Publications by authors named "Mohamad Hamad"

21 Publications

  • Page 1 of 1

Camptothecin's journey from discovery to WHO Essential Medicine: Fifty years of promise.

Eur J Med Chem 2021 Jun 17;223:113639. Epub 2021 Jun 17.

College of Pharmacy, University of Sharjah, 27272, Sharjah, United Arab Emirates; Sharjah Institute for Medical Research, 27272, Sharjah, United Arab Emirates. Electronic address:

Nature represents a rich source of compounds used for the treatment of many diseases. Camptothecin (CPT), isolated from the bark of Camptotheca acuminata, is a cytotoxic alkaloid that attenuates cancer cell replication by inhibiting DNA topoisomerase 1. Despite its promising and wide spectrum antiproliferative activity, its use is limited due to low solubility, instability, acquired tumour cell resistance, and remarkable toxicity. This has led to the development of numerous CPT analogues with improved pharmacodynamic and pharmacokinetic profiles. Three natural product-inspired drugs, namely, topotecan, irinotecan, and belotecan, are clinically approved and prescribed drugs for the treatment of several types of cancer, whereas other derivatives are in clinical trials. In this review, which covers literature from 2015 to 2020, we aim to provide a comprehensive overview and describe efforts that led to the development of a variety of CPT analogues. These efforts have led to the discovery of potent, first-in-class chemotherapeutic agents inspired by CPT. In addition, the mechanism of action, SAR studies, and recent advances of novel CPT drug delivery systems and antibody drug conjugates are discussed.
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http://dx.doi.org/10.1016/j.ejmech.2021.113639DOI Listing
June 2021

Systematic Review and Meta-analysis of Herpes Zoster Vaccine in Patients With CKD.

Kidney Int Rep 2021 May 3;6(5):1254-1264. Epub 2021 Mar 3.

Department of Nephrology, Wollongong Hospital, Wollongong, New South Wales, Australia.

Introduction: Chronic kidney disease (CKD) is a risk factor for herpes zoster (HZ) infection. Few studies have examined HZ vaccine (HZV) in this population. We conducted a systematic review and meta-analysis investigating the efficacy and safety of HZV in patients with renal disease (CKD, dialysis, and transplant).

Methods: MEDLINE, Embase, and Cochrane Central Register of Controlled Trials (CENTRAL) databases (up to May 2020) were searched for randomized controlled trials and nonrandomized controlled studies evaluating HZV in patients with CKD for effectiveness and adverse event risks. Studies without a control group (placebo or no vaccine) were excluded. Extraction of prespecified data and risk of bias assessments using the Newcastle-Ottawa scale for cohort studies and the Cochrane Risk of Bias Tool for randomized controlled trials were done by 3 authors. Random-effects meta-analysis was used to generate pooled treatment effects and 95% confidence intervals.

Results: Included were 404,561 individuals from 8 studies (3 randomized controlled trials and 5 nonrandomized). All 8 studies examined HZ as an outcome, with 3 reporting adverse events. Risk of HZ was lower in patients who received HZV compared with controls (hazard ratio, 0.55; 95% confidence interval, 0.37-0.82;  < 0.01); however, heterogeneity was high (  = 88%,  < 0.01). There was no significant difference in adverse events associated with HZV (hazard ratio, 1.03; 95% confidence interval, 0.54-1.28;  = 0.8).

Conclusions: HZV compared with control significantly lowers the risk of HZ without an increase in adverse events in CKD patients. However, significant heterogeneity was present. HZV should be actively considered in CKD patients because the prevalence of HZ is higher in this population.
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http://dx.doi.org/10.1016/j.ekir.2021.02.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116755PMC
May 2021

Drug development post COVID-19 pandemic: toward a better system to meet current and future global health challenges.

Expert Opin Drug Discov 2021 04 28;16(4):365-371. Epub 2020 Dec 28.

Director, Research Institute for Medical & Health Sciences, University of Sharjah, Sharjah, United Arab Emirates.

: Despite advances in drug research and development, our knowledge of the underlying molecular mechanisms of many diseases remains inadequate. This have led to limited effective medicines for several diseases. To address these challenges, efficient strategies, novel technologies, and policies are urgently needed. The main obstacles in drug discovery and development are the mounting cost, risk, and time frame needed to develop new medicines. Fair pricing and accessibility is another unmet global challenge.: Here, the authors cover the pace, risks, cost, and challenges facing drug development processes. Additionally, they introduce disease-associated data which demand global attention and propose solutions to overcome these challenges.: The massive challenges encountered during drug development urgently call for a serious global rethinking of the way this process is done. A partial solution might be if many consortiums of multi-nations, academic institutions, clinicians, pharma companies, and funding agencies gather at different fronts to crowdsource resources, share knowledge and risks. Such an ecosystem can rapidly generate first-in-class molecules that are safe, effective, and affordable. We think that this article represents a wake-up call for the scientific community to immediately reassess the current drug discovery and development procedures.
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http://dx.doi.org/10.1080/17460441.2021.1854221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784828PMC
April 2021

Divergent Strategy for Diastereocontrolled Synthesis of Small- and Medium-Ring Architectures.

J Org Chem 2020 08 10;85(16):10695-10708. Epub 2020 Aug 10.

Sharjah Institute for Medical Research, University of Sharjah, P.O. Box 27272, Sharjah, UAE.

Nitrogen and oxygen medium rings, in particular nine-membered rings, epitomize a unique area of chemical space that occurs in many natural products and biologically appealing compounds. The scarcity of 8- to 12-membered rings among clinically approved drugs is indicative of the difficulties associated with their synthesis, principally owing to the unfavorable entropy and transannular strain. We report here a scandium triflate-catalyzed reaction that allows for a modular access to a diverse collection of nine-membered ring heterocycles in a one-pot cascade and with complete diastereocontrol. This cascade features an intramolecular addition of an acyl group-derived enol to a α,β-unsaturated carbonyl moiety, leading to N- and O-derived medium-ring systems. Computational studies using the density functional theory support the proposed mechanism. Additionally, a one-pot cascade leading to hexacyclic chromeno[3',4':2,3]indolizino[8,7-]indole architectures, with six fused rings and four contiguous chiral centers, is reported. This novel cascade features many concerted events, including the formation of two azomethine ylides, [3 + 2]-cycloaddition, 1,3-sigmatropic rearrangement, Michael addition, and Pictet-Spengler reaction among others. Phenotypic screening of the resulting oxazonine collection identified chemical probes that regulate mitochondrial membrane potential, adenosine 5'-triphosphate contents, and reactive oxygen species levels in hepatoma cells (Hepa1-6), a promising approach for targeting cancer and metabolic disorders.
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http://dx.doi.org/10.1021/acs.joc.0c01244DOI Listing
August 2020

Superbugs but no drugs: steps in averting a post-antibiotic era.

Drug Discov Today 2019 12 16;24(12):2225-2228. Epub 2019 Aug 16.

Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates; College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates.

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http://dx.doi.org/10.1016/j.drudis.2019.08.004DOI Listing
December 2019

Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O-antigen ligases share similar membrane topology and biochemical properties.

Mol Microbiol 2018 10 3;110(1):95-113. Epub 2018 Oct 3.

Department of Microbiology and Immunology, University of Western Ontario, London, N6A 5C1, Canada.

WaaL is an inner membrane glycosyltransferase that catalyzes the transfer of O-antigen polysaccharide from its lipid-linked intermediate to a terminal sugar of the lipid A-core oligosaccharide, a conserved step in lipopolysaccharide biosynthesis. Ligation occurs at the periplasmic side of the bacterial cell membrane, suggesting the catalytic region of WaaL faces the periplasm. Establishing the membrane topology of the WaaL protein family will enable understanding its mechanism and exploit it as a potential antimicrobial target. Applying oxidative labeling of native methionine/cysteine residues, we previously validated a topological model for Escherichia coli WaaL, which differs substantially from the reported topology of the Pseudomonas aeruginosa WaaL, derived from the analysis of truncated protein reporter fusions. Here, we examined the topology of intact E. coli and P. aeruginosa WaaL proteins by labeling engineered cysteine residues with the membrane-impermeable sulfhydryl reagent polyethylene glycol maleimide (PEG-Mal). The accessibility of PEG-Mal to targeted engineered cysteine residues in both E. coli and P. aeruginosa WaaL proteins demonstrates that both ligases share similar membrane topology. Further, we also demonstrate that P. aeruginosa WaaL shares similar functional properties with E. coli WaaL and that E. coli WaaL may adopt a functional dimer conformation.
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http://dx.doi.org/10.1111/mmi.14085DOI Listing
October 2018

The Schizophrenia Coping Oral Health Profile. Development and Feasibility.

Transl Neurosci 2018 20;9:78-87. Epub 2018 Jun 20.

Clinical Research Unit, La Chartreuse Psychiatric Centre, 21033 Dijon, France.

Background: The aim of this work was to present the creation of appropriate tools to evaluate the coping strategies in Oral-Health-related Quality of Life (OHrQOL) implemented by persons with schizophrenia (PWS), the Schizophrenia Coping Oral Health Profile (SCOOHP), and the results of a feasibility study.

Methods: A qualitative investigation was conducted between June 2016 and May 2017.The first step included 26 semi-structured individual interviews, 20 with PWS and 6 with health professionals (HPs), and 2 focus groups (PWS and HPs) to explore the experiences of the participants and how they felt about coping strategies in OHrQOL. The second step was a feasibility study involving a statistical analysis to test the acceptability and internal consistency (Cronbach's α) of the SCOOHP.

Results: The analysis of these interviews allowed for us to obtain 277 items from 3545 verbatim transcriptions related to various dimensions of OHrQOL. We presented the items selected in coping concepts in this study. After selecting items in several stages, we drew up the SCOOHP scale with 23 items (15 items for positive coping and 8 items for negative coping). The feasibility study showed good acceptability, good understanding of the items and good consistency reliability (α = 0.59).

Conclusions: This is the first study that has enabled us to draw up a specific tool to assess coping strategies in OHrQOL of PWS. A multicentre study involving a larger sample of PWS is underway in order to perform the psychometric validation of the SCOOHP.

Trial Registration: Clinical Trials Gov NCT02730832. Date registered: 21 March 2016.
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http://dx.doi.org/10.1515/tnsci-2018-0014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6024692PMC
June 2018

A Rasch Analysis between Schizophrenic Patients and the General Population.

Transl Neurosci 2017 28;8:139-146. Epub 2017 Oct 28.

UMR 1246 INSERM - SPHERE "methodS in Patient-centered outcomes and HEalth ResEarch", Nantes, France.

Background: The aim of this study was to test the General Oral Health Assessment Index Questionnaire (GOHAI) items for differential item functioning (DIF) according to demographic characteristics (gender, age) and mental health status (schizophrenic disorders versus general population) using Rasch analysis.

Methods: This is a cross-sectional study using aggregated baseline data from a validation study of the GOHAI in the French general population (GP) and similar validation study in persons with schizophrenia (PWS). DIF was tested using the Partial Credit Model. DIF were estimated in different groups of patients.

Results: The cohort comprised 363 persons: 65% were female, 83% were 25-45 years old and 30% were PWS.Five of the 12 items exhibited DIF. DIF effects were observed with schizophrenia for 3 items, with age for 3 items and with gender for 1 item. The variable "age" gave a significant explanation of the latent variable: the latent variable decreased with age (-0.40±0.08-p<0.001 for each increase of the age of 10 years). This decrease represented an effect size of 0.27 which can be qualified of a small to medium effect. The status of the individuals (GP versus PWS) and the gender did not significantly explain differences in the values of the latent variable.

Conclusion: The GOHAI scores may not be comparable across sub-groups defined by health status, age and gender without accounting for DIF. In the future, other studies should explore this way with other Oral Health related Quality of Life assessment tools and populations with mental illness.
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http://dx.doi.org/10.1515/tnsci-2017-0020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662753PMC
October 2017

Psychometric characteristics of the "General Oral Health Assessment Index (GOHAI) » in a French representative sample of patients with schizophrenia.

BMC Oral Health 2017 Apr 11;17(1):75. Epub 2017 Apr 11.

EA 481 Integrative Neurosciences and Clinical, University Hospital of Besançon, F-25000, Besançon, France.

Background: The "General Oral Health Assessment Index" (GOHAI) was widely used in clinical or epidemiological studies worldwide, as it was available for use in different languages. Therefore, the aim of this study was to evaluate the psychometric characteristics of the GOHAI in a representative sample of patients with schizophrenia.

Methods: A total of 90 schizophrenic patients (in-patients and out-patients) were recruited from the participants of the "buccodor study" (NCT02167724) between March and September 2015. They were selected using a random stratified sampling method according to their age, sex, or residential area (urban/rural area). GOHAI validity (construct, predictive, concurrent and known group validity) and internal consistency (reliability) were tested. Test-retest reliability was evaluated in 32 subjects.

Results: The mean age was 47.34 (SD = 12.17). Internal consistency indicated excellent agreement, with a Cronbach's α value of 0.82 and average inter-item correlation of 0.65. Intraclass correlation coefficients for test-retest reliability with 95% confidence intervals were not significantly different (p > 0.05). Construct validity was supported by three factor that accounted for 60.94% of the variance observed. Predictive validity was corroborated as statistically significant differences were observed between a high GOHAI score, which was associated with self-perceived satisfaction with oral health, lower age and high frequency of toothbrushing. Concurrent validity was corroborated as statistically significant relationships were observed between the GOHAI scores and most objective measures of dental status. For known group validity, they was no significant difference of the mean GOHAI score between out or in-patients (p > 0.05).

Conclusion: Acceptable psychometric characteristics of the GOHAI could help caregivers to develop ways to improve the Oral Health related Quality Of Life of schizophrenic patients.

Trial Registration: Clinical Trials Gov NCT02167724 . Date registered 17 June, 2014.
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http://dx.doi.org/10.1186/s12903-017-0368-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387256PMC
April 2017

The LpxL acyltransferase is required for normal growth and penta-acylation of lipid A in Burkholderia cenocepacia.

Mol Microbiol 2017 04 27;104(1):144-162. Epub 2017 Jan 27.

Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, BT9 7BL, UK.

Lipid A anchors the lipopolysaccharide (LPS) to the outer membrane and is usually composed of a hexa-acylated diglucosamine backbone. Burkholderia cenocepacia, an opportunistic pathogen, produces a mixture of tetra- and penta-acylated lipid A. "Late" acyltransferases add secondary acyl chains to lipid A after the incorporation of four primary acyl chains to the diglucosamine backbone. Here, we report that B. cenocepacia has only one late acyltransferase, LpxL (BCAL0508), which adds a myristoyl chain to the 2' position of lipid A resulting in penta-acylated lipid A. We also identified PagL (BCAL0788), which acts as an outer membrane lipase by removing the primary β-hydroxymyristate (3-OH-C14:0) chain at the 3 position, leading to tetra-acylated lipid A. Unlike PagL, LpxL depletion caused reduced cell growth and defects in cell morphology, both of which were suppressed by overexpressing the LPS flippase MsbA (BCAL2408), suggesting that lipid A molecules lacking the fifth acyl chain contributed by LpxL are not good substrates for the flippase. We also show that intracellular B. cenocepacia within macrophages produced more penta-acylated lipid A, suggesting lipid A penta-acylation in B. cenocepacia is required not only for bacterial growth and morphology but also for adaptation to intracellular lifestyle.
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http://dx.doi.org/10.1111/mmi.13618DOI Listing
April 2017

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia.

J Biol Chem 2015 Aug 9;290(35):21305-19. Epub 2015 Jul 9.

From the Departments of Chemical Sciences and

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.
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http://dx.doi.org/10.1074/jbc.M115.649087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571861PMC
August 2015

A markerless deletion method for genetic manipulation of Burkholderia cenocepacia and other multidrug-resistant gram-negative bacteria.

Methods Mol Biol 2014 ;1197:311-27

Department of Microbiology and Immunology, Centre for Human Immunology, University of Western Ontario, London, ON, Canada, N6A 5C1.

Genetic manipulation of multidrug-resistant bacteria is often difficult and hinders progress in understanding their physiology and pathogenesis. This book chapter highlights advances in genetic manipulation of Burkholderia cenocepacia, which are also applicable to other members of the Burkholderia cepacia complex and multidrug-resistant gram-negative bacteria of other genera. The method detailed here is based on the I-SceI homing endonuclease system, which can be efficiently used for chromosomal integration, deletion, and genetic replacement. This system creates markerless mutations and insertions without leaving a genetic scar and thus can be reused successively to generate multiple modifications in the same strain.
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http://dx.doi.org/10.1007/978-1-4939-1261-2_18DOI Listing
May 2015

Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid-producing bacteria.

Environ Microbiol 2015 Mar 24;17(3):735-50. Epub 2014 Jun 24.

Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada.

Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. Burkholderia species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in Burkholderia cenocepacia by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by hpnH and hpnG were essential for production of all C35 extended hopanoids, including bacteriohopanetetrol (BHT), BHT glucosamine and BHT cyclitol ether. Deletion of hpnI resulted in BHT production, while ΔhpnJ produced only BHT glucosamine. Thus, HpnI is required for BHT glucosamine production while HpnJ is responsible for its conversion to the cyclitol ether. The ΔhpnH and ΔhpnG mutants could not grow under any stress condition tested, whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild-type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid-producing bacteria.
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http://dx.doi.org/10.1111/1462-2920.12509DOI Listing
March 2015

Structural-functional studies of Burkholderia cenocepacia D-glycero-β-D-manno-heptose 7-phosphate kinase (HldA) and characterization of inhibitors with antibiotic adjuvant and antivirulence properties.

J Med Chem 2013 Feb 22;56(4):1405-17. Epub 2013 Jan 22.

Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada.

As an essential constituent of the outer membrane of Gram-negative bacteria, lipopolysaccharide contributes significantly to virulence and antibiotic resistance. The lipopolysaccharide biosynthetic pathway therefore serves as a promising therapeutic target for antivirulence drugs and antibiotic adjuvants. Here we report the structural-functional studies of D-glycero-β-D-manno-heptose 7-phosphate kinase (HldA), an absolutely conserved enzyme in this pathway, from Burkholderia cenocepacia. HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270. Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites. Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.
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http://dx.doi.org/10.1021/jm301483hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585733PMC
February 2013

The Burkholderia cenocepacia sensor kinase hybrid AtsR is a global regulator modulating quorum-sensing signalling.

Environ Microbiol 2013 Feb 26;15(2):372-85. Epub 2012 Jul 26.

Centre for Human Immunology, Department of Microbiology, University of Western Ontario, London, Ontario, Canada.

Burkholderia cenocepacia is commonly found in the environment and also as an important opportunistic pathogen infecting patients with cystic fibrosis. Successful infection by this bacterium requires coordinated expression of virulence factors, which is achieved through different quorum sensing (QS) regulatory systems. Biofilm formation and Type 6 secretion system (T6SS) expression in B. cenocepacia K56-2 are positively regulated by QS and negatively regulated by the sensor kinase hybrid AtsR. This study reveals that in addition to affecting biofilm and T6SS activity, the deletion of atsR in B. cenocepacia leads to overproduction of other QS-regulated virulence determinants including proteases and swarming motility. Expression of the QS genes, cepIR and cciIR, was upregulated in the ΔatsR mutant and resulted in early and increased N-acylhomoserine lactone (AHL) production, suggesting that AtsR plays a role in controlling the timing and fine-tuning of virulence gene expression by modulating QS signalling. Furthermore, a ΔatsRΔcepIΔcciI mutant could partially upregulate the same virulence determinants indicating that AtsR also modulates the expression of virulence genes by a second mechanism, independently of any AHL production. Together, our results strongly suggest that AtsR is a global virulence regulator in B. cenocepacia.
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http://dx.doi.org/10.1111/j.1462-2920.2012.02828.xDOI Listing
February 2013

Aminoarabinose is essential for lipopolysaccharide export and intrinsic antimicrobial peptide resistance in Burkholderia cenocepacia(†).

Mol Microbiol 2012 Sep 18;85(5):962-74. Epub 2012 Jul 18.

Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.

One common mechanism of resistance against antimicrobial peptides in Gram-negative bacteria is the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires L-Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for L-Ara4N synthesis and transfer to the LPS. The absence of L-Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi-protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that L-Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.
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http://dx.doi.org/10.1111/j.1365-2958.2012.08154.xDOI Listing
September 2012

Adaptation and antibiotic tolerance of anaerobic Burkholderia pseudomallei.

Antimicrob Agents Chemother 2011 Jul 2;55(7):3313-23. Epub 2011 May 2.

Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.

The Gram-negative bacterium Burkholderia pseudomallei is the etiological agent of melioidosis and is remarkably resistant to most classes of antibacterials. Even after months of treatment with antibacterials that are relatively effective in vitro, there is a high rate of treatment failure, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. The pathology of melioidosis indicates that B. pseudomallei encounters host microenvironments that limit aerobic respiration, including the lack of oxygen found in abscesses and in the presence of nitric oxide produced by macrophages. We investigated whether B. pseudomallei could survive in a nonreplicating, oxygen-deprived state and determined if this physiological state was tolerant of conventional antibacterials. B. pseudomallei survived initial anaerobiosis, especially under moderately acidic conditions similar to those found in abscesses. Microarray expression profiling indicated a major shift in the physiological state of hypoxic B. pseudomallei, including induction of a variety of typical anaerobic-environment-responsive genes and genes that appear specific to anaerobic B. pseudomallei. Interestingly, anaerobic B. pseudomallei was unaffected by antibacterials typically used in therapy. However, it was exquisitely sensitive to drugs used against anaerobic pathogens. After several weeks of anaerobic culture, a significant loss of viability was observed. However, a stable subpopulation that maintained complete viability for at least 1 year was established. Thus, during the course of human infection, if a minor subpopulation of bacteria inhabited an oxygen-restricted environment, it might be indifferent to traditional therapy but susceptible to antibiotics frequently used to treat anaerobic infections.
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http://dx.doi.org/10.1128/AAC.00953-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122399PMC
July 2011

Construction of aminoglycoside-sensitive Burkholderia cenocepacia strains for use in studies of intracellular bacteria with the gentamicin protection assay.

Appl Environ Microbiol 2010 May 26;76(10):3170-6. Epub 2010 Mar 26.

Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.

Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.
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http://dx.doi.org/10.1128/AEM.03024-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869153PMC
May 2010

An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei.

Gene 2009 Feb 28;430(1-2):123-31. Epub 2008 Oct 28.

Department of Microbiology, University of Colorado School of Medicine, Mail Stop 8333, PO Box 6511, Aurora, CO 80045, USA.

Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.
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http://dx.doi.org/10.1016/j.gene.2008.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646673PMC
February 2009

Roles of YopN, LcrG and LcrV in controlling Yops secretion by Yersinia pestis.

Adv Exp Med Biol 2007 ;603:225-34

Department of Microbiology and Immunology, School of Medicine and Health Sciences, University of North Dakota, USA.

Control of Yops secretion in pathogenic Yersinia is achieved at several levels. These levels likely include transcriptional, post-transcriptional, translational and secretional controls. Secretion control appears to be mediated by two pathways. One pathway involves YopN and proteins that interact with YopN. The second pathway consists of LcrG and its interaction with LcrV. LcrV is a postive regulator of Yops secretion that exerts control over Yops secretion by negating the secretion blocking role of LcrG. However, the intersection of these two control pathways is not understood. Recent work has allowed the development of a speculative model that brings YopN-mediated and LcrG-LcrV-mediated control together in the context of the ability of the needle complex to respond to Ca2+.
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http://dx.doi.org/10.1007/978-0-387-72124-8_20DOI Listing
December 2007

Structure-function analysis of the C-terminal domain of LcrV from Yersinia pestis.

J Bacteriol 2007 Sep 20;189(18):6734-9. Epub 2007 Jul 20.

University of North Dakota, Dept. of Microbiol. and Immunol. Room 4700, School of Medicine and Health Sciences, 501 N. Columbia Road, Stop 9037, Grand Forks, ND 58203-9037, USA.

LcrV, a multifunctional protein, acts as a positive regulator of effector protein secretion for the type III secretion system (T3SS) in Yersinia pestis by interaction with the negative regulator LcrG. In this study, LcrV was analyzed to identify regions required for LcrG interaction. Random-linker insertion mutagenesis, deletion analysis, and site-directed mutagenesis of hydrophobic amino acids between residues 290 and 311 allowed the isolation of an LcrV mutant (LcrV L291R F308R) defective for LcrG interaction. The new residues identified in LcrG interaction lie in helix 12 of LcrV; residues in helix 7 of LcrV are known to be involved in LcrG interaction. Helix 7 and helix 12 of LcrV interact to form an intramolecular coiled coil; these new results suggest that the intramolecular coiled coil in LcrV is required for LcrG interaction and activation of the T3SS.
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http://dx.doi.org/10.1128/JB.00539-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045164PMC
September 2007
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