GacA regulation of luminescence in the symbiotic bacterium Vibrio fischeri

The symbiosis of Euprymna scolopes and Vibrio fischeri depends on the ability of the bacterium to colonize the squid and produce light. GacA is an activator of symbiosis phenotypes including luminescence though it is not understood how GacA functions. My hypothesis, based on other bacterial models, is that GacA activates expression of CsrBs, functional RNAs, which sequester and repress a translational repressor, CsrA. I approached this question with mutagenesis and complementation techniques. We found mutation of CsrA or addition of a CsrB in trans could complement the GacA mutant for symbiosis and luminescence phenotypes. The inability of other luminescence regulators including a constitutively expressed lux operon to complement GacADelta indicated that luminescence was regulated post-transcriptionally. A putative CsrA binding site found in the sequence of the lux mRNA increased light production when deleted. These data demonstrate that GacA regulates luminescence post-transcriptionally via CsrB/CsrA antagonism, supporting my hypothesis

Spontaneous phenotypic suppression of GacA-defective Vibrio fischeri is achieved via mutation of csrA and ihfA

Background: Symbiosis defective GacA-mutant derivatives of Vibrio fischeri are growth impaired thereby creating a selective advantage for growth-enhanced spontaneous suppressors. Suppressors were isolated and characterized for effects of the mutations on gacA-mutant defects of growth, siderophore activity and luminescence. The mutations were identified by targeted and whole genome sequencing. Results: Most mutations that restored multiple phenotypes were non-null mutations that mapped to conserved domains in or altered expression of CsrA, a post-transcriptional regulator that mediates GacA effects in a number of bacterial species. These represent an array of unique mutations compared to those that have been described previously. Different substitutions at the same amino acid residue were identified allowing comparisons of effects such as at the R6 residue, which conferred relative differences in luminescence and siderophore levels. The screen revealed residues not previously identified as critical for function including a single native alanine. Most csrA mutations enhanced luminescence more than siderophore activity, which was especially evident for mutations predicted to reduce the amount of CsrA. Although CsrA mutations compensate for many known GacA mutant defects, not all CsrA suppressors restore symbiotic colonization. Phenotypes of a suppressor allele of ihfA that encodes one subunit of the integration host factor (IHF) heteroduplex indicated the protein represses siderophore and activates luminescence in a GacA-independent manner. Conclusions: In addition to its established role in regulation of central metabolism, the CsrA regulator represses luminescence and siderophore as an intermediate of the GacA regulatory hierachy. Siderophore regulation was less sensitive to stoichiometry of CsrA consistent with higher affinity for the targets of this trait. The lack of CsrA null-mutant recovery implied these mutations do not enhance fitness of gacA mutants and alluded to this gene being conditionally essential. This study also suggests a role for IHF in the GacA-CsrB-CsrA regulatory cascade by potentially assisting with the binding of repressors of siderohphore and activators of luminescence. As many phosphorelay proteins reduce fitness when mutated, the documented instability used in this screen also highlights a potentially universal and underappreciated problem that, if not identified and strategically avoided, could introduce confounding variability during experimental study of these regulatory pathways

Pouring Salt on a Wound: Pseudomonas aeruginosa Virulence Factors Alter Na+ and Cl− Flux in the Lung

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen with multiple niches in the human body, including the lung. P. aeruginosa infections are particularly damaging or fatal for patients with ventilator-associated pneumonia, chronic obstructive pulmonary disease, and cystic fibrosis (CF). To establish an infection, P. aeruginosa relies on a suite of virulence factors, including lipopolysaccharide, phospholipases, exoproteases, phenazines, outer membrane vesicles, type III secreted effectors, flagella, and pili. These factors not only damage the epithelial cell lining but also induce changes in cell physiology and function such as cell shape, membrane permeability, and protein synthesis. While such virulence factors are important in initial infection, many become dysregulated or nonfunctional during the course of chronic infection. Recent work on the virulence factors alkaline protease (AprA) and CF transmembrane conductance regulator inhibitory factor (Cif) show that P. aeruginosa also perturbs epithelial ion transport and osmosis, which may be important for the long-term survival of this microbe in the lung. Here we discuss the literature regarding host physiology-altering virulence factors with a focus on Cif and AprA and their potential roles in chronic infection and immune evasion

Synthesis of Bioinspired Carbohydrate Amphiphiles that Promote and Inhibit Biofilms

The synthesis and characterization of a new class of bioinspired carbohydrate amphiphiles that modulate Pseudomonas aeruginosa biofilm formation are reported. The carbohydrate head is an enantiopure poly-amido-saccharide (PAS) prepared by a controlled anionic polymerization of β-lactam monomers derived from either glucose or galactose. The supramolecular assemblies formed by PAS amphiphiles are investigated in solution using fluorescence assays and dynamic light scattering. Dried samples are investigated using X-ray, infrared spectroscopy, and transmission electron microscopy. Additionally, the amphiphiles are evaluated for their ability to modulate biofilm formation by the Gram-negative bacterium Pseudomonas aeruginosa. Remarkably, from a library of eight amphiphiles, we identify a structure that promotes biofilm formation and two structures that inhibit biofilm formation. Using biological assays and electron microscopy, we relate the chemical structure of the amphiphiles to the observed activity. Materials that modulate the formation of biofilms by bacteria are important both as research tools for microbiologists to study the process of biofilm formation and for their potential to provide new drug candidates for treating biofilm-associated infections

Epoxide-Mediated Differential Packaging of Cif and Other Virulence Factors into Outer Membrane Vesicles

Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epox

Defining Prostatic Vascular Pedicle Recurrence and the Anatomy of Local Recurrence of Prostate Cancer on Prostate-specific Membrane Antigen Positron Emission Tomography/Computed Tomography.

Background The term local recurrence in prostate cancer is considered to mean persistent local disease in the prostatic bed, most commonly at the site of the vesicourethral anastomosis (VUA). Since the introduction of prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT) and magnetic resonance imaging for assessment of early biochemical recurrence (BCR), we have found histologically confirmed prostate cancer in the prostatic vascular pedicle (PVP). If a significant proportion of local recurrences are distant to the VUA, it may be possible to alter adjuvant and salvage radiation fields in order to reduce the potential morbidity of radiation in selected patients. Objective To describe PVP local recurrence and to map the anatomic pattern of prostate bed recurrence on PSMA PET/CT. Design setting and participants This was a retrospective multicentre study of 185 patients imaged with PSMA PET/CT following radical prostatectomy (RP) between January 2016 and November 2018. All patient data and clinical outcomes were prospectively collected. Recurrences were documented according to anatomic location. For patients presenting with local recurrence, the precise location of the recurrence within the prostate bed was documented. Intervention PSMA PET/CT for BCR following RP. Results and limitations A total of 43 local recurrences in 41/185 patients (22%) were identified. Tumour recurrence at the PVP was found in 26 (63%), VUA in 15 (37%), and within a retained seminal vesicle and along the anterior rectal wall in the region of the neurovascular bundle in one (2.4%) each. Histological and surgical evidence of PVP recurrence was acquired in two patients. The study is limited by its retrospective nature with inherent selection bias. This is an observational study reporting on the anatomy of local recurrence and does not include follow-up for patient outcomes. Conclusions Our study showed that prostate cancer can recur in the PVP and is distant to the VUA more commonly than previously thought. This may have implications for RP technique and for the treatment of selected patients in the local recurrence setting. Patient summary We investigated more precise identification of the location of tumour recurrence after removal of the prostate for prostate cancer. We describe a new definition of local recurrence in an area called the prostatic vascular pedicle. This new concept may alter the treatment recommended for recurrent disease

Epoxide-Mediated CifR Repression of cif Gene Expression Utilizes Two Binding Sites in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees

B cell and/or autoantibody deficiency do not prevent neuropsychiatric disease in murine systemic lupus erythematosus

Background: Neuropsychiatric lupus (NPSLE) can be one of the earliest clinical manifestations in human lupus. However, its mechanisms are not fully understood. In lupus, a compromised blood-brain barrier may allow for the passage of circulating autoantibodies into the brain, where they can induce neuropsychiatric abnormalities including depression-like behavior and cognitive abnormalities. The purpose of this study was to determine the role of B cells and/or autoantibodies in the pathogenesis of murine NPSLE. Methods: We evaluated neuropsychiatric manifestations, brain pathology, and cytokine expression in constitutively (JhD/MRL/lpr) and conditionally (hCD20-DTA/MRL/lpr, inducible by tamoxifen) B cell-depleted mice as compared to MRL/lpr lupus mice. Results: We found that autoantibody levels were negligible (JhD/MRL/lpr) or significantly reduced (hCD20-DTA/MRL/lpr) in the serum and cerebrospinal fluid, respectively. Nevertheless, both JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice showed profound depression-like behavior, which was no different from MRL/lpr mice. Cognitive deficits were also observed in both JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice, similar to those exhibited by MRL/lpr mice. Furthermore, although some differences were dependent on the timing of depletion, central features of NPSLE in the MRL/lpr strain including increased blood-brain barrier permeability, brain cell apoptosis, and upregulated cytokine expression persisted in B cell-deficient and B cell-depleted mice. Conclusions: Our study surprisingly found that B cells and/or autoantibodies are not required for key features of neuropsychiatric disease in murine NPSLE