Truncation of type IV pilin induces mucoidy in Pseudomonas aeruginosa strain PAO579

Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host\u27s defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ22). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the conversion to mucoidy. Pseudomonas aeruginosastrain PAO579, a derivative of the nonmucoid strain PAO1, is mucoid due to an unidentified mutation (muc-23). Using whole genome sequencing, we identified 16 nonsynonymous and 15 synonymous single nucleotide polymorphisms (SNP). We then identified three tandem single point mutations in the pilA gene (PA4525), as the cause of mucoidy in PAO579. These tandem mutations generate a premature stop codon resulting in a truncated version of PilA (PilA108), with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF). Inactivation of pilA108 confirmed it was required for mucoidy. Additionally, algW and algU were also required for mucoidy of PAO579. Western blot analysis indicated that MucA was less stable in PAO579 than nonmucoid PAO1 or PAO381. The mucoid phenotype and high PalgU and PalgD promoter activities of PAO579 require pilA108, algW, algU, and rpoN encoding the alternative sigma factor σ54. We also observed that RpoN regulates expression of algW and pilA in PAO579. Together, these results suggest that truncation in type IV pilin in P. aeruginosa strain PAO579 can induce mucoidy through an AlgW/AlgU-dependent pathway

The Influence of Chemical Modification on Linker Rotational Dynamics in Metal–Organic Frameworks

The robust synthetic flexibility of metal–organic frameworks (MOFs) offers a promising class of tailorable materials, for which the ability to tune specific physicochemical properties is highly desired. This is achievable only through a thorough description of the consequences for chemical manipulations both in structure and dynamics. Magic angle spinning solid‐state NMR spectroscopy offers many modalities in this pursuit, particularly for dynamic studies. Herein, we employ a separated‐local‐field NMR approach to show how specific intraframework chemical modifications to MOF UiO‐66 heavily modulate the dynamic evolution of the organic ring moiety over several orders of magnitude.Intraframework ring rotations in metal–organic frameworks have been sensitively detected by dipolar dephasing over the rotor period in magic angle spinning solid‐state NMR experiments. Information on the dynamics within MOFs is important, because the rate of rotational motions of linkers affects sorption and separation properties of MOFs.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144616/1/anie201805004.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144616/2/anie201805004-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144616/3/anie201805004_am.pd

Role of Anomalous Water Constraints in the Efficacy of Pharmaceuticals Probed by 1H Solid‐State NMR

Water plays a complex and central role in determining the structural and reactive properties in numerous chemical systems. In crystalline materials with structural water, the primary focus is often to relate hydrogen bonding motifs to functional properties such as solubility, which is highly relevant in pharmaceutical applications. Nevertheless, understanding the full electrostatic landscape is necessary for a complete structure‐function picture. Herein, a combination of tools including 1H magic angle spinning NMR and X‐ray crystallography are employed to evaluate the local landscape of water in crystalline hydrates. Two hydrates of an anti‐leukemia drug mercaptopurine, which exhibit dramatically different dehydration temperatures (by 90 °C) and a three‐fold difference in the in vivo bioavailability, are compared. The results identify an electrosteric caging mechanism for a kinetically trapped water in the hemihydrate form, which is responsible for the dramatic differences in properties.1H chemical shift tensors are valuable in the structural and dynamical studies of a variety of materials, and are directly measurable with fast MAS spinning experiments. The use of these novel techniques to reveal the structural differences water can adopt in pharmaceutical hydrates is demonstrated.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138433/1/slct201701547_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138433/2/slct201701547-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138433/3/slct201701547.pd

Barriers and facilitators experienced in collaborative prospective research in orthopaedic oncology

Recerca col·laborativa; Grup focal; Oncologia ortopèdicaCollaborative research; Focus group; Orthopaedic oncologyInvestigación colaborativa; Grupo focal; Oncología ortopédicaObjectives As tumours of bone and soft tissue are rare, multicentre prospective collaboration is essential for meaningful research and evidence-based advances in patient care. The aim of this study was to identify barriers and facilitators encountered in large-scale collaborative research by orthopaedic oncological surgeons involved or interested in prospective multicentre collaboration. Methods All surgeons who were involved, or had expressed an interest, in the ongoing Prophylactic Antibiotic Regimens in Tumour Surgery (PARITY) trial were invited to participate in a focus group to discuss their experiences with collaborative research in this area. The discussion was digitally recorded, transcribed and anonymised. The transcript was analysed qualitatively, using an analytic approach which aims to organise the data in the language of the participants with little theoretical interpretation. Results The 13 surgeons who participated in the discussion represented orthopaedic oncology practices from seven countries (Argentina, Brazil, Italy, Spain, Denmark, United States and Canada). Four categories and associated themes emerged from the discussion: the need for collaboration in the field of orthopaedic oncology due to the rarity of the tumours and the need for high level evidence to guide treatment; motivational factors for participating in collaborative research including establishing proof of principle, learning opportunity, answering a relevant research question and being part of a collaborative research community; barriers to participation including funding, personal barriers, institutional barriers, trial barriers, and administrative barriers and facilitators for participation including institutional facilitators, leadership, authorship, trial set-up, and the support of centralised study coordination. Conclusions Orthopaedic surgeons involved in an ongoing international randomised controlled trial (RCT) were motivated by many factors to participate. There were a number of barriers to and facilitators for their participation. There was a collective sense of fatigue experienced in overcoming these barriers, which was mirrored by a strong collective sense of the importance of, and need for, collaborative research in this field. The experiences were described as essential educational first steps to advance collaborative studies in this area. Knowledge gained from this study will inform the development of future large-scale collaborative research projects in orthopaedic oncology

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa

Background Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ22) to initiate transcription of the alginate biosynthetic operon. Results In the current study, we mapped the mucE transcriptional start site, and determined that PmucE activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in PmucE activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. Conclusions Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa

Analysis of the In Vivo Transcriptome of Bordetella pertussis during Infection of Mice

Bordetella pertussis causes the disease whooping cough through coordinated control of virulence factors by the Bordetella virulence gene system. Microarrays and, more recently, RNA sequencing (RNA-seq) have been used to describe in vitro gene expression profiles of B. pertussis and other pathogens. In previous studies, we have analyzed the in vitro gene expression profiles of B. pertussis, and we hypothesize that the infection transcriptome profile in vivo is significantly different from that under laboratory growth conditions. To study the infection transcriptome of B. pertussis, we developed a simple filtration technique for isolation of bacteria from infected lungs. The work flow involves filtering the bacteria out of the lung homogenate using a 5-μm-pore-size syringe filter. The captured bacteria are then lysed to isolate RNA for Illumina library preparation and RNA-seq analysis. Upon comparing the in vitro and in vivo gene expression profiles, we identified 351 and 255 genes as activated and repressed, respectively, during murine lung infection. As expected, numerous genes associated with virulent-phase growth were activated in the murine host, including pertussis toxin (PT), the PT secretion apparatus, and the type III secretion system. A significant number of genes encoding iron acquisition and heme uptake proteins were highly expressed during infection, supporting iron acquisition as critical for B. pertussis survival in vivo. Numerous metabolic genes were repressed during infection. Overall, these data shed light on the gene expression profile of B. pertussisduring infection, and this method will facilitate efforts to understand how this pathogen causes infection

Bordetella pertussis Whole Cell Immunization, Unlike Acellular Immunization, Mimics Naïve Infection by Driving Hematopoietic Stem and Progenitor Cell Expansion in Mice

Hematopoietic stem and progenitor cell (HSPC) compartments are altered to direct immune responses to infection. Their roles during immunization are not well-described. To elucidate mechanisms for waning immunity following immunization with acellular vaccines (ACVs) against Bordetella pertussis (Bp), we tested the hypothesis that immunization with Bp ACVs and whole cell vaccines (WCVs) differ in directing the HSPC characteristics and immune cell development patterns that ultimately contribute to the types and quantities of cells produced to fight infection. Our data demonstrate that compared to control and ACV-immunized CD-1 mice, immunization with an efficacious WCV drives expansion of hematopoietic multipotent progenitor cells (MPPs), increases circulating white blood cells (WBCs), and alters the size and composition of lymphoid organs. In addition to MPPs, common lymphoid progenitor (CLP) proportions increase in the bone marrow of WCV-immunized mice, while B220+ cell proportions decrease. Upon subsequent infection, increases in maturing B cell populations are striking in WCV-immunized mice. RNAseq analyses of HSPCs revealed that WCV and ACV-immunized mice vastly differ in developing VDJ gene segment diversity. Moreover, gene set enrichment analyses demonstrate WCV-immunized mice exhibit unique gene signatures that suggest roles for interferon (IFN) induced gene expression. Also observed in naïve infection, these IFN stimulated gene (ISG) signatures point toward roles in cell survival, cell cycle, autophagy, and antigen processing and presentation. Taken together, these findings underscore the impact of vaccine antigen and adjuvant content on skewing and/or priming HSPC populations for immune response

Intranasal Peptide-Based FpvA-KLH Conjugate Vaccine Protects Mice From Pseudomonas aeruginosa Acute Murine Pneumonia

Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic respiratory infections associated with morbidity and mortality, especially in patients with cystic fibrosis. Vaccination against P. aeruginosa before colonization may be a solution against these infections and improve the quality of life of at-risk patients. To develop a vaccine against P. aeruginosa, we formulated a novel peptide-based P. aeruginosa subunit vaccine based on the extracellular regions of one of its major siderophore receptors, FpvA. We evaluated the effectiveness and immunogenicity of the FpvA peptides conjugated to keyhole limpet hemocyanin (KLH) with the adjuvant curdlan in a murine vaccination and challenge model. Immunization with the FpvA-KLH vaccine decreased the bacterial burden and lung edema after P. aeruginosa challenge. Vaccination with FpvA-KLH lead to antigen-specific IgG and IgM antibodies in sera, and IgA antibodies in lung supernatant. FpvA-KLH immunized mice had an increase in recruitment of CD11b+ dendritic cells as well as resident memory CD4+ T cells in the lungs compared to non-vaccinated challenged mice. Splenocytes isolated from vaccinated animals showed that the FpvA-KLH vaccine with the adjuvant curdlan induces antigen-specific IL-17 production and leads to a Th17 type of immune response. These results indicate that the intranasal FpvA-KLH conjugate vaccine can elicit both mucosal and systemic immune responses. These observations suggest that the intranasal peptide-based FpvA-KLH conjugate vaccine with curdlan is a potential vaccine candidate against P. aeruginosa pneumonia

Intranasal Acellular Pertussis Vaccine Provides Mucosal Immunity and Protects Mice from Bordetella Pertussis

Current acellular pertussis vaccines fall short of optimal protection against the human respiratory pathogen Bordetella pertussis resulting in increased incidence of a previously controlled vaccine- preventable disease. Natural infection is known to induce a protective mucosal immunity. Therefore, in this study, we aimed to use acellular pertussis vaccines to recapitulate these mucosal immune responses. We utilized a murine immunization and challenge model to characterize the efficacy of intranasal immunization (IN) with DTaP vaccine or DTaP vaccine supplemented with curdlan, a known Th1/Th17 promoting adjuvant. Protection from IN delivered DTaP was compared to protection mediated by intraperitoneal injection of DTaP and whole-cell pertussis vaccines. We tracked fluorescently labeled DTaP after immunization and detected that DTaP localized preferentially in the lungs while DTaP with curdlan was predominantly in the nasal turbinates. IN immunization with DTaP, with or without curdlan adjuvant, resulted in anti-B. pertussis and anti-pertussis toxin IgG titers at the same level as intraperitoneally administered DTaP. IN immunization was able to protect against B. pertussis challenge and we observed decreased pulmonary pro-inflammatory cytokines, neutrophil infiltrates in the lung, and bacterial burden in the upper and lower respiratory tract at day 3 post challenge. Furthermore, IN immunization with DTaP triggered mucosal immune responses such as production of B. pertussis-specific IgA, and increased IL-17A. Together, the induction of a mucosal immune response and humoral antibody-mediated protection associated with an IN administered DTaP and curdlan adjuvant warrant further exploration as a pertussis vaccine candidate formulation