Julie Chang - Academia.edu (original) (raw)

Papers by Julie Chang

mBio

F. nucleatum colonizes various human tissues, including oral cavity, placenta, and colon. How thi... more F. nucleatum colonizes various human tissues, including oral cavity, placenta, and colon. How this obligate anaerobe withstands oxidative stress in host immune cells has not been described.

Molecular Oral Microbiology, 2022

Actinomyces oris plays an important role in oral biofilm development. Like many Gram-positive bac... more Actinomyces oris plays an important role in oral biofilm development. Like many Gram-positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA - a genetic suppressor of srtA deletion lethality - not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild-type cells, under elevated concentrations of mono- and divalent cations formation of mono- and multi-species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, as compared to the wild-type strain. Altogether, the results support the notion that the cell wall-anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris. This article is protected by copyright. All rights reserved.

Proceedings of the National Academy of Sciences, 2021

Significance Fusobacterium nucleatum interacts with many oral microbes and has the ability to spr... more Significance Fusobacterium nucleatum interacts with many oral microbes and has the ability to spread to the placenta and amniotic fluid, promoting preterm birth. Yet, the molecular mechanisms underlying polymicrobial interactions, termed coaggregation, by Fusobacteria are poorly understood. Here, we revealed that the two-component signal transduction system CarRS regulates expression of genes encoding lysine utilization factors (e.g., KamA) and the coaggregation factor RadD. Extracellular lysine blocks RadD-mediated coaggregation by binding to RadD. Significantly, mutants lacking KamA or CarR (which up-regulates RadD) are attenuated in virulence in a preterm birth model, while mutants devoid of RadD or CarS (which down-regulates RadD) exhibit increased virulence. Our findings unveiled a molecular linkage between coaggregation and lysine metabolism via CarRS-mediated gene regulation that modulates bacterial virulence.

Scientific Reports, 2020

Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the su... more Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of...

Journal of Bacteriology, 1999

The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic... more The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic and acidic residues, with the overall net charge predicted to be positive. The C-terminal domain of λ S was found to be cytoplasmic, as defined by protease accessibility in spheroplasts and inverted membrane vesicles. C-terminal nonsense mutations were constructed in S and found to be lysis proficient, as long as at least one basic residue is retained at the C terminus. In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis. However, the capacity of each truncated lytic allele for inhibition by the S107 inhibitor product of S is retained. The K97am allele, when incorporated into the phage context, confers a plaque-forming defect because its early lysis significantly reduces the burst size. Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis defect of the mutant S A52G, which causes lysis at o...

Proceedings of the National Academy of Sciences, 2019

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enz... more Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris , a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA- deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili...

Journal of the American Chemical Society, Jan 11, 2018

Proteins that are site-specifically modified with peptides and chemicals can be used as novel the... more Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (SrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created SrtA, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. SrtA mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that SrtA can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.

mBio, Jan 20, 2017

The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral b... more The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans-acting factors contributing to these processes. We report here a large-scale Tn5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis, and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn...

Infection and immunity, May 29, 2016

Enterococcus faecium is an important cause of hospital-associated infections, including urinary t... more Enterococcus faecium is an important cause of hospital-associated infections, including urinary tract infections (UTIs), bacteremia and infective endocarditis. Pili have been shown to play a role in the pathogenesis of gram-positive bacteria, including E. faecium. We previously demonstrated that a non-piliated ΔempABC::cat derivative of E. faecium TX82 was attenuated in biofilm formation and in a UTI model. Here, we studied the contribution of the individual pilus subunits EmpA, EmpB and EmpC to pilus architecture, biofilm formation, adherence to extracellular matrix (ECM) proteins and infection. We identified EmpA as the tip of the pili and found that deletion of empA reduced biofilm formation to the same level as deletion of the empABC operon, a phenotype that was restored by reconstituting in situ the empA gene. Deletion of empB also caused reduction in biofilm, while EmpC was found to be dispensable. Significant reduction in adherence to fibrinogen and collagen type I was observ...

Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2016

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as c... more Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker interm...

Molecular Microbiology, 2015

The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many ext... more The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

Disease models & mechanisms, 2015

Oral-facial-digital syndrome (OFD) is a ciliopathy that is characterized by oral-facial abnormali... more Oral-facial-digital syndrome (OFD) is a ciliopathy that is characterized by oral-facial abnormalities, including cleft lip and/or palate, broad nasal root, dental anomalies, micrognathia and glossal defects. In addition, these individuals have several other characteristic abnormalities that are typical of a ciliopathy, including polysyndactyly, polycystic kidneys and hypoplasia of the cerebellum. Recently, a subset of OFD cases in humans has been linked to mutations in the centriolar protein C2 Ca(2+)-dependent domain-containing 3 (C2CD3). Our previous work identified mutations in C2CD3 as the causal genetic lesion for the avian talpid(2) mutant. Based on this common genetic etiology, we re-examined the talpid(2) mutant biochemically and phenotypically for characteristics of OFD. We found that, as in OFD-affected individuals, protein-protein interactions between C2CD3 and oral-facial-digital syndrome 1 protein (OFD1) are reduced in talpid(2) cells. Furthermore, we found that all com...

Journal of Biological Chemistry, 2015

Background: Gram-positive bacteria secrete pilins through the Sec translocon in unfolded states. ... more Background: Gram-positive bacteria secrete pilins through the Sec translocon in unfolded states. Results: Disruption of pilus disulfide bonds or genetic disruption of oxidoreductase-encoding genes mdbA and vkor abrogates pilus assembly in Actinomyces oris. Conclusion: MdbA and VKOR constitute a disulfide bond-forming machine in A. oris. Significance: Oxidative protein folding may be common in Actinobacteria and an attractive target for antimicrobials. Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membranebound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the ⌬vkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.

Biophysical Journal, 2014

Acta crystallographica. Section D, Biological crystallography, 2014

The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expres... more The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys-Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys-Asn isopeptide-bond cross-link, assumed to giv...

Molecular microbiology, 2014

Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall ... more Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpre...

Journal of Bacteriology, 2014

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous... more The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix ...

Proceedings of the National Academy of Sciences, 2014

Significance The development of dental plaque biofilm requires specific and sequential molecular ... more Significance The development of dental plaque biofilm requires specific and sequential molecular interactions between oral bacteria-colonizing host surfaces. Coaggregation between early colonizers is crucial to establish an environment suitable for late colonizers. Here, we describe that a surface protein in a Gram-positive bacterium that is not genetically linked to the fimbrial gene clusters hijacks a specific fimbrial polymerization apparatus to be displayed on the fimbrial tip. This tip-localized protein not only functions as the bona fide cell-to-cell adhesion factor for mediating coaggregation between the early colonizers Actinomyces oris and Streptococcus oralis but also serves as an initiator of fimbrial assembly.

PLoS ONE, 2013

The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of E... more The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, nonsignificant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.

The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic... more The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic and acidic residues, with the overall net charge predicted to be positive. The C-terminal domain of l S was found to be cytoplasmic, as defined by protease accessibility in spheroplasts and inverted membrane vesicles. C-terminal nonsense mutations were constructed in S and found to be lysis proficient, as long as at least one basic residue is retained at the C terminus. In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis. However, the capacity of each truncated lytic allele for inhibition by the S107 inhibitor product of S is retained. The K97am allele, when incorporated into the phage context, confers a plaque-forming defect because its early lysis significantly reduces the burst size. Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis defect of the mutant SA52G, which causes lysis at or...

mBio

Molecular Oral Microbiology, 2022

Proceedings of the National Academy of Sciences, 2021

Scientific Reports, 2020

Journal of Bacteriology, 1999

Proceedings of the National Academy of Sciences, 2019

Journal of the American Chemical Society, Jan 11, 2018

mBio, Jan 20, 2017

Infection and immunity, May 29, 2016

Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2016

Molecular Microbiology, 2015

Disease models & mechanisms, 2015

Journal of Biological Chemistry, 2015

Biophysical Journal, 2014

Acta crystallographica. Section D, Biological crystallography, 2014

Molecular microbiology, 2014

Journal of Bacteriology, 2014

Proceedings of the National Academy of Sciences, 2014

PLoS ONE, 2013

The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic... more The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic and acidic residues, with the overall net charge predicted to be positive. The C-terminal domain of l S was found to be cytoplasmic, as defined by protease accessibility in spheroplasts and inverted membrane vesicles. C-terminal nonsense mutations were constructed in S and found to be lysis proficient, as long as at least one basic residue is retained at the C terminus. In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis. However, the capacity of each truncated lytic allele for inhibition by the S107 inhibitor product of S is retained. The K97am allele, when incorporated into the phage context, confers a plaque-forming defect because its early lysis significantly reduces the burst size. Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis defect of the mutant SA52G, which causes lysis at or...