Functional characterization of AmpC β-lactamase and role of LMM-PBPs in peptidoglycan composition, β-lactam resistance and ampC regulation in Pseudomonas aeruginosa

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-11-2014Pseudomonas aeruginosa is one of the most problematic versatile Gram-negative bacteria in causing opportunistic human infections which are particularly difficult to treat because of its intrinsic resistance to antibiotics, as a consequence of many intervening resistance mechanisms involving the ability to overproduce the chromosomally encoded cephalosporinases, Pae-AmpC, which are periplasmic enzymes, belong to group I class C serine β-lactamases and are also responsible of bacterial resistance in many bacteria. In P. aeruginosa, ampC expression is regulated mainly by AmpG permeases, AmpD amidases, AmpR, NagZ, and two competing AmpR-binding muropeptides [UDP-MurNAc-pentapeptides (ampC suppressor) and 1,6-anhydromuropeptides (ampC inducer)]. Low molecular mass penicillin-binding proteins [LMM-PBP; e.g. PBP4 (DacB), PBP5 (DacC), PBP7 (PbpG)] are a group of periplasmic enzymes that have DD-carboxypeptidase and/or DD-endopeptidase activities which participate in cell separation, peptidoglycan (PG) maturation and recycling. Binding of β-lactams (e.g. penicillin) with LMM-PBPs causes an increase in anhydromuropeptides and periplasmic AmpC overproduction to hydrolyze that external unwelcome inducer. This study aims to highlight and to characterize the functions of Pae-AmpC and the role of LMM-PBPs PBP4, PBP5 and PBP7 in PG composition and bacterial resistance in P. aeruginosa; also, to study the role of these LMM-PBPs in Pae-ampC regulation and to see if they are needed for the recovery of rod shape of imipenem-induced round cells in P. aeruginosa. To fulfill this study we characterized several Pae-AmpC forms (wild type and mutants) in wild type and mutants of E. coli and in P. aeruginosa PAO1 strain which were tested for their PG composition by HPLC analysis and for bacterial resistance by disc diffusion method. Also, we constructed single and combined mutants of dacB, dacC, pbpG and ampC in PAO1 strain which were tested for their PG composition, ampC expression by RT-PCR, β-lactams susceptibility and their PBPs pattern by Bocillin-FL binding test. We analyzed PG composition and PBPs pattern in imipenem-induced round cells and their rod shape recovered cells in PAO1. We found that some Pae-AmpC mutants had a very low β-lactamase activity (AmpC-F4:C3 and AmpC-F4:C6); the mature form of Pae-AmpC had a high β-lactamase activity and a secondary DD-endopeptidase and DD-carboxypeptidase activities; only dacB single and combined mutations produced high ampC expression and β-lactam resistance; only dacC single and combined mutations produced maximum increase of PG pentapeptides. The triple mutant of dacB, dacC and pbpG displayed the largest increase in ampC expression and β-lactams resistance. Microscopic examination of all the constructed Pae mutants showed that they still retain their rod shape morphology similar to their parental PAO1 strain. Also, we found that activities of DacB, DacC and PbpG are not essential for recovery of rod shape in imipenem-induced spheres in P. aeruginosa.P. aeruginosa es una de las bacterias Gram-negativas versátiles y más problemáticas causantes de infecciones oportunistas en humanos y que son particularmente difíciles de curar debido a su resistencia intrínseca a los antibióticos, como consecuencia de los muchos mecanismos de resistencia intervinientes, y que implica la capacidad de sobreproducir las cefalosporinas codificados cromosómicamente, Pae-AmpC. En P. aeruginosa, la expresión de ampC se rige principalmente por la permeasa AmpG, la amidasa AmpD, AmpR, NagZ y dos muropéptidos competidores fijadores de AmpR [UDP-MurNAc-pentapéptido (supresor de ampC) y 1,6-anhidro-muropéptidos (inductor de ampC)]. Las proteínas (de baja masa molecular) fijadoras de penicilina [LMM-PBP; por ejemplo PBP4 (DacB), PBP5 (DacC), PBP7 (PbpG)] son un grupo de enzimas periplásmicas que tienen actividades DD-carboxipeptidasa/DD-endopeptidasa y que participan en la separación celular, y en la maduración y reciclaje del peptidoglicano (PG). La unión de un β-lactámicos (por ejemplo, penicilina) a las LMM-PBP provoca un aumento en anhidro-muropéptidos y sobreproducción de AmpC en periplasma para hidrolizar este inductor externo no deseado. Este estudio tiene como objetivo destacar y caracterizar las funciones de Pae-AmpC y la implicación de PBP4, PBP5 y PBP7 en la composición del PG y la resistencia bacteriana en P. aeruginosa y además, analizar el papel de estas LMM-PBPs en la regulación de Pae-ampC y determinar si son necesarias para la recuperación de la forma bacilar a partir de esferoplastos de P. aeruginosa inducidos por imipenem. Para realizar este estudio hemos caracterizado varias formas de Pae-AmpC en E. coli y P. aeruginosa PAO1 que se ensayaron mediante la determinación de su composición del PG por análisis de HPLC y su resistencia bacteriana por el método de difusión en disco en agar. Además, hemos construido mutantes individuales y combinados de dacB, dacC, pbpG y ampC en la cepa PAO1. Además, se analizó la composición del PG y los patrones de PBP en esferoplastos inducidos por imipenem y de sus formas bacilares recuperadas en PAO1. Hemos encontrado que algunos mutantes de Pae-AmpC tenían una actividad muy baja β-lactamasa (AmpC-F4: C3 y AmpC-F4: C6); que el tipo silvestre de Pae-AmpC (AmpC-F4) tenía una alta actividad β-lactamasa y unas actividades secundarias de DD-endopeptidasa y DD-carboxipeptidasa; que sólo mutaciones individuales en dacB y combinadas producen una alta expresión de ampC y resistencia a β-lactámicos; que sólo mutaciones individuales en dacC y combinadas producen un aumento máximo de pentapéptidos en el PG. El triple mutante de dacB, dacC y pbpG mostró el mayor aumento en la resistencia a β-lactámicos y en la expresión de ampC. El examen microscópico de todos los mutantes construidos en PAO1 mostró que todavía conservan sus formas morfologías bacilares similar a su cepa parental PAO1. Las actividades de DacB, DacC y PbpG no son esenciales para la recuperación de la forma bacilar en los esferoplastos de P. aeruginosa inducidos por imipene

Role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins in AmpC expression, β-lactam resistance, and peptidoglycan structure

This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, β-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of β-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major DD-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as DD-carboxypeptidase only if PBP5 is absent, and their DD-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the β-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic β-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on β-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, β-lactam trapping, and DD-carboxypeptidase/β-lactamase activity.This work was supported by the Ministerio de Economía y Competitividad of Spain and the Instituto de Salud Carlos III through the Spanish Network for the Research in Infectious Diseases (grants RD06/0008 and RD12/0015) and grants PS09/00033 and PI12/00103. The work at CBMSO was supported by grants BFU2009-09200 from the Ministerio de Economía y Competitividad of Spain and 223431 DIVINOCELL from the European Union. We acknowledge the predoctoral grant JAE/Predoc from the Consejo Superior de Investigaciones Cientificas to A.R. The study is also supported by the Direcció General d’Universitats, Recerca i Transferència del Coneixement del Govern de les Illes Balears, Spain.Peer Reviewe

Coatings containing molybdenum trisulphide QDs to protect oil paintings against different environmental factors

Purpose This study aims to investigate profoundly the protection of oil painting from deterioration using molybdenum trisulphide quantum dots (MoS3 QDs) against microbe, dirt accumulation and ultraviolet (UV) degradation. Design/methodology/approach The protection of painting against different deterioration factors necessitates the sustainable methods and advanced techniques. Scanning electron microscopy and transmission electron microscopy have been used to investigate the morphological structure of the painting and MoS3 QDs, respectively, and optical microscopy was used to examine antibacterial activity of MoS3 QDs towards different types of bacteria. To investigate the protection of painting against deterioration, the Fourier transform IR spectroscopy (FTIR) was used to investigate the paintings left in open air for a year. Chemical composition and crystal structure of MoS3 QDs have been studied using X-ray diffraction and X-ray photoelectron spectroscopy analysis, respectively. Findings The addition of MoS3 nanoparticles into painted coatings enhances the durability of linseed oil-based paintings toward UV ageing regarding the change in colour which confirmed by FTIR analysis. The protection of oil painting opposed to various deterioration factors was developed by involving of MoS3 QDs in the coating of the painting. Antibacterial effect of MoS3 QDs was tested against different types of bacteria such as Pseudomonas aeruginosa confirming that the MoS3 QDs involved in the coatings of oil paintings produces a high protection layer for the paintings against several microbial attacks. In addition, coatings containing MoS3 QDs reduce the accumulation of dirt on oil paintings when subjected to open air for a year. Originality/value The novel MoS3 QDs was used to form a protective and transparent coating layer for the oil painting to overcome the deterioration, displays the promising protection and can be applied for different oil paintings. </jats:sec