Expressão dos receptores Toll-like em carcinoma espinocelular de orofaringe

Nos últimos anos, notou-se aumento da incidência de carcinoma espinocelular de orofaringe (CECOF) associado ao HPV. Sabe-se que CECOF associado ao HPV apresenta melhor prognóstico do que CECOF não infectado por HPV. Inúmeros estudos em carcinoma cervical demonstram alterações de TLRs, isto provavelmente devido às associações das oncoproteínas E6 e E7 com estes receptores. Em humanos, existem 10 TLRs identificados, os quais colaboram na resposta imune contra bactérias, fungos e vírus, bem como colaboram na promoção ou regressão do tumor. Esta influência do TLR na carcinogênese tem sido alvo de inúmeros estudos devido à ligação entre inflamação e o câncer. O presente trabalho teve como objetivo verificar diferenças na expressão e função de receptores Toll-like em carcinoma espinocelular de orofaringe (CECOF). Para tal, foram utilizados trinta e sete espécimes diagnosticados como CECOF e a expressão imuno-histoquímica das proteínas p16 e TLR4 analisadas. Duas linhagens de CECOF HPV16 + e duas CECOF HPV-. foram utilizadas para análise da expressão de TLR1-10, IL-6 e IL-8, por qPCR. A detecção dos principais TLRs (TLR1, TLR2, TLR6 e TLR4) foi feita por citometria de fluxo. Para ativação da via de sinalização de TLR2, e posterior análise da expressão de IL6 e IL8, as células foram estimuladas com peptidoglicano. Para verificar a expressão e função de TLR4, as células foram estimuladas com LPS e LPS UP para posterior análise de IL-6 e IL-8, por ELISA. Os resultados demonstraram diferenças na expressão gênica de TLR1 e TLR6 entre as linhagens HPV- e o grupo HPV+ e diferenças na expressão proteica de TLR9. TLR2 apresentou aumento da expressão proteica em todas as linhagens e demonstra desencadeamento da resposta imune, com secreção de IL6 e IL8 nas linhagens HPV- (SCC72 e SCC89) e em uma das linhagens HPV+ (SCC2). Interessantemente, TLR4 não apresentou diferenças significativas na expressão gênica e proteica. Entretanto, as linhagens HPV+ não demonstraram resposta pró-inflamatória mesmo quando estimuladas com LPS e LPS ultra puro, agonista específico de TLR4. Assim, este trabalho contribui para estabelecer o perfil da expressão dos receptores Toll-like em linhagens celulares de CECOF HPV- e HPV+, e aponta para alterações ocorridas na via de sinalização mediada por TLR4. Além disso, nossos resultados abrem portas para futuros estudos na avaliação de alterações causadas no sistema imune inato pelo HPV, em carcinomas espinocelulares de orofaringe.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric pepticloglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity. (C) 2004 Wiley Periodicals, Inc.

Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica

The planctomycetes are a phylum of bacteria that have a unique cell compartmentalisation and yeast-like budding cell division and peptidoglycan-less proteinaceous cell walls. We wished to further our understanding of these unique organisms at the molecular level by searching for conserved amino acid sequence motifs and domains in the proteins encoded by Rhodopirellula baltica. Using BLAST and single-linkage clustering, we have discovered several new protein domains and sequence motifs in this planctomycete. R. baltica has multiple members of the newly discovered GEFGR protein family and the ASPIC C-terminal domain family, whilst most other organisms for which whole genome sequence is available have no more than one. Many of the domains and motifs appear to be restricted to the planctomycetes. It is possible that these protein domains and motifs may have been lost or replaced in other phyla, or they may have undergone multiple duplication events in the planctomycete lineage. One of the novel motifs probably represents a novel N-terminal export signal peptide. With their unique cell biology, it may be that the planctomycete cell compartmentalisation plan in particular needs special membrane transport mechanisms. The discovery of these new domains and motifs, many of which are associated with secretion and cell-surface functions, will help to stimulate experimental work and thus enhance further understanding of this fascinating group of organisms. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Intracellular compartmentation in planctomycetes

The phylum Planctomycetes of the domain Bacteria consists of budding, peptidoglycan-less organisms important for understanding the origins of complex cell organization. Their significance for cell biology lies in their possession of intracellular membrane compartmentation. All planctomycetes share a unique cell plan, in which the cell cytoplasm is divided into compartments by one or more membranes, including a major cell compartment containing the nucleoid. Of special significance is Gemmata obscuriglobus, in which the nucleoid is enveloped in two membranes to form a nuclear body that is analogous to the structure of a eukaryotic nucleus. Planctomycete compartmentation may have functional physiological roles, as in the case of anaerobic ammonium-oxidizing anammox planctomycetes, in which the anammoxosome harbors specialized enzymes and is wrapped in an envelope possessing unique ladderane lipids. Organisms in phyla other than the phylum Planctomycetes may possess compartmentation similar to that of some planctomycetes, as in the case of members of the phylum Poribacteria from marine sponges.

Induction of inflammatory cytokines and nitric oxide in J774.2 cells and murine macrophages by lipoteichoic acid and related cell wall antigens from Staphylococcus epidermidis

Staphylococcus epidermidis causes infections associated with medical devices including central venous catheters, orthopaedic prosthetic joints and artificial heart valves. This coagulase-negative Staphylococcus produces a conventional cellular lipoteichoic acid (LTA) and also releases a short-glycerophosphate-chain-length form of LTA (previously termed lipid S) into the medium during growth. The relative pro-inflammatory activities of cellular and short-chain-length exocellular LTA were investigated in comparison with peptidoglycan and wall teichoic acid from S. epidermidis and LPS from Escherichia coli O111. The ability of these components to stimulate the production of proinflammatory cytokines [interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α] and nitric oxide was investigated in a murine macrophage-like cell line (J774.2), and in peritoneal and splenic macrophages. On a weight-for-weight basis the short-chain-length exocellular LTA was the most active of the S. epidermidis products, stimulating significant amounts of each of the inflammatory cytokines and nitric oxide, although it was approximately 100-fold less active than LPS from E. coli. By comparison the full-chain-length cellular LTA and peptidoglycan were less active and the wall teichoic acid had no activity. As an exocellular product potentially released from S. epidermidis biofilms, the short-chain-length exocellular LTA may act as the prime mediator of the host inflammatory response to device-related infection by this organism and act as the Gram-positive equivalent of LPS in Gram-negative sepsis. The understanding of the role of short-chain-length exocellular LTA in Gram-positive sepsis may lead to improved treatment strategies. © 2005 SGM.

Mediators of gram-positive septic shock

Septic shock can occur as a result of Gram-negative or Gram-positive infection and involves a complex interaction between bacterial factors and the host immune system producing a systemic inflammatory state that may progress to multiple organ failure and death. Gram-positive bacteria are increasingly becoming more prevalent especially Staphylococcus epidermidis in association with indwelling devices. Lipopolysaccaride (LPS) is the key Gram-negative component involved in this process, but it is not clear which components of Gram-positive bacteria are responsible for progression of this often fatal disease. The aim of this thesis was to investigate the effect of bacterial components on the immune systems. Lipid S, a short chain form of lipoteichoic acid (LTA) found to be excreted from bacteria during growth in culture medium was examined along with other Gram-positive cell wall components: LTA, peptidoglycan (PG) and wall teichoic acids (WTA) and LPS from Gram-negative bacteria. Lipid S, LTA, PG and LPS but not WTA all stimulated murine macrophages and cell lines to produce significant amounts of NO, TNF-a, IL-6 and IL-1 and would induce fever and tissue damage seen in inflammatory diseases. Lipid S proved to be the most potent out of the Gram-positive samples tested. IgG antibodies in patients serum were found to bind to and cross react with lipid S and LTA. Anti-inflammatory antibiotics, platelet activating factor (PAF), PAF receptor antagonists and monoclonal antibodies (mAbs) directed to LTA, CD14 and toll-like receptors were utilised to modulate cytokine and NO production. In cell culture the anti-LTA and the anti-CD14 mAbs failed to markedly attenuate the production of NO, TNF-a, IL-6 or IL-1, the anti-TLR4 antibody did greatly inhibit the ability of LPS to stimulate cytokine production but not lipid S. The tetracyclines proved to be the most effective compounds, many were active at low concentrations and showed efficacy to inhibit both lipid S and LPS stimulated macrophages to produce NO.

Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria

Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of antimicrobials. However, resistance due to restricted penetration can occur, as illustrated by vancomycin-intermediate resistant Staphylococcus aureus strains (VISA) which produce a markedly thickened cell wall. Alterations in these strains include increased amounts of nonamidated glutamine residues in the peptidoglycan and it is suggested that the resistance mechanism involves 'affinity trapping' of vancomycin in the thickened cell wall. VISA strains have reduced doubling times, lower sensitivity to lysostaphin and reduced autolytic activity, which may reflect changes in the D-alanyl ester content of the wall and membrane teichoic acids. Mycobacterial cell walls have a high lipid content, which is assumed to act as a major barrier to the penetration of antimicrobial agents. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may be able to cross the cell wall by diffusion through the hydrophobic bilayer composed of long chain length mycolic acids and glycolipids. Hydrophilic antibiotics and nutrients cannot diffuse across this layer and are thought to use porin channels which have been reported in many species of mycobacteria. The occurrence of porins in a lipid bilayer supports the view that the mycobacterial wall has an outer membrane analogous to that of gram-negative bacteria. However, mycobacterial porins are much less abundant than in the gram-negative outer membrane and allow only low rates of uptake for small hydrophilic nutrients and antibiotics.

Modes of action of antibacterial agents

This chapter describes the modes of action of the major antibiotics and synthetic agents used to treat bacterial infections. Particular attention is given to the biochemical mechanisms by which the agents interfere with biosynthetic processes and the basis for their selective antibacterial action. Interference with the biosynthesis and assembly of structural components of the bacterial cell wall provides the basis for many important groups of antibiotics, including the agents targeting steps in peptidoglycan synthesis. Other agents exploit more subtle differences between bacteria and mammalian cells in fundamental processes such as DNA, RNA and protein synthesis.

An assessment of the microbial contribution to aquatic dissolved organic nitrogen using amino acid enantiomeric ratios

There is increasing evidence that certain microbially-derived compounds may account for part of the aquatic dissolved organic nitrogen (DON) pool. Enantiomeric ratios of amino acids were used to assess the microbial input to the DON pool in the Florida Everglades, USA. Elevated levels of d-alanine, d-aspartic acid, d-glutamic acid and d-serine indicated the presence of peptidoglycan in the samples. The estimated peptidoglycan contribution to amino acid nitrogen ranged from 2.8 ± 0.1% to 6.4 ± 0.9%, increasing with salinity from freshwater to coastal waters. The distribution of individual d-amino acids in the samples suggests additional inputs to DON, possibly from archaea or from abiotic racemization of l-amino acids.

Channel-forming activity of syringopeptin 25 A in mercury-supported phospholipid monolayers and negatively charged bilayers

Interactions of the cationic lipodepsipeptide syringopeptin 25 A (SP25A) with mercury-supported dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioeleoylphosphatidic acid (DOPA) self-assembled monolayers (SAMs) were investigated by AC voltammetry in 0.1 M KCl at pH 3, 5.4 and 6.8. SP25A targets and penetrates the DOPS SAM much more effectively than the other SAMs not only at pH 6.8, where the DOPS SAM is negatively charged, but also at pH 3, where it is positively charged just as SP25A. Similar investigations at tethered bilayer lipid membranes (tBLMs) consisting of a thiolipid called DPTL anchored to mercury, with a DOPS, DOPA or DOPC distal monolayer on top of it, showed that, at physiological transmembrane potentials, SP25A forms ion channels spanning the tBLM only if DOPS is the distal monolayer. The distinguishing chemical feature of the DOPS SAM is the ionic interaction between the protonated amino group of a DOPS molecule and the carboxylate group of an adjacent phospholipid molecule. Under the reasonable assumption that SP25A preferentially interacts with this ion pair, the selective lipodepsipeptide antimicrobial activity against Gram-positive bacteria may be tentatively explained by its affinity for similar protonated amino-carboxylate pairs, which are expected to be present in the peptide moieties of peptidoglycan strands.

The cellular and mechanistic study of nisin inhibition of bacillus anthracis spore outgrowth, nisin alteration of infection, and native producer immunity

A critical step during Bacillus anthracis infection is the outgrowth of germinated spores into vegetative bacilli that proliferate and disseminate rapidly within the host. An important challenge exists for developing chemotherapeutic agents that act upon and kill B. anthracis immediately after germination initiation when antibiotic resistance is lost, but prior to the outgrowth into vegetative bacilli, which is accompanied by toxin production. Chemical agents must also function in a manner refractive to the development of antimicrobial resistance. In this thesis we have identified the lantibiotics as a class of chemotherapeutics that are predicted to satisfy these two criteria. The objective of this thesis was to evaluate the efficacy of nisin, a prototypical lantibiotic, in prevention of outgrowth of germinated B. anthracis spores. Like all lantibiotics, nisin is a ribosomally translated peptide that undergoes post-translational modification to form (methyl)lanthionine rings that are critical for antimicrobial activity. Our studies indicate that nisin rapidly inhibits the in vitro outgrowth of germinated B. anthracis Sterne 7702 spores. Although germination initiation was shown to be essential for nisin-dependent antimicrobial activity, nisin did not inhibit or promote germination initiation. Nisin irreversibly killed germinated spores by blocking the establishment of a membrane potential and oxidative metabolism, while not affecting the dissolution of the outer spore structures. The membrane permeability of the spore was increased by nisin, but germinated spores did not undergo full lysis. Nisin was demonstrated to localize to lipid II, which is the penultimate precursor for cell wall biogenesis. This localization suggests two possible independent mechanisms of action, membrane pore formation and inhibition of peptidoglycan synthesis. Structure-activity studies with a truncated form of nisin lacking the two C-terminal (methyl)lanthionine rings and with non-pore forming mutants indicated that membrane disruption is essential for nisin-dependent inhibition of spore outgrowth to prevent membrane potential establishment. Finally, utilizing an in vitro infection model, it was shown that nisin reduced the viability of B. anthracis spores within an infection resulting in increased survival of immune cells while reducing infection-mediated cytokine expression. Fluorescence microscopy indicated that nisin localizes with spores within phagosomes of peritioneal macrophages in germinating conditions. These data demonstrate the effectiveness of nisin, as a model lantibiotic, for preventing spore outgrowth. It is speculated that nisin targeting of lipid II, resulting in membrane perturbations, may be effective at inhibiting the outgrowth of spores prepared from bacteria across a number of species.

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) or the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs.

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development

Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. IMPORTANCE Determining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host.

Comparative Genomics of Field Isolates of Mycobacterium bovis and M. caprae Provides Evidence for Possible Correlates with Bacterial Viability and Virulence

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to develop tools to evaluate risks for TB disease caused by M. bovis/M.caprae and for TB control in humans and animals.