Cationic antimicrobial peptides (CAPs) are an important component of the innate immune system and are instrumental in the elimination of bacteria, viruses, protozoa, yeast, fungi and cancerous cells from the body. CAPs are comprised of less than 100 amino acids and have a net positive charge due to a multitude of basic residues in their primary sequences. CAPs exert their antimicrobial activity primarily through the formation of pores in microbial membranes, but also play important immunostimulatory roles in the body. Glycosaminoglycans (GAGs) are negatively charged, polydisperse linear polysaccharides found at cellular surfaces. Although many protein-binding interactions of the GAG family, including heparin and heparan sulfate, have been well-characterized, it is not known to what extent endogenous GAGs affect the innate immune system. In the studies here the modulatory activities of GAGs and other polyanionic polysaccharides (PPSs) on CAPs were probed. Initial studies focused on interactions between a short peptide derived from bovine lactoferricin and GAGs. GAGs and other PPSs were then tested for their ability to modulate the antimicrobial activities of a number of CAPs against Gram-positive and -negative organisms. GAGs were also tested for the ability to modulate CAPs binding to bacterial lipopolysaccharide. CAP affinities for the GAGs were determined from lipopolysaccharide competition binding assays. Finally GAGs were evaluated for the ability to protect CAPs from proteolytic degradation. The modulatory activities of GAGs and other PPSs are largely dependent upon all components of the test system and, to a lesser extent, the charge of the molecule.

Lipopolysaccharide or LPS is localized to the outer leaflet of the outer membrane and serves as the major surface component of the bacterial cell envelope. This remarkable glycolipid is essential for virtually all Gram-negative organisms and represents one of the conserved microbial structures responsible for activation of the innate immune system. For these reasons, the structure, function, and biosynthesis of LPS has been an area of intense research. The LPS of a number of bacteria is composed of three distinct regions - lipid A, a short core oligosaccharide, and the O-antigen polysaccharide. The lipid A domain, also known as endotoxin, anchors the molecule in the outer membrane and is the bioactive component recognized by TLR4 during human infection. Overall, the biochemical synthesis of lipid A is a highly conserved process; however, investigation of the lipid A structures of various organisms shows an impressive amount of diversity. These differences can be attributed to the action of latent enzymes that modify the canonical lipid A molecule. Variation of the lipid A domain of LPS serves as one strategy utilized by Gram-negative bacteria to promote survival by providing resistance to components of the innate immune system and helping to evade recognition by TLR4. This review summarizes the biochemical machinery required for the production of diverse lipid A structures of human pathogens and how structural modification of endotoxin impacts pathogenesis.

Modification of the lipid A domain of lipopolysaccharide (LPS) is important for the pathogenesis and virulence of various Gram-negative bacteria. The major lipid A species of Helicobacter pylori is significantly different from that of Escherichia coli. H. pylori lipid A contains fewer acyl chains and phosphate groups with only one Kdo sugar attached to the disaccharide backbone. However, H. pylori produces a minor lipid A species that resembles E. coli lipid A, suggesting that the major lipid A species results from the action of specific modifying enzymes. This work describes two enzymes, a lipid A phosphatase and a phosphoethanolamine (pEtN) transferase, involved in modifying the 1-position of H. pylori lipid A. H. pylori lipid A contains a pEtN unit directly linked to the 1-position of the disaccharide backbone. This is in contrast to the pEtN units found in other pathogens, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. Using in-vitro assay systems, we demonstrate that the modification of the 1-position of H. pylori lipid A is a two-step process involving the removal of the 1-phosphate group by LpxEHP followed by the addition of a pEtN residue catalyzed by EptAHP. As compared to wild-type H. pylori, lpxEHP mutants are extremely sensitive to the cationic peptide polymyxin, thus, demonstrating the importance of modifying the 1-position of lipid A. Furthermore, this work describes another enzyme, YeiU (renamed LpxT), which specifically utilizes the carrier lipid undecaprenyl pyrophsphate (C55-PP) to modify the 1-position of E. coli lipid A. Typically, E. coli lipid A is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'; however, a small fraction contains a diphosphate moiety at the 1-position (lipid A 1-diphosphate). 32P-labeled lipid A obtained from lpxT deficient mutants produces only lipid A, and complementation with a plasmid expressing LpxT restores lipid A 1-diphosphate formation. Inhibition of lipid A 1-diphosphate synthesis was demonstrated by sequestering C55-PP with the cyclic polypeptide antibiotic bacitracin. In conclusion, this work describes two novel pathways for lipid A modification at the 1-position in Gram-negative bacteria.

Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-07Bitstream added on 2014-06-13T19:40:48Z : No. of bitstreams: 1 lopesfilho_fc_dr_sjrp_parcial.pdf: 183424 bytes, checksum: 8601f6f72a7635a3c9ada79092a5873d (MD5) Bitstreams deleted on 2015-06-25T13:01:06Z: lopesfilho_fc_dr_sjrp_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:24Z : No. of bitstreams: 1 000694954_20160706.pdf: 183130 bytes, checksum: 1526b9f9e1347a4fb71fe218102cf0ba (MD5) Bitstreams deleted on 2016-07-25T13:17:36Z: 000694954_20160706.pdf,. Added 1 bitstream(s) on 2016-07-25T13:18:45Z : No. of bitstreams: 1 000694954.pdf: 1071220 bytes, checksum: ead8820e5de7c1e29fdd2ec0459005b1 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Peptídeos antimicrobianos são moléculas biologicamente ativas que, geralmente, tem as membranas fosfolipídicas como alvo primário. Resultados de diferentes técnicas experimentais têm sugerido que esses peptídeos permeabilizam as membranas pela formação de poros. Parte dos peptídeos caracterizados apresentam especificidade de disrupção para membranas de bactérias, em detrimento das membranas dos hospedeiros. Essa característica tem atraído a atenção da comunidade científica internacional, porque indica que estas moléculas podem ser modelos para o desenvolvimento de novos antibióticos, portanto o entendimento do mecanismo de ação, ou seja, do mecanismo de formação de poro, tem extrema importância. Simulações por Dinâmica Molecular foram produzidas para investigarmos o impacto que peptídeos antimicrobianos da família Mastoparano tem sobre membranas lipídicas modelo. Dois cenários foram explorados: (i) de baixa concentração peptídeo/lipídeo, P/L=1/128, que consistia de simulações fine-grained das interações de um peptídeo com uma bicamada pura de 128 lipídeos aniônicos (POPG) ou zwiteriônicos (POPC); (ii) de alta concentração, P/L=1/21, que abordava as interações de seis peptídeos com uma bicamada mista de 128 lipídeos POPC/POPG (1/1) usando uma modelagem coarse-grained. Tomando o peptídeo MP1 como caso paradigmático, verificamos que em baixo P/L é possível sugerir que sua característica seletiva surge da capacidade de coordenar e perturbar maior número de lipídeos em membrana aniônica comparada à neutra. Essa capacidade fica acentuada nas simulações com membrana mista, onde a atração dos lipídeos aniônicos pelos peptídeos catiônicos guiou a separação local e a formação de domínios de lipídeos aniônicos, o que facilitou o afinamento local da membrana e a formação de poro transmembrânico. Esses achados ajudam a explicar como peptídeos / Antimicrobial peptides are biologically active molecules that, usually, have the phospholipid membranes as a primary target. Results from different experimental techniques have suggested these peptides permeabilize membranes by the pore formation. Part of the characterized peptides have specificity of disruption for bacterial membranes, instead of host membrane. This feature has attracted the attention of the international scientific community, because it indicates that these molecules can be models for the development of novel antibiotics, so understanding the mechanism of action, ie, the mechanism of pore formation, is extremely important. Molecular dynamics simulations were performed to investigate the impact of antimicrobial peptides from the Mastoparano family have on model lipid membranes. Two scenarios were explored: (i) of low peptide/lipid concentration, P/L=1/128, which consisted of fine-grained simulations of the interactions of a peptide with a pure bilayer of 128 anionic (POPG) or zwitterionic (POPC) lipids; (ii) of high concentration, P/L=1/21, which addressed the interactions of six peptides with a mixed bilayer of 128 POPC/POPG (1/1) lipids, using a coarse-grained modeling. Taking the MP1 peptide as a paradigmatic case, we found that in low P/L is possible to suggest that its selective feature arises of its ability to coordinate and disturb large number of lipids in the anionic membrane compared to neutral one. This ability is accentuated in simulations with mixed membrane, where the attraction of the anionic lipids by the cationic peptides led to the local segregation and formation of POPG lipid domains, which facilitated the local thinning of the membrane and the formation of transmembrane pore. These findings help to explain how short peptides, such as MP1, are able of forming pores in a membrane whose thickness is larger than the length of the peptide

Orientador: José Roberto Ruggiero / Banca: Pedro Geraldo Pascutti / Banca: José Maria Pires / Banca: Alexandre Suman de Araújo / Banca: Sabrina Thais Broggio Costa / Resumo: Peptídeos antimicrobianos são moléculas biologicamente ativas que, geralmente, tem as membranas fosfolipídicas como alvo primário. Resultados de diferentes técnicas experimentais têm sugerido que esses peptídeos permeabilizam as membranas pela formação de poros. Parte dos peptídeos caracterizados apresentam especificidade de disrupção para membranas de bactérias, em detrimento das membranas dos hospedeiros. Essa característica tem atraído a atenção da comunidade científica internacional, porque indica que estas moléculas podem ser modelos para o desenvolvimento de novos antibióticos, portanto o entendimento do mecanismo de ação, ou seja, do mecanismo de formação de poro, tem extrema importância. Simulações por Dinâmica Molecular foram produzidas para investigarmos o impacto que peptídeos antimicrobianos da família Mastoparano tem sobre membranas lipídicas modelo. Dois cenários foram explorados: (i) de baixa concentração peptídeo/lipídeo, P/L=1/128, que consistia de simulações fine-grained das interações de um peptídeo com uma bicamada pura de 128 lipídeos aniônicos (POPG) ou zwiteriônicos (POPC); (ii) de alta concentração, P/L=1/21, que abordava as interações de seis peptídeos com uma bicamada mista de 128 lipídeos POPC/POPG (1/1) usando uma modelagem coarse-grained. Tomando o peptídeo MP1 como caso paradigmático, verificamos que em baixo P/L é possível sugerir que sua característica seletiva surge da capacidade de coordenar e perturbar maior número de lipídeos em membrana aniônica comparada à neutra. Essa capacidade fica acentuada nas simulações com membrana mista, onde a atração dos lipídeos aniônicos pelos peptídeos catiônicos guiou a separação local e a formação de domínios de lipídeos aniônicos, o que facilitou o afinamento local da membrana e a formação de poro transmembrânico. Esses achados ajudam a explicar como peptídeos / Abstract: Antimicrobial peptides are biologically active molecules that, usually, have the phospholipid membranes as a primary target. Results from different experimental techniques have suggested these peptides permeabilize membranes by the pore formation. Part of the characterized peptides have specificity of disruption for bacterial membranes, instead of host membrane. This feature has attracted the attention of the international scientific community, because it indicates that these molecules can be models for the development of novel antibiotics, so understanding the mechanism of action, ie, the mechanism of pore formation, is extremely important. Molecular dynamics simulations were performed to investigate the impact of antimicrobial peptides from the Mastoparano family have on model lipid membranes. Two scenarios were explored: (i) of low peptide/lipid concentration, P/L=1/128, which consisted of fine-grained simulations of the interactions of a peptide with a pure bilayer of 128 anionic (POPG) or zwitterionic (POPC) lipids; (ii) of high concentration, P/L=1/21, which addressed the interactions of six peptides with a mixed bilayer of 128 POPC/POPG (1/1) lipids, using a coarse-grained modeling. Taking the MP1 peptide as a paradigmatic case, we found that in low P/L is possible to suggest that its selective feature arises of its ability to coordinate and disturb large number of lipids in the anionic membrane compared to neutral one. This ability is accentuated in simulations with mixed membrane, where the attraction of the anionic lipids by the cationic peptides led to the local segregation and formation of POPG lipid domains, which facilitated the local thinning of the membrane and the formation of transmembrane pore. These findings help to explain how short peptides, such as MP1, are able of forming pores in a membrane whose thickness is larger than the length of the peptide / Doutor