Involvement of Chromosomally Encoded Homologs of the RRNPP Protein Family in Enterococcus faecalis Biofilm Formation and Urinary Tract Infection Pathogenesis.

Enterococcus faecalis is an opportunistic pathogen capable of causing infections, including endocarditis and urinary tract infections (UTI). One of the well-characterized quorum-sensing pathways in E. faecalis involves coordination of the conjugal transfer of pheromone-responsive plasmids by PrgX, a member of the RRNPP protein family. Members of this protein family in various Firmicutes have also been shown to contribute to numerous cellular processes, including sporulation, competence, conjugation, nutrient sensing, biofilm formation, and virulence. As PrgX is a plasmid-encoded RRNPP family member, we surveyed the genome of the multidrug-resistant strain V583 for additional RRNPP homologs using computational searches and refined those identified hits for predicted structural similarities to known RRNPP family members. This led us to investigate the contribution of the chromosomally encoded RRNPP homologs to biofilm processes and pathogenesis in a catheter-associated urinary tract infection (CAUTI) model. In this study, we identified five such homologs and report that 3 of the 5 homologs, EF0073, EF1599, and EF1316, affect biofilm formation as well as outcomes in the CAUTI model.IMPORTANCEEnterococcus faecalis causes health care-associated infections and displays resistance to a variety of broad-spectrum antibiotics by acquisition of resistance traits as well as the ability to form biofilms. Even though a growing number of factors related to biofilm formation have been identified, mechanisms that contribute to biofilm formation are still largely unknown. Members of the RRNPP protein family regulate a diverse set of biological reactions in low-G+C Gram-positive bacteria (Firmicutes). Here, we identify three predicted structural homologs of the RRNPP family, EF0073, EF1599, and EF1316, which affect biofilm formation and CAUTI pathogenesis.

Confined Mobility of TonB and FepA in Escherichia coli Membranes.

The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E.coli, to take into account both the highly curved geometry of the cell and artifactual effects expected due to finite exposure time imaging. We find that both molecules perform confined lateral diffusion in their respective membranes in the absence of ligand with FepA confined to a region [Formula: see text] µm in radius in the outer membrane and TonB confined to a region [Formula: see text] µm in radius in the inner membrane. The diffusion coefficient of these molecules on millisecond time-scales was estimated to be [Formula: see text] µm2/s and [Formula: see text] µm2/s for FepA and TonB, respectively, implying that each molecule is free to diffuse within its domain. Disruption of the inner membrane potential, deletion of ExbB/D from the inner membrane, presence of ligand or antibody to FepA and disruption of the MreB cytoskeleton was all found to further restrict the mobility of both molecules. Results are analyzed in terms of changes in confinement size and interactions between the two proteins.

High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport.

The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of TonB activity and off in its absence. It functions in 96-well microtiter plates, in a variety of conditions, with Z factors of 0.8-1.0. TonB-dependent iron transport is energy dependent, and the inhibitory effects of the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, cyanide, and arsenate on FeEnt uptake were readily detected by the assay. Because iron acquisition is a determinant of bacterial pathogenesis, HTS with this method may identify inhibitors that block TonB function and constitute novel therapeutics against infectious disease caused by Gram-negative bacteria.

Concerted loop motion triggers induced fit of FepA to ferric enterobactin.

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles.

Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane.

Gram-negative bacteria acquire iron with TonB-dependent uptake systems. The TonB-ExbBD inner membrane complex is hypothesized to transfer energy to outer membrane (OM) iron transporters. Fluorescence microscopic characterization of green fluorescent protein (GFP)-TonB hybrid proteins revealed an unexpected, restricted localization of TonB in the cell envelope. Fluorescence polarization measurements demonstrated motion of TonB in living cells, which likely was rotation. By determining the anisotropy of GFP-TonB in the absence and presence of inhibitors, we saw the dependence of its motion on electrochemical force and on the actions of ExbBD. We observed higher anisotropy for GFP-TonB in energy-depleted cells and lower values in bacteria lacking ExbBD. However, the metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings demonstrate that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism.

Role of catecholate siderophores in gram-negative bacterial colonization of the mouse gut.

We investigated the importance of the production of catecholate siderophores, and the utilization of their iron (III) complexes, to colonization of the mouse intestinal tract by Escherichia coli. First, a ΔtonB strain was completely unable to colonize mice. Next, we compared wild type E. coli MG1655 to its derivatives carrying site-directed mutations of genes for enterobactin synthesis (ΔentA::Cm; strain CAT0), ferric catecholate transport (Δfiu, ΔfepA, Δcir, ΔfecA::Cm; CAT4), or both (Δfiu, ΔfepA, ΔfecA, Δcir, ΔentA::Cm; CAT40) during colonization of the mouse gut. Competitions between wild type and mutant strains over a 2-week period in vivo showed impairment of all the genetically engineered bacteria relative to MG1655. CAT0, CAT4 and CAT40 colonized mice 10(1)-, 10(5)-, and 10(2)-fold less efficiently, respectively, than MG1655. Unexpectedly, the additional inability of CAT40 to synthesize enterobactin resulted in a 1000-fold better colonization efficiency relative to CAT4. Analyses of gut mucus showed that CAT4 hyperexcreted enterobactin in vivo, effectively rendering the catecholate transport-deficient strain iron-starved. The results demonstrate that, contrary to prior reports, iron acquisition via catecholate siderophores plays a fundamental role in bacterial colonization of the murine intestinal tract.

Evidence of herpesvirus infection in Woodland Caribou in Saskatchewan.

Sera were collected from 40 female and two male woodland caribou (Rangifer tarandus caribou) in Saskatchewan (Canada) from March 1992 to January 1995, inclusive. The samples were examined for antibodies against smooth Brucella spp., five serovars of Leptospira interrogans, bovine viral diarrhea virus, and bovine herpesvirus 1 (BHV-1). Twenty-two (52%) of 42 sera exhibited positive reactions to BHV-1 by a modified serum neutralization test, and the prevalence correlated positively with the age of the animals. No antibodies were detected against the other pathogens. This is the first reported evidence of herpesvirus infection in isolated populations of woodland caribou in western Canada.

A blocking ELISA with improved sensitivity for the detection of passively acquired maternal antibodies to BHV-1.

Four serological tests were evaluated for their ability to detect passively acquired maternal antibodies to Bovine herpesvirus 1. A blocking enzyme-linked immunosorbent assay demonstrated superior sensitivity in the detection of such antibodies in calves up to 9-11 months old, versus calves up to 7 months old for other tests.