HilE regulates HilD by blocking DNA binding in Salmonella enterica serovar Typhimurium

ABSTRACT The Salmonella type three secretion system (T3SS), encoded in the Salmonella pathogenicity island 1 (SPI1) locus, mediates the invasion of the host intestinal epithelium. SPI1 expression is dependent upon three AraC-like regulators: HilD, HilC, and RtsA. These regulators act in a complex feed-forward loop to activate each other and hilA , which encodes the activator of the T3SS structural genes. HilD has been shown to be the major integration point of most signals known to activate the expression of the SPI1 T3SS, acting as a switch to control induction of the system. HilE is a negative regulator that acts upon HilD. Here we provide genetic and biochemical data showing that HilE specifically binds to HilD but not to HilC or RtsA. This protein-protein interaction blocks the ability of HilD to bind DNA as shown by both an in vivo reporter system and an in vitro gel shift assay. HilE does not affect HilD dimerization, nor does it control the stability of the HilD protein. We also investigated the role of HilE during the infection of mice using competition assays. Although deletion of hilE does not confer a phenotype, the hilE mutation does suppress the invasion defect conferred by loss of FliZ, which acts as a positive signal controlling HilD protein activity. Together, these data suggest that HilE functions to restrict low-level HilD activity, preventing premature activation of SPI1 until positive inputs reach a threshold required to fully induce the system. IMPORTANCE Salmonella is a leading cause of gastrointestinal and systemic disease throughout the world. The SPI1 T3SS is required for Salmonella to induce inflammatory diarrhea and to gain access to underlying tissue. A complex regulatory network controls expression of SPI1 in response to numerous physiological inputs. Most of these signals impinge primarily on HilD translation or activity. The system is triggered when HilD activity crosses a threshold that allows efficient activation of its own promoter. This threshold is set by HilE, which binds to HilD to prevent the inevitable minor fluctuations in HilD activity from inappropriately activating the system. The circuit also serves as a paradigm for systems that must integrate numerous environmental parameters to control regulatory output. </jats:p

A novel pair of inducible expression vectors for use in Methylobacterium extorquens

Background: Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used γ-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the α-proteobacterium Methylobacterium extorquens, a model methylotroph. Findings: We found that the P R promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P R/cmtO and P R/tetO, were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P R/cmtO and P R/tetO have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL - essential for growth on methanol - where we show P R/tetO is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts. Conclusions: We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P R/tetO promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria

Quantification of cervical elasticity during pregnancy based on transvaginal ultrasound imaging and stress measurement

Objective: Strain elastography and shear wave elastography are two commonly used methods to quantify cervical elasticity; however, they have limitations. Strain elastography is effective in showing tissue elasticity distribution in a single image, but the absence of stress information causes difficulty in comparing the results acquired from different imaging sessions. Shear wave elastography is effective in measuring shear wave speed (an intrinsic tissue property correlated with elasticity) in relatively homogeneous tissue, such as in the liver. However, for inhomogeneous tissue in the cervix, the shear wave speed measurement is less robust. To overcome these limitations, we develop a quantitative cervical elastography system by adding a stress sensor to an ultrasound imaging system. Methods: In an imaging session for quantitative cervical elastography, we use the transvaginal ultrasound imaging system to record B-mode images of the cervix showing its deformation and use the stress sensor to record the probe-surface stress simultaneously. We develop a correlation-based automatic feature tracking algorithm to quantify the deformation, from which the strain is quantified. After each imaging session, we calibrate the stress sensor and transform its measurement to true stress. Applying a linear regression to the stress and strain, we obtain an approximation of the cervical Young's modulus. Results: We validate the accuracy and robustness of this elastography system using phantom experiments. Applying this system to pregnant participants, we observe significant softening of the cervix during pregnancy (p-value < 0.001) with the cervical Young's modulus decreasing 3.95% per week. We estimate that geometric mean values of cervical Young's moduli during the first (11 to 13 weeks), second, and third trimesters are 13.07 kPa, 7.59 kPa, and 4.40 kPa, respectively.Comment: 26 pages, 8 figures, 1 tabl

Breast cancer risk, worry, and anxiety: Effect on patient perceptions of false-positive screening results

OBJECTIVE: The impact of mammography screening recall on quality-of-life (QOL) has been studied in women at average risk for breast cancer, but it is unknown whether these effects differ by breast cancer risk level. We used a vignette-based survey to evaluate how women across the spectrum of breast cancer risk perceive the experience of screening recall. METHODS: Women participating in mammography or breast MRI screening were recruited to complete a vignette-based survey. Using a numerical rating scale (0-100), women rated QOL for hypothetical scenarios of screening recall, both before and after benign results were known. Lifetime breast cancer risk was calculated using Gail and BRCAPRO risk models. Risk perception, trait anxiety, and breast cancer worry were assessed using validated instruments. RESULTS: The final study cohort included 162 women at low (n = 43, 26%), intermediate (n = 66, 41%), and high-risk (n = 53, 33%). Actual breast cancer risk was not a predictor of QOL for any of the presented scenarios. Across all risk levels, QOL ratings were significantly lower for the period during diagnostic uncertainty compared to after benign results were known (p \u3c 0.05). In multivariable regression analyses, breast cancer worry was a significant predictor of decreased QoL for all screening scenarios while awaiting results, including scenarios with non-invasive imaging alone or with biopsy. High trait anxiety and family history predicted lower QOL scores after receipt of benign test results (p \u3c 0.05). CONCLUSIONS: Women with high trait anxiety and family history may particularly benefit from discussions about the risk of recall when choosing a screening regimen

Enabling precision medicine in neonatology, an integrated repository for preterm birth research.

Preterm birth, or the delivery of an infant prior to 37 weeks of gestation, is a significant cause of infant morbidity and mortality. In the last decade, the advent and continued development of molecular profiling technologies has enabled researchers to generate vast amount of 'omics' data, which together with integrative computational approaches, can help refine the current knowledge about disease mechanisms, diagnostics, and therapeutics. Here we describe the March of Dimes' Database for Preterm Birth Research (http://www.immport.org/resources/mod), a unique resource that contains a variety of 'omics' datasets related to preterm birth. The database is open publicly, and as of January 2018, links 13 molecular studies with data across tens of thousands of patients from 6 measurement modalities. The data in the repository are highly diverse and include genomic, transcriptomic, immunological, and microbiome data. Relevant datasets are augmented with additional molecular characterizations of almost 25,000 biological samples from public databases. We believe our data-sharing efforts will lead to enhanced research collaborations and coordination accelerating the overall pace of discovery in preterm birth research

In vivo characterization of connective tissue remodeling using infrared photoacoustic spectra

Premature cervical remodeling is a critical precursor of spontaneous preterm birth, and the remodeling process is characterized by an increase in tissue hydration. Nevertheless, current clinical measurements of cervical remodeling are subjective and detect only late events, such as cervical effacement and dilation. Here, we present a photoacoustic endoscope that can quantify tissue hydration by measuring near-infrared cervical spectra. We quantify the water contents of tissue-mimicking hydrogel phantoms as an analog of cervical connective tissue. Applying this method to pregnant women in vivo, we observed an increase in the water content of the cervix throughout pregnancy. The application of this technique in maternal healthcare may advance our understanding of cervical remodeling and provide a sensitive method for predicting preterm birth

Intestinal Long-Chain Fatty Acids Act as a Direct Signal To Modulate Expression of the Salmonella Pathogenicity Island 1 Type III Secretion System

Salmonella enterica serovar Typhimurium uses the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS) to induce inflammatory diarrhea and bacterial uptake into intestinal epithelial cells. The expression of hilA, encoding the transcriptional activator of the T3SS structural genes, is directly controlled by three AraC-like regulators, HilD, HilC, and RtsA, each of which can activate hilD, hilC, rtsA, and hilA genes, forming a complex feed-forward regulatory loop. Expression of the SPI1 genes is tightly controlled by numerous regulatory inputs to ensure proper timing in production of the T3SS apparatus. Loss of FadD, an acyl coenzyme A (acyl-CoA) synthetase required for degradation of long-chain fatty acids (LCFAs), was known to decrease hilA expression. We show that free external LCFAs repress expression of hilA independently of FadD and the LCFA degradation pathway. Genetic and biochemical evidence suggests that LCFAs act directly to block primarily HilD activity. Further analyses show that in the absence of FadD, hilA expression is downregulated due to endogenous production of free LCFAs, which are excreted into the culture medium via TolC and then transported back into the bacterial cell via FadL. A fadL mutant is more virulent than the wild-type strain in mouse oral competition assays independently of LCFA degradation, showing that, in the host, dietary LCFAs serve as a signal for proper regulation of SPI1 expression, rather than an energy source

FliZ Regulates Expression of the Salmonella Pathogenicity Island 1 Invasion Locus by Controlling HilD Protein Activity in Salmonella enterica Serovar Typhimurium ▿ †

A prerequisite for Salmonella enterica to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals through a variety of global regulatory systems. We have previously shown that three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control the expression of the hilA gene, which encodes the direct regulator of the SPI1 structural genes. In this work, we characterize a major positive regulator of this system, the flagellar protein FliZ. Through genetic and biochemical analyses, we show that FliZ posttranslationally controls HilD to positively regulate hilA expression. This mechanism is independent of other flagellar components and is not mediated through the negative regulator HilE or through FliZ-mediated RpoS regulation. We demonstrate that FliZ controls HilD protein activity and not stability. FliZ regulates HilD in the absence of Lon protease, previously shown to degrade HilD. Indeed, it appears that FliZ, rather than HilD, is the most relevant target of Lon as it relates to SPI1 expression. Mutants lacking FliZ are significantly attenuated in their ability to colonize the intestine but are unaffected during systemic infection. The intestinal attenuation is partially dependent on SPI1, but FliZ has additional pleiotropic effects