Sortase-assembled pili promote extracellular electron transfer and iron acquisition inEnterococcus faecalisbiofilm

Abstract

24Enterococcus faecalis is an opportunistic human pathogen and the cause of biofilm-25 associated infections of the heart, catheterized urinary tract, wounds, and the dysbiotic gut 26 where it can expand to high numbers upon microbiome perturbations. The E. faecalis sortase-27 assembled endocarditis and biofilm associated pilus (Ebp) is involved in adhesion and 28 biofilm formation in vitro and in vivo. Extracellular electron transfer (EET) also promotes E. 29 faecalis biofilm formation in iron-rich environments, however neither the mechanism 30 underlying EET nor its role in virulence was previously known. Here we show that iron 31 associated with Ebp serve as a terminal electron acceptor for EET, leading to extracellular 32 iron reduction and intracellular iron accumulation. We found that a MIDAS motif within the 33 EbpA tip adhesin is required for interaction with iron, EET, and FeoB-mediated iron uptake. 34 We demonstrate that MenB and Ndh3, essential components of the aerobic respiratory chain 35 and a specialized flavin-mediated electron transport chain, respectively, are required for iron-36 mediated EET. In addition, using a mouse gastrointestinal (GI) colonization model, we show 37 that EET is essential for colonization of the GI tract, and Ebp is essential for augmented E. 38 faecalis GI colonization when dietary iron is in excess. Taken together, our findings show 39 that pilus mediated capture of iron within biofilms enables EET-mediated iron acquisition in 40 E. faecalis, and that these processes plays an important role in E. faecalis expansion in the GI 41 tract. 42 43 Significance 44 Understanding enterococcal biofilm development is the first step towards improved 45 therapeutics for the often antimicrobial resistant infections caused by these bacteria. Here we 46 report a role for Enterococcus faecalis endocarditis and biofilm associated pili (Ebp) in 47 (EET) which in turn promotes iron acquisition. Furthermore, we characterize the mechanisms 49 underlying electron transfer in the E. faecalis biofilm. Our findings support a model in which 50 E. faecalis use EET to drive the reduction of pilus-associated ferric iron, leading to iron 51 acquisition in E. faecalis biofilm, and contributing to enterococcal virulence in the GI tract.52 53 Introduction 54 Enterococcus faecalis is an important human opportunistic pathogen that causes a variety of 55 diseases including endocarditis, urinary tract infections (UTI), bacteremia, wound infection, 56 and medical device-associated infections 1 . Many of these infections are polymicrobial and 57 biofilm-associated, rendering them more tolerant to antimicrobial and immune clearance, and 58 contributing to their persistent nature 1 . Therefore, a detailed understanding of enterococcal 59 biofilm development is a critical step towards advancing new therapeutics for treatment of 60 these often antimicrobial resistant infections 1,2 . 61 62One important factor contributing to E. faecalis biofilm formation and virulence is the 63 sortase-assembled endo...

Two Routes for Extracellular Electron Transfer in Enterococcus faecalis

“…The isolated mutants were investigated for their ability to transfer electrons generated in glucose catabolism to a graphite electrode depending on type of provided redox mediator. In parallel to our work, Lam et al (17) have studied properties of E. faecalis strains with transposon insertion in genes corresponding to those known to be important for ferric reductase activity in L. monocytogenes (13) and for DMK synthesis in E. faecalis. Although the approaches in our research groups were different and EET was not measured using the same type of The collected data for E. faecalis (this work and (17)) and L. monocytogenes (13) indicate that an atypical NADH dehydrogenase (Ndh3 in E. faecalis) with the FAD containing domain presumably on the cytoplasmic side of the membrane, EetA which is mainly exposed to the periplasm, and EetB in the membrane, are required for ferric reductase activity dependent on DMK.…”

“…These recent findings together prompted us to investigate to what extent EET to electrodes and ferric reductase activity are connected processes in E. faecalis. While our research work was in progress it was reported that EET to ferric iron in E. faecalis depends on genes corresponding to those in the gene cluster originally found in L. monocytogenes and on a gene encoding the tip adhesion protein EbpA (17). To identify proteins involved in EET to ferric ions and determine if these also are crucial for EET we have here screened a collection of mutants for ferric reductase deficiency, identified the responsible mutations, and analyzed bioelectrochemical properties of the mutants.…”

Enterococcus faecalismanganese exporter MntE alleviates manganese toxicity and is required for mouse gastrointestinal colonization

“…Instead, increasing glycerol concentrations in the growth media enhanced biofilm formation in both the wild type control and mntE∷Tn mutant, regardless of Fe supplementation ( Figure S5 ). By contrast, these three glycerol catabolic genes ( glpF2, glpO, glpK ) were not upregulated under Mn supplemented conditions in wild type E. faecalis biofilm (Figure 4) and global transcriptional analysis showed that these genes are not Fe responsive in wild type OG1RF biofilm grown in Fe-supplemented media (38). Taken together, these results indicate that upregulation of glycerol catabolic genes is specifically observed in the absence of mntE when intracellular Fe levels are high and that glycerol supplementation contributes to biofilm growth.…”

Electroactivity across the cell wall of Gram-positive bacteria

“…Filaments of the type IV pili were also reported for some Gram-positive bacteria [86] . Although most of the functions of type IV pili in Gram-positive bacteria have been associated with mobility and adherence to host cells [86] , [87] , pili-like appendages have been observed in C. ferrireducens grown on iron oxides [74] , in Paenibacillus dendritiformis when growing on an electrode [28] and in E. faecalis pili revealed to contribute for EET [88] . It was demonstrated that E. faecalis biofilm matrix harboring iron sinks promotes EET and augments biofilm growth, increasing current generation in MFC [89] .…”

“…It was demonstrated that E. faecalis biofilm matrix harboring iron sinks promotes EET and augments biofilm growth, increasing current generation in MFC [89] . The biofilm associated pilus (Ebp) was demonstrated to be a key player in this process by sequestering iron in close proximity to the cells, either as surface attached pili or within the biofilm matrix, enabling E. faecalis to use it as terminal electron acceptor [88] .…”