Contribution of a ZIP-family protein to manganese uptake and infective endocarditis virulence in Streptococcus sanguinis.

Streptococcus sanguinis is an important cause of infective endocarditis. In strain SK36, the ABC-family manganese transporter, SsaACB, is essential for virulence. We have now identified a ZIP-family protein, TmpA, as a secondary manganese transporter. A tmpA mutant had no phenotype, but a ΔssaACB ΔtmpA mutant was more attenuated for serum growth and for virulence in a rabbit model than its ΔssaACB parent. The growth of both mutants was restored by supplemental manganese, but the ΔssaACB ΔtmpA mutant required twenty-fold more and accumulated less. Although ZIP-family proteins are known for zinc and iron transport, TmpA-mediated transport of either metal was minimal. While ssaACB appears ubiquitous in St. sanguinis, tmpA was present in a majority of strains and a mntH gene encoding an NRAMP-family transporter was identified in relatively few, including VMC66. As in SK36, deletion of ssaACB greatly diminished VMC66 endocarditis virulence and serum growth, and deletion of tmpA from this mutant diminished virulence further. Virulence was not significantly altered by deletion of mntH from either VMC66 or its ΔssaACB mutant. This and the accompanying paper together suggest that SsaACB is of primary importance for endocarditis virulence while secondary transporters TmpA and MntH contribute to growth under differing conditions.

Manganese transport by Streptococcus sanguinis in acidic conditions and its impact on growth in vitro and in vivo.

Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a ΔssaACB deletion mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP-family manganese transporter. Transcriptomic analysis of fermentor-grown cultures of SK36 WT and ΔssaACB strains identified pH-dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its ΔssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis.

A novel sialic acid-binding adhesin present in multiple species contributes to the pathogenesis of Infective endocarditis.

Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP- isolates also bind the cryptic receptor ß-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.

Intracellular Metal Speciation in Streptococcus sanguinis Establishes SsaACB as Critical for Redox Maintenance.

Streptococcus sanguinis is an oral commensal bacterium, but it can colonize pre-existing heart valve vegetations if introduced into the bloodstream, leading to infective endocarditis. Loss of Mn- or Fe-cofactored virulence determinants are thought to result in weakening of this bacterium. Indeed, intracellular Mn accumulation mediated by the lipoprotein SsaB, a component of the SsaACB transporter complex, has been shown to promote virulence for endocarditis and O2 tolerance. To delineate intracellular metal-ion abundance and redox speciation within S. sanguinis, we developed a protocol exploiting two spectroscopic techniques, Inductively coupled plasma-optical emission spectrometry (ICP-OES) and electron paramagnetic resonance (EPR) spectroscopy, to respectively quantify total intracellular metal concentrations and directly measure redox speciation of Fe and Mn within intact whole-cell samples. Addition of the cell-permeable siderophore deferoxamine shifts the oxidation states of accessible Fe and Mn from reduced-to-oxidized, as verified by magnetic moment calculations, aiding in the characterization of intracellular metal pools and metal sequestration levels for Mn2+ and Fe. We have applied this methodology to S. sanguinis and an SsaACB knockout strain (ΔssaACB), indicating that SsaACB mediates both Mn and Fe uptake, directly influencing the metal-ion pools available for biological inorganic pathways. Mn supplementation of ΔssaACB returns total intracellular Mn to wild-type levels, but it does not restore wild-type redox speciation or distribution of metal cofactor availability for either Mn or Fe. Our results highlight the biochemical basis for S. sanguinis oxidative resistance, revealing a dynamic role for SsaACB in controlling redox homeostasis by managing the intracellular Fe/Mn composition and distribution.

Variants in nicotinic acetylcholine receptors alpha5 and alpha3 increase risks to nicotine dependence.

Nicotinic acetylcholine receptors bind to nicotine and initiate the physiological and pharmacological responses to tobacco smoking. In this report, we studied the association of alpha5 and alpha3 subunits with nicotine dependence and with the symptoms of alcohol and cannabis abuse and dependence in two independent epidemiological samples (n = 815 and 1,121, respectively). In this study, seven single nucleotide polymorphisms were genotyped in the CHRNA5 and CHRNA3 genes. In both samples, we found that the same alleles of rs16969968 (P = 0.0068 and 0.0028) and rs1051730 (P = 0.0237 and 0.0039) were significantly associated with the scores of Fagerström test for nicotine dependence (FTND). In the analyses of the symptoms of abuse/dependence of alcohol and cannabis, we found that rs16969968 and rs1051730 were significantly associated with the symptoms of alcohol abuse or dependence (P = 0.0072 and 0.0057) in the combined sample, but the associated alleles were the opposite of that of FTND. No association with cannabis abuse/dependence was found. These results suggested that the alpha5 and alpha3 subunits play a significant role in both nicotine dependence and alcohol abuse/dependence. However, the opposite effects with nicotine dependence and alcohol abuse/dependence were puzzling and future studies are necessary to resolve this issue.

Association study of CREB1 with Major Depressive Disorder and related phenotypes.

Cyclic AMP response element binding protein (CREB) has been implicated in behavioral models of anxiety and depression, antidepressant response in humans, and suicide. One group reported a female-specific association of the CREB1 gene in early-onset Major Depressive Disorder (MDD), while another found no evidence of association with this phenotype. In this study, we sought to examine the evidence for association of the CREB1 gene to MDD and related phenotypes. We used multivariate structural equation modeling to identify and select twin pairs that scored at the extremes of a latent genetic risk factor shared by MDD, neuroticism, and several anxiety disorders from the Virginia Twin Registry. Using one member from each of these pairs, the resulting sample of 589 cases (including 473 subjects with lifetime MDD) and 539 controls were entered into a 2-stage association study in which genetic markers were screened in stage 1, the positive results of which were tested for replication in stage 2. Eight SNP markers selected to capture the major allelic variation across the haplotype block containing CREB1 were analyzed for differences between cases and controls. Several markers showed criterion differences between cases and controls in the stage 1 sample with some evidence of sex specific effects. However, none of these markers were significant in stage 2 in either sex individually or combined. Our data suggests that common variations in the CREB1 gene do not appear to increase susceptibility for MDD or related phenotypes.

Fabrication, characterization, and in vitro evaluation of silver-containing arabinoxylan foams as antimicrobial wound dressing.

Arabinoxylan ferulate (AXF) foams were fabricated via enzymatic peroxidase/hydrogen peroxide crosslinking reaction followed by freeze-drying and studied as a potential wound dressing material. The AXF foam's rheological, morphological, porous, and swelling properties were examined. AXF foams were found to be a viscoelastic material that proved to be highly porous and water absorbent. AXF foams possessed low endotoxin levels and were cytocompatible with fibroblasts. Silver was successfully integrated into AXF foams and slowly released over 48 h. AXF foams impregnated with silver demonstrated efficacy inhibiting bacterial growth according to a modified Kirby-Bauer disk diffusion susceptibility test. Overall, AXF foams possess appropriate material properties and the silver-loaded AXF foams showed antimicrobial activity necessary to be a candidate material in wound dressing development. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2456-2465, 2016.

Genetic association between RGS1 and internalizing disorders.

OBJECTIVE: Quantitative trait loci identified in animal models provide potential candidate susceptibility loci for human disorders. In this study, we investigated whether internalizing disorders (anxiety disorders, major depression, and neuroticism) were associated with a region on human chromosome 1 syntenic with a quantitative trait locus for rodent emotionality. METHODS: We genotyped 31 single-nucleotide polymorphisms in genes located on chromosome 1q31.2 in a two-stage association study of 1128 individuals chosen for a high or a low genetic risk for internalizing disorders from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. RESULTS: None of the individual single-nucleotide polymorphisms showed consistent association across stages. A four-marker haplotype in the regulator of G-protein signaling 1 gene (RGS1) was significantly associated with decreased internalizing risk in both stages, whereas another showed a nominal association with a higher risk. CONCLUSION: Our data suggest that markers in the RGS1 gene might be in linkage disequilibrium with a protective allele that reduces the risk of anxiety and depressive disorders.

Prioritization and association analysis of murine-derived candidate genes in anxiety-spectrum disorders.

BACKGROUND: Anxiety disorders are common psychiatric conditions that are highly comorbid with each other and related phenotypes such as depression, likely due to a shared genetic basis. Fear-related behaviors in mice have long been investigated as potential models of anxiety disorders, making integration of information from both murine and human genetic data a powerful strategy for identifying potential susceptibility genes for these conditions. METHODS: We combined genome-wide association analysis of fear-related behaviors with strain distribution pattern analysis in heterogeneous stock mice to identify a preliminary list of 52 novel candidate genes. We ranked these according to three complementary sources of prior anxiety-related genetic data: 1) extant linkage and knockout studies in mice, 2) a meta-analysis of human linkage scans, and 3) a preliminary human genome-wide association study. We genotyped tagging single nucleotide polymorphisms covering the nine top-ranked regions in a two-stage association study of 1316 subjects from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders chosen for high or low genetic loading for anxiety-spectrum phenotypes (anxiety disorders, neuroticism, and major depression). RESULTS: Multiple single nucleotide polymorphisms in the PPARGC1A gene demonstrated association in both stages that survived gene-wise correction for multiple testing. CONCLUSIONS: Integration of genetic data across human and murine studies suggests PPARGC1A as a potential susceptibility gene for anxiety-related disorders.

Catechol-O-methyltransferase contributes to genetic susceptibility shared among anxiety spectrum phenotypes.

BACKGROUND: Catechol-O-methyltransferase (COMT) has been investigated for its possible role in a wide range of psychiatric phenotypes. In particular, several studies support association of this gene with panic disorder and other anxiety-related traits. METHODS: We examined the COMT gene for association with genetic risk across a range of anxiety spectrum phenotypes. We used multivariate structural equation modeling to select twin pairs scoring at the extremes of a latent genetic risk factor shared by neuroticism, several anxiety disorders, and major depression from a large population-based twin sample. With one member from each of these pairs, the resulting sample of 589 cases and 539 control subjects were entered into a two-stage association study in which genetic markers were screened in stage 1, the positive results of which were tested for replication in stage 2. RESULTS: The functional val158met polymorphism (rs4680) plus nine other single nucleotide polymorphism markers selected to capture the major allelic variation across the COMT locus were analyzed for differences between cases and control subjects. Although the val (G) allele of rs4680 showed marginally significant association in our combined stage 1 plus stage 2 sample, a high-risk haplotype of this allele with the A allele of rs165599 was significantly over-represented in cases (p = 1.97e-5, odds ratio = 1.95). This haplotype also predicted individual differences in neuroticism and risk for several anxiety disorders and major depression. Consistent with prior studies, our findings are female-specific. CONCLUSIONS: Variations in the COMT gene contribute to genetic risk shared across a range of anxiety-related phenotypes.

Cannabinoid receptor 1 gene association with nicotine dependence.

CONTEXT: The endogenous cannabinoid system has been implicated in drug addiction in animal models. The cannabinoid receptor 1 (CNR1) gene is 1 of the 2 receptors expressed in the brain. It has been reported to be associated with alcoholism and multiple drug abuse and dependence. OBJECTIVE: To test the hypothesis that the CNR1 gene is associated with nicotine dependence. DESIGN: Genotype-phenotype association study. Ten single-nucleotide polymorphisms were genotyped in the CNR1 gene in 2 independent samples. For the first sample (n = 688), a 3-group case-control design was used to test allele association with smoking initiation and nicotine dependence. For the second sample (n = 961), association was assessed with scores from the Fagerström Test for Nicotine Dependence (FTND). Settings Population samples selected from the Mid-Atlantic Twin Registry. PARTICIPANTS: White patients aged 18 to 65 years who met the criteria of inclusion. MAIN OUTCOME MEASURES: Fagerström Tolerance Questionnaire and FTND scores. RESULTS: Significant single-marker and haplotype associations were found in both samples, and the associations were female specific. Haplotype 1-1-2 of markers rs2023239-rs12720071-rs806368 was associated with nicotine dependence and FTND score in the 2 samples (P < .001 and P = .009, respectively). CONCLUSION: Variants and haplotypes in the CNR1 gene may alter the risk for nicotine dependence, and the associations are likely sex specific.

Lack of association between the amiloride-sensitive cation channel 2 (ACCN2) gene and anxiety spectrum disorders.

OBJECTIVE: Ion channels are involved in a wide range of central nervous system functions and have been implicated in several neuropsychiatric disorders. Rodent studies suggest that the acid-sensing ion channel, ASIC1, may play a role in fear conditioning, a model for human anxiety disorders. In this study, we examined, for the first time, the human analog of ASIC1, the amiloride-sensitive cation channel 2 (ACCN2) gene, for its association with genetic risk across a range of anxiety spectrum phenotypes. METHODS: Using multivariate structural equation modeling, we selected twin pairs scoring at the extremes of a latent genetic risk factor that underlies susceptibility to neuroticism, major depression, generalized anxiety disorder, panic disorder, agoraphobia, and social phobia, from the population-based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. One member from each selected pair for whom DNA was available was entered into a 2-stage, case-control association study for the ACCN2 gene. In the resulting sample of 589 cases and 539 controls, a total of seven single nucleotide polymorphisms that represented the major allelic variation across the ACCN2 locus were screened in stage 1, the positive results of which were tested for replication in stage 2. RESULTS: Although several markers or haplotypic combinations met threshold significance criteria in stage 1, their association was not replicated in stage 2. Post hoc analyses did not reveal significant association to the specific psychiatric phenotypes. CONCLUSIONS: Although the ACCN2 gene may play a role in rodent fear conditioning, we could not detect association with genetic risk shared among human anxiety spectrum disorders.