Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of "cryptic" SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter-and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.The genus Pasteuria, a group of endospore-producing Grampositive bacteria, belongs to the Bacillus-Clostridium clade (9). Of those characterized, all are parasites of free-living and plant parasitic nematodes, with the exception of Pasteuria ramosa (24), a parasite of the water flea (Cladocera, Daphnia spp.).Pasteuria penetrans is an obligate parasite of plant parasitic nematodes belonging to Meloidogyne spp., which are responsible worldwide for more than $100 billion worth of annual crop damage (30). Populations, defined here as collections of endospores obtained from field isolates of particular host female nematodes in distinct geographic locations, have been isolated from Meloidogyne spp. from around the globe, but there is very little understanding of the genetic variation between and within populations. Previous studies have focused on the position of P. penetrans in the bacterial kingdom (9, 31, 34) and the use of consensus 16S rRNA gene sequences (6,7,18) to differentiate between species and populations of Pasteuria (5, 16, 33). However, this information has not been extensively supported with sequence data from protein-encoding genes, which potentially provide an additional source of discrimination, with the exception of one study examining partial coding sequences of some genes in P. ramosa populations (32), as well as a recent study of genes encoding collagen-like proteins in this species (22a).Geographically distinct populations of P. penetrans display various degrees of host specificity (11) through mechanisms that are not fully understood. It has been shown that cuticle heterogeneity, as exhibited by endospore attachment, is not linked...

The goal of this study was to identify correlations between interpersonal genetic variability and alterations in the intracellular glucocorticoid receptor (GR) signaling response to mild cardiovascular stress. Circulating cortisol concentrations in healthy volunteers varied significantly among participants, both before and after HPA axis stimulation. These differences could not be correlated with biobehavioral survey responses or targeted Sanger sequencing of genes encoding the GR, hsp90, or CRHR1. We consequently hypothesized that whole exome sequencing (WES) would provide a feasible approach for determining variability among phenotypic groups and revealing causal gene candidates that had not been previously considered. WES was conducted using genomic DNA from four participants, one showing the canonical high cortisol response and three showing an enigmatic negative response. Using an Agilent V4 exome enrichment platform and Illumina HiSeq2000 paired end sequencing, >44 million reads per sample were aligned to the hg19 reference build. Comparative analysis of opposing phenotypes showed a variance profile of mutations associated with the negative cortisol response that warrant further investigation. We predict that the statistically significant genetic variants shared among negative cortisol responders may be associated with critical changes in the GR‐mediated signaling pathway and alter a patient's response to stress and glucocorticoid‐based pharmacotherapies.

Hsp90 is a highly conserved eukaryotic chaperone protein responsible for mediating a multitude of intracellular signaling molecules, including those associated with the glucocorticoid receptor (GR) and stress response. The goal of this study was to identify the extent to which single nucleotide polymorphisms (SNPs) in genes encoding hsp90 correlated with previously observed phenotypic variations in circulating cortisol and hsp90 subcellular localization. Salivary cortisol was measured in healthy volunteers before and after HPA stimulation via mild cardiovascular stress. Whole exome sequencing of a matched participant pair identified HSP90AB1, encoding the stress‐inducible isoform of hsp90, as a potential causal candidate gene warranting further examination. Exons of HSP90AB1 and HSP90AA1 were amplified and sequenced in 20 participants with known differences in cortisol response and hsp90 localization using PCR and the Sanger method. Out of the twenty participants, three HSP90AB1 and eight HSP90AA1 SNPs were found, all of which were registered in the NCBI SNP database. Interestingly, a discrepancy in results was observed between SNPs called by whole exome analysis and those detected by Sanger sequencing. Although no correlation was found between analyzed SNPs, cortisol response, and hsp90 localization, further work utilizing this methodology and consideration of phenotypic polygenicity will likely provide a more comprehensive understanding of hsp90‐mediated GR signaling.

The goal of our study was to identify correlations between interpersonal genetic variability in healthy volunteers and alterations in the glucocorticoid receptor‐associated signaling pathway. Salivary cortisol (CORT) concentrations were measured in healthy volunteers (n=66) before and after CORT stimulation in order to determine variability in circulating CORT levels and CORT response. Statistical analysis revealed inter‐participant CORT variability in concentration or percent change were not exclusively correlated to participant demographics, health history, or glucocorticoid receptor (GR) single nucleotide polymorphisms (SNPs). Interestingly, nuclear localization of the GR‐associated molecular chaperone protein hsp90 correlated with an elevated pre‐stimulus CORT concentration. To investigate the observed CORT response‐hsp90 localization association further, coding and proximal coding regions of the constitutively expressed hsp90 gene (HSP90AA) were sequenced to identify a possible genetic linkage. Eight hsp90 SNPs were identified, four of which were not reported in the NCBI database. All participants expressed at least two of the hsp90 SNPs, with some participants exhibiting as many as five. These data demonstrate inter‐person differences in CORT response, nuclear hsp90 concentrations, and GR‐associated SNPs are readily detected and potentially interrelated.

The goal of this study was to identify correlations between interpersonal genetic variability and alterations in the intracellular glucocorticoid receptor (GR)‐mediated signaling pathway. Salivary cortisol concentrations were measured in healthy volunteers (n=66) in order to determine differences in circulating cortisol concentrations and cortisol response to HPA stimulation. Statistical analysis indicated inter‐participant cortisol variability was not correlated to participant demographics, health history, or SNPs in genes encoding the GR (NR3C1) or the essential GR chaperone protein hsp90 (HSP90AA1). Two participants with highly similar demographic profiles and opposing responses to HPA stimulation were selected for matched exome sequencing. Filtering analysis identified candidate SNPs in approximately 30 genes involved in mediating the cortisol response and GR signaling. Interestingly, corticotropin releasing hormone receptor 1 (CRHR1) showed a difference of over 20 SNPs between the matched exome pair. CRHR1 regions were sequenced from the larger participant group, and participants with average cortisol concentrations that slightly decreased following stimulation most commonly exhibited a unique SNP profile. We speculate participants with SNP profiles associated with altered cortisol response may share a corresponding sensitivity to corticosteroid‐based drug therapies. Grant Funding Source: Supported by NIH AREA grant GM086822