Periodic Reporting for period 1 - AgainstResistance (Novel Strategy to cope with Multidrug Resistant Pathogens)
Période du rapport: 2021-04-01 au 2023-03-31
Antimicrobial resistance has become a major global health and economic issue in recent years, and finding new ways to combat multidrug-resistant bacteria is crucial. Our study focused on Acinetobacter baumannii, classified as a priority 1 (critical) pathogen for research and development of new antibiotics by the World Health Organization (WHO). During the infection process, bacterial pathogens must gather resources from the host while simultaneously evading the immune system. To do so, they possess well-adapted mechanisms that ensure their survival within the host. In our study we specifically examined A. baumannii's adaptation to nitrosative stress, with a focus on the role of heme metabolism. Nitrosative stress is a common type of stress generated by the immune system to combat bacterial infections. Hence, protection against reactive nitrogen species is key for bacterial survival during the infection process. Moreover, heme metabolism is one of the most relevant processes within the prokaryotic cell, as it is involved in several essential mechanisms such as bacterial respiration and defense against the host immune response, including protection against oxidative and nitrosative stress. Therefore, understanding how heme metabolism protects A. baumannii from nitrosative stress will be crucial to identify essential mechanisms for its development during infection. Our main objectives for this study were: 1) Identification of the A. baumannii HSP pathway, their enzymes and intermediates, 2) Uncover how A. baumannii heme homeostasis related genes respond to the conditions imposed by the infected host. 3) Pinpoint Small Colony Variant (SCV) formation and their contribution to A. baumannii virulence. 4) Establish the contribution of heme homeostasis systems to A. baumannii virulence .
This action provided an excellent opportunity for the researcher to acquire scientific and transversal skills that will be beneficial. All the work conducted during this period met the expectations and will be published in peer-reviewed, open access journals.
This action provided an excellent opportunity for the researcher to acquire scientific and transversal skills that will be beneficial. All the work conducted during this period met the expectations and will be published in peer-reviewed, open access journals.
The first objective of the project was to identify and reconstitute the heme biosynthesis pathway in A. baumannii (WP1). Using bioinformatics, we found that A. baumannii encodes for a full protoporphyrin-dependent, also called canonical pathway. With a more extended analysis, we have also seen that different A. baumannii strains encode for different heme uptake systems. With these unexpected results we wanted to perform a more extensive and exhaustive study on the evolution of heme uptake systems in bacteria which was done in collaboration with an external laboratory with expertise in the study of genome evolution. As such, a three-month secondment took place in their laboratory in Vienna to do the tasks related with this work package. Moreover, we also identified some putative heme chaperones that could mediate heme trafficking within the cell establishing the bridge between the heme biosynthesis pathway and heme binding proteins. We have performed some protein-protein interactions to validate this hypothesis and future work will focus on the study of the heme delivery driven by these heme chaperones.
Our second objective was to uncover how A. baumannii heme homeostasis related genes respond to the conditions imposed by the infected host (WP2). We analysed the susceptibility of A. baumannii strains to nitric oxide (NO). We chose the multidrug-resistant (MDR) A. baumannii AB5075-UW strain to follow our physiological studies. After tuning the NO concentrations and time of exposure, we did an RNA-seq transcriptomic analysis under NO stress conditions compared to non-stress conditions. We identified different uncharacterized systems involved in NO detoxification, heme uptake, iron uptake, acinetobactin synthesis, Fe-S biogenesis and cysteine biosynthesis. After confirmation of these results by qRT-PCR, we tested the contribution of these systems on the protection to NO sensitivity. Our results indicated that only a few heme-binding proteins had a direct role in NO stress protection. To better characterise these newly discovered factors involved in NO resistance, we produced and purified the specific components to do a biochemical characterisation of the enzymes.
Our third objective was to pinpoint SCV formation and their contribution to A. baumannii virulence (WP3). We studied the physiological consequences of lacking heme oxygenase and heme metabolism related genes. However, we did not observe the formation of SCV or a growth defect.
Our last objective was to establish the contribution of heme homeostasis systems to A. baumannii virulence (WP4). After elucidating specific mechanisms involved in NO protection, we sought to determine the role of these mechanisms to other stresses encountered by the bacteria during invasion of the host. We tested the role of nitric oxide defense mechanisms, heme uptake systems and iron uptake systems under iron starvation and upon extracellular heme exposure. Our work in this package is in its final stage and we will perform infection studies with murine macrophages and with the model organism Galleria melonella to accomplish the characterisation and determination of the role of these mechanisms in the infection process of A. baumannii.
During the period of this project I also developed a variety of soft skills, including project management, teaching, mentoring and supervision. These skills were honed through participation in various courses such as the StartUp Research course, and the Innovation Management and Intellectual Property course, as well as through lecturing PhD students and supervising of Master students.
With AgainstResistance I also engaged in science communication with a general audience by participating in events such as the European Researchers' Night in Oeiras, Portugal, where we informed and shared the relevance and outcomes of our work for the society.
The work from this project was presented at several conferences and meetings, including the NOVAhealth online meeting in 2021; the Microbiotec online meeting in 2021; the Microbial Stress Response Gordon Research Conference in Massachusetts, US in 2022; the TIMB3 Final Meeting in Tomar, Portugal in 2022; and the International Hybrid Conference on Oxygen Binding and Sensing in Rome, Italy in 2022 and the Tetrapyrrole Discussion Group in Canterbury, UK in 2023.
By the end of the project I published one journal article, one review article and one book chapter.
Furthermore, all the results of this project will be published in peer-reviewed open access journals.
Our second objective was to uncover how A. baumannii heme homeostasis related genes respond to the conditions imposed by the infected host (WP2). We analysed the susceptibility of A. baumannii strains to nitric oxide (NO). We chose the multidrug-resistant (MDR) A. baumannii AB5075-UW strain to follow our physiological studies. After tuning the NO concentrations and time of exposure, we did an RNA-seq transcriptomic analysis under NO stress conditions compared to non-stress conditions. We identified different uncharacterized systems involved in NO detoxification, heme uptake, iron uptake, acinetobactin synthesis, Fe-S biogenesis and cysteine biosynthesis. After confirmation of these results by qRT-PCR, we tested the contribution of these systems on the protection to NO sensitivity. Our results indicated that only a few heme-binding proteins had a direct role in NO stress protection. To better characterise these newly discovered factors involved in NO resistance, we produced and purified the specific components to do a biochemical characterisation of the enzymes.
Our third objective was to pinpoint SCV formation and their contribution to A. baumannii virulence (WP3). We studied the physiological consequences of lacking heme oxygenase and heme metabolism related genes. However, we did not observe the formation of SCV or a growth defect.
Our last objective was to establish the contribution of heme homeostasis systems to A. baumannii virulence (WP4). After elucidating specific mechanisms involved in NO protection, we sought to determine the role of these mechanisms to other stresses encountered by the bacteria during invasion of the host. We tested the role of nitric oxide defense mechanisms, heme uptake systems and iron uptake systems under iron starvation and upon extracellular heme exposure. Our work in this package is in its final stage and we will perform infection studies with murine macrophages and with the model organism Galleria melonella to accomplish the characterisation and determination of the role of these mechanisms in the infection process of A. baumannii.
During the period of this project I also developed a variety of soft skills, including project management, teaching, mentoring and supervision. These skills were honed through participation in various courses such as the StartUp Research course, and the Innovation Management and Intellectual Property course, as well as through lecturing PhD students and supervising of Master students.
With AgainstResistance I also engaged in science communication with a general audience by participating in events such as the European Researchers' Night in Oeiras, Portugal, where we informed and shared the relevance and outcomes of our work for the society.
The work from this project was presented at several conferences and meetings, including the NOVAhealth online meeting in 2021; the Microbiotec online meeting in 2021; the Microbial Stress Response Gordon Research Conference in Massachusetts, US in 2022; the TIMB3 Final Meeting in Tomar, Portugal in 2022; and the International Hybrid Conference on Oxygen Binding and Sensing in Rome, Italy in 2022 and the Tetrapyrrole Discussion Group in Canterbury, UK in 2023.
By the end of the project I published one journal article, one review article and one book chapter.
Furthermore, all the results of this project will be published in peer-reviewed open access journals.
Our project, AgainstResistance, represents the first study on the role of heme metabolism in the infection process of the multidrug-resistant A. baumannii. Although there are some phylogenetic studies in the literature on relevant bacterial processes such as Fe-S biogenesis, to date, there are no other studies on the evolution of heme uptake systems in bacteria, which are essential mechanisms for successful host colonization and iron acquisition. Overall, our research will bring novel and relevant insights into A. baumannii's physiology and bacterial pathogen infection mechanisms.