Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea among children, human immunodeficiency virus-infected patients, and travelers to developing regions of the world. The pathogenesis of EAEC strains involves the production of biofilm. In this study, we determined the association between presence of putative EAEC virulence genes and biofilm formation in 57 EAEC isolates (as defined by HEp-2 adherence) from travelers with diarrhea and in 18 EAEC isolates from travelers without diarrhea. Twelve nondiarrheagenic E. coli isolates from healthy travelers were used as controls. Biofilm formation was measured by using a microtiter plate assay with the crystal violet staining method, and the presence of the putative EAEC virulence genes aap, aatA, aggR, astA, irp2, pet, set1A, and shf was determined by PCR. EAEC isolates were more likely to produce biofilm than nondiarrheagenic E. coli isolates (P = 0.027), and the production of biofilm was associated with the virulence genes aggR, set1A, aatA, and irp2, which were found in 16 (40%), 17 (43%), 10 (25%), and 27 (68%) of the biofilm producers versus only 4 (11%), 6 (6%), 2 (6%), and 15 (43%) in non-biofilm producers (P = 0.008 for aggR, P = 0.0004 for set1A, P = 0.029 for aatA, and P = 0.04 for irp2). Although the proportion of EAEC isolates producing biofilm in patients with diarrhea (51%) was similar to that in patients without diarrhea (61%), biofilm production was related to the carriage of aggR (P = 0.015), set1A (P = 0.001), and aatA (P = 0.025). Since aggR is a master regulator of EAEC, the presence of aap (P = 0.004), astA (P = 0.001), irp2 (P = 0.0006), pet (P = 0.002), and set1A (P = 0.014) in an aggR versus an aggR-lacking background was investigated and was also found to be associated with biofilm production. This study suggests that biofilm formation is a common phenomenon among EAEC isolates derived from travelers with or without diarrhea and that multiple genes associated with biofilm formation are regulated by aggR.

Crystal structure of the Anabaena sensory rhodopsin transducer.

We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed.

Vancomycin-resistant Enterococcus faecalis endocarditis: linezolid failure and strain characterization of virulence factors.

Infective endocarditis due to vancomycin-resistant (VR) Enterococcus faecalis has only rarely been reported. We report a case of VR E. faecalis endocarditis that failed to respond to linezolid therapy, outline the virulence traits of the isolate, and review previously published cases of VR E. faecalis endocarditis.

Laser-induced transient grating analysis of dynamics of interaction between sensory rhodopsin II D75N and the HtrII transducer.

The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII transducer protein HtrII (DeltaHtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K(1)-K(4)) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the DeltaHtrII portion was found to cause a DeltaHtrII-dependent increase in volume rising in 8 micros in the K(4) state and a drastic decrease in the diffusion coefficient (D) of K(4) relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of DeltaHtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the protein and the solvent.

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

Chronic administration of methylphenidate produces neurophysiological and behavioral sensitization.

The electrophysiological properties of acute and chronic methylphenidate (MPD) on neurons of the prefrontal cortex (PFC) and caudate nucleus (CN) have not been studied in awake, freely behaving animals. The present study was designed to investigate the dose-response effects of MPD on sensory evoked potentials recorded from the PFC and CN in freely behaving rats previously implanted with permanent electrodes, as well as their behavioral (locomotor) activities. On experimental day 1, locomotor behavior of rats was recorded for 2 h post-saline injection, and sensory evoked field potentials were recorded before and after saline and 0.6, 2.5, and 10 mg/kg, i.p., MPD administration. Animals were injected for the next five days with daily 2.5 mg/kg MPD to elicit behavioral sensitization. Locomotor recording was resumed on experimental days 2 and 6 after the MPD maintenance dose followed by 3 days of washout. On experimental day 10, rats were connected again to the electrophysiological recording system and rechallenged with saline and the identical MPD doses as on experimental day 1. On experimental day 11, rat's locomotor recording was resumed before and after 2.5 mg/kg MPD administration. Behavioral results showed that repeated administration of MPD induced behavioral sensitization. Challenge doses (0.6, 2.5, and 10.0 mg/kg) of MPD on experimental day 1 elicited dose-response attenuation in the response amplitude of the average sensory evoked field potential components recorded from the PFC and CN. Chronic MPD administration resulted in attenuation of the PFC's baseline recorded on experimental day 10, while the same treatment did not modulate the baseline recorded from the CN. Treatment of MPD on experimental day 10 resulted in further decrease of the average sensory evoked response compared to that obtained on experimental day 1. This observation of further decrease in the electrophysiological responses after chronic administration of MPD suggests that the sensory evoked responses on experimental day 10 represent neurophysiological sensitization. Moreover, two different response patterns were obtained from PFC and CN following chronic methylphenidate administration. In PFC, the baseline and effect of methylphenidate expressed electrophysiological sensitization on experimental day 10, while recording from CN did not exhibit any electrophysiological sensitization.

Interaction between cell division proteins FtsE and FtsZ.

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.

All in the family: atypical Hsp70 chaperones are conserved modulators of Hsp70 activity.

Divergent relatives of the Hsp70 protein chaperone such as the Hsp110 and Grp170 families have been recognized for some time, yet their biochemical roles remained elusive. Recent work has revealed that these "atypical" Hsp70s exist in stable complexes with classic Hsp70s where they exert a powerful nucleotide-exchange activity that synergizes with Hsp40/DnaJ-type cochaperones to dramatically accelerate Hsp70 nucleotide cycling. This represents a novel evolutionary transition from an independent protein-folding chaperone to what appears to be a dedicated cochaperone. Contributions of the atypical Hsp70s to established cellular roles for Hsp70 now must be deciphered.

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P < 0.0001). Blocking Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

EbpR is important for biofilm formation by activating expression of the endocarditis and biofilm-associated pilus operon (ebpABC) of Enterococcus faecalis OG1RF.

We identify ef1090 (renamed ebpR) and show its importance for the transcriptional regulation of expression of the Enterococcus faecalis pilus operon, ebpABC. An ebpR deletion (DeltaebpR) mutant was found to have reduced ebpABC expression with loss of pilus production and a defect in primary adherence with, as a consequence, reduced biofilm formation.

Genome sequence of Fusobacterium nucleatum subspecies polymorphum - a genetically tractable fusobacterium.

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum, nucleatum, vincentii, fusiforme, and animalis. F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus. A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the CdISt IStrons described in Clostridium difficile. IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes, particularly the Clostridia, is common.

Neurogranin controls the spatiotemporal pattern of postsynaptic Ca2+/CaM signaling.

Neurogranin (Ng) is a postsynaptic IQ-motif containing protein that accelerates Ca(2+) dissociation from calmodulin (CaM), a key regulator of long-term potentiation and long-term depression in CA1 pyramidal neurons. The exact physiological role of Ng, however, remains controversial. Two genetic knockout studies of Ng showed opposite outcomes in terms of the induction of synaptic plasticity. To understand its function, we test the hypothesis that Ng could regulate the spatial range of action of Ca(2+)/CaM based on its ability to accelerate the dissociation of Ca(2+) from CaM. Using a mathematical model constructed on the known biochemistry of Ng, we calculate the cycle time that CaM molecules alternate between the fully Ca(2+) saturated state and the Ca(2+) unbound state. We then use these results and include diffusion of CaM to illustrate the impact that Ng has on modulating the spatial profile of Ca(2+)-saturated CaM within a model spine compartment. Finally, the first-passage time of CaM to transition from the Ca(2+)-free state to the Ca(2+)-saturated state was calculated with or without Ng present. These analyses suggest that Ng regulates the encounter rate between Ca(2+) saturated CaM and its downstream targets during postsynaptic Ca(2+) transients.

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).