Publications by year
2020
Kovacs-Simon A, Metters G, Norville I, Hemsley C, Titball RW (2020). Coxiella burnetii replicates in Galleria mellonella hemocytes and transcriptome mapping reveals in vivo regulated genes. Virulence, 11(1), 1268-1278.
Jitprasutwit S, Jitprasutwit N, Hemsley CM, Onlamoon N, Withatanung P, Muangsombut V, Vattanaviboon P, Stevens JM, Ong C, Stevens MP, et al (2020). Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction.
Front Microbiol,
11Abstract:
Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction.
Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.
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2019
Kovacs-Simon A, Hemsley CM, Scott AE, Prior JL, Titball RW (2019). Burkholderia thailandensis strain E555 is a surrogate for the investigation of Burkholderia pseudomallei replication and survival in macrophages.
BMC Microbiol,
19(1).
Abstract:
Burkholderia thailandensis strain E555 is a surrogate for the investigation of Burkholderia pseudomallei replication and survival in macrophages.
BACKGROUND: Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells. RESULTS: We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei. CONCLUSIONS: a weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms.
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Hemsley CM, O'Neill PA, Essex-Lopresti A, Norville IH, Atkins TP, Titball RW (2019). Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups.
BMC Genomics,
20(1).
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Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups.
BACKGROUND: Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. An improved understanding of the genetic diversity of C. burnetii is essential for the development of diagnostics, vaccines and therapeutics, but genotyping data is lacking from many parts of the world. Sporadic outbreaks of Q fever have occurred in the United Kingdom, but the local genetic make-up of C. burnetii has not been studied in detail. RESULTS: Here, we report whole genome data for nine C. burnetii sequences obtained in the UK. All four genomes of C. burnetii from cattle, as well as one sheep sample, belonged to Multi-spacer sequence type (MST) 20, whereas the goat samples were MST33 (three genomes) and MST32 (one genome), two genotypes that have not been described to be present in the UK to date. We established the phylogenetic relationship between the UK genomes and 67 publically available genomes based on single nucleotide polymorphisms (SNPs) in the core genome, which confirmed tight clustering of strains within genomic groups, but also indicated that sub-groups exist within those groups. Variation is mainly achieved through SNPs, many of which are non-synonymous, thereby confirming that evolution of C. burnetii is based on modification of existing genes. Finally, we discovered genomic-group specific genome content, which supports a model of clonal expansion of previously established genotypes, with large scale dissemination of some of these genotypes across continents being observed. CONCLUSIONS: the genetic make-up of C. burnetii in the UK is similar to the one in neighboring European countries. As a species, C. burnetii has been considered a clonal pathogen with low genetic diversity at the nucleotide level. Here, we present evidence for significant variation at the protein level between isolates of different genomic groups, which mainly affects secreted and membrane-associated proteins. Our results thereby increase our understanding of the global genetic diversity of C. burnetii and provide new insights into the evolution of this emerging zoonotic pathogen.
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Metters G, Norville IH, Titball RW, Hemsley CM (2019). From cell culture to cynomolgus macaque: infection models show lineage-specific virulence potential of Coxiella burnetii.
J Med Microbiol,
68(10), 1419-1430.
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From cell culture to cynomolgus macaque: infection models show lineage-specific virulence potential of Coxiella burnetii.
Coxiella burnetii is an obligate intracellular pathogen that causes the zoonotic disease Q fever in humans, which can occur in either an acute or a chronic form with serious complications. The bacterium has a wide host range, including unicellular organisms, invertebrates, birds and mammals, with livestock representing the most significant reservoir for human infections. Cell culture models have been used to decipher the intracellular lifestyle of C. burnetii, and several infection models, including invertebrates, rodents and non-human primates, are being used to investigate host-pathogen interactions and to identify bacterial virulence factors and vaccine candidates. However, none of the models replicate all aspects of human disease. Furthermore, it is becoming evident that C. burnetii isolates belonging to different lineages exhibit differences in their virulence in these models. Here, we compare the advantages and disadvantages of commonly used infection models and summarize currently available data for lineage-specific virulence.
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2016
Champion OL, Gourlay LJ, Scott AE, Lassaux P, Conejero L, Perletti L, Hemsley C, Prior J, Bancroft G, Bolognesi M, et al (2016). Immunisation with proteins expressed during chronic murine melioidosis provides enhanced protection against disease.
Vaccine,
34(14), 1665-1671.
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Immunisation with proteins expressed during chronic murine melioidosis provides enhanced protection against disease.
There is an urgent need for an effective vaccine against human disease caused by Burkholderia pseudomallei, and although a wide range of candidates have been tested in mice none provide high level protection. We considered this might reflect the inability of these vaccine candidates to protect against chronic disease. Using Q-RT PCR we have identified 6 genes which are expressed in bacteria colonising spleens and lungs of chronically infected mice. Three of the genes (BPSL1897, BPSL3369 and BPSL2287) have been expressed in Escherichia coli and the encoded proteins purified. We have also included BPSL2765, a protein known to induce immune responses associated with a reduced incidence of chronic/recurrent disease in humans. Immunisation of mice with a combination of these antigens resulted in the induction of antibody responses against all of the proteins. Compared with mice immunised with capsular polysaccharide or LolC protein, mice immunised with the combination of chronic stage antigens showed enhanced protection against experimental disease in mice.
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Anutrakunchai C, Hemsley CM, Sermswan RW, Titball RW, Chareonsudjai S, Taweechaisupapong S (2016). Role of RelA and SpoT in Burkholderia pseudomallei survival, biofilm formation and ceftazidime tolerance during nutritional stress.
Tropical Biomedicine,
33(4), 786-798.
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Role of RelA and SpoT in Burkholderia pseudomallei survival, biofilm formation and ceftazidime tolerance during nutritional stress
Burkholderia pseudomallei a saprophyte found in soil and stagnant water is the causative agent of human melioidosis, an often cause fatal disease. B. pseudomallei is intrinsically resistant to many antibiotics. The stringent response is a global bacterial adaptation process in response to nutritional limitation and is mediated by the alarmone (p)ppGpp, which is produced by two proteins, RelA and SpoT. In order to test whether the stringent response is involved in ceftazidime tolerance, biofilm formation, and bacterial survival in the soil microcosm, B. pseudomallei strain K96243 and its isogenic ΔrelA and ΔrelAΔspoT mutants were grown in rich and nutrient-limited media. In nutrient-limiting conditions, both the wild type and mutants were found to be up to 64-times more tolerant to ceftazidime than when grown in rich culture conditions. Moreover, the biofilm formation of all bacterial isolates tested were significantly higher under nutrient-limiting conditions than under nutrient-rich conditions. The ΔrelAΔspoT mutant produced less biofilm than its wild type or ΔrelA mutant under nutrient-limiting conditions. The survival of the ΔrelAΔspoT double mutant cultured in 1% moisture content soil was significantly decreased compared to the wild type and the ΔrelA mutant. Therefore, the RelA/SpoT protein family might represent a promising target for the development of novel antimicrobial agents to combat B. pseudomallei.
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2014
Hemsley CM, Luo JX, Andreae CA, Butler CS, Soyer OS, Titball RW (2014). Bacterial drug tolerance under clinical conditions is governed by anaerobic adaptation but not anaerobic respiration.
Antimicrob Agents Chemother,
58(10), 5775-5783.
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Bacterial drug tolerance under clinical conditions is governed by anaerobic adaptation but not anaerobic respiration.
Noninherited antibiotic resistance is a phenomenon whereby a subpopulation of genetically identical bacteria displays phenotypic tolerance to antibiotics. We show here that compared to Escherichia coli, the clinically relevant genus Burkholderia displays much higher levels of cells that tolerate ceftazidime. By measuring the dynamics of the formation of drug-tolerant cells under conditions that mimic in vivo infections, we show that in Burkholderia bacteria, oxygen levels affect the formation of these cells. The drug-tolerant cells are characterized by an anaerobic metabolic signature and can be eliminated by oxygenating the system or adding nitrate. The transcriptome profile suggests that these cells are not dormant persister cells and are likely to be drug tolerant as a consequence of the upregulation of anaerobic nitrate respiration, efflux pumps, β-lactamases, and stress response proteins. These findings have important implications for the treatment of chronic bacterial infections and the methodologies and conditions that are used to study drug-tolerant and persister cells in vitro.
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Moule MG, Hemsley CM, Seet Q, Guerra-Assunção JA, Lim J, Sarkar-Tyson M, Clark TG, Tan PBO, Titball RW, Cuccui J, et al (2014). Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development.
mBio,
5(1).
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Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development
Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. © 2014 Moule et al.
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Moule MG, Hemsley CM, Seet Q, Guerra-Assunção JA, Lim J, Sarkar-Tyson M, Clark TG, Tan PBO, Titball RW, Cuccui J, et al (2014). Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development.
mBio,
5(1), e00926-e00913.
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Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development.
UNLABELLED: Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 10(6) transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE: Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.
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BUTT A, HIGMAN VA, WILLIAMS C, CRUMP MP, HEMSLEY C, HARMER N, TITBALL RW (2014). NMR structure of the HicA toxin from Burkholderia pseudomallei.
Butt A, Higman VA, Williams C, Crump MP, Hemsley CM, Harmer N, Titball RW (2014). The HicA toxin from Burkholderia pseudomallei has a role in persister cell formation.
Biochem J,
459(2), 333-344.
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The HicA toxin from Burkholderia pseudomallei has a role in persister cell formation.
TA (toxin-antitoxin) systems are widely distributed amongst bacteria and are associated with the formation of antibiotic tolerant (persister) cells that may have involvement in chronic and recurrent disease. We show that overexpression of the Burkholderia pseudomallei HicA toxin causes growth arrest and increases the number of persister cells tolerant to ciprofloxacin or ceftazidime. Furthermore, our data show that persistence towards ciprofloxacin or ceftazidime can be differentially modulated depending on the level of induction of HicA expression. Deleting the hicAB locus from B. pseudomallei K96243 significantly reduced persister cell frequencies following exposure to ciprofloxacin, but not ceftazidime. The structure of HicA(H24A) was solved by NMR and forms a dsRBD-like (dsRNA-binding domain-like) fold, composed of a triple-stranded β-sheet, with two helices packed against one face. The surface of the protein is highly positively charged indicative of an RNA-binding protein and His24 and Gly22 were functionality important residues. This is the first study demonstrating a role for the HicAB system in bacterial persistence and the first structure of a HicA protein that has been experimentally characterized.
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Wagley S, Hemsley C, Thomas R, Moule MG, Vanaporn M, Andreae C, Robinson M, Goldman S, Wren BW, Butler CS, et al (2014). The twin arginine translocation system is essential for aerobic growth and full virulence of Burkholderia thailandensis.
J Bacteriol,
196(2), 407-416.
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The twin arginine translocation system is essential for aerobic growth and full virulence of Burkholderia thailandensis.
The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some β-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated.
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Jitprasutwit S, Ong C, Juntawieng N, Ooi WF, Hemsley CM, Vattanaviboon P, Titball RW, Tan P, Korbsrisate S (2014). Transcriptional profiles of Burkholderia pseudomallei reveal the direct and indirect roles of Sigma E under oxidative stress conditions.
BMC Genomics,
15(1).
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Transcriptional profiles of Burkholderia pseudomallei reveal the direct and indirect roles of Sigma E under oxidative stress conditions
Background: Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative bacterium widely distributed in soil and water in endemic areas. This soil saprophyte can survive harsh environmental conditions, even in soils where herbicides (containing superoxide generators) are abundant. Sigma factor E (σ ) is a key regulator of extra-cytoplasmic stress response in Gram-negative bacteria. In this study, we identified the B. pseudomallei σ. regulon and characterized the indirect role that σ. plays in the regulation of spermidine, contributing to the successful survival of B. pseudomallei in stressful environments. Results: Changes in the global transcriptional profiles of B. pseudomallei wild type and σ. mutant under physiological and oxidative stress (hydrogen peroxide) conditions were determined. We identified 307 up-regulated genes under oxidative stress condition. Comparison of the transcriptional profiles of B. pseudomallei wild type and σ. mutant under control or oxidative stress conditions identified 85 oxidative-responsive genes regulated by σ , including genes involved in cell membrane repair, maintenance of protein folding and oxidative stress response and potential virulence factors such as a type VI secretion system (T6SS). Importantly, we identified that the speG gene, encoding spermidine-acetyltransferase, is a novel member of the B. pseudomallei σ. regulon. The expression of speG was regulated by σ , implying that σ. plays an indirect role in the regulation of physiological level of spermidine to protect the bacteria during oxidative stress.Conclusion: This study identified B. pseudomallei genes directly regulated by σ. in response to oxidative stress and revealed the indirect role of σ. in the regulation of the polyamine spermidine (via regulation of speG) for bacterial cell protection during oxidative stress. This study provides new insights into the regulatory mechanisms by which σ. contributes to the survival of B. pseudomallei under stressful conditions. E E E E E E E E E E E E
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Jitprasutwit S, Ong C, Juntawieng N, Ooi WF, Hemsley CM, Vattanaviboon P, Titball RW, Tan P, Korbsrisate S (2014). Transcriptional profiles of Burkholderia pseudomallei reveal the direct and indirect roles of Sigma E under oxidative stress conditions.
BMC Genomics,
15Abstract:
Transcriptional profiles of Burkholderia pseudomallei reveal the direct and indirect roles of Sigma E under oxidative stress conditions.
BACKGROUND: Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative bacterium widely distributed in soil and water in endemic areas. This soil saprophyte can survive harsh environmental conditions, even in soils where herbicides (containing superoxide generators) are abundant. Sigma factor E (σE) is a key regulator of extra-cytoplasmic stress response in Gram-negative bacteria. In this study, we identified the B. pseudomallei σE regulon and characterized the indirect role that σE plays in the regulation of spermidine, contributing to the successful survival of B. pseudomallei in stressful environments. RESULTS: Changes in the global transcriptional profiles of B. pseudomallei wild type and σE mutant under physiological and oxidative stress (hydrogen peroxide) conditions were determined. We identified 307 up-regulated genes under oxidative stress condition. Comparison of the transcriptional profiles of B. pseudomallei wild type and σE mutant under control or oxidative stress conditions identified 85 oxidative-responsive genes regulated by σE, including genes involved in cell membrane repair, maintenance of protein folding and oxidative stress response and potential virulence factors such as a type VI secretion system (T6SS). Importantly, we identified that the speG gene, encoding spermidine-acetyltransferase, is a novel member of the B. pseudomallei σE regulon. The expression of speG was regulated by σE, implying that σE plays an indirect role in the regulation of physiological level of spermidine to protect the bacteria during oxidative stress. CONCLUSION: This study identified B. pseudomallei genes directly regulated by σE in response to oxidative stress and revealed the indirect role of σE in the regulation of the polyamine spermidine (via regulation of speG) for bacterial cell protection during oxidative stress. This study provides new insights into the regulatory mechanisms by which σE contributes to the survival of B. pseudomallei under stressful conditions.
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2013
Thomas RJ, Hamblin KA, Armstrong SJ, Müller CM, Bokori-Brown M, Goldman S, Atkins HS, Titball RW (2013). Galleria mellonella as a model system to test the pharmacokinetics and efficacy of antibiotics against Burkholderia pseudomallei.
Int J Antimicrob Agents,
41(4), 330-336.
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Galleria mellonella as a model system to test the pharmacokinetics and efficacy of antibiotics against Burkholderia pseudomallei.
Mammalian models of infection are paramount to elucidating the mechanisms of bacterial pathogenesis and are also used for evaluating the efficacy of novel antimicrobials before the commencement of human trials. In this study, Galleria mellonella was used to determine the efficacy of antibiotics towards a Burkholderia thailandensis infection in G. mellonella larvae. Kanamycin, imipenem, ceftazidime, doxycycline and ciprofloxacin could all provide some protection when given 1 h before challenge with B. thailandensis; however, at 2 h or 6 h post challenge, imipenem and kanamycin were unable to rescue larvae. The most effective antibiotic for the prevention or treatment of disease was ceftazidime. Pharmacokinetic properties of a single dose of these antibiotics in G. mellonella larvae were also determined, and it was demonstrated that this model is useful for approximating the antibiotic response in humans. The G. mellonella model was used to screen a panel of novel antimicrobials for activity towards B. thailandensis and Burkholderia pseudomallei, and three novel compounds with antibiotic activity were identified. These results support the hypothesis that G. mellonella can be used to screen antimicrobial efficacy. This is the first study to determine the pharmacokinetic parameters of clinically relevant antibiotics in this model system.
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Butt A, Müller C, Harmer N, Titball RW (2013). Identification of type II toxin-antitoxin modules in Burkholderia pseudomallei.
FEMS Microbiol Lett,
338(1), 86-94.
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Identification of type II toxin-antitoxin modules in Burkholderia pseudomallei.
Type II toxin-antitoxin (TA) systems are believed to be widely distributed amongst bacteria although their biological functions are not clear. We have identified eight candidate TA systems in the genome of the human pathogen Burkholderia pseudomallei. Five of these were located in genome islands. of the candidate toxins, BPSL0175 (RelE1) or BPSS1060 (RelE2) caused growth to cease when expressed in Escherichia coli, whereas expression of BPSS0390 (HicA) or BPSS1584 (HipA) (in an E. coli ΔhipBA background) caused a reduction in the number of culturable bacteria. The cognate antitoxins could restore growth and culturability of cells.
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Ooi WF, Ong C, Nandi T, Kreisberg JF, Chua HH, Sun G, Chen Y, Mueller C, Conejero L, Eshaghi M, et al (2013). The condition-dependent transcriptional landscape of Burkholderia pseudomallei.
PLoS Genet,
9(9).
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The condition-dependent transcriptional landscape of Burkholderia pseudomallei.
Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes--Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes--quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an "accidental pathogen", where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts.
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2012
Müller CM, Conejero L, Spink N, Wand ME, Bancroft GJ, Titball RW (2012). Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity.
Infect. Immun.,
80(9), 3247-3255.
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Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity
Burkholderia pseudomallei is a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created a relA spoT double mutant in B. pseudomallei strain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotype in vitro and in vivo. The B. pseudomallei ΔrelA ΔspoT mutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in the Galleria mellonella insect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ΔrelA ΔspoT mutant resulted in partial protection against infection with wild-type B. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation in B. pseudomallei, and the ΔrelA ΔspoT mutant may be a promising live-attenuated vaccine candidate.
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2011
Debieux CM, Dridge EJ, Mueller CM, Splatt P, Paszkiewicz K, Knight I, Florance H, Love J, Titball RW, Lewis RJ, et al (2011). A bacterial process for selenium nanosphere assembly.
Proc Natl Acad Sci U S A,
108(33), 13480-13485.
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A bacterial process for selenium nanosphere assembly.
During selenate respiration by Thauera selenatis, the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed no cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor a (SefA), and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichia coli, and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coli when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication.
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Wand ME, Müller CM, Titball RW, Michell SL (2011). Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis.
BMC Microbiol,
11(1).
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Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis.
BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella) infection models. RESULTS: B. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD) values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis. CONCLUSIONS: We have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.
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2010
Müller CM, Schneider G, Dobrindt U, Emödy L, Hacker J, Uhlin BE (2010). Differential effects and interactions of endogenous and horizontally acquired H-NS-like proteins in pathogenic Escherichia coli.
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75(2), 280-293.
Abstract:
Differential effects and interactions of endogenous and horizontally acquired H-NS-like proteins in pathogenic Escherichia coli
Summary the nucleoid-associated protein H-NS is important for gene regulation in Escherichia coli. We have studied H-NS interaction with StpA and an uncharacterized H-NS-like protein, Hfp, in the uropathogenic E. coli isolate 536 that expresses all three nucleoid-associated proteins. We found distinct interactions of the three proteins at the protein level, resulting in the formation of heteromers, as well as differences in their gene expression at the transcriptional level. Mutants lacking either StpA or Hfp alone did not exhibit a phenotype at 37°C, which is consistent with a low level of expression at that temperature. Expression of the hfp and stpA genes was found to be induced by apparently diametrical conditions, and StpA and Hfp levels could be correlated to modulatory effects on the expression of different H-NS targets, the bgl operon and operons for virulence factors such as fimbriae and capsular polysaccharide. The hns/hfp and hns/stpA double mutants displayed severe growth defects at low and high temperatures respectively. Our findings demonstrated different requirements for the alternative H-NS/Hfp/StpA combinations under these growth conditions. We propose that Hfp and StpA have distinct functions and roles in a dynamic pool of nucleoid-associated proteins that is adapting to requirements in a particular environment.
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2009
Sjöström AE, Sondén B, Müller C, Rydström A, Dobrindt U, Wai SN, Uhlin BE (2009). Analysis of the sfaXII locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region of the main S fimbrial operon.
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46, 150-158.
Abstract:
Analysis of the sfaXII locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region of the main S fimbrial operon
We describe the expression and regulation of the gene sfaXII located near the SfaII fimbrial determinant in the newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaXII belongs to a gene family, the 17-kDa genes, typically located downstream (300-3000 bp) of different fimbrial operons found in E. coli isolates of uropathogenic and newborn meningitis origin. Using transcriptional sfaXII reporter gene fusions we found that different environmental conditions commonly affecting expression of fimbrial genes also affected sfaXII expression. Analysis of the sfaXII transcripts showed that the gene is part of the main fimbrial operon as it is transcribed together with the rest of the fimbrial genes. In addition, the sfaXII gene can be expressed from a more proximal promoter and is found to be subject to strong down-regulation by the nucleoid protein H-NS. Studies with an sfaXII mutant derivative of IHE3034 did not reveal effects on SfaII fimbrial biogenesis as monitored by e.g. immunofluorescence microscopy. Nevertheless, a mutation in sfaXII resulted in altered expression of other surface components. Moreover, we define a new gene, sfaYII, coding for a putative phosphodiesterase that is located in between the sfaXII gene and the fimbrial biogenesis genes. Our studies by ectopic expression of sfaYII in Vibrio cholerae showed that the gene product caused reduced biofilm formation and it is proposed that sfaYII can influence cyclic-di-GMP turnover in the bacteria. Our findings demonstrate that the operons typical for S-fimbriae of extraintestinal pathogenic E. coli include previously unrecognized novel regulatory genes.
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Müller CM, Åberg A, Straseviçiene J, Emődy L, Uhlin BE, Balsalobre C (2009). Type 1 Fimbriae, a Colonization Factor of Uropathogenic Escherichia coli, Are Controlled by the Metabolic Sensor CRP-cAMP.
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5(2).
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Type 1 Fimbriae, a Colonization Factor of Uropathogenic Escherichia coli, Are Controlled by the Metabolic Sensor CRP-cAMP
Attachment of bacteria to the surface of host tissues is a crucial initial step in the establishment of bacterial infections. This process is mediated by adhesins, such as the type 1 fimbriae of Escherichia coli, which play a key role during urinary tract infections by mediating adhesion to the uroepithelium. The expression of type 1 fimbriae is finely regulated attending to environmental signals and is under phase variation control, which determines the percentage of fimbriated cells in the population. In this report, we show that the expression of type 1 fimbriae is repressed by a metabolic sensor of the cell, the global regulatory complex CRP-cAMP. We demonstrate that CRP-cAMP affects the switching outcome by selectively inhibiting the recombination process in one direction only, resulting in a lower percentage of fimbriated cells. Such a switch to the non-fimbriated state after successful adhesion might be advantageous in the urinary tract, where the immune mechanisms of the host favor the removal of bacteria expressing immunogenic surface structures. Understanding the regulatory networks that govern regulation of virulence and colonization factors is both of basic interest and might help to develop novel strategies to treat bacterial infections.
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2006
Müller CM, Dobrindt U, Nagy G, Emődy L, Uhlin BE, Hacker J (2006). Role of Histone-Like Proteins H-NS and StpA in Expression of Virulence Determinants of Uropathogenic Escherichia coli.
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188(15), 5428-5438.
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Role of Histone-Like Proteins H-NS and StpA in Expression of Virulence Determinants of Uropathogenic Escherichia coli
The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.
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