Introduction: The mechanism of cell activation by LPS in the absence of surface Toll-like receptor 4 (TLR4) is unclear. We hypothesize that ICAM-1 binds LPS on the cell surface, mediating cell activation independent of TLR4. Methods: The interaction between murine ICAM-1 and LPS was measured in a binding assay. Alveolar macrophages (AMs) isolated from TLR4 deficient mice were stimulated with LPS. Cell activation was measured by flow cytometry and cytokine production. The role of ICAM-1 in cell activation was determined by siRNA transfection. Results: Murine ICAM-1 binds LPS. TLR4 deficient AMs respond to LPS stimulation by upregulation of LPS binding sites, ICAM-1 expression and cytokine release. Cell activation is attenuated by treatment with polymyxin B and ICAM-1 gene silencing. Conclusions: ICAM-1 binds LPS and is important in TLR4-independent cell activation. Strategies devised to target ICAM-1 may have the potential to block the excessive inflammatory response seen in gram-negative sepsis.

Introduction: The mechanism of cell activation by LPS in the absence of surface Toll-like receptor 4 (TLR4) is unclear. We hypothesize that ICAM-1 binds LPS on the cell surface, mediating cell activation independent of TLR4. Methods: The interaction between murine ICAM-1 and LPS was measured in a binding assay. Alveolar macrophages (AMs) isolated from TLR4 deficient mice were stimulated with LPS. Cell activation was measured by flow cytometry and cytokine production. The role of ICAM-1 in cell activation was determined by siRNA transfection. Results: Murine ICAM-1 binds LPS. TLR4 deficient AMs respond to LPS stimulation by upregulation of LPS binding sites, ICAM-1 expression and cytokine release. Cell activation is attenuated by treatment with polymyxin B and ICAM-1 gene silencing. Conclusions: ICAM-1 binds LPS and is important in TLR4-independent cell activation. Strategies devised to target ICAM-1 may have the potential to block the excessive inflammatory response seen in gram-negative sepsis.

<p>Enterotoxigenic <italic>Escherichia coli</italic> (ETEC) is a leading cause of morbidity and mortality worldwide. The causative agent of traveler's diarrhea, ETEC is often associated with cholera-like disease, especially in developing countries. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which upsets the balance of electrolytes in the intestine. LT is highly similar to cholera toxin (CT) produced by <italic>Vibrio cholerae</italic>, both in structure and function. The toxin consists of a single catalytically active A subunit and a ring of five B subunits mediating its binding and secretion. Previous work from our lab has shown that, after export by the type II secretion (T2S) system, LT associates with lipopolysaccharide (LPS) on the bacterial surface. However, little is known about what identifies LT as a T2S substrate, and the portion of the toxin that mediates LPS binding has not previously been defined. Site-directed mutagenesis of residues in a peripheral sugar binding pocket of the toxin was performed, revealing mutations that affect its binding to LPS, as determined by an in vitro cell surface binding assay. One binding mutant, which is expressed and secreted at wild-type levels from ETEC, holds particular promise for further studies of the role of the LT-LPS interaction. Interestingly, some mutations made affected the secretion of the toxin as detected by ganglioside-binding ELISAs of cell-free supernatant, and several mutations affected both secretion and LPS binding. These mutations identify residues of the toxin that are involved in its secretion and association with LPS. In addition, we introduced mutations affecting the secretion of LT into CT, due to the high similarity between the two toxins. While one mutation affects the secretion of each, other mutations affect one toxin but not the other. These results demonstrate that LT and CT are recognized in different ways during T2S. Combined with an analysis of the effects of secretion mutations on the stability of the toxin, the results described here highlight the delicate balance between structure and function of the LT B subunit.</p> / Dissertation

The studies described herein were designed to determine the influence of temperament on stress hormones and the immune system in response to various stressors. These stressors included transportation, lipopolysaccharide (LPS) challenge, and adrenocorticotropin (ACTH) challenge. In the first transportation study, bulls (8 Calm, 8 Intermediate, and 8 Temperamental) were loaded into a trailer and transported for 9 hr. Rectal temperature (monitored via indwelling recorders) increased within 0.5 hr of transportation, with greater peak rectal temperature in Temperamental than Calm bulls. Pre- and post-transport concentrations of cortisol and epinephrine were not affected by transportation, but were greater in Temperamental than Calm bulls. A second transportation study utilized 2 automatic sampling devices to allow the recording of rectal temperature and collection of blood samples, respectively. Rectal temperature was not affected by transportation or temperament in response to 4-hr of transport. Average heart rate oscillated between 60 and 130 bpm in Temperamental bulls, but remained around 100 bpm in Calm bulls. Transportation did not affect concentrations of epinephrine, although concentrations were greater in Temperamental bulls than Calm bulls. Cortisol concentrations increased in Calm bulls but not in Temperamental bulls in response to transportation. Additionally, there were limited effects of transportation on peripheral blood mononuclear cell proliferation, IgM production, and cytokine gene expression. Specifically, proliferation tended to be greater post-transport. Expression of the glucocorticoid receptor was, and the expression of toll-like receptor 4 tended to be, reduced post-transport, as analyzed by quantitative real-time RT-PCR. In a study utilizing a LPS challenge, basal stress hormone concentrations during the pre-challenge period were greater in Temperamental bulls than Calm bulls. However, in response to the LPS challenge, only the epinephrine response was influenced by temperament. Additionally, Temperamental bulls exhibited a smaller increase in rectal temperature and sickness behavior than Calm bulls. In the last study, change in gene expression in peripheral blood mononuclear cells in response to acute increases in cortisol was assessed. Plasma cortisol and gene expression of cytokines and the glucocorticoid receptor tended to increase in response to placement of jugular cannula. Additionally, administration of ACTH significantly increased plasma concentrations of cortisol and the gene expression of some cytokines (interleukin-4 and interleukin-10). This suggests that acute increases in cortisol may have positive effects on immune function in Brahman calves. Through an increased understanding of the interaction between the stress response and animal temperament, as well as how stress hormones and temperament influence immune function, animal management practices can be modified to reduce negative impacts on growth and productivity.

­^ ¤å ºK ­n We investigated the role of heat shock proteins (HSPs), particularly HSP60, HSP70 or HSP90 in E. coli lipopolysaccharide (LPS)-induced na&#x00EF;ve pheochrommocytoma cell (PC12) death. PC12 cells seeded at a density of 1x105 cells per poly-L-lysine-coated 3.5 cm diameter polystyrene dish were incubated with LPS (1 mg/ml; serotype O55:B5) for 3, 6, 12, or 24 hr. Cell viability was measured by trypan blue test, and expression of HSP60, HSP70, and HSP90 were detected by Western blot analysis. We found that the viability of PC12 cell decreased significantly after treatment with LPS for 12 hr, and viability was only 30% at 24 hr post-treatment. Western blot analysis revealed that LPS-induced PC12 cell death was associated with an increase in HSP70 or HSP60. HSP70 was markedly up-regulation at 12 hr; and both HSP70 and HSP60 increased significantly by over 1000% and 200%, respectively, 24 hr after administration of LPS. There was no significant change in HSP90 level 3, 6, 12, or 24 hr after LPS treatment. To further investigate the role of HSP70, 60, or HSP90 in LPS-induced PC12 cell death, we treated PC12 cells with hsps antisense oligonucleotide (AODN) for 24 hr. The effects of LPS on cell viability and HSP60, HSP70, or HSP90 expression were again tested. We found that suppression of HSP70 or HSP60 expression accelerated the process of LPS-induced cell death. A reduction in HSP90 level, however, had little effect. The study revealed that HSP70 and HSP60 played an anti-death role during LPS-induced PC12 cell death, and HSP90 did not appear to be involved.

Lipopolysaccharide (LPS) is the glycolipid that comprises the outer leaflet of the Gram-negative outer membrane (OM). Because it is essential in nearly all Gram-negative species, and because it is responsible for making these bacteria impervious to many types of antibiotics, LPS biogenesis has become an important area of research. While its biosynthesis at the cytoplasmic face of the inner membrane (IM) is well studied, the process by which it is removed from the IM, transported across the aqueous periplasmic compartment, and specifically inserted into the outer leaflet of the OM is only beginning to be understood. This transport process is mediated by the essential seven-protein LPS transport (Lpt) complex, LptA/B/C/D/E/F/G. The OM portion of the exporter, LptD/E, is a unique plug-and-barrel protein complex in which LptE, a lipoprotein, sits inside of LptD, a β-barrel integral membrane protein. LptD is of particular interest, as it is the target of an antibiotic in Pseudomonas aeruginosa. Part I of this thesis investigates how the cell forms the two non-consecutive disulfide bonds that connect LptD's C-terminal β-barrel to its N-terminal soluble domain. These disulfides, one of which is almost universally conserved among Gram-negatives, are essential for cell viability. Here, we show that an intermediate oxidation state with non-native disulfide bonds accumulates in the absence of LptE and in strains defective in either LptE or LptD. We then demonstrate that this observed intermediate is on-pathway and part of the native LptD oxidative folding pathway. Using a defective mutant of DsbA, the protein that introduces disulfide bonds into LptD, we are able to identify additional intermediates in the LptD oxidative folding pathway. We ultimately demonstrate that the disulfide rearrangement that activates the LptD/E complex occurs following an exceptionally slow β-barrel assembly step and is dependent on the presence of LptE. Part II describes work towards obtaining X-ray crystal structures of the LptD N-terminal domain and LptD/E complex. Expression construct and purification optimization enabled the production of stable LptD/E in quantities that make crystallography feasible. Numerous precipitants, detergents, and additives were screened, ultimately resulting in protein crystals that diffract to a resolution of 3.85 Å. / Chemistry and Chemical Biology

No description available.

Universiẗat, Diss., 2007--Giessen.

Orientador: Edson Antunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T20:19:41Z (GMT). No. of bitstreams: 1 Morganti_RafaelPrada_D.pdf: 740260 bytes, checksum: c6b0779cd2f8af7471aefb0da921a0a6 (MD5) Previous issue date: 2008 / Resumo: A sepsis é a segunda causa de morte em pacientes internados em unidades de tratamento intensivo não coronariano. Pacientes com sepsis apresentam anormalidades plaquetárias como trombocitopenia, prolongamento do tempo de coagulação, coagulação intravascular disseminada, trombose microvascular maciça e sangramentos. Entretanto, os mecanismos envolvidos nestas alterações plaquetárias ainda são mal compreendidos. O objetivo deste trabalho foi investigar as ações do lipopolissacarídeo (LPS) de E. coli, e de enterotoxinas de Staphylococcus aureus (SEA e SEB), na adesão e na agregação de plaquetas humanas, bem como os mecanismos envolvidos nesses fenômenos. As plaquetas foram expostas ao LPS, SEA ou SEB em diferentes concentrações e tempos de incubação para avaliação in vitro da adesão ou agregação. Além disso, realizou-se experimentos para verificar o papel do óxido nítrico (NO) e de espécies reativas de oxigênio nas ações induzidas pelo LPS, SEA e SEB, assim como a sinalização intracelular (mobilização de Ca+2) e ativação do receptor do fibrinogênio glicoproteína IIb/IIIa (GPIIb/IIIa). A incubação de plaquetas com LPS, SEA e SEB por períodos 5 a 120 min inibiu a adesão espontânea das plaquetas ao fibrinogênio, sendo esta inibição dependente da concentração e do tempo de incubação. A adesão de plaquetas ativadas com trombina também foi inibida após incubação com LPS, SEA ou SEB por 5 a 120 min. Porém, a inibição em plaquetas ativadas foi menor quando comparada à inibição da adesão espontânea. Esta inibição ocorreu de forma independente de aumentos intraplaquetários de GMPc e AMPc. Com o objetivo de se entender o mecanismo de ação envolvido na inibição da adesão plaquetária pelo LPS, SEA e SEB as plaquetas foram pré-tratadas com os seguintes agentes farmacológicos: superóxido dismutase (seqüestrador de ânion superóxido), polietilino glicol superóxido dismutase (seqüestrador de ânion superóxido intracelular), polietileno glicol catalase (degrada peróxido de hidrogênio), puromicina e ciclohexamida (inibidores de síntese protéica). Em conjunto, nossos dados mostraram que o efeito inibitório do LPS não envolve aumento de anion superóxido (O2 -) e de peróxido de hidrogênio (H2O2), nem a formação de proteínas neo-sintetizadas, e alterações na ativação da GPIIb/IIIa. Por outro lado, o LPS reduziu significativamente o influxo externo de cálcio, sem afetar a mobilização intracelular deste cátion. Em relação às enterotoxinas estafilocócicas, a síntese de neo-proteínas e aumento de O2 - não tem papel direto sobre a inibição plaquetária, embora um aumento nas concentrações de H2O2 intracelulares esteja ligado a esta resposta. Em conclusão, a exposição ao LPS inibiu a adesão a agregação plaquetária, dosee tempo-dependente, inibindo a influxo de cálcio; da mesma forma, a incubação com SEA ou SEB inibiu a adesão de plaquetas ao fibrinogênio, por um mecanismo que envolveria a formação de H2O2 / Abstract: Sepsis is the second major cause of death in intensive care unit. Septic patients show abnormalities in platelet like thrombocytopenia, prolongation in coagulation time, disseminated intravascular coagulation, deep venous thrombosis and bleedings. However, the mechanisms involved in these platelet alterations are not totally clear. The objective of this work was investigate the actions of lipopolysaccharide (LPS) from E. coli and enterotoxins from Staphylococcus aureus (SEA and SEB), in human platelet adhesion and aggregation, likewise the mechanism involved in these phenomenon. The platelets were exposed to LPS, SEA or SEB in different concentrations and times of incubation in vitro evaluations of adhesion and aggregation. Moreover, experiments to elucidate the participation of nitric oxide (NO) and reactive oxygen species in the actions of LPS, SEA and SEB, likewise the intracellular sinalization (calcium mobilization) and activation of fibrinogen receptor glycoprotein IIb/IIIa (GPIIb/IIIa) were carry out. The platelets incubation with LPS, SEA and SEB for 5 to 120 min, inhibited the spontaneous adhesion to fibrinogen and this inhibition was dependent of concentration and time of incubation. The thrombin activated adhesion of platelets was concentration and time dependently also inhibited after incubation with LPS, SEA and SEB for 5 to 120 min. However, the activated platelets inhibition was lower when compared to spontaneous adhesion inhibition. This inhibition was not dependent of intracellular levels increase of cGMP and cAMP. To understand the mechanism of action involved in the platelet adhesion inhibition by LPS, SEA and SEB, the platelets were previously treated with pharmacological agents like: superoxide dismutase (superoxide anion scavenger), superoxide dismutase-polyethylene glycol (superoxide anion intracellular scavenger), catalase-polyethylene glycol (Hydrogen peroxide intracellular scavenger), puromycin and cyclohexamide (protein synthesis inhibitors). Our data suggests that the inhibitory effect of LPS neither involve superoxide anion (O2 -) and hydrogen peroxide (H2O2) increase, nor the formation of neo-proteins, and alterations on activation of glycoprotein IIb/IIIa. In contrast, LPS significantly reduce the external calcium influx, without effecting calcium internal mobilization. With regard to staphylococcal enterotoxin, the protein synthesis and rise in O2 - do not play a role in the platelet inhibition; however the increase of H2O2 intracellular concentration was involved in this response. In conclusion, platelet aggregation and adhesion was inhibited after addition of LPS, concentration and time-dependently, inhibiting calcium influx; similary, incubation with SEA or SEB inhibited platelets adhesion to fibrinogen, by a mechanism involving H2O2 formation / Doutorado / Doutor em Farmacologia

The widespread emergence of antibiotic resistance determinants for nearly all drug classes threatens human health on a global scale. It is therefore essential to discover antibiotics with novel functions that are less likely to be influenced by pre-existing resistance mechanisms. An emerging approach to identify inhibitors of investigator-defined cellular processes involves screening compounds for antimicrobial activity under non-standard growth conditions. Indeed, by growing cells under conditions of stress, inhibitors of specific cellular targets can be enriched, thereby allowing for the identification of molecules with predictable activities in the complex environment of the cell. Here, I exploit cold stress in Escherichia coli to identify molecules targeting ribosome biogenesis and outer membrane biosynthesis. First, through a screen of 30,000 small molecules for growth inhibition exclusively at 15°C, I was able to identify the first small molecule inhibitor of bacterial ribosome biogenesis, lamotrigine. Second, by leveraging the idiosyncratic cold sensitivity of E. coli to vancomycin, I developed a novel screening technology designed to enrich for non-lethal inhibitors of Gram- negative outer membrane biosynthesis. From this platform, I identified pentamidine as an efficient outer membrane perturbant that was able to potentiate Gram-positive antibiotics against Gram-negative pathogens, similar to the polymyxins. Remarkably, however, this compound was able to overcome mcr-1 mediated polymyxin resistance. Together, this thesis highlights the utility of exploiting the bacterial cold stress response in antibiotic discovery. / Thesis / Doctor of Philosophy (PhD)