Characterization of Adenylyl Cyclase Isoform 6 Residues Interacting with Forskolin.

BACKGROUND: The adenylyl cyclase (AC) pathway, crucial for pulmonary vasodilation, is inhibited by hypoxia. Forskolin (FSK) binds allosterically to AC, stimulating ATP catalysis. As AC6 is the primary AC isoform in the pulmonary artery, selective reactivation of AC6 could provide targeted reinstatement of hypoxic AC activity. This requires elucidation of the FSK binding site in AC6. METHODS: HEK293T cells stably overexpressing AC 5, 6, or 7 were incubated in normoxia (21% O2) or hypoxia (10% O2) or exposed to s-nitrosocysteine (CSNO). AC activity was measured using terbium norfloxacin assay; AC6 structure built by homology modeling; ligand docking to examine FSK-interacting amino acids; roles of selected residues determined by site-directed mutagenesis; FSK-dependent cAMP generation measured in wild-type and FSK-site mutants by biosensor-based live cell assay. RESULTS: Only AC6 is inhibited by hypoxia and nitrosylation. Homology modeling and docking revealed residues T500, N503, and S1035 interacting with FSK. Mutation of T500, N503, or S1035 decreased FSK-stimulated AC activity. FSK site mutants were not further inhibited by hypoxia or CSNO; however, mutation of any of these residues prevented AC6 activation by FSK following hypoxia or CSNO treatment. CONCLUSIONS: FSK-interacting amino acids are not involved in the hypoxic inhibition mechanism. This study provides direction to design FSK derivatives for selective activation of hypoxic AC6.

Sphingosine kinase activity and sphingosine-1-phosphate in the inflamed human periodontium.

OBJECTIVES: This study evaluated changes in the levels of Sphingosine-1-Phosphate (S1P) and Sphingosine Kinase (SPHK) activity in response to non-surgical periodontal treatment in humans. METHODS: Diseased (n = 65) and healthy sites (n = 72) were screened in 18 patients with localized periodontitis stage II or III. Periodontal clinical parameters were recorded, and the gingival crevicular fluid (GCF) collected at baseline, 30 and 90 days of non-surgical treatment. Internal control sites without attachment loss/bleeding were sampled at baseline and after 90 days of treatment. SPHK activity and S1P levels and SPHK 1/2 isoforms were determined in the GCF at different time points using ELISA. RESULTS: Non-surgical treatment caused significant improvement in all periodontal clinical parameters (p < 0.01). Activity of SPHK and S1P levels was decreased (p < 0.05) 30 days after treatment and continued up to 90 days (p < 0.01); control sites remained unchanged throughout the study and resembled treated sites at 3 months (p > 0.05). SPHK1 levels presented decrease after periodontal treatment (p < 0.001). SPHK2 levels were lower than SPHK1 (p < 0.001) and remained unchanged. CONCLUSIONS: S1P levels and SPHK activity decreased within 3 months of non-surgical periodontal treatment, which were correlated with improvements in periodontal parameters. Only SPHK1 levels varied significantly in the states of health and disease.

Critical cysteines in the functional interaction of adenylyl cyclase isoform 6 with Gαs.

Activation of adenylyl cyclases (ACs) by G-protein Gαs catalyzes the production of cyclic adenosine monophosphate (cAMP), a key second messenger that regulates diverse physiological responses. There are 10 AC isoforms present in humans, with AC5 and AC6 proposed to play vital roles in cardiac function. We have previously shown that under hypoxic conditions, AC6 is amenable to post-translational modification by nitrosylation, resulting in decreased AC catalytic activity. Using a computational model of the AC6-Gαs complex, we predicted key nitrosylation-amenable cysteine residues involved in the interaction of AC6 with Gαs and pursued a structure-function analysis of these cysteine residues in both AC6 and Gαs. Our results based on site-directed mutagenesis of AC6 and Gαs, a constitutively active Gαs, AC activity, and live cell intracellular cAMP assays suggest that Cys1004 in AC6 (subunit C2) and Cys237 in Gαs are present at the AC-Gαs interface and are important for the activation of AC6 by Gαs. We further provide mechanistic evidence to show that mutating Cys 1004 in the second catalytic domain of AC6 makes it amenable to inhibition by Gαi, which may account for decreased functional activity of AC6 when this residue is unavailable.

Pharmacology of T2R Mediated Host-Microbe Interactions.

Bitter taste receptors (T2Rs) belong to the G protein-coupled receptor superfamily. Humans express 25 T2Rs that are known to detect several bitter compounds including bacterial quorum sensing molecules (QSM). Primarily found to be key receptors for bitter sensation T2Rs are known to play an important role in mediating innate immune responses in oral and extraoral tissues. Several studies have led to identification of Gram-negative and Gram-positive bacterial QSMs as agonists for T2Rs in airway epithelial cells and immune cells. However, the pharmacological characterization for many of the QSM-T2R interactions remains poorly defined. In this chapter, we discuss the extraoral roles including localization of T2Rs in extracellular vesicles, molecular pharmacology of QSM-T2R interactions, role of T2Rs in mediating innate immune responses, and some of the challenges in understanding T2R pharmacology.

Role of Maternal Infections and Inflammatory Responses on Craniofacial Development.

Pregnancy is a tightly regulated immunological state. Mild environmental perturbations can affect the developing fetus significantly. Infections can elicit severe immunological cascades in the mother's body as well as the developing fetus. Maternal infections and resulting inflammatory responses can mediate epigenetic changes in the fetal genome, depending on the developmental stage. The craniofacial development begins at the early stages of embryogenesis. In this review, we will discuss the immunology of pregnancy and its responsive mechanisms on maternal infections. Further, we will also discuss the epigenetic effects of pathogens, their metabolites and resulting inflammatory responses on the fetus with a special focus on craniofacial development. Understanding the pathophysiological mechanisms of infections and dysregulated inflammatory responses during prenatal development could provide better insights into the origins of craniofacial birth defects.

ClpV3 of the H3-Type VI Secretion System (H3-T6SS) Affects Multiple Virulence Factors in Pseudomonas aeruginosa.

The type VI secretion system (T6SS) is a toxic effector delivery apparatus widely distributed in Gram-negative bacteria. The opportunistic pathogen Pseudomonas aeruginosa encodes three T6SSs, namely H1-, H2-, and H3-T6SS. Each T6SS possesses its own effectors and their roles are not yet fully understood. Here, we report that an H3-T6SS deletion mutant PAO1(ΔclpV3) significantly affected the virulence-related phenotypes including pyocyanin production, biofilm formation, proteolytic activity, and motilities. Most interestingly, the expression of T3SS genes was markedly affected, indicating a link between H3-T6SS and T3SS. RNA-Sequencing was performed to globally identify the genes differentially expressed when H3-T6SS was inactivated and the results obtained correlated well with the observed phenotypes. Interestingly, the expressions of T2SS, T3SS, H2-T6SS, and H3-T6SS were all significantly decreased, while H1-T6SS was increased in the PAO1(ΔclpV3) strain. We also observed that the intracellular concentration of secondary messenger cAMP was reduced in PAO1(ΔclpV3), and the c-di-GMP level was also decreased as indicated by the decreased cdrA reporter activity. Finally, by using a Galleria mellonella infection model, we show that H3-T6SS plays a key role in the pathogenicity of P. aeruginosa in vivo. Overall, our study highlights the unique connection of H3-T6SS in P. aeruginosa with T3SS, pyocyanin production, biofilm formation and in vivo pathogenicity.

Effects of Post-translational Modifications on Membrane Localization and Signaling of Prostanoid GPCR-G Protein Complexes and the Role of Hypoxia.

G protein-coupled receptors (GPCRs) play a pivotal role in the adaptive responses to cellular stresses such as hypoxia. In addition to influencing cellular gene expression profiles, hypoxic microenvironments can perturb membrane protein localization, altering GPCR effector scaffolding and altering downstream signaling. Studies using proteomics approaches have revealed significant regulation of GPCR and G proteins by their state of post-translational modification. The aim of this review is to examine the effects of post-translational modifications on membrane localization and signaling of GPCR-G protein complexes, with an emphasis on vascular prostanoid receptors, and to highlight what is known about the effect of cellular hypoxia on these mechanisms. Understanding post-translational modifications of protein targets will help to define GPCR targets in treatment of disease, and to inform research into mechanisms of hypoxic cellular responses.

Two Component Regulatory Systems and Antibiotic Resistance in Gram-Negative Pathogens.

Gram-negative pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are the leading cause of nosocomial infections throughout the world. One commonality shared among these pathogens is their ubiquitous presence, robust host-colonization and most importantly, resistance to antibiotics. A significant number of two-component systems (TCSs) exist in these pathogens, which are involved in regulation of gene expression in response to environmental signals such as antibiotic exposure. While the development of antimicrobial resistance is a complex phenomenon, it has been shown that TCSs are involved in sensing antibiotics and regulating genes associated with antibiotic resistance. In this review, we aim to interpret current knowledge about the signaling mechanisms of TCSs in these three pathogenic bacteria. We further attempt to answer questions about the role of TCSs in antimicrobial resistance. We will also briefly discuss how specific two-component systems present in K. pneumoniae, A. baumannii, and P. aeruginosa may serve as potential therapeutic targets.

CmpX Affects Virulence in Pseudomonas aeruginosa Through the Gac/Rsm Signaling Pathway and by Modulating c-di-GMP Levels.

Pseudomonas aeruginosa is an ubiquitous organism which is able to infect and colonize many types of hosts including humans. Colonization of P. aeruginosa in chronic infections leads to the formation of biofilms, which are difficult to eradicate. P. aeruginosa is capable of regulating its virulence factors in response to external environment triggers and its signaling mechanism involves two-component regulatory systems and small molecules such as bis-(3'-5')-cyclic dimeric guanosine monophosphate. PA1611-RetS-GacS/A-RsmA/Y/Z is a key regulatory pathway in P. aeruginosa that controls several virulence factors and biofilm formation. We have previously identified a conserved cytoplasmic membrane protein cmpX (PA1775), as a regulator for PA1611 expression. In this study, we demonstrate that cmpX regulates virulence, and controls biofilm formation in P. aeruginosa as well as provide evidence showing that cmpX affects Gac/Rsm pathway, possibly by modulating intra-cellular c-di-GMP levels. A cmpX knockout showed significantly decreased promoter activity of exoS (PA1362) and increased activity of small RNA, RsmY. As compared to the wild-type PAO1, cmpX mutant had elevated intracellular c-di-GMP level as measured indirectly by cdrA (PA4625) activity, as well as increased expression of wspR (PA3702), a c-di-GMP synthase. The transcription of the major outer membrane porin gene oprF (PA1777), and sigma factor sigX (PA1776) was also significantly decreased in the cmpX mutant. Biolog phenotype microarray experiments further indicated that the cmpX knockout mutant had increased sensitivity to membrane detergents and antibiotics such as lauryl sulfobetaine, tobramycin, and vancomycin. These results point to a significant role of cmpX in P. aeruginosa virulence and colonization.

Antibacterial Properties of Chitosan Nanoparticles and Propolis Associated with Calcium Hydroxide against Single- and Multispecies Biofilms: An In Vitro and In Situ Study.

INTRODUCTION: The aim of this study was to evaluate the efficacy of chitosan nanoparticles (CNPs) and ethanolic propolis extract (EPE) incorporated into a calcium hydroxide paste (Ca[OH]2) to kill bacterial biofilms. METHODS: Human root canal dentin was infected with Enterococcus faecalis for 21 days and also intraorally for 48 hours followed by incubation in brain-heart infusion for 48 hours to standardize biofilm growth. Ca(OH)2 pastes associated or not with CNPs or EPE were tested on biofilms for 7 and 14 days. Distilled water was used for control purposes. After the treatment procedures, microbiological analysis was performed to determine the reduction in E. faecalis colonies. Confocal microscopy was used to determine the percentage of cell viability in polymicrobial biofilms before and after the exposure to the experimental intracanal medications. RESULTS: All experimental pastes were able to significantly reduce the E. faecalis colony-forming units (CFU) after 7 or 14 days (P < .05). However, the CFU reduction was significantly improved when CNPs were incorporated into the Ca(OH)2 paste (P < .05). The multispecies biofilms treated with Ca(OH)2 showed similar percentages of bacterial viability to the control regardless of the exposure time (P > .05). The viable cell count significantly dropped in the Ca(OH)2/CNPs groups for both 7 and 14 days (P < .05), whereas the Ca(OH)2/EPE groups were only effective in eliminating bacteria during the first 7 days of treatment (P < .05). CONCLUSIONS: Incorporating CNPs into pastes of Ca(OH)2 could potentially be beneficial when using interappointment intracanal medications because of their ability to kill bacteria in short- and long-term exposure.

Characterization of the Direct Interaction between Hybrid Sensor Kinases PA1611 and RetS That Controls Biofilm Formation and the Type III Secretion System in Pseudomonas aeruginosa.

One of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients is pulmonary infection with Pseudomonas aeruginosa, and the pathophysiology of pulmonary infection in CF is affected by the lifestyle of this micro-organism. RetS-GacS/A-RsmA is a key regulatory pathway in P. aeruginosa that determines the bacterium's lifestyle choice. Previously, we identified PA1611, a hybrid sensor kinase, as a new player in this pathway that interacts with RetS and influences biofilm formation and type III secretion system. In this study, we explored the structural and mechanistic basis of the interaction between PA1611 and RetS. We identified the amino acid residues critical for PA1611-RetS interactions by molecular modeling. These residues were then targeted for site-directed mutagenesis. Amino acid substitutions were carried out at seven key positions in PA1611 and at six corresponding key positions in RetS. The influence of such substitutions in PA1611 on the interaction was analyzed by bacterial two-hybrid assays. We carried out functional analysis of these mutants in P. aeruginosa for their effect on specific phenotypes. Two residues, F269 and E276, located within the histidine kinase A and histidine kinase-like ATPase domains of PA1611 were found to play crucial roles in the PA1611-RetS interaction and had profound effects on phenotypes. Corresponding mutations in RetS demonstrated similar results. We further confirmed that these mutations in PA1611 function through the GacS/GacA-RsmY/Z signaling pathway. Collectively, our findings provide a noncognate sensor kinase direct interaction model for a signaling pathway, key for lifestyle selection in P. aeruginosa, and targeting such interaction may serve as a novel way of controlling infections with P. aeruginosa.

Cystic fibrosis lung environment and Pseudomonas aeruginosa infection.

BACKGROUND: The airways of patients with cystic fibrosis (CF) are highly complex, subject to various environmental conditions as well as a distinct microbiota. Pseudomonas aeruginosa is recognized as one of the most important pulmonary pathogens and the predominant cause of morbidity and mortality in CF. A multifarious interplay between the host, pathogens, microbiota, and the environment shapes the course of the disease. There have been several excellent reviews detailing CF pathology, Pseudomonas and the role of environment in CF but only a few reviews connect these entities with regards to influence on the overall course of the disease. A holistic understanding of contributing factors is pertinent to inform new research and therapeutics. DISCUSSION: In this article, we discuss the deterministic alterations in lung physiology as a result of CF. We also revisit the impact of those changes on the microbiota, with special emphasis on P. aeruginosa and the influence of other non-genetic factors on CF. Substantial past and current research on various genetic and non-genetic aspects of cystic fibrosis has been reviewed to assess the effect of different factors on CF pulmonary infection. A thorough review of contributing factors in CF and the alterations in lung physiology indicate that CF lung infection is multi-factorial with no isolated cause that should be solely targeted to control disease progression. A combinatorial approach may be required to ensure better disease outcomes. CONCLUSION: CF lung infection is a complex disease and requires a broad multidisciplinary approach to improve CF disease outcomes. A holistic understanding of the underlying mechanisms and non-genetic contributing factors in CF is central to development of new and targeted therapeutic strategies.