Novos antibióticos antiparietais

Os antibióticos β-lactâmicos, pertencentes ao grupo dos antibióticos antiparietais são, simultaneamente, a família mais numerosa de antibacterianos e a mais utilizada na prática clínica, actuando por inibição da última etapa da síntese da parede celular bacteriana, nomeadamente na fase parietal da biossíntese do peptidoglicano. Após a descoberta da penicilina, em 1928, verificou-se um desenvolvimento exponencial de novos compostos capazes de combater as doenças infecciosas que se revelavam, não raras vezes, fatais. Contudo, este desenvolvimento foi acompanhado pelo surgimento, a um ritmo ainda mais acelerado, de novos mecanismos de resistência por parte das bactérias que representam, actualmente, um elevado foco de apreensão temendo-se, a curto prazo, um cenário de inexistência de moléculas eficazes no combate a infecções, mesmo aquelas consideradas relativamente corriqueiras e triviais. Ao longo desta monografia pretende-se apurar o actual panorama do armamento terapêutico no que diz respeito à sua eficácia, ao flagelo das resistências e às potenciais soluções alternativas com novos compostos que se revelam promissores.β-lactam antibiotics, which belong to the group of antiparietal antibiotics are, simultaneously, the largest family of antibacterial agents and the most widely used in clinical practice, acting by inhibition of the last step of bacterial cell wall synthesis, particularly in the parietal phase of peptidoglycan biosynthesis. After the discovery of penicillin, in 1928, there was an exponential development of new compounds capable of fighting infectious diseases that were usually fatal. However, this development was followed by the emergence, at an even faster pace, of new resistance mechanisms by bacteria, representing a high level of apprehension and, in the short term, fears of a lack of effective molecules in the fight against infections, even those considered relatively commonplace and trivial. Throughout this monograph it is intended to investigate the current panorama of therapeutic armaments with regard to their effectiveness, the scourge of resistances and the potential alternative solutions with new compounds that are proving promising

Novos antibióticos antiparietais

Os antibióticos β-lactâmicos, pertencentes ao grupo dos antibióticos antiparietais são, simultaneamente, a família mais numerosa de antibacterianos e a mais utilizada na prática clínica, actuando por inibição da última etapa da síntese da parede celular bacteriana, nomeadamente na fase parietal da biossíntese do peptidoglicano. Após a descoberta da penicilina, em 1928, verificou-se um desenvolvimento exponencial de novos compostos capazes de combater as doenças infecciosas que se revelavam, não raras vezes, fatais. Contudo, este desenvolvimento foi acompanhado pelo surgimento, a um ritmo ainda mais acelerado, de novos mecanismos de resistência por parte das bactérias que representam, actualmente, um elevado foco de apreensão temendo-se, a curto prazo, um cenário de inexistência de moléculas eficazes no combate a infecções, mesmo aquelas consideradas relativamente corriqueiras e triviais. Ao longo desta monografia pretende-se apurar o actual panorama do armamento terapêutico no que diz respeito à sua eficácia, ao flagelo das resistências e às potenciais soluções alternativas com novos compostos que se revelam promissores.β-lactam antibiotics, which belong to the group of antiparietal antibiotics are, simultaneously, the largest family of antibacterial agents and the most widely used in clinical practice, acting by inhibition of the last step of bacterial cell wall synthesis, particularly in the parietal phase of peptidoglycan biosynthesis. After the discovery of penicillin, in 1928, there was an exponential development of new compounds capable of fighting infectious diseases that were usually fatal. However, this development was followed by the emergence, at an even faster pace, of new resistance mechanisms by bacteria, representing a high level of apprehension and, in the short term, fears of a lack of effective molecules in the fight against infections, even those considered relatively commonplace and trivial. Throughout this monograph it is intended to investigate the current panorama of therapeutic armaments with regard to their effectiveness, the scourge of resistances and the potential alternative solutions with new compounds that are proving promising

Understanding the Structure–Function Relationship of Lysozyme Resistance in Staphylococcus aureus by Peptidoglycan O‑Acetylation Using Molecular Docking, Dynamics, and Lysis Assay

Lysozyme is an important component of the host innate defense system. It cleaves the β-1,4 glycosidic bonds between <i>N</i>-acetylmuramic acid and <i>N</i>-acetylglucosamine of bacterial peptidoglycan and induce bacterial lysis. Staphylococcus aureus (S. aureus), an opportunistic commensal pathogen, is highly resistant to lysozyme, because of the O-acetylation of peptidoglycan by <i>O</i>-acetyl transferase (<i>oatA</i>). To understand the structure–function relationship of lysozyme resistance in S. aureus by peptidoglycan O-acetylation, we adapted an integrated approach to (i) understand the effect of lysozyme on the growth of S. aureus parental and the <i>oatA</i> mutant strain, (ii) study the lysozyme induced lysis of exponentially grown and stationary phase of both the S. aureus parental and <i>oatA</i> mutant strain, (iii) investigate the dynamic interaction mechanism between normal (de-O-acetylated) and O-acetylated peptidoglycan substrate in complex with lysozyme using molecular docking and molecular dynamics simulations, and (iv) quantify lysozyme resistance of S. aureus parental and the <i>oatA</i> mutant in different human biological fluids. The results indicated for the first time that the active site cleft of lysozyme binding with O-acetylated peptidoglycan in S. aureus was sterically hindered and the structural stability was higher for the lysozyme in complex with normal peptidoglycan. This could have conferred reduced survival of the S. aureus <i>oatA</i> mutant in different human biological fluids. Consistent with this computational analysis, the experimental data confirmed decrease in the growth, lysozyme induced lysis, and lysozyme resistance, due to peptidoglycan O<i>-</i>acetylation in S. aureus

The Role of the MSAABCR Operon in Cell Wall Integrity and Programmed Cell Death During Biofilm Development

Staphylococcus aureus is an important human pathogen in both community and health care settings. Biggest challenges with S. aureus as a pathogen is its ability to acquire antibiotic resistance and produce robust biofilms. In this work, we investigated the nature of the cell wall defect in the msaABCR operon mutant in the Mu50 (VISA) and USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA) strains. Results showed that msaABCR-mutant cells had decreased cell wall thickness and cell wall crosslinking in both strains. These defects are most likely due to increased murein hydrolase activity and/or nonspecific processing of murein hydrolases mediated by increased protease activity in mutant cells. The defect was enhanced by a decrease in teichoic acid content in the msaABCR mutant cell wall. Moreover, we also observed significantly downregulated transcription of early cell wall-synthesizing genes, supporting the finding that msaABCR-mutant cells have decreased peptidoglycan synthesis. Thus, we conclude that the msaABCR operon controls the balance between cell wall synthesis and cell wall hydrolysis, which is required for maintaining a robust cell wall and acquiring resistance to cell wall-targeting antibiotics, such as vancomycin and the β-lactams. This dissertation also elucidated the mechanism of the cell death phenomenon regulated by the msaABCR operon at the molecular level in the USA300 LAC strain. This study showed that msaABCR represses weak acid-dependent cell death. Rate of glucose consumption, and acetate and acetoin production in msaABCR mutant was higher than the USA300 LAC (wild type) strain in the biofilm microenvironment, which caused increased intracellular acidification and led to increased cell death. We showed that MsaB binds directly to the LysR-type transcriptional regulator, cidR promoter and represses expression of the cidR regulon. We also showed role of MsaB in indirect repression of pyruvate catabolism via cidR to play an important role in overflow metabolism and programmed cell death during biofilm development in S. aureus. In addition, pyruvate was also shown to induce expression of the msaABCR operon. This article has thus deciphered the role of the msaABCR operon in staphylococcal metabolic adaption during biofilm development

Influência da amidação do peptidoglicano na resistência à lisozima em Staphylococcus aureus

Staphylococcus aureus é uma bactéria patogénica oportunista com elevada capacidade de adquirir resistência a antibióticos, sendo uma das principais causas de infeções hospitalares, especialmente as estirpes MRSA. A amidação do resíduo D-iso-glutamato do pentapéptido do peptidoglicano, pelo complexo enzimático MurT-GatD, é uma modificação secundária que ocorre na parede celular desta bactéria. Esta reação é essencial para o crescimento ótimo e para a expressão da resistência aos antibióticos β-lactâmicos e à lisozima, muramidase à qual S. aureus é intrinsecamente resistente. Contudo, estudos anteriores sugerem que existem outros determinantes genéticos, para além dos genes murT e gatD, envolvidos no fenótipo de resistência à lisozima, de forma dependente da amidação do peptidoglicano. Deste modo, foi construída uma biblioteca de 494 mutantes no background genético de COLpCadmurT-gatD, mutante condicional do operão murT-gatD da estirpe COL, com recurso ao transposão Tn551, de modo a identificar determinantes genéticos envolvidos na resistência à lisozima. A caracterização de 415 mutantes foi realizada através de ensaios de resistência à lisozima, permitindo selecionar 273 mutantes em que a inserção do transposão resultou num fenótipo alterado de resistência à lisozima do mutante COLpCadmurT-gatD, de forma dependente da expressão de murT-gatD. A integridade do promotor induzível foi confirmada para 241 dos mutantes selecionados, por análise da resistência aos antibióticos β-lactâmicos. O DNA genómico de 187 mutantes foi extraído, digerido com uma enzima de restrição de corte frequente e o mapeamento da inserção do transposão foi obtido por Southern blotting, com recurso a uma sonda específica para o Tn551. Dos 138 mutantes que apresentaram apenas uma inserção do transposão, foram observados 31 padrões de hibridação diferentes. Por sequenciação total do genoma foram identificados o gene miaA, que codifica para uma tRNA transferase, e o gene SACOL0710, que codifica para uma proteína hipotética, que poderão ser novos alvos para o desenvolvimento de estratégias antimicrobianas.Staphylococcus aureus is an opportunistic pathogen with high capacity to acquire resistance to antibiotics, being one of the main causes of nosocomial infections, especially MRSA strains. Amidation of the D-iso-glutamate residue of the peptidoglycan pentapeptide by the MurT-GatD enzymatic complex is a secondary modification that occurs in the cell wall of this bacteria. This reaction is essential for optimal growth and for the expression of resistance to β-lactam antibiotics and lysozyme, a muramidase to which S. aureus is intrinsically resistant. However, previous studies showed that other genetic determinants, in addition to the murT and gatD genes, are involved in the lysozyme resistance phenotype, in a way that is dependent on peptidoglycan amidation. To identify these genetic determinants, a transposon mutant library with 494 mutants was constructed in the genetic background of COLpCadmurT-gatD, murT-gatD conditional mutant of strain COL, using the Tn551 transposon. The characterization of 415 mutants was performed by lysozyme resistance assays, allowing the selection of 273 mutants in which the transposon insertion resulted in an altered phenotype of lysozyme resistance of COLpCadmurT-gatD, in a mechanism that is dependent on the expression of murT-gatD. The integrity of the inducible promoter was confirmed for 241 of the selected mutants, through the analysis of resistance to β-lactam antibiotics. The genomic DNA of 187 mutants was extracted, digested with a high frequency restriction enzyme and the mapping of the transposon insertion was analyzed by Southern blotting using a Tn551-internal probe. Of the 138 mutants that showed a single transposon insertion, 31 different hybridization patterns were observed. Whole genome sequencing allowed the identification of the genes miaA (encoding a tRNA transferase) and SACOL0710 (hypothetical protein), which can ultimately be used as targets for the development of new antimicrobial strategies

A new bacterial peptidoglycan peptidase LytU and insights into substrate recognition by lysostaphin family

Staphylococcus aureus is a pervasive pathogen, whose infections frequently result in serious medical complications and death. Its encounters are yet more perilous in clinical settings where professional care and financial resources alone do not suffice to ensure successful treatment results. The virulence of the bacteria is enforced by numerous cellular mechanisms that have allowed it to develop resistance to every drug used to this date. The bacterial cell wall (CW) is the primary line of defense, the most common target in treatment strategies, and is likely to remain the prioritized candidate for future therapeutic solutions. The main structural component of bacterial CW is peptidoglycan (PG) that forms protective layers. PG is administered by a large number of enzymes that are involved in its synthesis, maintenance, and cleavage. One family of enzymes, M23 peptidases, cleaves pentaglycine bridges that link chains of PG and are specific to S. aureus. These enzymes can be used by the bacteria to manage its own PG in a controlled manner or, alternatively, by hostile microorganisms and cause cell death. Therefore, M23 peptidases of S. aureus are important as potential targets for drugs as well as pharmacological tools themselves that are already employed by nature. Substrate recognizing SH3b domains enhance the effectiveness of M23 endopeptidases. Previous research had identified a putative M23 peptidase gene, transcription of which is upregulated under S. aureus exposure to compounds harmful to cell wall. We examined and characterized the product of the gene. The protein, which we named LytU, is an M23 family zinc-dependent enzyme that cleaves pentaglycine. It is anchored in plasma membrane and is extracytoplasmic, residing in a periplasm-like space. The physiological role of LytU is not confirmed, but evidence suggest it can recycle PG fragments and participate in daughter cell separation. A distinct feature of the enzyme is its ability to strongly bind a second zinc ion, which incapacitates catalytic residues. We propose that together with pH, the binding of second ion serves a regulatory function in situ. Solution structure of the LytU catalytic domain has been determined. Binding of substrate pentaglycine to catalytic M23 domain is very transient at least in vitro. The binding, nevertheless, is accomplished by SH3b domain of enzymes bearing it. Contrarily to previous beliefs, we found that SH3b domain binding to substrate is primarily driven by interactions with PG branching peptides, rather than by weaker interaction with pentaglycine. The binding of SH3b to substrate is independent of catalytic domain and it targets and binds the PG peptide moieties that are proximal to but different from the pentaglycine cleaved by catalytic domain. In summary, we have introduced and characterized a new M23 family endopeptidase, proposed a regulation mechanism, and changed the paradigm of substrate binding by M23 peptidases. Our results are expected to contribute to a better understanding of S. aureus physiology and provide means for the development of cures.Staphylococcus aureus on yleinen bakteeri, joka aiheuttaa usein vakavia, jopa hengenvaarallisia infektioita. Sairaalaympäristön ammattitaidosta tai hoitoon käytettävistä resursseista huolimatta infektioihin liittyy huomattava kuolleisuus. Bakteeri tuottaa useita mekanistisesti erilaisia virulenssitekijöitä, minkä seurauksena bakteeri on kehittänyt vastustuskyvyn kaikille nykyisille antibiooteille. Bakteerin soluseinä on sen ensisijainen suojautumiskeino. Soluseinä on myös tavallisin lääkehoidon kohde ja todennäköisesti jatkossa edelleen etusijalla lääkekehityksen kohteista. Bakteerin soluseinän päärakennekomponentti on peptidoglykaani, joka muodostaa bakteeria suojaavia kerroksia. Tätä peptidoglykaania tuottaa, ylläpitää ja hajottaa lukuisa joukko entsyymejä, kuten M23-entsyymiperheen peptidaasit, jotka pilkkovat S. aureus-bakteerille ominaisia, peptidoglykaaniketjuja yhdistäviä pentaglysiinisiltoja. Bakteerit voivat käyttää näitä entsyymejä oman soluseinänsä hallittuun ylläpitämiseen tai vaihtoehtoisesti aiheuttamaan kilpailevan mikro-organismin solukuoleman. Tämän takia S. aureus-bakteerin M23-peptidaasit ovat sekä merkittäviä lääkekehityksen kohteita että luonnossakin käytössä oleva farmakologinen työkalu. Substraatin tunnistava SH3b-domeeni tehostaa M23-endopeptidaasien vaikutusta. Aikaisempi tutkimus on tunnistanut mahdollisen M23-peptidaasigeenin, jonka transkriptio aktivoituu kun S. aureus altistuu soluseinää vahingoittaville yhdisteille. Me tutkimme ja karakterisoimme tämän geenituotteen. Proteiini, jonka nimesimme LytU:ksi, on M23-perheen sinkistä riippuvainen pentaglysiiniä pilkkova entsyymi. Se on ankkuroitunut solukalvoon ja on ekstrasytoplasminen, sijoittuen periplasman kaltaiseen tilaan. LytU:n fysiologista roolia ei ole varmistettu, mutta tutkimustulokset viittaavat tehtävään peptidoglykaanifragmenttien kierrätyksessä sekä tytärsolujen erottamisessa toisistaan. Entsyymille omaleimaista on sen kyky sitoa voimakkaasti toinen sinkki-ioni, minkä seurauksena entsyymin katalyyttiset histidiinit muuttuvat toimintakyvyttömiksi. Esitämme, että toisen sinkin sitominen ja pH yhdessä säätelevät entsyymiä in situ. Entsyymin katalyyttisen M23-domeenin sitoutuminen pentaglysiinisubstraattiin on hyvin lyhytaikaista ainakin in vitro. SH3b-domeenin sisältävien entsyymien ja substraatin välinen vuorovaikutus on kuitenkin todennettu. Toisin kuin aiemmin oletettiin, me osoitimme, että SH3b-domeenin sitoutumista substraattiin ohjaa ensisijaisesti sen voimakkaampi vuorovaikutus peptidoglykaanin haarapeptidien kanssa eikä niinkään heikompi vuorovaikutus pentaglysiinin kanssa. SH3b:n sitoutuminen substraattiin ei riipu katalyyttisesta domeenista ja se tunnistaa ja sitoutuu peptidoglykaanin peptidiosaan, joka on proksimaalinen, mutta eri kuin se pentaglysiini, jonka katalyyttinen domeeni pilkkoo. Yhteenvetona väitöskirjassa karakterisoitiin uusi M23-perheen entsyymi, ehdotettiin säätelymekanismi sekä muutettiin näkemystä M23-peptidaasien substraatin sitomisesta. Tuloksemme edistävät S. aureus-bakteerin fysiologian tuntemusta sekä tarjoavat keinoja hoidon kehittämiselle