TamAB is regulated by PhoPQ and functions in outer membrane homeostasis during Salmonella pathogenesis.

Salmonella survive and replicate in macrophages, which normally kill bacteria by exposing them to a variety of harsh conditions and antimicrobial effectors, many of which target the bacterial cell envelope. The PhoPQ two-component system responds to the phagosome environment and induces factors that protect the outer membrane, allowing adaptation and growth in the macrophage. We show that PhoPQ induces the transcription of the tamAB operon both in vitro and in macrophages. The TamA protein is structurally similar to BamA, an essential protein in the Bam complex that assembles ß-barrel proteins in the outer membrane, while TamB is an AsmA-family protein implicated in lipid transport between the inner and outer membranes. We show that the Bam machinery is stressed in vitro under low Mg2+, low pH conditions that mimic the phagosome. Not surprisingly, mutations affecting Bam function confer significant virulence defects. Although loss of TamAB alone confers no virulence defect, a tamAB deletion confers a synthetic phenotype in bam mutant backgrounds in animals and macrophages, and in vitro upon treatment with vancomycin or sodium dodecyl sulfate. Mutations affecting YhdP, which functions in partial redundancy with TamB, also confer synthetic phenotypes with bam mutations in the animal, but this interaction is not evident in vitro. Thus, in the harsh phagocytic environment of the macrophage, the outer membrane Bam machinery is compromised, and the TamAB system, and perhaps other PhoPQ-regulated factors, is induced to compensate. It is most likely that TamAB and other systems assist the Bam complex indirectly by affecting outer membrane properties. IMPORTANCE The TamAB system has been implicated in both outer membrane protein localization and phospholipid transport between the inner and outer membranes. We show that the ß-barrel protein assembly complex, Bam, is stressed under conditions thought to mimic the macrophage phagosome. TamAB expression is controlled by the PhoPQ two-component system and induced in macrophages. This system somehow compensates for the Bam complex as evidenced by the fact that mutations affecting the two systems confer synthetic phenotypes in animals, macrophages, and in vitro in the presence of vancomycin or SDS. This study has implications concerning the role of TamAB in outer membrane homeostasis. It also contributes to our understanding of the systems necessary for Salmonella to adapt and reproduce within the macrophage phagosome.

Envelope Stress and Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System.

Salmonella enterica serovar Typhimurium uses a type three secretion system (T3SS) encoded on the Salmonella pathogenicity island 1 (SPI1) to invade intestinal epithelial cells and induce inflammatory diarrhea. The SPI1 T3SS is regulated by numerous environmental and physiological signals, integrated to either activate or repress invasion. Transcription of hilA, encoding the transcriptional activator of the SPI1 structural genes, is activated by three AraC-like regulators, HilD, HilC, and RtsA, that act in a complex feed-forward loop. Deletion of bamB, encoding a component of the ß-barrel assembly machinery, causes a dramatic repression of SPI1, but the mechanism was unknown. Here, we show that partially defective ß-barrel assembly activates the RcsCDB regulon, leading to decreased hilA transcription. This regulation is independent of RpoE activation. Though Rcs has been previously shown to repress SPI1 when disulfide bond formation is impaired, we show that activation of Rcs in a bamB background is dependent on the sensor protein RcsF, whereas disulfide bond status is sensed independently. Rcs decreases transcription of the flagellar regulon, including fliZ, the product of which indirectly activates HilD protein activity. Rcs also represses hilD, hilC, and rtsA promoters by an unknown mechanism. Both dsbA and bamB mutants have motility defects, though this is simply regulatory in a bamB background; motility is restored in the absence of Rcs. Effector secretion assays show that repression of SPI1 in a bamB background is also regulatory; if expressed, the SPI1 T3SS is functional in a bamB background. This emphasizes the sensitivity of SPI1 regulation to overall envelope homeostasis.IMPORTANCESalmonella causes worldwide foodborne illness, leading to massive disease burden and an estimated 600,000 deaths per year. Salmonella infects orally and invades intestinal epithelial cells using a type 3 secretion system that directly injects effector proteins into host cells. This first step in invasion is tightly regulated by a variety of inputs. In this work, we demonstrate that Salmonella senses the functionality of outer membrane assembly in determining regulation of invasion machinery, and we show that Salmonella uses distinct mechanisms to detect specific perturbations in envelope assembly.

PhoP-Mediated Repression of the SPI1 Type 3 Secretion System in Salmonella enterica Serovar Typhimurium.

Salmonella must rapidly adapt to various niches in the host during infection. Relevant virulence factors must be appropriately induced, and systems that are detrimental in a particular environment must be turned off. Salmonella infects intestinal epithelial cells using a type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The system is controlled by three AraC-like regulators, HilD, HilC, and RtsA, which form a complex feed-forward loop to activate expression of hilA, encoding the main transcriptional regulator of T3SS structural genes. This system is tightly regulated, with many of the activating signals acting at the level of hilD translation or HilD protein activity. Once inside the phagosomes of epithelial cells, or in macrophages during systemic stages of disease, the SPI1 T3SS is no longer required or expressed. Here, we show that the PhoPQ two-component system, critical for intracellular survival, appears to be the primary mechanism by which Salmonella shuts down the SPI1 T3SS. PhoP negatively regulates hilA through multiple distinct mechanisms: direct transcriptional repression of the hilA promoter, indirect transcriptional repression of both the hilD and rtsA promoters, and activation of the small RNA (sRNA) PinT. Genetic analyses and electrophoretic mobility shift assays suggest that PhoP specifically binds the hilA promoter to block binding of activators HilD, HilC, and RtsA as a mechanism of repression.IMPORTANCESalmonella is one of the most common foodborne pathogens, causing an estimated 1.2 million illnesses per year in the United States. A key step in infection is the activation of the bacterial invasion machinery, which induces uptake of the bacterium into epithelial cells and leads to induction of inflammatory diarrhea. Upon entering the vacuolar compartments of host cells, Salmonella senses an environmental transition and represses the invasion machinery with a two-component system relevant for survival within the vacuole. This adaptation to specific host niches is an important example of how signals are integrated for survival of the pathogen.

The Small RNA PinT Contributes to PhoP-Mediated Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System in Salmonella enterica Serovar Typhimurium.

Salmonella enterica serovar Typhimurium induces inflammatory diarrhea and bacterial uptake into intestinal epithelial cells using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). HilA activates transcription of the SPI1 structural components and effector proteins. Expression of hilA is activated by HilD, HilC, and RtsA, which act in a complex feed-forward regulatory loop. Many environmental signals and other regulators are integrated into this regulatory loop, primarily via HilD. After the invasion of Salmonella into host intestinal epithelial cells or during systemic replication in macrophages, the SPI T3SS is no longer required or expressed. We have shown that the two-component regulatory system PhoPQ, required for intracellular survival, represses the SPI1 T3SS mostly by controlling the transcription of hilA and hilD Here we show that PinT, one of the PhoPQ-regulated small RNAs (sRNAs), contributes to this regulation by repressing hilA and rtsA translation. PinT base pairs with both the hilA and rtsA mRNAs, resulting in translational inhibition of hilA, but also induces degradation of the rts transcript. PinT also indirectly represses expression of FliZ, a posttranslational regulator of HilD, and directly represses translation of ssrB, encoding the primary regulator of the SPI2 T3SS. Our in vivo mouse competition assays support the concept that PinT controls a series of virulence genes at the posttranscriptional level in order to adapt Salmonella from the invasion stage to intracellular survival.IMPORTANCESalmonella is one of the most important food-borne pathogens, infecting over one million people in the United States every year. These bacteria use a needle-like device to interact with intestinal epithelial cells, leading to invasion of the cells and induction of inflammatory diarrhea. A complex regulatory network controls expression of the invasion system in response to numerous environmental signals. Here we explore the molecular mechanisms by which the small RNA PinT contributes to this regulation, facilitating inactivation of the system after invasion. PinT controls several important virulence systems in Salmonella, tuning the transition between different stages of infection.

Genetic variation underlying resistance to infectious hematopoietic necrosis virus in a steelhead trout (Oncorhynchus mykiss) population.

Understanding the mechanisms of host resistance to pathogens will allow insights into the response of wild populations to the emergence of new pathogens. Infectious hematopoietic necrosis virus (IHNV) is endemic to the Pacific Northwest and infectious to Pacific salmon and trout (Oncorhynchus spp.). Emergence of the M genogroup of IHNV in steelhead trout O. mykiss in the coastal streams of Washington State, between 2007 and 2011, was geographically heterogeneous. Differences in host resistance due to genetic change were hypothesized to be a factor influencing the IHNV emergence patterns. For example, juvenile steelhead trout losses at the Quinault National Fish Hatchery (QNFH) were much lower than those at a nearby facility that cultures a stock originally derived from the same source population. Using a classical quantitative genetic approach, we determined the potential for the QNFH steelhead trout population to respond to selection caused by the pathogen, by estimating the heritability for 2 traits indicative of IHNV resistance, mortality (h² = 0.377 (0.226 - 0.550)) and days to death (h² = 0.093 (0.018 - 0.203)). These results confirm that there is a genetic basis for resistance and that this population has the potential to adapt to IHNV. Additionally, genetic correlation between days to death and fish length suggests a correlated response in these traits to selection. Reduction of genetic variation, as well as the presence or absence of resistant alleles, could affect the ability of populations to adapt to the pathogen. Identification of the genetic basis for IHNV resistance could allow the assessment of the susceptibility of other steelhead populations.

Final 1-Year Results of the TUTOR Randomized Trial Comparing Carpal Tunnel Release with Ultrasound Guidance to Mini-open Technique.

Background: Studies comparing carpal tunnel release with ultrasound guidance (CTR-US) to mini-open CTR (mOCTR) are limited. This randomized trial compared the efficacy and safety of these techniques. Methods: In this multicenter randomized trial, patients were randomized (2:1) to unilateral CTR-US or mOCTR. Outcomes included Boston Carpal Tunnel Questionnaire Symptom Severity Scale (BCTQ-SSS) and Functional Status Scale (BCTQ-FSS), numeric pain scale (0-10), EuroQoL-5 Dimension 5-Level (EQ-5D-5L), scar outcomes, and complications over 1 year. Results: Patients received CTR-US (n = 94) via wrist incision (mean 6 mm) or mOCTR (n = 28) via palmar incision (mean 22 mm). Comparing CTR-US with mOCTR, the mean changes in BCTQ-SSS (-1.8 versus -1.8; P = 0.96), BCTQ-FSS (-1.0 versus -1.0; P = 0.75), numeric pain scale (-3.9 versus -3.8; P = 0.74), and EQ-5D-5L (0.13 versus 0.12; P = 0.79) over 1 year were comparable between groups. Freedom from scar sensitivity or pain favored CTR-US (95% versus 74%; P = 0.005). Complications occurred in 2.1% versus 3.6% of patients (P = 0.55), all within 3 weeks postprocedure. There was one revision surgery in the CTR-US group, and no revisions for persistent or recurrent symptoms in either group. Conclusions: CTR-US and mOCTR demonstrated similar improvement in carpal tunnel syndrome symptoms and quality of life with comparable low complication rates over 1 year of follow-up. CTR-US was performed with a smaller incision and associated with less scar discomfort.

Social Synchronization of Conditioned Fear in Mice Requires Ventral Hippocampus Input to the Amygdala.

BACKGROUND: Social organisms synchronize behaviors as an evolutionary-conserved means of thriving. Synchronization under threat, in particular, benefits survival and occurs across species, including humans, but the underlying mechanisms remain unknown because of the scarcity of relevant animal models. Here, we developed a rodent paradigm in which mice synchronized a classically conditioned fear response and identified an underlying neuronal circuit. METHODS: Male and female mice were trained individually using auditory fear conditioning and then tested 24 hours later as dyads while allowing unrestricted social interaction during exposure to the conditioned stimulus under visible or infrared illumination to eliminate visual cues. The synchronization of the immobility or freezing bouts was quantified by calculating the effect size Cohen's d for the difference between the actual freezing time overlap and the overlap by chance. The inactivation of the dorsomedial prefrontal cortex, dorsal hippocampus, or ventral hippocampus was achieved by local infusions of muscimol. The chemogenetic disconnection of the hippocampus-amygdala pathway was performed by expressing hM4D(Gi) in the ventral hippocampal neurons and infusing clozapine N-oxide in the amygdala. RESULTS: Mice synchronized cued but not contextual fear. It was higher in males than in females and attenuated in the absence of visible light. Inactivation of the ventral but not dorsal hippocampus or dorsomedial prefrontal cortex abolished fear synchronization. Finally, the disconnection of the hippocampus-amygdala pathway diminished fear synchronization. CONCLUSIONS: Mice synchronize expression of conditioned fear relying on the ventral hippocampus-amygdala pathway, suggesting that the hippocampus transmits social information to the amygdala to synchronize threat response.

Multicenter randomized trial of carpal tunnel release with ultrasound guidance versus mini-open technique.

BACKGROUND: Comparative studies of carpal tunnel release with ultrasound guidance (CTR-US) vs. mini-open CTR (mOCTR) are limited, prompting development of this randomized trial to compare efficacy and safety of these techniques. RESEARCH DESIGN AND METHODS: Patients were randomized (2:1) to CTR-US or mOCTR, treated by experienced hand surgeons (median previous cases: 12 CTR-US; 1000 mOCTR), and followed for 3 months. RESULTS: Among 149 randomized patients, 122 received CTR-US (n = 94) or mOCTR (n = 28). Mean incision length was 6 ± 2 mm in the wrist (CTR-US) vs. 22 ± 7 mm in the palm (mOCTR) (p < 0.001). Median time to return to daily activities (2 vs. 2 days; p = 0.81) and work (3 vs. 4 days; p = 0.61) were similar. Both groups reported statistically significant and clinically important improvements in Boston Carpal Tunnel Questionnaire Symptom Severity and Functional Status Scales, Numeric Pain Scale, and EuroQoL-5 Dimension 5-Level, with no statistical differences between groups. Freedom from wound sensitivity and pain favored CTR-US (61.1% vs. 17.9%; p < 0.001). Adverse event rates were low in each group (2.1% vs. 3.6%; p = 0.55). CONCLUSIONS: The efficacy and safety of CTR-US were comparable to mOCTR despite less previous surgical experience with CTR-US. The choice of CTR technique should be determined by shared decision-making between patient and physician. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov identifier is NCT05405218.

Trial of ultrasound guided carpal tunnel release versus traditional open release (TUTOR).

BACKGROUND: Carpal tunnel release (CTR) is a surgical treatment option for patients with carpal tunnel syndrome (CTS) symptoms that are unresponsive to conservative treatment. Most patients experience symptomatic relief after CTR regardless of the surgical technique. However, direct comparisons of the safety and effectiveness between CTR surgical techniques are limited. The purpose of this randomized controlled trial is to compare the safety and effectiveness of CTR with ultrasound guidance (CTR-US) versus mini-open CTR (mOCTR) in subjects with symptomatic CTS. DESIGN AND METHODS: TUTOR (Trial of Ultrasound guided CTR versus Traditional Open Release) is a randomized controlled trial in which 120 subjects at up to 12 sites in the United States will be randomized (2:1) to receive CTR-US or mOCTR. The primary endpoint of the study is the percentage of patients who return to normal daily activities within 3 days of the procedure. Secondary endpoints of the study are median time to return to normal daily activities, percentage of patients who return to work within 3 days of the procedure, median time to return to work, Boston Carpal Tunnel Questionnaire Symptom Severity Scale (BCTQ-SSS) change score at 3 months, BCTQ Functional Status Scale (BCTQ-FSS) change score at 3 months, Numeric Pain Scale change score at 3 months, EuroQoL-5 Dimension 5-Level (EQ-5D-5L) change score at 3 months, and the incidence of device- or procedure-related adverse events at 3 months. Patient follow-up in this trial will continue for 1 year. ETHICS AND DISSEMINATION: This study was approved by a central institutional review board and ongoing trial oversight will be provided by a data safety monitoring board (DSMB). The authors intend to report the results of this trial at medical conferences and peer-reviewed journals. The outcomes of TUTOR will have important clinical and economic implications for all stakeholders involved in treating patients with CTS. STUDY REGISTRATION: ClinicalTrials.gov (https://clinicaltrials.gov): NCT05405218. LEVEL OF EVIDENCE: 1.

XoxF is required for expression of methanol dehydrogenase in Methylobacterium extorquens AM1.

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ∼50% identical to MxaF and ∼90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes.

A survey of culturable aerobic and anaerobic marine bacteria in de novo biofilm formation on natural substrates in St. Andrews Bay, Scotland.

This study reports a novel study of marine biofilm formation comprising aerobic and anaerobic bacteria. Samples of quartz and feldspar, minerals commonly found on the earth, were suspended 5 m deep in the North Sea off the east coast of St. Andrews, Scotland for 5 weeks. The assemblage of organisms attached to these stones was cultivated under aerobic and anaerobic conditions in the laboratory. Bacteria isolated on Marine Agar 2216 were all Gram-negative and identified to genus level by sequencing the gene encoding 16S rRNA. Colwellia, Maribacter, Pseudoaltermonas and Shewanella were observed in aerobically-grown cultures while Vibrio was found to be present in both aerobic and anaerobic cultures. The obligate anaerobic bacterium Psychrilyobacter atlanticus, a recently defined genus, was identified as a close relative of isolates grown anaerobically. The results provide valuable information as to the main players that attach and form de novo biofilms on common minerals in sea water.

Cell-type-specific profiling of loaded miRNAs from Caenorhabditis elegans reveals spatial and temporal flexibility in Argonaute loading.

Multicellularity has coincided with the evolution of microRNAs (miRNAs), small regulatory RNAs that are integrated into cellular differentiation and homeostatic gene-regulatory networks. However, the regulatory mechanisms underpinning miRNA activity have remained largely obscured because of the precise, and thus difficult to access, cellular contexts under which they operate. To resolve these, we have generated a genome-wide map of active miRNAs in Caenorhabditis elegans by revealing cell-type-specific patterns of miRNAs loaded into Argonaute (AGO) silencing complexes. Epitope-labelled AGO proteins were selectively expressed and immunoprecipitated from three distinct tissue types and associated miRNAs sequenced. In addition to providing information on biological function, we define adaptable miRNA:AGO interactions with single-cell-type and AGO-specific resolution. We demonstrate spatial and temporal dynamicism, flexibility of miRNA loading, and suggest miRNA regulatory mechanisms via AGO selectivity in different tissues and during ageing. Additionally, we resolve widespread changes in AGO-regulated gene expression by analysing translatomes specifically in neurons.

Doxorubicin induces wide-spread transcriptional changes in the myocardium of hearts distinguishing between mice with preserved and impaired cardiac function.

AIMS: Doxorubicin (DOX) is an important drug for the treatment of various tumor entities. However, the occurrence of heart failure limits its application. This study investigated differential gene expression profiles in the left and right ventricles of DOX treated mice with either preserved or impaired myocardial function. We provide new mechanistic insights into the pathophysiology of DOX-induced heart failure and have discovered pathways that counteract DOX-induced cardiotoxicity. MAIN METHODS: We used in total 48 male mice and applied a chronic low dose DOX administration (5 mg/kg per injection, in total 20 mg/kg over 4 weeks) to induce heart failure. Echocardiographic parameters were evaluated one week after the final dose and mice were separated according to functional parameters into doxorubicin responding and non-responding animals. Post mortem, measurements of reactive oxygen species (ROS) and gene expression profiling was performed in separated right and left hearts. KEY FINDINGS: We detected significant ROS production in the left heart of the mice in response to DOX treatment, although interestingly, not in the right heart. We found that transcriptional changes differ between right and left heart correlating with the occurrence of myocardial dysfunction. SIGNIFICANCE: Doxorubicin induces changes in gene expression in the entire heart of animals without necessarily impairing cardiac function. We identified a set of transcripts that are associated with DOX cardiotoxicity. These might represent promising targets to ameliorate DOX-induced heart failure. Moreover, our results emphasize that parameters of left and right heart function should be evaluated during standardized echocardiography in patients undergoing DOX therapy.

Mechanisms of Salmonella pathogenesis in animal models.

Animal models play an important role in understanding the mechanisms of bacterial pathogenesis. Here we review recent studies of Salmonella infection in various animal models. Although mice are a classic animal model for Salmonella, mice do not normally get diarrhea, raising the question of how well the model represents normal human infection. However, pretreatment of mice with oral streptomycin, which apparently reduces the normal microbiota, leads to an inflammatory diarrheal response upon oral infection with Salmonella. This has led to a re-evaluation of the role of various Salmonella virulence factors in colonization of the intestine and induction of diarrhea. Indeed, it is now clear that Salmonella purposefully induces inflammation, which leads to the production of both carbon sources and terminal electron acceptors by the host that allow Salmonella to outgrow the normal intestinal microbiota. Overall use of this modified mouse model provides a more nuanced understanding of Salmonella intestinal infection in the context of the microbiota with implications for the ability to predict human risk.

Susceptibility of Koi and Yellow Perch to infectious hematopoietic necrosis virus by experimental exposure.

Infectious hematopoietic necrosis virus (IHNV) is a novirhabdoviral pathogen that originated in western North America among anadromous Pacific salmonids. Severe disease epidemics in the late 1970s resulting from IHNV's invasion into farmed Rainbow Trout Oncorhynchus mykiss in North America, Asia, and Europe emphasized IHNV's ability to adapt to new hosts under varying rearing conditions. Yellow Perch Perca flavescens and Koi Carp Cyprinus carpio (hereafter, "Koi") are aquaculture-reared fish that are highly valued in sport fisheries and the ornamental fish trade, respectively, but it is unknown whether these fish species are vulnerable to IHNV infection. In this study, we exposed Yellow Perch, Koi, and steelhead (anadromous Rainbow Trout) to IHNV by intraperitoneal injection (10(6) PFU/fish) and by immersion (5.7×10(5) PFU/mL) for 7 h, and monitored fish for 28 d. The extended immersion exposure and high virus concentrations used in the challenges were to determine if the tested fish had any level of susceptibility. After experimental exposure, Yellow Perch and Koi experienced low mortality (<6%) compared with steelhead (>35%). Virus was found in dead fish of all species tested and in surviving Yellow Perch by plaque assay and quantitative reverse transcription polymerase chain reaction (qPCR), with a higher prevalence in Yellow Perch than Koi. Infectious virus was also detected in Yellow Perch out to 5 d after bath challenge. These findings indicate that Yellow Perch and Koi are highly resistant to IHNV disease under the conditions tested, but Yellow Perch are susceptible to infection and may serve as possible virus carriers.