Publications by authors named "Yaoqin Hong"

12 Publications

  • Page 1 of 1

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

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

Two extremely divergent sequence forms of the genes that define Escherichia coli group 3 capsules suggest a very long history since their common ancestor.

FEMS Microbiol Lett 2019 04;366(8)

School of Life and Environmental Sciences, The University of Sydney, Camperdown, NSW 2006, Australia.

Capsules are a critical virulence factor in many pathogenic Escherichia coli, of which groups 2 and 3 capsules are synthesised by the ABC transporter pathway. The well-studied forms are in group 2 and much of our knowledge of group 3 is inferred from our understanding of group 2. We analyse six group 3 gene clusters including representatives of K10, K11 and K96, and find unexpected diversity. Groups 2 and 3 both have gene clusters with terminal regions 1 and 3 containing mostly genes shared by all members of both groups, plus a central region 2, that in group 2 has the genes for synthesising the serotype-specific repeat unit. We find that in all but one case group 3 gene clusters include, in addition to serotype-specific genes, a previously unrecognised set of shared genes in region 2 that probably codes for an additional structural element. Also, the six shared genes in regions 1 and 3 of group 3 exist in two very different sequence forms. It appears that the E. coli ABC transporter capsules have a very long history, with more fundamental diversity present in group 3, but greater diversity in the exposed strongly antigenic serotype-specific component encoded by region 2.
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http://dx.doi.org/10.1093/femsle/fnz091DOI Listing
April 2019

Development and retention of a primordial moonlighting pathway of protein modification in the absence of selection presents a puzzle.

Proc Natl Acad Sci U S A 2018 01 16;115(4):647-655. Epub 2018 Jan 16.

Department of Biochemistry, University of Illinois at Urbana-Champagne, Urbana, IL 61801;

Lipoic acid is synthesized by a remarkably atypical pathway in which the cofactor is assembled on its cognate proteins. An octanoyl moiety diverted from fatty acid synthesis is covalently attached to the acceptor protein, and sulfur insertion at carbons 6 and 8 of the octanoyl moiety form the lipoyl cofactor. Covalent attachment of this cofactor is required for function of several central metabolism enzymes, including the glycine cleavage H protein (GcvH). In , GcvH is the sole substrate for lipoate assembly. Hence lipoic acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the cofactor only by transfer from lipoylated GcvH. Lipoyl transfer has been argued to be the primordial pathway of OADH lipoylation. The pathway where lipoate is directly assembled on both its GcvH and OADH proteins, is proposed to have arisen later. Because roughly 3 billion years separate the divergence of these bacteria, it is surprising that GcvH functionally substitutes for the protein in lipoyl transfer. Known and putative GcvHs from other bacteria and eukaryotes also substitute for GcvH in OADH modification. Because glycine cleavage is the primary GcvH role in ancestral bacteria that lack OADH enzymes, lipoyl transfer is a "moonlighting" function: that is, development of a new function while retaining the original function. This moonlighting has been conserved in the absence of selection by some, but not all, GcvH proteins. Moreover, encodes five putative GcvHs, two of which have the moonlighting function, whereas others function only in glycine cleavage.
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http://dx.doi.org/10.1073/pnas.1718653115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789953PMC
January 2018

Progress in Our Understanding of Wzx Flippase for Translocation of Bacterial Membrane Lipid-Linked Oligosaccharide.

J Bacteriol 2018 01 5;200(1). Epub 2017 Dec 5.

School of Life and Environmental Sciences, University of Sydney, Camperdown, NSW, Australia

Translocation of lipid-linked oligosaccharides is a common theme across prokaryotes and eukaryotes. For bacteria, such activity is used in cell wall construction, polysaccharide synthesis, and the relatively recently discovered protein glycosylation. To the best of our knowledge, the Gram-negative inner membrane flippase Wzx was the first protein identified as being involved in oligosaccharide translocation, and yet we still have only a limited understanding of this protein after 3 decades of research. At present, Wzx is known to be a multitransmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varied degrees of preference. In this review, we provide an overview of the major findings for this protein, with a particular focus on substrate preference.
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http://dx.doi.org/10.1128/JB.00154-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717161PMC
January 2018

Model for the Controlled Synthesis of O-Antigen Repeat Units Involving the WaaL Ligase.

mSphere 2016 Jan-Feb;1(1). Epub 2015 Dec 30.

School of Molecular Bioscience (D17), The University of Sydney, New South Wales, Australia.

The Wzx/Wzy O-antigen pathway involves synthesis of a repeat unit (O unit) consisting of 3 to 8 sugars on an inner-membrane-embedded lipid carrier. These O units are translocated across the membrane to its periplasmic face by Wzx, while retaining linkage to the carrier, and then polymerized by Wzy to O-antigen polymer, which WaaL ligase transfers to a lipopolysaccharide precursor to complete lipopolysaccharide synthesis, concomitantly releasing the lipid carrier. This lipid carrier is also used for peptidoglycan assembly, and sequestration is known to be toxic. Thus, O-unit synthesis must involve precise regulation to meet demand but avoid overproduction. Here we show that loss of WaaL reverses a known growth defect in a Salmonella mutant that otherwise accumulates O-unit intermediates and propose that WaaL is also involved in a novel feedback mechanism to regulate O-unit synthesis, based on the availability of O units on the periplasmic face of the membrane.
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http://dx.doi.org/10.1128/mSphere.00074-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863624PMC
June 2016

Three Wzy polymerases are specific for particular forms of an internal linkage in otherwise identical O units.

Microbiology (Reading) 2015 Aug 18;161(8):1639-1647. Epub 2015 May 18.

School of Molecular Bioscience, Building D17, University of Sydney, NSW 2006, Australia.

The Wzx/Wzy-dependent pathway is the predominant pathway for O-antigen production in Gram-negative bacteria. The O-antigen repeat unit (O unit) is an oligosaccharide that is assembled at the cytoplasmic face of the membrane on undecaprenyl pyrophosphate. Wzx then flips it to the periplasmic face for polymerization by Wzy, which adds an O unit to the reducing end of a growing O-unit polymer in each round of polymerization. Wzx and Wzy both exhibit enormous sequence diversity. It has recently been shown that, contrary to earlier reports, the efficiency of diverse Wzx forms can be significantly reduced by minor structural variations to their native O-unit substrate. However, details of Wzy substrate specificity remain unexplored. The closely related galactose-initiated Salmonella O antigens present a rare opportunity to address these matters. The D1 and D2 O units differ only in an internal mannose-rhamnose linkage, and D3 expresses both in the same chain. D1 and D2 polymerases were shown to be specific for O units with their respective α or β configuration for the internal mannose-rhamnose linkage. The Wzy encoded by D3 gene cluster polymerizes only D1 O units, and deleting the gene does not eliminate polymeric O antigen, both observations indicating the presence of an additional wzy gene. The levels of Wzx and Wzy substrate specificity will affect the ease with which new O units can evolve, and also our ability to modify O antigens, capsules or secreted polysaccharides by glyco-engineering, to generate novel polysaccharides, as the Wzx/Wzy-dependent pathway is responsible for much of the diversity.
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http://dx.doi.org/10.1099/mic.0.000113DOI Listing
August 2015

Inefficient translocation of a truncated O unit by a Salmonella Wzx affects both O-antigen production and cell growth.

FEMS Microbiol Lett 2015 May 2;362(9). Epub 2015 Apr 2.

School of Molecular Bioscience, Charles Perkins Centre (D17), the University of Sydney, Camperdown, NSW 2006, Australia

Bacterial Wzx flippases translocate (flip) short oligosaccharide repeat units (O units) across the inner membrane into the periplasm, which is a critical step in the assembly of many O antigens, capsules and other surface polysaccharides. There is enormous diversity in O antigens and capsules in particular, even within species. Wzx proteins are similarly diverse, but it has been widely accepted that they have significant specificity only for the first sugar of an O unit. In this study, we analysed the Wzx from the Salmonella enterica group C2 O antigen gene cluster, which is a unique and divergent member of a set of gene clusters that produce galactose-initiated O antigens. We demonstrate that this Wzx has a strong preference for the presence of an abequose side-branch, which manifests in a reduction of long-chain O antigen and a major growth defect. This contributes to a growing body of evidence that, contrary to earlier proposals, Wzx flippases commonly exhibit a strong preference for the structure of their native O unit.
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http://dx.doi.org/10.1093/femsle/fnv053DOI Listing
May 2015

Diversity of o-antigen repeat unit structures can account for the substantial sequence variation of wzx translocases.

J Bacteriol 2014 May 14;196(9):1713-22. Epub 2014 Feb 14.

School of Molecular Bioscience (G08), University of Sydney, New South Wales, Australia.

The most common system for synthesis of cell surface polysaccharides is the Wzx/Wzy-dependent pathway, which involves synthesis, on the cytoplasmic face of the cell membrane, of repeat units, which are then translocated to the periplasmic face by a Wzx translocase and then polymerized by Wzy to generate the polysaccharide. One such polysaccharide is O antigen, which is incorporated into lipopolysaccharide (LPS). The O antigen is extremely variable, with over 186 forms in Escherichia coli. Wzx proteins are also very diverse, but they have been thought to be specific only for the first sugar of the repeat units. However, recent studies demonstrated examples in which Wzx translocases have considerable preference for their native repeat unit, showing that specificity can extend well beyond the first sugar. These results appear to be in conflict with the early conclusions, but they involved specificity for side branch residues and could be a special case. Here we take six Wzx translocases that were critical in the earlier studies on the importance of the first sugar and assess their ability to translocate the Escherichia coli O16 and O111 repeat units. We use gene replacements to optimize maintenance of expression level and show that under these conditions the native translocases are the most effective for their native repeat unit, being, respectively, 64-fold and 4-fold more effective than the next best. We conclude that Wzx translocases are commonly adapted to their native repeat unit, which provides an explanation for the great diversity of wzx genes.
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http://dx.doi.org/10.1128/JB.01323-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993327PMC
May 2014

The WbaK acetyltransferase of Salmonella enterica group E gives insights into O antigen evolution.

Microbiology (Reading) 2013 Nov 6;159(Pt 11):2316-2322. Epub 2013 Sep 6.

School of Molecular Bioscience, University of Sydney, New South Wales 2006, Australia.

O antigens are polysaccharides consisting of repeat units of three to eight sugars, generally assembled by genes in a discrete O antigen gene cluster. Salmonella enterica produces 46 forms of O antigen, and most of the variation is determined by genes in the gene cluster. However in some cases the structures are modified by enzymes encoded outside of the gene cluster, and several such modifications have been reported for Salmonella enterica group E, some with the genes on bacteriophages and one gene at a distant chromosomal site. We identified the enzyme, WbaK, that is responsible for O-acetylating the subgroup E1 O antigen, and found that the gene is located just downstream of the gene cluster as currently known. The wbaK gene appears to have been imported by a recombination event that also replaced the last 37 bp of the wbaP gene, indicating that homologous recombination was involved. Some of the group E strains we studied must have the original gene cluster, as they lack wbaK and the sequence downstream of wbaP is very similar to that in several other S. enterica O antigen gene clusters. In effect the gene cluster was extended by one gene in subgroup E1. It appears that a function that is usually encoded by a gene outside of the gene cluster has been added to the gene cluster, in this case giving an example of how such gene clusters can evolve.
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http://dx.doi.org/10.1099/mic.0.069823-0DOI Listing
November 2013

The Wzx translocases for Salmonella enterica O-antigen processing have unexpected serotype specificity.

Mol Microbiol 2012 May 13;84(4):620-30. Epub 2012 Apr 13.

School of Molecular Bioscience, The University of Sydney, Sydney, NSW, Australia.

Most Gram-negative bacteria have an O antigen, a polysaccharide with many repeats of a short oligosaccharide that is a part of the lipopolysaccharide, the major lipid in the outer leaflet of the outer membrane. Lipopolysaccharide is variable with 46 forms in Salmonella enterica that underpin the serotyping scheme. Repeat units are assembled on a lipid carrier that is embedded in the cell membrane, and are then translocated by the Wzx translocase from the cytoplasmic face to the outer face of the cell membrane, followed by polymerization. The O antigen is then incorporated into lipopolysaccharide and exported to the outer membrane. The Wzx translocase is widely thought to be specific only for the first sugar of the repeat unit, despite extensive variation in both O antigens and Wzx translocases. However, we found for S. enterica groups B, D2 and E that Wzx translocation exhibits significant specificity for the repeat-unit structure, as variants with single sugar differences are translocated with lower efficiency and little long-chain O antigen is produced. It appears that Wzx translocases are specific for their O antigen for normal levels of translocation.
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http://dx.doi.org/10.1111/j.1365-2958.2012.08048.xDOI Listing
May 2012