Differential Screening in Immunodeficient Mice Reveals Bacterial Enzymes With Unexpected Roles in Host-Pathogen Interactions

Over centuries of co-existence with their hosts, microbes that are exclusively vertebrate pathogens have evolved mechanisms for manipulation of host cells and evasion of the immune system. Some pathogens dodge the immune response by altering the functions of and hiding within the very cells that should be consuming and digesting them: macrophages. The aim of these studies was to explore the dynamic interactions the host with two microbes that opt to live within the macrophage phagosome: Mycobacterium tuberculosis (Mtb) and Salmonella typhimurium. In order to identify bacterial genes that provide protection against specific host immune pathways, we have developed the strategy of differential signature-tagged transposon mutagenesis (STM). By evaluating bacterial mutants for differential virulence in strains of immuno-deficient mice, we identified five enzymes required for in vivo survival in the face of specific immune stresses: four Mtb enzymes essential for growth and rapid lethality in NOS2- deficient mice but not in IFN-y-deficient mice, and one S. typhimurium enzyme that participates in resistance to phagocyte oxidase. The Mtb enzymes suggest novel pathways for countering IFN-y-dependent immune mechanisms other than NOS2. Analysis of the sequence of the S. typhimurium enzyme suggests the existence in prokaryotes of an alternative signaling pathway that complements classical phospho-relay 2-component regulatory systems

Autogenic-feedback training improves pilot performance during emergency flying conditions

Studies have shown that autonomous mode behavior is one cause of aircraft fatalities due to pilot error. In such cases, the pilot is in a high state of psychological and physiological arousal and tends to focus on one problem, while ignoring more critical information. The effect of training in physiological self-recognition and regulation, as a means of improving crew cockpit performance was examined. Seventeen pilots were assigned to the treatment and control groups matched for accumulated flight hours. The treatment group comprised four pilots of HC-130 Hercules aircraft and four HH-65 Dolphin helicopter pilots; the control group comprised three pilots of HC-130's and six Dolphin helicopter pilots. During an initial flight physiological data were recorded for each crewmember and individual crew performance and rated by an instructor pilot. Eight crewmembers were then taught to regulate their own physiological response levels using Autogenic-Feedback Training (AFT). The remaining subjects received no training. During a second flight, treatment subjects showed significant improvement in performance, while controls did not improve. The results indicate that AFT management of high states of physiological arousal may improve pilot performance during emergency flying conditions

The Relationship between Pre- and Post-Matriculation Factors and College Student Retention in a Small, Technical, Public Institution

Despite the volume of research on college retention, additional investigation is warranted as many institutions continue to be plagued by poor retention rates. This study investigated the impact of pre- and post-matriculation factors on student retention at SUNY Cobleskill. The focus of the study was on the associate degree students and includes variables such as financial aid type and amount, and college choice. Statistical procedures such as Chi Square, Kendall’s Tau and Cramer’s V were employed during the data analysis. This research adds to the existing body of research by studying a small, regional, state-assisted institution that focuses on teaching. Most of the currently available studies have been conducted at large or mid-size research-based institutions. The researcher did not find statistically significant findings when analyzing relationships between college choice, type of financial aid and expected family contribution, and college student retention. A significant relationship was found when looking at the total amount of aid received and college student retention. This relationship warrants further research into the ability to pay versus willingness to pay, as well as how financial aid packaging may impact college student retention

Restoring Cystic Fibrosis Transmembrane Conductance Regulator Function Reduces Airway Bacteria and Inflammation in People with Cystic Fibrosis and Chronic Lung Infections

Rationale: Previous work indicates that ivacaftor improves cystic fibrosis transmembrane conductance regulator (CFTR) activity and lung function in people with cystic fibrosis and G551D-CFTR mutations but does not reduce density of bacteria or markers of inflammation in the airway. These findings raise the possibility that infection and inflammation may progress independently of CFTR activity once cystic fibrosis lung disease is established. Objectives: To better understand the relationship between CFTR activity, airway microbiology and inflammation, and lung function in subjects with cystic fibrosis and chronic airway infections. Methods: We studied 12 subjects with G551D-CFTR mutations and chronic airway infections before and after ivacaftor. We measured lung function, sputum bacterial content, and inflammation, and obtained chest computed tomography scans. Measurements and Main Results: Ivacaftor produced rapid decreases in sputum Pseudomonas aeruginosa density that began within 48 hours and continued in the first year of treatment. However, no subject eradicated their infecting P. aeruginosa strain, and after the first year P. aeruginosa densities rebounded. Sputum total bacterial concentrations also decreased, but less than P. aeruginosa. Sputum inflammatory measures decreased significantly in the first week of treatment and continued to decline over 2 years. Computed tomography scans obtained before and 1 year after ivacaftor treatment revealed that ivacaftor decreased airway mucous plugging. Conclusions: Ivacaftor caused marked reductions in sputum P. aeruginosa density and airway inflammation and produced modest improvements in radiographic lung disease in subjects with G551D-CFTR mutations. However, P. aeruginosa airway infection persisted. Thus, measures that control infection may be required to realize the full benefits of CFTR-targeting treatments

Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis

Previously, we showed that serum and monocytes from patients with CF exhibit an enhanced NLRP3-inflammasome signature with increased IL-18, IL-1β, caspase-1 activity and ASC speck release (Scambler et al. eLife 2019). Here we show that CFTR modulators down regulate this exaggerated proinflammatory response following LPS/ATP stimulation. In vitro application of ivacaftor/lumacaftor or ivacaftor/tezacaftor to CF monocytes showed a significant reduction in IL-18, whereas IL-1β was only reduced with ivacaftor/tezacaftor. Thirteen adults starting ivacaftor/lumacaftor and eight starting ivacaftor/tezacaftor were assessed over three months. Serum IL-18 and TNF decreased significantly with treatments, but IL-1β only declined following ivacaftor/tezacaftor. In (LPS/ATP-stimulated) PBMCs, IL-18/TNF/caspase-1 were all significantly decreased and IL-10 was increased with both combinations. Ivacaftor/tezacaftor alone showed a significant reduction in IL-1β and pro-IL-1β mRNA. This study demonstrates that these CFTR modulator combinations have potent anti-inflammatory properties, in addition to their ability to stimulate CFTR function, which could contribute to improved clinical outcomes

The bacterial stressosome:a modular system that has been adapted to control secondary messenger signaling

SummaryThe stressosome complex regulates downstream effectors in response to environmental signals. In Bacillus subtilis, it activates the alternative sigma factor σB, leading to the upregulation of the general stress regulon. Herein, we characterize a stressosome-regulated biochemical pathway in Moorella thermoacetica. We show that the presumed sensor, MtR, and the scaffold, MtS, form a pseudo-icosahedral structure like that observed in B. subtilis. The N-terminal domain of MtR is structurally homologous to B. subtilis RsbR, despite low sequence identity. The affinity of the switch kinase, MtT, for MtS decreases following MtS phosphorylation and not because of structural reorganization. Dephosphorylation of MtS by the PP2C type phosphatase MtX permits the switch kinase to rebind the stressosome to reset the response. We also show that MtT regulates cyclic di-GMP biosynthesis through inhibition of a GG(D/E)EF-type diguanylate cyclase, demonstrating that secondary messenger levels are regulated by the stressosome

Cyclic Diguanylate Signaling Proteins Control Intracellular Growth of Legionella pneumophila

Proteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle of Legionella pneumophila, the causative agent of Legionnaires’ disease, has not previously been examined. The L. pneumophila genome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes as cdgS (cyclic diguanylate signaling) genes. Strains of L. pneumophila containing deletions of all individual cdgS genes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, several cdgS genes strongly decreased the ability of L. pneumophila to grow inside host cells. Expression of these cdgS genes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth in L. pneumophila. L. pneumophila strains overexpressing these cdgS genes were less cytotoxic to THP-1 macrophages than wild-type L. pneumophila but retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused by cdgS gene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of the cdgS genes in a Salmonella enterica serovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that several cdgS genes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability of L. pneumophila to grow in host cells