Efecto de la sobre-expresión de los sistemas de bombeo múltiple de drogas, MexAB-OprM y MexCD-OprJ, sobre el sistema de señalización por quorum-sensing de Pseudomonas aeruginosa

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-09-2017Pseudomonas aeruginosa is among the most relevant opportunistic pathogen involved in infections at hospitals, as well as in immunocompromised and cystic fibrosis patients. In addition, this bacterial species displays a characteristic low susceptibility to antibiotics and is able to acquire increased levels of resistance upon selection with antibiotics during treatment. All these circumstances make that P. aeruginosa infections are currently of special concern. One of the most important causes of resistance in this bacteria is the expression of multidrug efflux pumps, able to extrude disinfectants and antibiotics among others. In this PhD Thesis we analyse the physiological effects, with a special focus on the quorum sensing (QS) regulation network, of the acquisition of high level of resistance due to mutations in the mexR or nfxB genes, which leads to the overexpression of the MexAB-OprM or MexCD-OprJ efflux pumps respectively. In addition, as an independent objective, we have analysed, using experimental evolution and whole genome sequence tools, the potential mechanisms of resistance of P. aeruginosa against the new antibiotic MDN-57. In this work, we demonstrate using transcriptomic analyses, that both the over-expression and the deletion of the MexAB-OprM or MexCD-OprJ efflux systems produce a high impact over the expression of a large number of genes, evidencing the importance of an appropriate expression of these RND systems for cellular homeostasis. We also show that NfxB, the negative regulator of MexCD-OprJ efflux pump, has a potential role as global regulator controlling the expression of a set of genes in a direct or indirect way, beyond the mexCD-oprJ operon. Further, we specially focus in the comparison between the transcriptomes of the mexR* and nfxB* mutants, which overproduce the MexAB-OprM and MexCD-OprJ efflux system respectively in order to decipher the differences and similarities observed in gene expression between these two strains. In this sense, we have found that the main similarity observed when each one of the mexR* and nfxB* transcriptomes are compared with that of the wild-type parental strain is the alteration in the expression of a large set QS-regulated genes. These results could be initially considered as a consequence of an unspecific burden associated to the hyperactivity of whatever these two RND systems. However, we show that these changes are dependent of the specific efflux system overexpressed, being different the underlying mechanism by which mexR* and nfxB* mutant present an impaired QS signalling network. In contrast with previous claims, we demonstrate that defective QS response associated with the overexpression of MexAB-OprM, rather than being a consequence of an excessive 3-oxo-C12-HSL extrusion, is due to an impaired production of both PQS and HHQ, probably caused by a decreased production of its immediate precursor, octanoic acid. On the other hand, we show that the impaired QS-response observed in the nfxB* strain is mainly caused by an excessive non-physiological extrusion of the autoinducer signal HHQ, which produce a decrease its own production and in the synthesis of PQS. Despite we also demonstrate that MexCD-OprJ is able to extrude kynurenine, another precursor of AQs, our results indicate that this extrusion is not the main cause of the low AQs production observed in the nfxB* mutant. Altogether, these results highlight the relevance of these two RND efflux systems in the modulation of the QS-response. Finally, we have showed that prolonged exposition of P. aeruginosa cultures to the new antibiotic MDN-57 leads to selection of resistant mutants containing mutations in hemA and/or hemD genes. Since these two genes are implicated in heme biosynthesis, it is possible that this pathway would be the target of MDN-57. In addition, we also show that the MexAB-OprM, MexCD-OprJ and MexEF-OprN efflux pumps contribute to the intrinsic and the acquired (when overexpressed) resistance to MDN-57

Multidrug Efflux Pumps at the Crossroad between Antibiotic Resistance and Bacterial Virulence

Multidrug efflux pumps can be involved in bacterial resistance to antibiotics at different levels. Some efflux pumps are constitutively expressed at low levels and contribute to intrinsic resistance. In addition, their overexpression may allow higher levels of resistance. This overexpression can be transient, in the presence of an effector (phenotypic resistance), or constitutive when mutants in the regulatory elements of the expression of efflux pumps are selected (acquired resistance). Efflux pumps are present in all cells, from human to bacteria and are highly conserved, which indicates that they are ancient elements in the evolution of different organisms. Consequently, it has been suggested that, besides antibiotic resistance, bacterial multidrug efflux pumps would likely contribute to other relevant processes of the microbial physiology. In the current article, we discuss some specific examples of the role that efflux pumps may have in the bacterial virulence of animals’ and plants’ pathogens, including the processes of intercellular communication. Based in these evidences, we propose that efflux pumps are at the crossroad between resistance and virulence of bacterial pathogens. Consequently, the comprehensive study of multidrug efflux pumps requires addressing these functions, which are of relevance for the bacterial–host interactions during infection.Work in our laboratory is supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2014-54507-R and JPI Water StARE JPIW2013-089-C02-01); from Madrid Autonomous Community [S2010/BMD2414 (PROMPT)]; and from the Instituto de Salud Carlos III [Spanish Network for Research on Infectious Diseases (REIPI RD12/0015)]. MA-R and PB are recipients of FPI fellowships from MINECO.Peer reviewedPeer Reviewe

The impaired quorum sensing response of MexAB?OprM efflux pump overexpressing mutants is not due to non?physiological efflux of 3?oxo?C12?HSL

Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analyzed efflux pump. Herein, we performed an in‐depth analysis of the QS‐mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB‐OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3‐oxo‐C12‐HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This ‘apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals

Role of the Multidrug Resistance Efflux Pump MexCD-OprJ in the Pseudomonas aeruginosa Quorum Sensing Response

Multidrug efflux pumps constitute a category of antibiotic resistance determinants that are a part of the core bacterial genomes. Given their conservation, it is conceivable that they present functions beyond the extrusion of antibiotics currently used for therapy. Pseudomonas aeruginosa stands as a relevant respiratory pathogen, with a high prevalence at hospitals and in cystic fibrosis patients. Part of its success relies on its low susceptibility to antibiotics and on the production of virulence factors, whose expression is regulated in several cases by quorum sensing (QS). We found that overexpression of the MexCD-OprJ multidrug efflux pump shuts down the P. aeruginosa QS response. Our results support that MexCD-OprJ extrudes kynurenine, a precursor of the alkyl-quinolone signal (AQS) molecules. Anthranilate and octanoate, also AQS precursors, do not seem to be extruded by MexCD-OprJ. Kynurenine extrusion is not sufficient to reduce the QS response in a mutant overexpressing this efflux pump. Impaired QS response is mainly due to the extrusion of 4-hydroxy-2-heptylquinoline (HHQ), the precursor of the Pseudomonas Quinolone Signal (PQS), leading to low PQS intracellular levels and reduced production of QS signal molecules. As the consequence, the expression of QS-regulated genes is impaired and the production of QS-regulated virulence factors strongly decreases in a MexCD-OprN P. aeruginosa overexpressing mutant. Previous work showed that MexEF-OprJ, another P. aeruginosa efflux pump, is also able of extruding kynurenine and HHQ. However, opposite to our findings, the QS defect in a MexEF-OprN overproducer is due to kynurenine extrusion. These results indicate that, although efflux pumps can share some substrates, the affinity for each of them can be different. Although the QS response is triggered by population density, information on additional elements able of modulating such response is still scarce. This is particularly important in the case of P. aeruginosa lung chronic infections, a situation in which QS-defective mutants are accumulated. If MexCD-OprJ overexpression alleviates the cost associated to triggering the QS response when un-needed, it could be possible that MexCD-OprJ antibiotic resistant overproducer strains might be selected even in the absence of antibiotic selective pressure, acting as antibiotic resistant cheaters in heterogeneous P. aeruginosa populations

Dynamics of the MRSA Population in a Chilean Hospital: a Phylogenomic Analysis (2000-2016)

La diseminación mundial de Staphylococcus aureus resistente a meticilina (SARM) está asociada a la aparición y el establecimiento de clones en zonas geográficas específicas. El clon chileno-cordobés (ChC) (ST5-SCCmecI) ha sido el clon de SARM predominante en Chile desde su primera descripción en 1998, a pesar del informe de otros clones de SARM emergentes en los últimos años. Aquí, caracterizamos la historia evolutiva de MRSA desde 2000 hasta 2016 en un centro de salud terciario chileno utilizando análisis filogenómicos. Secuenciamos 469 aislamientos de SARM recogidos entre 2000 y 2016. Evaluamos las tendencias temporales de los clones circulantes y realizamos una reconstrucción filogenómica para caracterizar la dinámica clonal. Encontramos un aumento significativo en la diversidad y riqueza de tipos de secuencia (STs; Spearman r = 0,8748, P , 0,0001) con un índice de diversidad de Shannon que aumentó de 0,221 en el año 2000 a 1,33 en 2016, y una diversidad efectiva (número de Hill; q = 2) que aumentó de 1,12 a 2,71. El análisis de la tendencia temporal reveló que en el periodo de 2000 a 2003 la mayoría de los aislados (94,2%; n = 98) pertenecían al clon ChC. Sin embargo, desde entonces, la frecuencia del clon ChC ha disminuido con el tiempo, representando el 52% de la colección en el período de 2013 a 2016. Este descenso estuvo acompañado por el aumento de dos linajes emergentes de SARM, ST105-SCCmecII y ST72-SCCmecVI. En conclusión, el clon ChC sigue siendo el linaje MRSA más frecuente, pero este linaje está siendo reemplazado gradualmente por varios clones emergentes, el más importante de los cuales es el clon ST105-SCCmecII. Hasta donde sabemos, éste es el mayor estudio de la dinámica clonal del SARM realizado en Sudamérica. © 2023 Martínez et al.The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P , 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. © 2023 Martínez et al

Dynamics of the Mrsa Population in a Chilean Hospital: a Phylogenomic analysis (2000-2016)

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P \u3c 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. to the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or more limited typing techniques that lack the resolution to represent an accurate description of the genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000 and 2016 in Chile providing the largest and most detailed study of clonal dynamics of MRSA in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing in frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America

The MexJK Multidrug Efflux Pump Is Not Involved in Acquired or Intrinsic Antibiotic Resistance in Pseudomonas aeruginosa, but Modulates the Bacterial Quorum Sensing Response

Multidrug efflux pumps are critical elements in both intrinsic and acquired antibiotic resistance of bacterial populations. Consequently, most studies regarding these protein machineries focus on this specific phenotype. Nevertheless, different works show that efflux pumps participate in other aspects of bacterial physiology too. Herein, we study the Pseudomonas aeruginosa multidrug efflux pump MexJK. Previous studies, using model strains lacking MexAB-OprM and MexCD-OprJ efflux pumps, support that MexJK can extrude erythromycin, tetracycline, and triclosan. However, the results here reported indicate that this potential increased extrusion, in a mutant overexpressing mexJK, does not alter the antibiotics susceptibility in a wild-type genetic background where all intrinsic multidrug efflux pumps remain functional. Nevertheless, a clear impact on the quorum sensing (QS) response, mainly in the Pqs-dependent QS regulation network and in the expression of Pqs-regulated virulence factors, was observed linked to mexJK overexpression. The production of the siderophore pyoverdine strongly depended on the level of mexJK expression, suggesting that MexJK might participate in P. aeruginosa pyoverdine-dependent iron homeostasis. All in all, the results presented in the current article support that the functions of multidrug efflux pumps, as MexJK, go beyond antibiotic resistance and can modulate other relevant aspects of bacterial physiology

Novel inducers of the expression of multidrug efflux pumps that trigger pseudomonas aeruginosa transient antibiotic resistance

The study of the acquisition of antibiotic resistance (AR) has mainly focused on inherited processes, namely, mutations and acquisition of AR genes. However, inducible, noninheritable AR has received less attention, and most information in this field derives from the study of antibiotics as inducers of their associated resistance mechanisms. Less is known about nonantibiotic compounds or situations that can induce AR during infection. Multidrug resistance efflux pumps are a category of AR determinants characterized by the tight regulation of their expression. Their contribution to acquired AR relies in their overexpression. Here, we analyzed potential inducers of the expression of the chromosomally encoded Pseudomonas aeruginosa clinically relevant efflux pumps, MexCD-OprJ and MexAB-OprM. For this purpose, we developed a set of luxCDABE-based P. aeruginosa biosensor strains, which allows the high-throughput analysis of compounds able to modify the expression of these efflux pumps. Using these strains, we analyzed a set of 240 compounds present in Biolog phenotype microarrays. Several inducers of the expression of the genes that encode these efflux pumps were found. The study focused in dequalinium chloride, procaine, and atropine, compounds that can be found in clinical settings. Using real-time PCR, we confirmed that these compounds indeed induce the expression of the mexCD-oprJ operon. In addition, P. aeruginosa presents lower susceptibility to ciprofloxacin (a MexCD-OprJ substrate) when dequalinium chloride, procaine, or atropine are present. This study emphasizes the need to study compounds that can trigger transient AR during antibiotic treatment, a phenotype difficult to discover using classical susceptibility tests.This study was supported by grants from the Instituto de Salud Carlos III (Spanish Network for Research on Infectious Diseases [RD16/0016/0011]); the Spanish Ministry of Economy, Industry, and Competitivity (BIO2017-83128-R); JPI Water StARE (JPIW2013-089-C02-01); and the Autonomous Community of Madrid (B2017/BMD-3691)

Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic- Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps

ABSTRACT It is generally assumed that the acquisition of antibiotic resistance is as- sociated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar sys- tems. To answer this question, we studied a set of P. aeruginosa mutants that inde- pendently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intra- cellular H accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consump- tion, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs de- rived from overexpression of P. aeruginosa multidrug efflux pumps. The develop- ment of drugs that block this metabolic “reaccommodation” might help in reducing the persistence and spread of antibiotic resistance elements among bacterial popu- lations. IMPORTANCE It is widely accepted that the acquisition of resistance confers a fit- ness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been proposed in the belief that they will reduce the persistence and spread of resistance among bacterial pathogens. Unfortunately, trials testing this possibility have frequently failed, indicating that resistant microorganisms are not al- ways outcompeted by susceptible ones. Indeed, some mutations do not result in a fitness cost, and in case they do, the cost may be compensated for by a secondary mutation. Here we describe an alternative nonmutational mechanism for compensat- ing for fitness costs, which consists of the metabolic rewiring of resistant mutants. Deciphering the mechanisms involved in the compensation of fitness costs of antibiotic-resistant mutants may help in the developPeer reviewe

Naringenin Inhibition of the Pseudomonas aeruginosa Quorum Sensing Response Is Based on Its Time-Dependent Competition With N-(3-Oxo-dodecanoyl)-L-homoserine Lactone for LasR Binding

© 2020 Hernando-Amado, Alcalde-Rico, Gil-Gil, Valverde and Martínez.Bacterial quorum sensing (QS) is a cell-to-cell communication system that governs the expression of a large set of genes involved in bacterial–host interactions, including the production of virulence factors. Conversely, the hosts can produce anti-QS compounds to impair virulence of bacterial pathogens. One of these inhibitors is the plant flavonoid naringenin, which impairs the production of QS-regulated Pseudomonas aeruginosa virulence factors. In the present work, we analyze the molecular basis for such inhibition. Our data indicate that naringenin produces its effect by directly binding the QS regulator LasR, hence competing with its physiological activator, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12-HSL). The in vitro analysis of LasR binding to its cognate target DNA showed that the capacity of naringenin to outcompete 3OC12-HSL, when the latter is previously bound to LasR, is low. By using an E. coli LasR-based biosensor strain, which does not produce 3OC12-HSL, we determined that the inhibition of LasR is more efficient when naringenin binds to nascent LasR than when this regulator is already activated through 3OC12-HSL binding. According to these findings, at early exponential growth phase, when the amount of 3OC12-HSL is low, naringenin should proficiently inhibit the P. aeruginosa QS response, whereas at later stages of growth, once 3OC12-HSL concentration reaches a threshold enough for binding LasR, naringenin would not efficiently inhibit the QS response. To test this hypothesis, we analyze the potential effect of naringenin over the QS response by adding naringenin to P. aeruginosa cultures at either time zero (early inhibition) or at stationary growth phase (late inhibition). In early inhibitory conditions, naringenin inhibited the expression of QS-regulated genes, as well as the production of the QS-regulated virulence factors, pyocyanin and elastase. Nevertheless, in late inhibitory conditions, the P. aeruginosa QS response was not inhibited by naringenin. Therefore, this time-dependent inhibition may compromise the efficiency of this flavonoid, which will be effective just when used against bacterial populations presenting low cellular densities, and highlight the importance of searching for QS inhibitors whose mechanism of action does not depend on the QS status of the population.Work has been supported by grants from the Instituto de Salud Carlos III (Spanish Network for Research on Infectious Diseases [RD16/0016/0011]), from the Spanish Ministry of Economy, Industry and Competitivity (BIO2017-83128-R) and from the Autonomous Community of Madrid (B2017/BMD-3691)