Differential Contributions of Outer Membrane Permeability and Active Efflux in Physiology and Drug Susceptibility of Gram-Negative Bacteria

The increased frequency of antibiotic resistant bacterial isolates is of great concern for public health. It is currently estimated that infections caused by multidrug resistant pathogens will surpass cancer as a leading cause of death by 2050. Of particular concern are gram-negative bacteria, due to their robust intrinsic resistance provided by a low permeability outer membrane barrier in combination with active efflux. Active efflux is mainly mediated by Resistance-Nodulation cell Division (RND) transporters, which associate as tripartite complexes that facilitate the export of substrates across the outer membrane. These two mechanisms of resistance work in synergy to efficiently limit intracellular accumulation of antibiotics. This directly results in very low hit rates during antimicrobial screenings and presents unique challenges in the development of antibiotics. In addition to providing resistance, RND transporters are also involved in the physiology of bacteria. Pseudomonas aeruginosa possesses twelve RND transporters of which three are indicated to be involved in its quorum sensing networks. Overexpressing or deleting the genes of these transporters has a great physiological impact and can result in a loss of virulence or extended lag phases during growth. In many cases, the endogenous substrates of these transporters are not known and their impact on the physiology of the cell is not well understood. This dissertation focuses on the interplay between the low permeability outer membrane and active efflux in drug resistance and their contribution to physiology of P. aeruginosa. Current methods to investigate the contributions of the outer membrane barrier in drug resistance, like the use of polymyxins to permeabilize the outer membrane, have many disadvantages. Here, we developed a novel approach to separate the contributions of efflux and the outer membrane in antimicrobial susceptibility by introducing a genetically modified pore that non-selectively increases the permeability of the outer membrane. In combination with the removal of efflux transporters, this hyperporination approach highlights the synergy of the outer membrane and efflux, and has potential implications for the development of new antibiotics. It could provide the means to discover new rules in drug design that predict the uptake of a compound into the gram-negative cell according to its structural features. Furthermore, this dissertation will characterize the contributions of the RND transporter MexGHI-OpmD from P. aeruginosa in the physiology and antimicrobial resistance of the bacterium. Our results suggest that the transporter is involved in establishing the steady-state concentrations of the important virulence factor pyocyanin. In addition, we showed that the unusual fourth component MexG physically interacts with the transporter and that its presence results in inhibition of the efflux activity towards certain substrates. Our results suggest that the activity of RND transporters are masked by the presence of the outer membrane and that hyperporination highlights the actual transport efficiencies of these pumps. RND transporters heavily rely on the outer membrane barrier, and this hyperporination approach could be used to re-evaluate their substrate specificities

Resection of the mesopancreas (RMP): a new surgical classification of a known anatomical space

BACKGROUND: Prognosis after surgical therapy for pancreatic cancer is poor and has been attributed to early lymph node involvement as well as to a strong tendency of cancer cells to infiltrate into the retropancreatic tissue and to spread along the peripancreatic neural plexuses. The objective of our study was to classify the anatomical-surgical layer of the mesopancreas and to describe the surgical principles relevant for resection of the mesopancreas (RMP). Immunohistochemical investigation of the mesopancreatic-perineural lymphogenic structures was carried out with the purpose of identifying possible routes of metastatic spread. METHODS: Resection of the mesopancreas (RMP) was performed in fresh corpses. Pancreas and mesopancreas were separated from each other and the mesopancreas was immunohistochemically investigated. RESULTS: The mesopancreas strains itself dorsally of the mesenteric vessels as a whitish-firm, fatty tissue-like layer. Macroscopically, in the dissected en-bloc specimens of pancreas and mesopancreas nerve plexuses were found running from the dorsal site of the pancreatic head to the mesopancreas to establish a perineural plane. Immunohistochemical examinations revealed the lymphatic vessels localized in direct vicinity of the neuronal plexuses between pancreas and mesopancreas. CONCLUSION: The mesopancreas as a perineural lymphatic layer located dorsally to the pancreas and reaching beyond the mesenteric vessels has not been classified in the anatomical or surgical literature before. The aim to ensure the greatest possible distance from the retropancreatic lymphatic tissue which drains the carcinomatous focus can be achieved in patients with pancreatic cancer only by complete resection of the mesopancreas (RMP)

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13–1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13–1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation

Microenvironmental adaptation of experimental tumours to chronic vs acute hypoxia

This study investigated long-term microenvironmental responses (oxygenation, perfusion, metabolic status, proliferation, vascular endothelial growth factor (VEGF) expression and vascularisation) to chronic hypoxia in experimental tumours. Experiments were performed using s.c.-implanted DS-sarcomas in rats. In order to induce more pronounced tumour hypoxia, one group of animals was housed in a hypoxic atmosphere (8% O2) for the whole period of tumour growth (chronic hypoxia). A second group was acutely exposed to inspiratory hypoxia for only 20 min prior to the measurements (acute hypoxia), whereas animals housed under normal atmospheric conditions served as controls. Acute hypoxia reduced the median oxygen partial pressure (pO2) dramatically (1 vs 10 mmHg in controls), whereas in chronically hypoxic tumours the pO2 was significantly improved (median pO2=4 mmHg), however not reaching the control level. These findings reflect the changes in tumour perfusion where acutely hypoxic tumours show a dramatic reduction of perfused tumour vessels (maybe the result of a simultaneous reduction in arterial blood pressure). In animals under chronic inspiratory hypoxia, the number of perfused vessels increased (compared to acute hypoxia), although the perfusion pattern found in control tumours was not reached. In the chronically hypoxic animals, tumour cell proliferation and tumour growth were significantly reduced, whereas no differences in VEGF expression and vascular density between these groups were observed. These results suggest that long-term adaptation of tumours to chronic hypoxia in vivo, while not affecting vascularity, does influence the functional status of the microvessels in favour of a more homogeneous perfusion

Pseudomonas aeruginosa mutants defective in glucose uptake have pleiotropic phenotype and altered virulence in non-mammal infection models

Pseudomonas spp. are endowed with a complex pathway for glucose uptake that relies on multiple transporters. In this work we report the construction and characterization of Pseudomonas aeruginosa single and multiple mutants with unmarked deletions of genes encoding outer membrane (OM) and inner membrane (IM) proteins involved in glucose uptake. We found that a triple \u394gltKGF \u394gntP \u394kguT mutant lacking all known IM transporters (named GUN for Glucose Uptake Null) is unable to grow on glucose as unique carbon source. More than 500 genes controlling both metabolic functions and virulence traits show differential expression in GUN relative to the parental strain. Consistent with transcriptomic data, the GUN mutant displays a pleiotropic phenotype. Notably, the genome-wide transcriptional profile and most phenotypic traits differ between the GUN mutant and the wild type strain irrespective of the presence of glucose, suggesting that the investigated genes may have additional roles besides glucose transport. Finally, mutants carrying single or multiple deletions in the glucose uptake genes showed attenuated virulence relative to the wild type strain in Galleria mellonella, but not in Caenorhabditis elegans infection model, supporting the notion that metabolic functions may deeply impact P. aeruginosa adaptation to specific environments found inside the host

Kinetic Control of Quorum Sensing in <i>Pseudomonas aeruginosa</i> by Multidrug Efflux Pumps

<i>Pseudomonas aeruginosa</i> is an important human pathogen, the physiology and virulence of which are under the control of quorum sensing signals. These signals often have dual roles, functioning as toxins to some cells and as oxidative-stress protectors for their producer cells. Hence, their internal and external concentrations should be tightly controlled. In this study, we analyzed the interplay between the multidrug efflux transporters MexEF-OprN and MexG/HI-OpmD in quorum sensing of <i>P. aeruginosa.</i> We found that the two transporters have overlapping substrate specificities but different efficiencies. When overproduced, both MexEF-OprN and MexG/HI-OpmD provide clinical levels of resistance to diverse fluoroquinolones and protect <i>P. aeruginosa</i> against toxic phenazines. However, this similarity is enabled by synergistic interactions with the outer membrane. In hyperporinated cells, MexG/HI-OpmD is saturated by much lower concentrations of fluoroquinolones but is more efficient than MexEF-OprN in efflux of phenazines. Unlike MexEF-OprN, mutational inactivation of MexG/HI-OpmD reduces the levels of pyocyanin and makes <i>P. aeruginosa</i> cells hypersusceptible to phenazines. Our results further show that MexG binds pyocyanin, physically associates with MexHI, and represses the activity of the transporter, revealing a negative regulatory role of this protein. We conclude that differences in kinetic properties of transporters are critical to maintain proper intra- and extracellular concentrations of phenazines and other signaling molecules and that MexG/HI-OpmD controls the steady state in the synthesis and secretion of phenazines

Circadian and dopaminergic regulation of fatty acid oxidation pathway genes in retina and photoreceptor cells

The energy metabolism of the retina might comply with daily changes in energy demand and is impaired in diabetic retinopathy-one of the most common causes of blindness in Europe and the USA. The aim of this study was to investigate putative adaptation of energy metabolism in healthy and diabetic retina. Hence expression analysis of metabolic pathway genes was performed using quantitative polymerase chain reaction, semi-quantitative western blot and immunohistochemistry. Transcriptional profiling of key enzymes of energy metabolism identified transcripts of mitochondrial fatty acid β-oxidation enzymes, i.e. carnitine palmitoyltransferase-1α (Cpt-1α) and medium chain acyl-CoA dehydrogenase (Acadm) to display daily rhythms with peak values during daytime in preparations of the whole retina and microdissected photoreceptors. The cycling of both enzymes persisted in constant darkness, was dampened in mice deficient for dopamine D4 (D4) receptors and was altered in db/db mice-a model of diabetic retinopathy. The data of the present study are consistent with circadian clock-dependent and dopaminergic regulation of fatty acid oxidation in retina and its putative disturbance in diabetic retina