Publications
90 results found
Bradley R, Simon D, Spiga L, et al., 2024, Laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS) demonstrates a direct impact of hypochlorous acid stress on PQS-mediated quorum sensing in Pseudomonas aeruginosa., mSystems, Vol: 9
UNLABELLED: To establish infections in human hosts, Pseudomonas aeruginosa must overcome innate immune-generated oxidative stress, such as the hypochlorous acid (HOCl) produced by neutrophils. We set out to find specific biomarkers of oxidative stress through the development of a protocol for the metabolic profiling of P. aeruginosa cultures grown in the presence of different oxidants using a novel ionization technique for mass spectrometry, laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS). We demonstrated the ability of LD-REIMS to classify samples as untreated or treated with a specific oxidant with 100% accuracy and identified a panel of 54 metabolites with significantly altered concentrations after exposure to one or more of the oxidants. Key metabolic changes were conserved in P. aeruginosa clinical strains isolated from patients with cystic fibrosis lung infections. These data demonstrated that HOCl stress impacted the Pseudomonas quinolone signal (PQS) quorum sensing system. Ten 2-alkyl-4-quinolones (AHQs) associated with the PQS system were significantly lower in concentration in HOCl-stressed P. aeruginosa cultures, including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), the most active signal molecule of the PQS system. The PQS system regulates the production of virulence factors, including pyocyanin and elastase, and their levels were markedly affected by HOCl stress. No pyocyanin was detectable and elastase concentrations were reduced by more than 75% in cultures grown with sub-lethal concentrations of HOCl, suggesting that this neutrophil-derived oxidant may disrupt the ability of P. aeruginosa to establish infections through interference with production of PQS-associated virulence factors. IMPORTANCE: This work demonstrates that a high-throughput ambient ionization mass spectrometry method can be used successfully to study a bacterial stress response. Its application to the opportunistic pathogen Pseudomonas aeruginosa led to the
Lougheed KE, Thomson M, Koziej LS, et al., 2022, A universal stress protein acts as a metabolic rheostat controlling carbon flux in mycobacteria
<jats:title>Abstract</jats:title><jats:p>Universal stress proteins (USPs) are ubiquitous amongst prokaryotes and have empirically determined roles in adaptation to stress, while their specific biochemical function in cellular processes is opaque. The most frequently encountered USP is a small ~15kDa protein comprised of a single domain (PF00582). In <jats:italic>Mycobacterium tuberculosis</jats:italic> Rv1636 is the sole single domain protein amongst its 10 USPs. We were unable to delete rv1636 from <jats:italic>M. tuberculosis</jats:italic>, supporting previous data that it is essential for growth. We deleted its orthologue, MS3811, from <jats:italic>M. smegmatis</jats:italic>, and in exponential phase competition experiments we observed that the advantage switched between wildtype and the mutant depending on the growth environment. In rich medium the wildtype had the competitive advantage, but in a defined medium with a single carbon source the competitive advantage switched, with the mutant rapidly taking over the culture. We hypothesised the USP is regulating metabolic flux, with its deletion leading to increased flux and more rapid turn-over of central-carbon metabolites. We tested this by performing <jats:sup>13</jats:sup>C-stable isotope tracing, using [U-<jats:sup>13</jats:sup>C<jats:sub>6</jats:sub>] glucose as sole carbon source for growth of the parental and deletion strains. The findings were unequivocal; deletion of the USP led to an increase in label incorporation in central carbon metabolites and a profound change in the isotopologue distribution. Furthermore, <jats:italic>in vivo</jats:italic> protein-crosslinking provided evidence that Rv1636 interacts directly with key central metabolic enzymes, including the glycolytic enzymes pyruvate kinase, pyruvate dehydrogenase, pyruvate synthase. We propose the mycobacterial single domain USP acts as a metab
Farrant KV, Spiga L, Davies JC, et al., 2021, Response of Pseudomonas aeruginosa to the innate immune system-derived oxidants hypochlorous acid and hypothiocyanous acid, Journal of Bacteriology, Vol: 203, ISSN: 0021-9193
Pseudomonas aeruginosa is a significant nosocomial pathogen and associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN, and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis and catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), that are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress, and responds to both oxidants by upregulating expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated) there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type III secretion system, sulphur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.
Price RL, Bugeon L, Mostowy S, et al., 2019, In vitro and in vivo properties of the bovine antimicrobial peptide, Bactenecin 5, PLoS ONE, Vol: 14, ISSN: 1932-6203
Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum.
Zeden MS, Schuster CF, Bowman L, et al., 2018, Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions, Journal of Biological Chemistry, Vol: 293, Pages: 3180-3200, ISSN: 0021-9258
Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus. However, the exact role of this molecule has not been identified. dacA, the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA and qoxB, coding for proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these finding reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus.
Smith WD, Bardin E, Cameron L, et al., 2017, Current and future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis, FEMS Microbiology Letters, Vol: 364, ISSN: 0378-1097
Pseudomonas aeruginosa opportunistically infects the airways of patients with cystic fibrosis and causes significant morbidity and mortality. Initial infection can often be eradicated though requires prompt detection and adequate treatment. Intermittent and then chronic infection occurs in the majority of patients. Better detection of P. aeruginosa infection using biomarkers may enable more successful eradication before chronic infection is established. In chronic infection P. aeruginosa adapts to avoid immune clearance and resist antibiotics via efflux pumps, β-lactamase expression, reduced porins and switching to a biofilm lifestyle. The optimal treatment strategies for P. aeruginosa infection are still being established, and new antibiotic formulations such as liposomal amikacin, fosfomycin in combination with tobramycin and inhaled levofloxacin are being explored. Novel agents such as the alginate oligosaccharide OligoG, cysteamine, bacteriophage, nitric oxide, garlic oil and gallium may be useful as anti-pseudomonal strategies, and immunotherapy to prevent infection may have a role in the future. New treatments that target the primary defect in cystic fibrosis, recently licensed for use, have been associated with a fall in P. aeruginosa infection prevalence. Understanding the mechanisms for this could add further strategies for treating P. aeruginosa in future.
Lund-Palau H, Turnbull AR, Bush A, et al., 2016, Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches, Expert Review of Respiratory Medicine, Vol: 10, Pages: 685-697, ISSN: 1747-6348
Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development.
Thompson AJ, Koziej L, Williams H, et al., 2016, Towards optical fibre based Raman spectroscopy for the detection of surgical site infection, BiOS, SPIE Photonics West, Publisher: SPIE
Surgical site infections (SSIs) are common post-surgical complications that remain significant clinical problems, as they are associated with substantial mortality and morbidity. As such, there is significant interest in the development of minimally invasive techniques that permit early detection of SSIs. To this end, we are applying a compact, clinically deployable Raman spectrometer coupled to an optical fibre probe to the study of bacteria, with the long term goal of using Raman spectroscopy to detect infection in vivo. Our system comprises a 785 nm laser diode for excitation and a commercial (Ocean Optics, Inc.) Raman spectrometer for detection. Here we discuss the design, optimisation and validation of this system, and describe our first experiences interrogating bacterial cells (Escherichia coli) in vitro. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
La Rosa R, Behrends V, Williams HD, et al., 2015, Influence of the Crc regulator on the hierarchical use of carbon sources from a complete medium in Pseudomonas, Environmental Microbiology, Vol: 18, Pages: 807-818, ISSN: 1462-2920
The Crc protein, together with the Hfq protein, participates in catabolite repression in pseudomonads, helping to coordinate metabolism. Little is known about how Crc affects the hierarchy of metabolite assimilation from complex mixtures. Using proton NMR spectroscopy, we carried out comprehensive metabolite profiling of culture supernatants (metabolic footprinting) over the course of growth of both Pseudomonas putida and P. aeruginosa, and compared the wild-type strains to deletion mutants for crc. A complex metabolite consumption hierarchy was observed, which was broadly similar between the two species, although with some important differences, for example in sugar utilisation. The order of metabolite utilisation changed upon inactivation of the crc gene, but even in the Crc-null strains some compounds were completely consumed before late metabolites were taken up. This suggests the presence of additional regulatory elements that determine the time and order of consumption of compounds. Unexpectedly, the loss of Crc led both species to excrete acetate and pyruvate as a result of unbalanced growth during exponential phase, compounds that were later consumed in stationary phase. This loss of carbon during growth helps to explain the contribution of the Crc/Hfq regulatory system to evolutionary fitness of pseudomonads.
Lougheed KEA, Bennett MH, Williams HD, 2014, An <i>in vivo</i> crosslinking system for identifying mycobacterial protein-protein interactions, JOURNAL OF MICROBIOLOGICAL METHODS, Vol: 105, Pages: 67-71, ISSN: 0167-7012
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- Citations: 9
Nair C, Shoemark A, Chan M, et al., 2014, Cyanide levels found in infected cystic fibrosis sputum inhibit airway ciliary function, European Respiratory Journal, ISSN: 0903-1936
Ryall B, Carrara M, Zlosnik JEA, et al., 2014, The Mucoid Switch in <i>Pseudomonas aeruginosa</i> Represses Quorum Sensing Systems and Leads to Complex Changes to Stationary Phase Virulence Factor Regulation, PLOS ONE, Vol: 9, ISSN: 1932-6203
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- Citations: 34
Nair CG, Ryall B, Williams HD, 2014, Cyanide measurements in bacterial culture and sputum., Methods Mol Biol, Vol: 1149, Pages: 325-336
Cyanide is produced by a few bacterial species, including Pseudomonas aeruginosa, and it has a role in the opportunistic infections of this bacterium including in cystic fibrosis lung infections. We describe two methods for determining cyanide in culture and patient sputum samples. One uses an ion-selective electrode to provide a convenient, rapid method of cyanide quantitation in culture or sputum, and the second is a semiquantitative method using Feigl-Anger paper that is useful for screening large numbers of bacterial strains for cyanide production.
Frangipani E, Perez-Martinez I, Williams HD, et al., 2014, A novel cyanide-inducible gene cluster helps protect <i>Pseudomonas aeruginosa</i> from cyanide, ENVIRONMENTAL MICROBIOLOGY REPORTS, Vol: 6, Pages: 28-34, ISSN: 1758-2229
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- Citations: 13
Behrends V, Williams HD, Bundy JG, 2014, Metabolic Footprinting: Extracellular Metabolomic Analysis, PSEUDOMONAS: METHODS AND PROTOCOLS, Vol: 1149, Pages: 281-292, ISSN: 1064-3745
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- Citations: 13
Behrends V, Bell TJ, Liebeke M, et al., 2013, Metabolite Profiling to Characterize Disease-related Bacteria <i>GLUCONATE EXCRETION BY PSEUDOMONAS AERUGINOSA MUTANTS AND CLINICAL ISOLATES FROM CYSTIC FIBROSIS PATIENTS</i>, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 15098-15109, ISSN: 0021-9258
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- Citations: 31
Behrends V, Geier B, Williams HD, et al., 2013, Direct Assessment of Metabolite Utilization by <i>Pseudomonas aeruginosa</i> during Growth on Artificial Sputum Medium, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 79, Pages: 2467-2470, ISSN: 0099-2240
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- Citations: 13
Nair CG, Chao C, Ryall B, et al., 2013, Sub-lethal concentrations of antibiotics increase mutation frequency in the cystic fibrosis pathogen <i>Pseudomonas aeruginosa</i>, LETTERS IN APPLIED MICROBIOLOGY, Vol: 56, Pages: 149-154, ISSN: 0266-8254
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- Citations: 29
Behrends V, Ryall B, Zlosnik JEA, et al., 2013, Metabolic adaptations of Pseudomonas aeruginosa during cystic fibrosis chronic lung infections, ENVIRONMENTAL MICROBIOLOGY, Vol: 15, Pages: 398-408, ISSN: 1462-2912
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- Citations: 57
Nair C, Shoemark A, Donovan S, et al., 2012, THE TOXIC <i>P</i>. <i>AERUGINOSA</i> EXOPRODUCTS CYANIDE AND PYOCYANIN INHIBIT CILIARY FUNCTION OF HUMAN RESPIRATORY EPITHELIA VIA DIFFERENT MECHANISMS, Publisher: WILEY-BLACKWELL, Pages: 329-329, ISSN: 8755-6863
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- Citations: 1
Behrends V, Ryall B, Zlosnik JEA, et al., 2012, Metabolic adaptations of Pseudomonas aeruginosa during cystic fibrosis lung infections, INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, Vol: 302, Pages: 109-109, ISSN: 1438-4221
Trauner A, Lougheed KEA, Bennett MH, et al., 2012, The dormancy regulator DosR controls ribosome stability in hypoxic mycobacteria, Journal of Biological Chemistry, Vol: 287, Pages: 24053-24063, ISSN: 0021-9258
It is thought that during latent infection, Mycobacterium tuberculosis bacilli are retained within granulomas in a low-oxygen environment. The dormancy survival (Dos) regulon, regulated by the response regulator DosR, appears to be essential for hypoxic survival in M. tuberculosis, but it is not known how the regulon promotes survival. Here we report that mycobacteria, in contrast to enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers) upon entry into stasis. Instead, ribosomes are stabilized in the associated form (70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during hypoxic stasis, we show that the dormancy regulator DosR is required for optimal ribosome stabilization. We present evidence that the majority of this effect is mediated by the DosR-regulated protein MSMEG_3935 (a S30AE domain protein), which is associated with the ribosome under hypoxic conditions. A Δ3935 mutant phenocopies the ΔdosR mutant during hypoxia, and complementation of ΔdosR with the MSMEG_3935 gene leads to complete recovery of dosR mutant phenotypes during hypoxia. We suggest that this protein is named ribosome-associated factor under hypoxia (RafH) and that it is the major factor responsible for DosR-mediated hypoxic survival in mycobacteria.
Williams HD, Davies JC, 2012, Basic science for the chest physician: <i>Pseudomonas aeruginosa</i> and the cystic fibrosis airway, THORAX, Vol: 67, Pages: 465-467, ISSN: 0040-6376
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- Citations: 35
Behrends V, Bundy JG, Williams HD, 2011, Differences in strategies to combat osmotic stress in <i>Burkholderia cenocepacia</i> elucidated by NMR-based metabolic profiling, LETTERS IN APPLIED MICROBIOLOGY, Vol: 52, Pages: 619-625, ISSN: 0266-8254
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- Citations: 15
Trauner A, Bennett MH, Williams HD, 2011, Isolation of Bacterial Ribosomes with Monolith Chromatography, PLOS ONE, Vol: 6, ISSN: 1932-6203
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- Citations: 25
Nair CG, Shoemark A, Donovan S, et al., 2011, POISON IN THE LUNG - CYANIDE, AT CONCENTRATIONS FOUND IN THE SPUTUM OF <i>PSEUDOMONAS AERUGINOSA</i> INFECTED CF PATIENTS INHIBITS THE BEATING OF HUMAN RESPIRATORY CILIA <i>IN VITRO</i>, Publisher: WILEY-BLACKWELL, Pages: 319-319, ISSN: 8755-6863
Williams HD, Behrends V, Bundy JG, et al., 2010, Hypertonic saline therapy in cystic fibrosis: do population shifts caused by the osmotic sensitivity of infecting bacteria explain the effectiveness of this treatment?, Frontiers in Microbiology, Vol: 1
Hingley-Wilson SM, Lougheed KEA, Ferguson K, et al., 2010, Individual <i>Mycobacterium tuberculosis</i> universal stress protein homologues are dispensable <i>in vitro</i>, TUBERCULOSIS, Vol: 90, Pages: 236-244, ISSN: 1472-9792
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- Citations: 50
Behrends V, Ryall B, Wang X, et al., 2010, Metabolic profiling of <i>Pseudomonas</i> <i>aeruginosa</i> demonstrates that the anti-sigma factor MucA modulates osmotic stress tolerance, MOLECULAR BIOSYSTEMS, Vol: 6, Pages: 562-569, ISSN: 1742-206X
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- Citations: 40
Ryall B, Mitchell H, Mossialos D, et al., 2009, Cyanogenesis by the entomopathogenic bacterium <i>Pseudomonas entomophila</i>, LETTERS IN APPLIED MICROBIOLOGY, Vol: 49, Pages: 131-135, ISSN: 0266-8254
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- Citations: 14
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