Human and murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorum-sensing

The pathogenic bacterium Pseudomonas aeruginosa uses acyl-HSL quorum-sensing signals to regulate genes controlling virulence and biofilm formation. We found that paraoxonase 1 (PON1), a mammalian lactonase with an unknown natural substrate, hydrolyzed the P. aeruginosa acyl-HSL 3OC12-HSL. In in vitro assays, mouse serum-PON1 was required and sufficient to degrade 3OC12-HSL. Furthermore, PON2 and PON3 also degraded 3OC12-HSL effectively. Serum-PON1 prevented P. aeruginosa quorum-sensing and biofilm formation in vitro by inactivating the quorum-sensing signal. Although 3OC12-HSL production by P. aeruginosa was important for virulence in a mouse sepsis model, Pon1-knock-out mice were paradoxically protected. These mice showed increased levels of PON2 and PON3 mRNA in epithelial tissues suggesting a possible compensatory mechanism. Thus, paraoxonase interruption of bacterial communication represents a novel mechanism to modulate quorum-sensing by bacteria. The consequences for host immunity are yet to be determined.     

Coexistence of bacterial sulfide oxidizers, sulfate reducers, and spirochetes in a gutless worm (Oligochaeta) from the Peru margin

Olavius crassitunicatus is a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distant O. crassitunicatus populations (separated by 385 km) on the Peru margin (water depth, approximately 300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the gamma-Proteobacteria (Gamma 1 and 2 symbionts), two members of the delta-Proteobacteria (Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the delta-Proteobacteria (Delta 3 symbiont) was found in only one of the two host populations. Three of the O. crassitunicatus bacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts of Olavius algarvensis, Olavius loisae, and Inanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts of O. algarvensis, and the spirochete is closely related to spirochetal symbionts of O. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.     

Weaknesses in reports of "fertility" for horses and other species

Apparent fertility of a male or group of females is considered frequently by veterinarians or animal scientists. Unfortunately, concepts of experimental design and statistics impacting validity and interpretation of values for average pregnancy rate frequently are ignored. The magnitude of this problem was documented by examination of published papers; 51 of 67 (76%) were considered flawed for one or more reasons. The discussion considers why: (a) conclusions from most published fertility studies reporting no significant difference due to treatment(s) are suspect, because too few males and/or females were used; (b) the experimental unit in an IVF study should be a droplet of co-incubating gametes rather than an ovum; (c) apparent fertility of a male is profoundly influenced by the range in actual fertility of the females with which he was mated, and thus might shift over a two- to three-fold range; and (d) scientists should refrain from conduct of studies destined to be inconclusive, and should be candid in reporting each fertility trial. It was emphasized that no fertility data were better than an imprecise average value for a given male or a conclusion based on an inadequate research study or incomplete report of what was done.     

In situ substrate conversion and assimilation by nitrifying bacteria in a model biofilm

Local nitrification and carbon assimilation activities were studied in situ in a model biofilm to investigate carbon yields and contribution of distinct populations to these activities. Immobilized microcolonies (related to Nitrosomonas europaea/eutropha, Nitrosomonas oligotropha, Nitrospira sp., and to other Bacteria) were incubated with [14C]-bicarbonate under different experimental conditions. Nitrifying activity was measured concomitantly with microsensors (oxygen, ammonium, nitrite, nitrate). Biofilm thin sections were subjected to fluorescence in situ hybridization (FISH), microautoradiography (MAR), and local quantification of [14C]-bicarbonate uptake (beta microimaging). Nitrifying activity and tracer assimilation were restricted to a surface layer of different thickness in the various experiments (substrate or oxygen limitation). Excess oxygen uptake under all conditions revealed heterotrophic activity fuelled by decay or excretion products during active nitrification. Depth limits and intensity of tracer incorporation profiles were in agreement with ammonia-oxidation activity (measured with microsensors), and distribution of incorporated tracer (detected with MAR). Microautoradiography revealed a sharp individual response of distinct populations in terms of in-/activity depending on the (local) environmental conditions within the biofilm. Net in situ carbon yields on N, expressed as e- equivalent ratios, varied between 0.005 and 0.018, and, thus, were in the lower range of data reported for pure cultures of nitrifiers.     

Graphical representation of ribosomal RNA probe accessibility data using ARB software package

Background  

Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments.     

Phenotypic variation resulting from a deficiency of epidermal growth factor receptor in mice is caused by extensive genetic heterogeneity that can be genetically and molecularly partitioned

The timing of lethality caused by homozygosity for a null allele of the epidermal growth factor receptor (Egfrtm1Mag) in mice is strongly dependent on genetic background. Initial attempts to genetically map background modifiers using Swiss-derived, outbred CD-1 mice were unsuccessful. To investigate the genetic architecture contributing to survival of Egfrtm1Mag homozygous embryos, the genetic variability segregating within the outbred population was partitioned by surveying viability of Egfrtm1Mag mutants using intercrosses between 129S6/SvEvTAC-Egfrtm1Mag and nine Swiss-derived, inbred strains: ALR/LtJ, ALS/LtJ, APN, APS, ICR/HaRos, NOD/LtJ, NON/LtJ, SJL/J, and SWR/J. The observations showed that these strains support varying levels of survival of Egfrtm1Mag homozygous embryos, suggesting that genetic heterogeneity within the CD-1 stock contributed to the original lack of Egfrtm1Mag modifier detection. Similar to the Swiss-derived intercrosses, nine congenic strains, derived from 129S6/SvEvTAC, AKR/J, APN, BALB/cJ, BTBR-T+ tf/tf, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ inbred backgrounds, also supported varying levels of survival of Egfrtm1Mag mutants. By intercrossing the congenic lines to create hybrid F1 embryos, different genetic backgrounds were found to have complementary modifiers. Analysis of the congenic lines argues against heterosis of outbred backgrounds contributing to Egfrtm1Mag phenotypic variability. A detailed analysis of the crosses suggests that modifiers function at three distinct stages of development. One class of modifiers supports survival of Egfrtm1Mag homozygous embryos to mid-gestation, another class supports development through the mid-gestation transition from yolk-sac to placental-derived nutrient sources, and a third class supports survival through later stages of gestation. Data from microarray analysis using RNA from wild-type and Egfrtm1Mag mutant placentas support the existence of extensive genetic heterogeneity and suggest that it can be molecularly partitioned. This method should be generally useful to partition heterogeneity contributing to other complex traits.     

Solution structure of a CCHHC domain of neural zinc finger factor-1 and its implications for DNA binding

The structure of a CCHHC zinc-binding domain from neural zinc finger factor-1 (NZF-1) has been determined in solution though the use of NMR methods. This domain is a member of a family of domains that have the Cys-X(4)-Cys-X(4)-His-X(7)-His-X(5)-Cys consensus sequence. The structure determination reveals a novel fold based around a zinc(II) ion coordinated to three Cys residues and the second of the two conserved His residues. The other His residue is stacked between the metal-coordinated His residue and a relatively conserved aromatic residue. Analysis of His to Gln sequence variants reveals that both His residues are required for the formation of a well-defined structure, but neither is required for high-affinity metal binding at a tetrahedral site. The structure suggests that a two-domain protein fragment and a double-stranded DNA binding site may interact with a common two-fold axis relating the two domains and the two half-sites of the DNA-inverted repeat.     

Identification and functional validation of novel autoantigens in equine uveitis

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.     

Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5'-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (oligochaeta) from Bermuda and the Bahamas

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5'-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.