Publications by authors named "Makrina Totsika"

78 Publications

Antivirulence DsbA inhibitors attenuate serovar Typhimurium fitness without detectable resistance.

FASEB Bioadv 2021 Apr 10;3(4):231-242. Epub 2021 Feb 10.

Institute of Health and Biomedical Innovation School of Biomedical Sciences Queensland University of Technology Herston QLD Australia.

Inhibition of the DiSulfide Bond (DSB) oxidative protein folding machinery, a major facilitator of virulence in Gram-negative bacteria, represents a promising antivirulence strategy. We previously developed small molecule inhibitors of DsbA from K-12 (EcDsbA) and showed that they attenuate virulence of Gram-negative pathogens by directly inhibiting multiple diverse DsbA homologues. Here we tested the evolutionary robustness of DsbA inhibitors as antivirulence antimicrobials against serovar Typhimurium under pathophysiological conditions in vitro. We show that phenylthiophene DsbA inhibitors slow . Typhimurium growth in minimal media, phenocopying . Typhimurium isogenic null mutants. Through passaging experiments, we found that DsbA inhibitor resistance was not induced under conditions that rapidly induced resistance to ciprofloxacin, an antibiotic commonly used to treat infections. Furthermore, no mutations were identified in the gene of inhibitor-treated . Typhimurium, and . Typhimurium virulence remained susceptible to DsbA inhibitors. Our work demonstrates that under in vitro pathophysiological conditions, DsbA inhibitors can have both antivirulence and antibiotic action. Importantly, our finding that DsbA inhibitors appear to be evolutionarily robust offers promise for their further development as next-generation antimicrobials against Gram-negative pathogens.
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http://dx.doi.org/10.1096/fba.2020-00100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019255PMC
April 2021

BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Antioxid Redox Signal 2021 Apr 12. Epub 2021 Apr 12.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia.

Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from termed bovine colonization factor protein H (BcfH) and defined its role in virulence. In the conserved bovine colonization factor () fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a proline instead of the conserved -proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to fimbrial biogenesis. Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity.
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http://dx.doi.org/10.1089/ars.2020.8218DOI Listing
April 2021

Chemoradiation therapy changes oral microbiome and metabolomic profiles in patients with oral cavity cancer and oropharyngeal cancer.

Head Neck 2021 Feb 2. Epub 2021 Feb 2.

The Saliva and Liquid Biopsy Translational Research Team, School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.

Background: Patients with oral cavity cancer (OCC) and oropharyngeal cancer (OPC) are often seen with locoregionally advanced disease requiring complex multimodality treatments. These treatments may have detrimental effects on the oral microbiome, which is critical to maintaining physiological balance and health.

Methods: The effects of different OCC and OPC treatment types on the oral microbiome and metabolomic profiles for 24-month post-treatment in patients with OCC and OPC were investigated using 16S rRNA gene amplicon next-generation sequencing and gas chromatography-mass spectrometry (GC-MS), respectively.

Results: Chemoradiation resulted in oral dysbiosis with specific depletion of genera which regulate the enterosalivary nitrate-nitrite-nitric oxide pathway. These data also correlate with the oral metabolomic profiles with nitric oxide-related precursor, modulator, or catalyst significantly downregulated in saliva samples from patients' postchemoradiation.

Conclusions: Together, we have shown that oral dysbiosis due to the effects of chemoradiation could potentially have an impact on OCC and OPC patient's quality of life post-treatment.
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http://dx.doi.org/10.1002/hed.26619DOI Listing
February 2021

A high-throughput cell-based assay pipeline for the preclinical development of bacterial DsbA inhibitors as antivirulence therapeutics.

Sci Rep 2021 Jan 15;11(1):1569. Epub 2021 Jan 15.

Institute of Health and Biomedical Innovation and Centre for Immunology and Infection Control, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, 4059, Australia.

Antibiotics are failing fast, and the development pipeline remains alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which inhibit bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors, previously identified by biophysical and biochemical assays. Inhibitors of DsbA block oxidative protein folding required for virulence factor folding in pathogens. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that the combination of a cell-based sulfotransferase assay and a motility assay (both DsbA reporter assays), modified for a higher throughput format, can provide a robust and target-specific platform for the identification and evaluation of DsbA inhibitors.
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http://dx.doi.org/10.1038/s41598-021-81007-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810732PMC
January 2021

Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiL).

J Med Chem 2020 07 24;63(13):6863-6875. Epub 2020 Jun 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00111DOI Listing
July 2020

An Reconstructed Human Skin Equivalent Model to Study the Role of Skin Integration Around Percutaneous Devices Against Bacterial Infection.

Front Microbiol 2020 14;11:670. Epub 2020 May 14.

Infection and Immunity Research Program, Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia.

Percutaneous devices are a key technology in clinical practice, used to connect internal organs to external medical devices. Examples include prosthesis, catheters and electrical drivelines. Percutaneous devices breach the skin's natural barrier and create an entry point for pathogens, making device infections a widespread problem. Modification of the percutaneous implant surface to increase skin integration with the aim to reduce subsequent infection is attracting a great deal of attention. While novel surfaces have been tested in various models used to study skin integration around percutaneous devices, no skin model has been reported, for the study of bacterial infection around percutaneous devices. Here, we report the establishment of an human skin equivalent model for driveline infections caused by , the most common cause of driveline-related infections. Three types of mock drivelines manufactured using melt electrowriting (smooth or porous un-seeded and porous pre-seeded with human fibroblasts) were implanted in human skin constructs and challenged with Our results show a high and stable load of in association with the skin surface and no signs of -induced tissue damage. Furthermore, our results demonstrate that bacterial migration along the driveline surface occurs in micro-gaps caused by insufficient skin integration between the driveline and the surrounding skin consistent with clinical reports from explanted patient drivelines. Thus, the human skin-driveline infection model presented here provides a clinically-relevant and versatile experimental platform for testing novel device surfaces and infection therapeutics.
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http://dx.doi.org/10.3389/fmicb.2020.00670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240036PMC
May 2020

Bacterial Biofilm Eradication Agents: A Current Review.

Front Chem 2019 28;7:824. Epub 2019 Nov 28.

School of Chemistry, Physics, and Mechanical Engineering, Queensland University of Technology, Brisbane, QLD, Australia.

Most free-living bacteria can attach to surfaces and aggregate to grow into multicellular communities encased in extracellular polymeric substances called biofilms. Biofilms are recalcitrant to antibiotic therapy and a major cause of persistent and recurrent infections by clinically important pathogens worldwide (e.g., , and ). Currently, most biofilm remediation strategies involve the development of biofilm-inhibition agents, aimed at preventing the early stages of biofilm formation, or biofilm-dispersal agents, aimed at disrupting the biofilm cell community. While both strategies offer some clinical promise, neither represents a direct treatment and eradication strategy for established biofilms. Consequently, the discovery and development of biofilm eradication agents as comprehensive, stand-alone biofilm treatment options has become a fundamental area of research. Here we review our current understanding of biofilm antibiotic tolerance mechanisms and provide an overview of biofilm remediation strategies, focusing primarily on the most promising biofilm eradication agents and approaches. Many of these offer exciting prospects for the future of biofilm therapeutics for a large number of infections that are currently refractory to conventional antibiotics.
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http://dx.doi.org/10.3389/fchem.2019.00824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893625PMC
November 2019

Nitroxide Functionalized Antibiotics Are Promising Eradication Agents against Staphylococcus aureus Biofilms.

Antimicrob Agents Chemother 2019 12 20;64(1). Epub 2019 Dec 20.

Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia

Treatment of biofilm-related infections represents an important medical challenge worldwide, as biofilms, even those involving drug-susceptible strains, are highly refractory to conventional antibiotic therapy. Nitroxides were recently shown to induce the dispersal of Gram-negative biofilms , but their action against Gram-positive bacterial biofilms remains unknown. Here, we demonstrate that the biofilm dispersal activity of nitroxides extends to , a clinically important Gram-positive pathogen. Coadministration of the nitroxide CTEMPO (4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl) with ciprofloxacin significantly improved the biofilm eradication activity of the antibiotic against Moreover, covalently linking the nitroxide to the antibiotic moiety further reduced the ciprofloxacin minimal biofilm eradication concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids could penetrate biofilms and enter cells localized at the surface and base of the biofilm structure. No toxicity to human cells was observed for the nitroxide CTEMPO or the nitroxide-antibiotic hybrids. Taken together, our results show that nitroxides can mediate the dispersal of Gram-positive biofilms and that dual-acting biofilm eradication antibiotics may provide broad-spectrum therapies for the treatment of biofilm-related infections.
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http://dx.doi.org/10.1128/AAC.01685-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187575PMC
December 2019

Eradicating uropathogenic biofilms with a ciprofloxacin-dinitroxide conjugate.

Medchemcomm 2019 May 25;10(5):699-711. Epub 2019 Feb 25.

Queensland University of Technology , School of Chemistry, Physics and Mechanical Engineering , 2 George St , Brisbane , Queensland 4001 , Australia . Email:

Urinary tract infections (UTIs) are amongst the most common and prevalent infectious diseases worldwide, with uropathogenic (UPEC) reported as the main causative pathogen. Fluoroquinolone antibiotics are commonly used to treat UTIs but for infections involving UPEC biofilms, which are commonly associated with catheter use and recurrent episodes, ciprofloxacin is often ineffective. Here we report the development of a ciprofloxacin-dinitroxide (CDN) conjugate with potent UPEC biofilm-eradication activity. CDN exhibited a 2-fold increase in potency over the parent antibiotic ciprofloxacin against UPEC biofilms. Moreover, CDN resulted in almost complete UPEC biofilm cell eradication (99.7%) at concentrations as low as 12.5 μM, and significantly potentiated ciprofloxacin's biofilm-eradication activity against UPEC upon co-administration. The biofilm-eradication activity of CDN highlights the potential of nitroxide functionalized antibiotics as a promising strategy for the treatment of biofilm-related UTIs.
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http://dx.doi.org/10.1039/c9md00062cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533797PMC
May 2019

Profluorescent Fluoroquinolone-Nitroxides for Investigating Antibiotic⁻Bacterial Interactions.

Antibiotics (Basel) 2019 Mar 4;8(1). Epub 2019 Mar 4.

Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4006, Australia.

Fluorescent probes are widely used for imaging and measuring dynamic processes in living cells. Fluorescent antibiotics are valuable tools for examining antibiotic⁻bacterial interactions, antimicrobial resistance and elucidating antibiotic modes of action. Profluorescent nitroxides are 'switch on' fluorescent probes used to visualize and monitor intracellular free radical and redox processes in biological systems. Here, we have combined the inherent fluorescent and antimicrobial properties of the fluoroquinolone core structure with the fluorescence suppression capabilities of a nitroxide to produce the first example of a profluorescent fluoroquinolone-nitroxide probe. Fluoroquinolone-nitroxide (FN) exhibited significant suppression of fluorescence (>36-fold), which could be restored via radical trapping (fluoroquinolone-methoxyamine ) or reduction to the corresponding hydroxylamine . Importantly, FN was able to enter both Gram-positive and Gram-negative bacterial cells, emitted a measurable fluorescence signal upon cell entry (switch on), and retained antibacterial activity. In conclusion, profluorescent nitroxide antibiotics offer a new powerful tool for visualizing antibiotic⁻bacterial interactions and researching intracellular chemical processes.
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http://dx.doi.org/10.3390/antibiotics8010019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466543PMC
March 2019

Author Correction: A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance.

Nat Commun 2019 03 1;10(1):976. Epub 2019 Mar 1.

Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD, 4072, Australia.

This Article contains errors in Fig. 1, Table 1 and the Methods section. In panel c, the labels for PmScsC and EcDsbC in the upper two curves are interchanged. In Table 1 and the Methods section entitled 'Extended structure', the space group of the extended PmScsC structure is incorrectly referred to as H3 and should read H32. Correct versions of Fig. 1 and Table 1 are presented below; the errors have not been corrected in the Article.
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http://dx.doi.org/10.1038/s41467-019-08920-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397206PMC
March 2019

Moraxella catarrhalis NucM is an entry nuclease involved in extracellular DNA and RNA degradation, cell competence and biofilm scaffolding.

Sci Rep 2019 02 22;9(1):2579. Epub 2019 Feb 22.

Institute for Glycomics, Griffith University, Gold Coast, Queensland, 4215, Australia.

Moraxella catarrhalis is a host-adapted bacterial pathogen that causes otitis media and exacerbations of chronic obstructive pulmonary disease. This study characterises the conserved M. catarrhalis extracellular nuclease, a member of the ββα metal finger family of nucleases, that we have named NucM. NucM shares conserved sequence motifs from the ββα nuclease family, including the DRGH catalytic core and Mg co-ordination site, but otherwise shares little primary sequence identity with other family members, such as the Serratia Nuc and pneumococcal EndA nucleases. NucM is secreted from the cell and digests linear and circular nucleic acid. However, it appears that a proportion of NucM is also associated with the cell membrane and acts as an entry nuclease, facilitating transformation of M. catarrhalis cells. This is the first example of a ββα nuclease in a Gram negative bacteria that acts as an entry nuclease. In addition to its role in competence, NucM affects cell aggregation and biofilm formation by M. catarrhalis, with ΔnucM mutants having increased biofilm biomass. NucM is likely to increase the ability of cells to survive and persist in vivo, increasing the virulence of M. catarrhalis and potentially affecting the behaviour of other pathogens that co-colonise the otorhinolaryngological niche.
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http://dx.doi.org/10.1038/s41598-019-39374-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384898PMC
February 2019

Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.

FASEB J 2019 02 27;33(2):2095-2104. Epub 2018 Sep 27.

Institute for Glycomics, Griffith University, Queensland, Australia.

Bacterial infection is one of the leading causes of death in young, elderly, and immune-compromised patients. The rapid spread of multi-drug-resistant (MDR) bacteria is a global health emergency and there is a lack of new drugs to control MDR pathogens. We describe a heretofore-unexplored discovery pathway for novel antibiotics that is based on self-targeting, structure-disrupting peptides. We show that a helical peptide, KFF- EcH3, derived from the Escherichia coli methionine aminopeptidase can disrupt secondary and tertiary structure of this essential enzyme, thereby killing the bacterium (including MDR strains). Significantly, no detectable resistance developed against this peptide. Based on a computational analysis, our study predicted that peptide KFF- EcH3 has the strongest interaction with the structural core of the methionine aminopeptidase. We further used our approach to identify peptide KFF- NgH1 to target the same enzyme from Neisseria gonorrhoeae. This peptide inhibited bacterial growth and was able to treat a gonococcal infection in a human cervical epithelial cell model. These findings present an exciting new paradigm in antibiotic discovery using self-derived peptides that can be developed to target the structures of any essential bacterial proteins.-Zhan, J., Jia, H., Semchenko, E. A., Bian, Y., Zhou, A. M., Li, Z., Yang, Y., Wang, J., Sarkar, S., Totsika, M., Blanchard, H., Jen, F. E.-C., Ye, Q., Haselhorst, T., Jennings, M. P., Seib, K. L., Zhou, Y. Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.
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http://dx.doi.org/10.1096/fj.201700613RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338635PMC
February 2019

The Performance of an Oral Microbiome Biomarker Panel in Predicting Oral Cavity and Oropharyngeal Cancers.

Front Cell Infect Microbiol 2018 3;8:267. Epub 2018 Aug 3.

The School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia.

The oral microbiome can play a role in the instigation and progression of oral diseases that can manifest into other systemic conditions. These associations encourage the exploration of oral dysbiosis leading to the pathogenesis of cancers. In this study, oral rinse was used to characterize the oral microbiome fluctuation associated with oral cavity cancer (OCC) and oropharyngeal cancers (OPC). The study cohort consists of normal healthy controls ( = 10, between 20 and 30 years of age; = 10, above 50 years of age), high-risk individuals ( = 11, above 50 years of age with bad oral hygiene and/or oral diseases) and OCC and OPC patients ( = 31, HPV-positive; = 21, HPV-negative). Oral rinse samples were analyzed using 16S rRNA gene amplicon sequencing on the MiSeq platform. Kruskal-Wallis rank test was used to identify genera associated with OCC and OPC. A logistic regression analysis was carried out to determine the performance of these genera as a biomarker panel to predict OCC and OPC. In addition, a two-fold cross-validation with a bootstrap procedure was carried out in R to investigate how well the panel would perform in an emulated clinical scenario. Our data indicate that the oral microbiome is able to predict the presence of OCC and OPC with sensitivity and specificity of 100 and 90%, respectively. With further validation, the panel could potentially be implemented into clinical diagnostic and prognostic workflows for OCC and OPC.
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http://dx.doi.org/10.3389/fcimb.2018.00267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085444PMC
August 2019

Intestinal Colonization Traits of Pandemic Multidrug-Resistant Escherichia coli ST131.

J Infect Dis 2018 08;218(6):979-990

Institute of Health and Biomedical Innovation, Queensland University of Technology, Queensland, Australia.

Background: Epidemiological studies point to the gut as a key reservoir of multidrug resistant Escherichia coli multilocus sequence type 131 (ST131), a globally dominant pathogenic clone causing urinary tract and bloodstream infections. Here we report a detailed investigation of its intestinal lifestyle.

Methods: Clinical ST131 isolates and type 1 fimbriae null mutants were assessed for colonization of human intestinal epithelia and in mouse intestinal colonization models. Mouse gut tissue underwent histologic analysis for pathology and ST131 localization. Key findings were corroborated in mucus-producing human cell lines and intestinal biopsy specimens.

Results: ST131 strains adhered to and invaded human intestinal epithelial cells more than probiotic and commensal strains. The reference ST131 strain EC958 established persistent intestinal colonization in mice, and expression of type 1 fimbriae mediated higher colonization levels. Bacterial loads were highest in the distal parts of the mouse intestine and did not cause any obvious pathology. Further analysis revealed that EC958 could bind to both mucus and underlying human intestinal epithelia.

Conclusions: ST131 strains can efficiently colonize the mammalian gut and persist long term. Type 1 fimbriae enhance ST131 intestinal colonization, suggesting that mannosides, currently developed as therapeutics for bladder infections and Crohn's disease, could also be used to limit intestinal ST131 reservoirs.
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http://dx.doi.org/10.1093/infdis/jiy031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093498PMC
August 2018

Inhibition of Diverse DsbA Enzymes in Multi-DsbA Encoding Pathogens.

Antioxid Redox Signal 2018 09 1;29(7):653-666. Epub 2018 Feb 1.

2 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University , Bundoora, Australia .

Aims: DsbA catalyzes disulfide bond formation in secreted and outer membrane proteins in bacteria. In pathogens, DsbA is a major facilitator of virulence constituting a target for antivirulence antimicrobial development. However, many pathogens encode multiple and diverse DsbA enzymes for virulence factor folding during infection. The aim of this study was to determine whether our recently identified inhibitors of Escherichia coli K-12 DsbA can inhibit the diverse DsbA enzymes found in two important human pathogens and attenuate their virulence.

Results: DsbA inhibitors from two chemical classes (phenylthiophene and phenoxyphenyl derivatives) inhibited the virulence of uropathogenic E. coli and Salmonella enterica serovar Typhimurium, encoding two and three diverse DsbA homologues, respectively. Inhibitors blocked the virulence of dsbA null mutants complemented with structurally diverse DsbL and SrgA, suggesting that they were not selective for prototypical DsbA. Structural characterization of DsbA-inhibitor complexes showed that compounds from each class bind in a similar region of the hydrophobic groove adjacent to the Cys30-Pro31-His32-Cys33 (CPHC) active site. Modeling of DsbL- and SrgA-inhibitor interactions showed that these accessory enzymes could accommodate the inhibitors in their different hydrophobic grooves, supporting our in vivo findings. Further, we identified highly conserved residues surrounding the active site for 20 diverse bacterial DsbA enzymes, which could be exploited in developing inhibitors with a broad spectrum of activity. Innovation and Conclusion: We have developed tools to analyze the specificity of DsbA inhibitors in bacterial pathogens encoding multiple DsbA enzymes. This work demonstrates that DsbA inhibitors can be developed to target diverse homologues found in bacteria. Antioxid. Redox Signal. 29, 653-666.
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http://dx.doi.org/10.1089/ars.2017.7104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067686PMC
September 2018

Oral Microbiome: A New Biomarker Reservoir for Oral and Oropharyngeal Cancers.

Theranostics 2017 26;7(17):4313-4321. Epub 2017 Sep 26.

The School of Biomedical Sciences, Institute of Health and Biomedical Innovations, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Brisbane, QLD 4059, Australia.

Current biomarkers (DNA, RNA and protein) for oral cavity and oropharyngeal cancers demonstrate biological variations between individuals, rendering them impractical for clinical translation. Whilst these biomarkers originate from the host, there is not much information in the literature about the influence of oral microbiota on cancer pathogenesis, especially in oral cancers. Oral microbiotas are known to participate in disease initiation and progression not only limited to the oral cavity, but also at other distant sites. Due to the close proximity of oral microbiota and oral cavity and oropharyngeal tumours, abundance changes in oral microbiota may provide useful information on tumourigenesis. This review aims to highlight information on the role of oral microbiota in oral cavity and oropharyngeal cancers. An in-depth analysis into the oral microbiota may provide a new avenue to diagnose and treat these patients.
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http://dx.doi.org/10.7150/thno.21804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695015PMC
July 2018

YeeJ is an inverse autotransporter from Escherichia coli that binds to peptidoglycan and promotes biofilm formation.

Sci Rep 2017 09 12;7(1):11326. Epub 2017 Sep 12.

Institut Pasteur, Unité de Génétique des Biofilms, 28 rue du Dr. Roux, 75724, Paris, CEDEX 15, France.

Escherichia coli is a commensal or pathogenic bacterium that can survive in diverse environments. Adhesion to surfaces is essential for E. coli colonization, and thus it is important to understand the molecular mechanisms that promote this process in different niches. Autotransporter proteins are a class of cell-surface factor used by E. coli for adherence. Here we characterized the regulation and function of YeeJ, a poorly studied but widespread representative from an emerging class of autotransporter proteins, the inverse autotransporters (IAT). We showed that the yeeJ gene is present in ~40% of 96 completely sequenced E. coli genomes and that YeeJ exists as two length variants, albeit with no detectable functional differences. We demonstrated that YeeJ promotes biofilm formation in different settings through exposition at the cell-surface. We also showed that YeeJ contains a LysM domain that interacts with peptidoglycan and thus assists its localization into the outer membrane. Additionally, we identified the Polynucleotide Phosphorylase PNPase as a repressor of yeeJ transcription. Overall, our work provides new insight into YeeJ as a member of the recently defined IAT class, and contributes to our understanding of how commensal and pathogenic E. coli colonise their environments.
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http://dx.doi.org/10.1038/s41598-017-10902-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595812PMC
September 2017

The saliva microbiome profiles are minimally affected by collection method or DNA extraction protocols.

Sci Rep 2017 08 17;7(1):8523. Epub 2017 Aug 17.

The School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Brisbane, QLD, 4059, Australia.

Saliva has attracted attention as a diagnostic fluid due to the association of oral microbiota with systemic diseases. However, the lack of standardised methods for saliva collection has led to the slow uptake of saliva in microbiome research. The aim of this study was to systematically evaluate the potential effects on salivary microbiome profiles using different methods of saliva collection, storage and gDNA extraction. Three types of saliva fractions were collected from healthy individuals with or without the gDNA stabilising buffer. Subsequently, three types of gDNA extraction methods were evaluated to determine the gDNA extraction efficiencies from saliva samples. The purity of total bacterial gDNA was evaluated using the ratio of human β-globin to bacterial 16S rRNA PCR while 16S rRNA gene amplicon sequencing was carried out to identify the bacterial profiles present in these samples. The quantity and quality of extracted gDNA were similar among all three gDNA extraction methods and there were no statistically significant differences in the bacterial profiles among different saliva fractions at the genus-level of taxonomic classification. In conclusion, saliva sampling, processing and gDNA preparation do not have major influence on microbiome profiles.
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http://dx.doi.org/10.1038/s41598-017-07885-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561025PMC
August 2017

A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance.

Nat Commun 2017 07 19;8:16065. Epub 2017 Jul 19.

Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.

Copper resistance is a key virulence trait of the uropathogen Proteus mirabilis. Here we show that P. mirabilis ScsC (PmScsC) contributes to this defence mechanism by enabling swarming in the presence of copper. We also demonstrate that PmScsC is a thioredoxin-like disulfide isomerase but, unlike other characterized proteins in this family, it is trimeric. PmScsC trimerization and its active site cysteine are required for wild-type swarming activity in the presence of copper. Moreover, PmScsC exhibits unprecedented motion as a consequence of a shape-shifting motif linking the catalytic and trimerization domains. The linker accesses strand, loop and helical conformations enabling the sampling of an enormous folding landscape by the catalytic domains. Mutation of the shape-shifting motif abolishes disulfide isomerase activity, as does removal of the trimerization domain, showing that both features are essential to foldase function. More broadly, the shape-shifter peptide has the potential for 'plug and play' application in protein engineering.
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http://dx.doi.org/10.1038/ncomms16065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524982PMC
July 2017

Disarming pathogens: benefits and challenges of antimicrobials that target bacterial virulence instead of growth and viability.

Authors:
Makrina Totsika

Future Med Chem 2017 03 16;9(3):267-269. Epub 2017 Feb 16.

Institute of Health & Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4059, Australia.

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http://dx.doi.org/10.4155/fmc-2016-0227DOI Listing
March 2017

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection.

Sci Rep 2016 10 21;6:35285. Epub 2016 Oct 21.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia.

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.
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http://dx.doi.org/10.1038/srep35285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073308PMC
October 2016

Differential Regulation of the Surface-Exposed and Secreted SslE Lipoprotein in Extraintestinal Pathogenic Escherichia coli.

PLoS One 2016 6;11(9):e0162391. Epub 2016 Sep 6.

Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, QLD 4072, Brisbane, Australia.

Extra-intestinal pathogenic Escherichia coli (ExPEC) are responsible for diverse infections including meningitis, sepsis and urinary tract infections. The alarming rise in anti-microbial resistance amongst ExPEC complicates treatment and has highlighted the need for alternative preventive measures. SslE is a lipoprotein secreted by a dedicated type II secretion system in E. coli that was first identified as a potential vaccine candidate using reverse genetics. Although the function and protective efficacy of SslE has been studied, the molecular mechanisms that regulate SslE expression remain to be fully elucidated. Here, we show that while the expression of SslE can be detected in E. coli culture supernatants, different strains express and secrete different amounts of SslE when grown under the same conditions. While the histone-like transcriptional regulator H-NS strongly represses sslE at ambient temperatures, the variation in SslE expression at human physiological temperature suggested a more complex mode of regulation. Using a genetic screen to identify novel regulators of sslE in the high SslE-expressing strain UTI89, we defined a new role for the nucleoid-associated regulator Fis and the ribosome-binding GTPase TypA as positive regulators of sslE transcription. We also showed that Fis-mediated enhancement of sslE transcription is dependent on a putative Fis-binding sequence located upstream of the -35 sequence in the core promoter element, and provide evidence to suggest that Fis may work in complex with H-NS to control SslE expression. Overall, this study has defined a new mechanism for sslE regulation and increases our understanding of this broadly conserved E. coli vaccine antigen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162391PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012682PMC
August 2017

Comprehensive analysis of type 1 fimbriae regulation in fimB-null strains from the multidrug resistant Escherichia coli ST131 clone.

Mol Microbiol 2016 09 15;101(6):1069-87. Epub 2016 Jul 15.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, 4072, Australia.

Uropathogenic Escherichia coli (UPEC) of sequence type 131 (ST131) are a pandemic multidrug resistant clone associated with urinary tract and bloodstream infections. Type 1 fimbriae, a major UPEC virulence factor, are essential for ST131 bladder colonization. The globally dominant sub-lineage of ST131 strains, clade C/H30-R, possess an ISEc55 insertion in the fimB gene that controls phase-variable type 1 fimbriae expression via the invertible fimS promoter. We report that inactivation of fimB in these strains causes altered regulation of type 1 fimbriae expression. Using a novel read-mapping approach based on Illumina sequencing, we demonstrate that 'off' to 'on' fimS inversion is reduced in these strains and controlled by recombinases encoded by the fimE and fimX genes. Unlike typical UPEC strains, the nucleoid-associated H-NS protein does not strongly repress fimE transcription in clade C ST131 strains. Using a genetic screen to identify novel regulators of fimE and fimX in the clade C ST131 strain EC958, we defined a new role for the guaB gene in the regulation of type 1 fimbriae and in colonisation of the mouse bladder. Our results provide a comprehensive analysis of type 1 fimbriae regulation in ST131, and highlight important differences in its control compared to non-ST131 UPEC.
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http://dx.doi.org/10.1111/mmi.13442DOI Listing
September 2016

Biofilm formation by multidrug resistant Escherichia coli ST131 is dependent on type 1 fimbriae and assay conditions.

Pathog Dis 2016 Apr 2;74(3). Epub 2016 Mar 2.

Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland 4059, Australia School of Biomedical Sciences, Queensland University of Technology, Kelvin Grove, Queensland 4059, Australia

Escherichia coli sequence type 131 (ST131) has emerged as a pandemic lineage of important multidrug resistant pathogens worldwide. Despite many studies examining the epidemiology of ST131, only a few studies to date have investigated the capacity of ST131 strains to form biofilms. Some of these studies have reported contrasting findings, with no specific ST131 biofilm-promoting factors identified. Here, we examined a diverse collection of ST131 isolates for in vitro biofilm formation in different media and assay conditions, including urine from healthy adult women. We found significant differences among strains and assay conditions, which offers an explanation for the contrasting findings reported by previous studies using a single condition. Importantly, we showed that expression of type 1 fimbriae is a critical determinant for biofilm formation by ST131 strains and that inhibition of the FimH adhesin significantly reduces biofilm formation. We also offer direct genetic evidence for the contribution of type 1 fimbriae in biofilm formation by the reference ST131 strain EC958, a representative of the clinically dominant H30-Rx ST131 subgroup. This is the first study of ST131 biofilm formation in biologically relevant conditions and paves the way for the application of FimH inhibitors in treating drug resistant ST131 biofilm infections.
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http://dx.doi.org/10.1093/femspd/ftw013DOI Listing
April 2016

Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli.

J Bacteriol 2016 05 28;198(10):1487-98. Epub 2016 Apr 28.

Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia

Unlabelled: The vacuolating autotransporter toxin (Vat) contributes to uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator; we termed this gene vatX The vat-vatX genes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, the hns gene was mutated in CFT073 (to generate CFT073 hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073 hns compared to that in wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein of Enterobacteriaceae (SPATE) secreted by UPEC during infection.

Importance: Uropathogenic Escherichia coli (UPEC) is the major cause of hospital- and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation of vat was demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream gene vatX (encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected with vat-positive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor.
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http://dx.doi.org/10.1128/JB.00791-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859599PMC
May 2016

The role of H4 flagella in Escherichia coli ST131 virulence.

Sci Rep 2015 Nov 9;5:16149. Epub 2015 Nov 9.

School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia.

Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug resistant clone associated with urinary tract and bloodstream infections. Most ST131 strains exhibit resistance to multiple antibiotics and cause infections associated with limited treatment options. The largest sub-clonal ST131 lineage is resistant to fluoroquinolones, contains the type 1 fimbriae fimH30 allele and expresses an H4 flagella antigen. Flagella are motility organelles that contribute to UPEC colonisation of the upper urinary tract. In this study, we examined the specific role of H4 flagella in ST131 motility and interaction with host epithelial and immune cells. We show that the majority of H4-positive ST131 strains are motile and are enriched for flagella expression during static pellicle growth. We also tested the role of H4 flagella in ST131 through the construction of specific mutants, over-expression strains and isogenic mutants that expressed alternative H1 and H7 flagellar subtypes. Overall, our results revealed that H4, H1 and H7 flagella possess conserved phenotypes with regards to motility, epithelial cell adhesion, invasion and uptake by macrophages. In contrast, H4 flagella trigger enhanced induction of the anti-inflammatory cytokine IL-10 compared to H1 and H7 flagella, a property that may contribute to ST131 fitness in the urinary tract.
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http://dx.doi.org/10.1038/srep16149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637896PMC
November 2015

Comparative proteomics of uropathogenic Escherichia coli during growth in human urine identify UCA-like (UCL) fimbriae as an adherence factor involved in biofilm formation and binding to uroepithelial cells.

J Proteomics 2016 Jan 3;131:177-189. Epub 2015 Nov 3.

Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia. Electronic address:

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infection (UTI) in humans. For the successful colonisation of the human urinary tract, UPEC employ a diverse collection of secreted or surface-exposed virulence factors including toxins, iron acquisition systems and adhesins. In this study, a comparative proteomic approach was utilised to define the UPEC pan and core surface proteome following growth in pooled human urine. Identified proteins were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Fourteen core surface proteins were identified, as well as eleven iron uptake receptor proteins and four distinct fimbrial types, including type 1, P, F1C/S and a previously uncharacterised fimbrial type, designated UCA-like (UCL) fimbriae in this study. These pathogenicity island (PAI)-associated fimbriae are related to UCA fimbriae of Proteus mirabilis, associated with UPEC and exclusively found in members of the E. coli B2 and D phylogroup. We further demonstrated that UCL fimbriae promote significant biofilm formation on abiotic surfaces and mediate specific attachment to exfoliated human uroepithelial cells. Combined, this study has defined the surface proteomic profiles and core surface proteome of UPEC during growth in human urine and identified a new type of fimbriae that may contribute to UTI.
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http://dx.doi.org/10.1016/j.jprot.2015.11.001DOI Listing
January 2016

Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition.

Infect Immun 2015 May 9;83(5):1749-64. Epub 2015 Feb 9.

Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.
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http://dx.doi.org/10.1128/IAI.02810-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399054PMC
May 2015

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

Angew Chem Int Ed Engl 2015 Feb 30;54(7):2179-84. Epub 2014 Dec 30.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052 (Australia) http://www.pharm.monash.edu.au.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.
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http://dx.doi.org/10.1002/anie.201410341DOI Listing
February 2015