Interbacterial interaction pathways play an important role in defining the structure and complexity of bacterial associations. A quantitative description of such pathways offers promise for understanding the forces that contribute to community composition. We developed time-lapse fluorescence microscopy methods for quantitation of interbacterial interactions and applied these to the characterization of type VI secretion (T6S) in Pseudomonas aeruginosa. Our analyses allowed a direct determination of the efficiency of recipient cell lysis catalyzed by this intercellular toxin delivery pathway and provided evidence that its arsenal extends beyond known effector proteins. Measurement of T6S apparatus localization revealed correlated activation among neighboring cells, which, taken together with genetic data, implicate the elaboration of a functional T6S apparatus with a marked increase in susceptibility to intoxication. This possibility was supported by the identification of T6S-inactivating mutations in a genome-wide screen for resistance to T6S-mediated intoxication and by time-lapse fluorescence microscopy analyses showing a decreased lysis rate of recipient cells lacking T6S function. Our discoveries highlight the utility of singlecell approaches for measuring interbacterial phenomena and provide a foundation for studying the contribution of a widespread bacterial interaction pathway to community structure.imaging | peptidoglycan | polymicrobial | protein secretion

T he corticosteroid hormones cortisol and aldosterone are important determinants of blood pressure and cardiovascular risk. Excess cortisol results in Cushing syndrome, associated with hypertension and accelerated atherogenesis, 1 whereas excess aldosterone, in primary aldosteronism, leads to severe hypertension with markedly increased risk of myocardial infarction, stroke, and left ventricular hypertrophy. 2 The frequency of primary aldosteronism in hypertensive patients is now accepted to be ≈5% to 10% 3,4 ; recent studies show that aldosterone is an important predictor of cardiovascular risk and outcome, with levels toward the high end of the normal range predicting blood pressure and development of hypertension. 5 Controlled release of corticosteroids from the adrenal cortex is achieved, in part, by strictly regulated expression of genes encoding the steroidogenic enzymes that catalyze their biosynthetic pathways. The final enzymes in this process are particularly important: 11β-hydroxylase (CYP11B1) is responsible for the terminal conversion that produces cortisol, whereas aldosterone synthase (CYP11B2) fulfills the equivalent role in aldosterone production. The CYP11B1 and CYP11B2 genes lie in tandem on human chromosome 8 and have ≈93% sequence similarity within their coding regions. 6 Changes in the expression of these genes have been observed in cases of aldosterone-producing adenoma (APA). 7,8 In addition, the rate of aldosterone production is known to be heritable, 9 and several polymorphisms in the CYP11B1 and CYP11B2 genes associate with altered 11β-hydroxylation, aldosterone production, or hypertension. 10 Although it is relatively straightforward to propose mechanisms by which polymorphisms in the 5′ regulatory regions of these genes, such as rs1799998, 11 could affect transcription, it is not immediately apparent how polymorphisms located in introns or the 3′ untranslated region (3′ UTR) of these genes could alter expression. However, in recent years, microRNAs (miRNAs) have emerged as key regulatory molecules that regulate ≈30% of human genes.12 They are endogenous, single-stranded noncoding RNA molecules of ≈22 nucleotides, produced through a series of maturation reactions mediated by the RNase III enzymes, Drosha and Dicer.13 These post-transcriptional regulatory molecules exert their effects by targeting the 3′ UTR of specific mRNAs. Through mRNA destabilization Abstract-Dysregulation of aldosterone or cortisol production can predispose to hypertension, as seen in aldosteroneproducing adenoma, a form of primary aldosteronism. We investigated the role of microRNA (miRNA) in their production, with particular emphasis on the CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) genes, which produce the enzymes responsible for the final stages of cortisol and aldosterone biosynthesis, respectively. Knockdown of Dicer1, a key enzyme in miRNA maturation, significantly altered CYP11B1 and CYP11B2 expression in a human adrenocortical cell line. Screening of nondiseased human adrenal and aldost...

The loss of normal regulation of corticosteroid secretion is important in the development of cardiovascular disease. We previously showed that microRNAs regulate the terminal stages of corticosteroid biosynthesis. Here, we assess microRNA regulation across the whole corticosteroid pathway. Knockdown of microRNA using Dicer1 siRNA in H295R adrenocortical cells increased levels of CYP11A1, CYP21A1, and CYP17A1 mRNA and the secretion of cortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycorticosterone, and aldosterone. Bioinformatic analysis of genes involved in corticosteroid biosynthesis or metabolism identified many putative microRNA-binding sites, and some were selected for further study. Manipulation of individual microRNA levels demonstrated a direct effect of miR-125a-5p and miR-125b-5p on CYP11B2 and of miR-320a-3p levels on CYP11A1 and CYP17A1 mRNA. Finally, comparison of microRNA expression profiles from human aldosterone-producing adenoma and normal adrenal tissue showed levels of various microRNAs, including miR-125a-5p to be significantly different. This study demonstrates that corticosteroidogenesis is regulated at multiple points by several microRNAs and that certain of these microRNAs are differentially expressed in tumorous adrenal tissue, which may contribute to dysregulation of corticosteroid secretion. These findings provide new insights into the regulation of corticosteroid production and have implications for understanding the pathology of disease states where abnormal hormone secretion is a feature.