Significance Fusobacterium nucleatum interacts with many oral microbes and has the ability to spread placenta amniotic fluid, promoting preterm birth. Yet, molecular mechanisms underlying polymicrobial interactions, termed coaggregation, by Fusobacteria are poorly understood. Here, we revealed that two-component signal transduction system CarRS regulates expression of genes encoding lysine utilization factors (e.g., KamA) coaggregation factor RadD. Extracellular blocks RadD-mediated binding...

The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome Enterococcus faecalis that has been shown to be important for biofilm formation, adherence host fibrinogen, collagen platelets, in experimental urinary tract infection models. Here, we created single double deletion mutants subunits sortases; next, by combining western blotting, immunoelectron microscopy, using ebpR trans increase production, identified EbpA as tip pilin EbpB anchor at base, latter...

Significance Bacteria colonizing the oropharynx must adhere despite mechanical challenges from coughing, sneezing, and chewing; however, little is known about how Gram-positive organisms achieve this feat. We studied pilus adhesive proteins two report a conserved mechanism for dissipating energy of perturbation. The are stable up to forces 525 pN 690 pN, respectively, making these most mechanically known. After perturbation, refold rapidly at low force, resulting in large hysteresis with...

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

Summary Sortase, a cysteine‐transpeptidase conserved in G ram‐positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of housekeeping sortase several pathogens reported thus far attenuates virulence, but not growth. Paradoxically, we discovered depletion SrtA was lethal for A ctinomyces oris ; yet, all its predicted wall‐anchored protein substrates ( AcaA ‐ N ) were individually dispensable viability. Using Tn 5...

The Gram-negative anaerobe Fusobacterium nucleatum is a major producer of hydrogen sulfide (H2S), volatile sulfur compound that causes halitosis. Here, we dissected the genetic determinants H2S production and its role in bacterial fitness virulence this important member oral microbiome. F. possesses four enzymes, CysK1, CysK2, Hly, MegL, presumably metabolize l-cysteine to H2S, CysK1 was previously shown account for most vitro, based on correlations enzymatic activities with gene expression...

Summary The Gram‐positive pathogen C orynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include key virulence factors diphtheria toxin and adhesive pili. How these attain their native conformations after translocation as unfolded precursors remains elusive. fact majority of exported contain multiple cysteine residues several membrane‐bound oxidoreductases are encoded in corynebacterial genome suggests existence an oxidative protein‐folding pathway...

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents materials. However, there few enzyme-catalyzed methods currently available to selectively conjugate internal sites within proteins. Here we show a pilus-specific sortase enzyme from Corynebacterium diphtheriae (

Fusobacterium nucleatum, an anaerobic Gram-negative bacterium frequently found in the human oral cavity and some extra-oral sites, is implicated several important diseases: periodontitis, adverse pregnancy outcomes, colorectal cancer. To date, how this obligate anaerobe copes with oxidative stress host immunity within multiple tissues remains unknown. Here, we uncovered a critical role process of multigene locus encoding single, fused methionine sulfoxide reductase (MsrAB), two-component...

ABSTRACT 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 Gram-positive bacteria, E. . We previously demonstrated that nonpiliated Δ empABC :: cat derivative TX82 was attenuated biofilm formation UTI model. Here, we studied contributions individual pilus subunits EmpA, EmpB, EmpC architecture, formation, adherence...

ABSTRACT The WxL domain recently has been identified as a novel cell wall binding found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of containing this domain. Here, we identify and characterize 6 Enterococcus faecium which, by reverse transcription-PCR (RT-PCR) genomic analyses, are located three similarly organized operons, deemed loci A, B, C. Western blotting, electron microscopy, enzyme-linked immunosorbent...

Pilus assembly in Gram-positive bacteria occurs by a two-step mechanism, whereby pilins are polymerized and then covalently anchored to the cell wall. In Corynebacterium diphtheriae, pilin-specific sortase SrtA catalyses polymerization of SpaA-type pilus, consisting shaft pilin SpaA, tip SpaC minor SpaB. Cell wall anchoring SpaA polymers is triggered when incorporates SpaB into pilus base via lysine-mediated transpeptidation; peptidoglycan subsequently catalysed housekeeping SrtF. Here we...

Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. SpaA are known to mediate bacterial adherence pharyngeal epithelial cells. However, date little is about expression of different various clinical isolates their importance pathogenesis. Here, we characterized a large collection for pilin gene pool by PCR respective pilins...

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located bacterial membrane catalyze a two-step protein ligation reaction, first cleaving LPXTG motif one pilin protomer to form an acyl-enzyme intermediate and then joining terminal Thr nucleophilic Lys residue residing within another protomer. To date, determinants enzymes that uniquely enable them construct pili remain...

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization pilin subunits and another anchoring to peptidoglycan. How this machine controls pilus length whether is critical cell-to-cell interactions remain unknown. We report here in Actinomyces oris , a key colonizer development oral biofilms, that genetic disruption its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by pilus-specific...

The Gram-negative anaerobe Fusobacterium nucleatum is an oral oncobacterium that promotes colorectal cancer (CRC) development with the amyloid-forming cell surface adhesin FadA integral to CRC tumorigenesis. We describe here molecular genetic studies uncovering a novel mode of metabolic regulation FadA-mediated tumor formation by highly conserved respiratory enzyme known as Rnf complex. First, we show disruption Rnf, via rnfC deletion, significantly reduces level fadA transcript, accompanied...

ABSTRACT The Gram-negative anaerobe Fusobacterium nucleatum is an oral oncobacterium that promotes colorectal cancer (CRC) development with the amyloid-forming cell surface adhesin FadA integral to CRC tumorigenesis. We describe here molecular genetic studies uncovering a novel mode of metabolic regulation FadA-mediated tumor formation by highly conserved respiratory enzyme known as Rnf complex. First, we show disruption Rnf, via rnfC deletion, significantly reduces level fadA transcript,...

The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization host. These are covalent protein polymers composed three types pilin subunit that assembled by specific sortase enzymes. A structural analysis major SpaD, forms polymeric backbone one pilus expressed C. is reported. Mass-spectral crystallographic shows SpaD contains internal Lys-Asn isopeptide bonds. One these, shown mass...

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation unfolded precursor polypeptides; however, how gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that FimA pilin Actinomyces oris contains two disulfide bonds. Alanine substitution cysteine residues forming C-terminal bridge abrogates pilus assembly, turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis A....

Abstract 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 discover a useful sortase inhibitor have relied upon an in vitro activity assay which enzyme is removed from its native site on truncated improve solubility. To inhibitors effective inactivating sortases vivo , we developed implemented novel cell-based screen using Actinomyces oris key...

ABSTRACT The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to inherent ability adhere receptor polysaccharides on surface streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting shaft FimA coaggregation factor CafA forming tip. While essential role sortase machine SrtC2 assembly, biofilm formation, has been established, little is known about...

Significance Bacteria display hair-like structures called pili that enable them to adhere host tissues, surfaces, and other bacteria. Understanding how microbes assemble is important, as these play a key role in host–pathogen interactions cause disease. In this study, we have gained insight into pathogenic Gram-positive bacteria by visualizing the intersubunit covalent crosslink stabilizes fibrous investigating are assembled specialized pilus-specific sortase enzymes. We propose via...

<em>Porphyromonas gingivalis</em> (<em>Pg</em>), one of the 'red‑complex' perio‑pathogens known to play a critical role in development periodontitis, has been used various animal models mimic human bacteria‑induced periodontitis. In order achieve more realistic model <em>Pg</em> infection, present study investigated whether repeated small‑volume topical applications directly into gingival pocket can induce local including periodontitis and systemic vascular inflammation wild‑type mice....

Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for transpeptidase that anchors many extracellular proteins to Gram-positive bacterial cell wall. Here, we uncover hitherto unknown function this class conserved proteins, which name SafA, as topological modulator in oral Actinobacterium Actinomyces oris . Genetic deletion safA induces cleavage and excretion otherwise predominantly membrane-bound SrtA wild-type...