vivo.
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http://dx.doi.org/10.1086/652807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872041PMC
June 2010

Activation of a dormant ClpX recognition motif of bacteriophage Mu repressor by inducing high local flexibility.

J Biol Chem 2008 Apr 28;283(14):9060-70. Epub 2008 Jan 28.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, D. C. 20057, USA.

The C-terminal domain (CTD) of bacteriophage Mu immunity repressor (Rep) regulates DNA binding by the N-terminal domain and degradation by ClpXP protease. Five residues at the Rep C terminus (CTD5) can serve as a ClpX recognition motif, but it is dormant unless activated, a state that can be induced by the presence of dominant-negative mutant repressors (Vir). Conversion of Rep to ClpXP-sensitive form was associated with not only increased exposure of CTD5 to solvent but also increased CTD motion or flexibility as measured by fluorescence anisotropy. CTD mutations (V183S, K193S, and V196S) promoting ClpXP resistance without destroying the recognition motif prevented increased CTD motion induced by Vir. Suppression of ClpXP protease resistance conferred by the V196S mutation also correlated with restoration of CTD motion. The temperature-sensitive R47Q mutation present in cis within the DNA-binding domain restored ClpXP protease sensitivity to the V196S mutant, and anisotropy analysis indicated that R47Q allows the V196S CTD to gain increased flexibility when Vir was present. The results indicate that the CTD functions to turn the recognition motif on and off, most likely by modulating flexibility of the domain that harbors the ClpX recognition motif, suggesting a general mechanism by which proteins can regulate their own degradation.
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http://dx.doi.org/10.1074/jbc.M705508200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2431027PMC
April 2008

Trans-targeting of protease substrates by conformationally activating a regulable ClpX-recognition motif.

Mol Microbiol 2008 Feb 7;67(4):920-33. Epub 2008 Jan 7.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Rm. 331 Basic Science Bldg., 3900 Reservoir Rd. NW, Washington, DC 20057-1455, USA.

Conversion of bacteriophage Mu repressor to ClpXP-sensitive form correlates with induced local flexibility at the ClpX recognition motif located at the C-terminal end. Changing the C-terminal valine to an alanine (RepV196A) caused the degradation tag to be constitutively active like that of mutant repressors called Vir, which have a dominant ClpXP-sensitive conformation. However, unlike Vir, RepV196A was unable to convert wild-type repressor (Rep) to the ClpXP-sensitive form. In mixtures with Rep, only RepV196A was rapidly degraded by ClpXP. Unlike Rep, RepV196A was ClpXP sensitive without induced C-terminal flexibility. And unlike adaptor proteins that tether and deliver substrates to ClpX for trans-targeting, Vir promoted rapid degradation of Rep by ClpX deleted for the tethering site that binds adaptor proteins. Therefore, Rep's ClpX recognition motif has regulable properties, allowing an alternative trans-targeting mechanism in which an inactive degradation tag is turned on by induced conformational changes.
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http://dx.doi.org/10.1111/j.1365-2958.2007.06099.xDOI Listing
February 2008

Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C-terminal domain.

Mol Microbiol 2003 Jan;47(1):171-82

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Rm. 331, Basic Science Bldg., 3900 Reservoir Road NW, Washington, DC 20057, USA.

Rapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease-hypersensitive repressors (Vir) with an altered C-terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild-type repressor CTD modulate Vir-induced degradation by Escherichia coli ClpXP protease and DNA binding by the N-terminal DNA-binding domain (DBD). Although deletions of the repressor C-terminus resulted in both resistance to ClpXP protease and suppression of a temperature-sensitive DBD mutation (cts62), some cysteine-replacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild-type repressor, and at least two mutant repressors protected Vir from proteolysis. One protease-resistant mutant became susceptible to Vir-induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease-sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.
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http://dx.doi.org/10.1046/j.1365-2958.2003.03286.xDOI Listing
January 2003

Trans-targeting of the phage Mu repressor is promoted by conformational changes that expose its ClpX recognition determinant.

J Biol Chem 2003 Jan 6;278(3):1612-7. Epub 2002 Nov 6.

Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20057, USA.

Dominant negative forms of the phage Mu repressor, including the mutant Vir repressors, are not only rapidly degraded by the ClpXP protease but also promote degradation of the unmodified, wild-type repressor. This trans-targeting of the wild-type repressor depends upon a determinant within its C-terminal domain, which is needed for recognition by ClpX. An environmentally sensitive fluorescent probe (2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS)) attached to the C terminus of the full-length repressor indicated that Vir induces the movement of this domain into a more exposed configuration. Vir also promoted attachment of MIANS to the C terminus of the repressor at an accelerated rate, and it greatly increased the rate of phosphorylation of a cAMP-dependent protein kinase motif attached to the repressor C terminus. While an excess of Vir was needed to promote repressor phosphorylation at maximal rates, the presence of ClpX could increase phosphorylation rates at lower Vir levels. trans-Targeting of the Mu repressor is therefore promoted by exposing its ClpX recognition determinant, and the action of ClpX can assist Vir in exposing these determinants.
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http://dx.doi.org/10.1074/jbc.M209352200DOI Listing
January 2003