Antibiotic Resistance and Cell-Wall Recycling in Pseudomonas aeruginosa

The threat of antibiotic resistance and the global rise of pan-resistant bacteria is a serious concern at present. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is frequently associated with multi and pan-drug resistant infections. This research delves into the mechanism of resistance to a class of drugs known as the β-lactams. AmpC β-lactamase encoded chromosomally in P. aeruginosa is one of the predominant causes of resistance to many β-lactams. Previous research on this pathway identified the AmpC regulatory protein - AmpR and elaborated on its regulon in P. aeruginosa. In this dissertation, further investigation in the mechanisms associated with AmpR regulation of AmpC and its connection with the cell-wall recycling pathway is explored. Cell-wall recycling, a common phenomenon in both Gram-positive and negative bacteria is investigated in some detail in P. aeruginosa for the first time. The identity of the cell-wall recycling products or muropeptides in P. aeruginosa is elucidated. Around 20 distinct muropeptides were identified through liquid chromatography/mass spectrometry analyses of bacterial extracts. Furthermore, iv the muropeptide effector of AmpR that is instrumental in the activation of this transcription factor is identified. The role of two permeases AmpG and AmpP in antibiotic resistance and cell-wall recycling are also investigated by comparing antibiotic susceptibility and muropeptide profile of the isogenic mutants of ampG and ampP with the wild-type PAO1. Along with investigating permeases, the role of a putative N-acetylglucosaminidase FlgJ is also investigated. Finally, keeping in mind the broad role of AmpR in regulating P. aeruginosa virulence and antibiotic resistance, we try to identify small -molecule inhibitors for AmpR. In our effort to identify inhibitors, a novel reporter-based screening assay is developed. In summary, this dissertation increases our understanding of cell-wall recycling and mechanisms of β-lactam resistance and attempts at establishing novel-antibacterial targets and inhibitors

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors

A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa is a metabolically versatile bacterium that is found in a wide range of biotic and abiotic habitats. It is a major human opportunistic pathogen causing numerous acute and chronic infections. The critical traits contributing to the pathogenic potential of P. aeruginosa are the production of a myriad of virulence factors, formation of biofilms and antibiotic resistance. Expression of these traits is under stringent regulation, and it responds to largely unidentified environmental signals. This review is focused on providing a global picture of virulence gene regulation in P. aeruginosa. In addition to key regulatory pathways that control the transition from acute to chronic infection phenotypes, some regulators have been identified that modulate multiple virulence mechanisms. Despite of a propensity for chaotic behaviour, no chaotic motifs were readily observed in the P. aeruginosa virulence regulatory network. Having a ‘birds-eye’ view of the regulatory cascades provides the forum opportunities to pose questions, formulate hypotheses and evaluate theories in elucidating P. aeruginosa pathogenesis. Understanding the mechanisms involved in making P. aeruginosa a successful pathogen is essential in helping devise control strategies

Structural Dynamics of L1 and L2 β-lactamase

Stenotrophomonas maltophilia is a Gram-negative bacterium, found in several different environments, such as soil, water and hospital. It can cause multiple infections but also has strong resistance to many antibiotics such as cephalosporins, carbapenems, and aminoglycosides. S. maltophilia confers antibiotic resistance through expression of two different β-lactamases: L1-metallo-β-lactamase (L1 MBL) and L2 β-lactamase. L1 MBL is a class B3 β-lactamase and is the only known tetrameric β-lactamase known to humans. L2 is a class A β-lactamase which has been recently identified. In L1 MBL, there are, two loops (α3-β7 and β12-α5) known as the gating loops, that enclose the active site. The “open” and “close” conformations of these two loops were observed in the molecular dynamic simulation. These two conformations allow the gate loops have the ability of controlling the volume of the zinc binding pocket. The pocket size affects the substrate binding and further influence the catalytic activity of the whole protein. Therefore, gating loops are thought to have an important role in substrate binding and catalysis. In this thesis, the dynamics of the gating loops is explored through Markov state models. The “open” and “closed” confirmations are defined and three key interactions (salt bridge between R236 and D150c, the π–π stacking between H151 and Y227 and the orientation of P225) were identified that play an important role in controlling the conformation of the gating loops. Furthermore, as a tetramer, the correlation between the four subunits was also explored through CVAE-based deep learning and network analysis. The results revealed a ‘dimer of dimer’ dynamics in L1 MBL. The second part of the thesis focuses on exploring the dynamics of L2 β-lactamase family consisting of L2a, L2b, L2c and L2d enzymes. Homology modelling, MDLovofit, Markov state models, dynamic cross-correlation analysis and CVAE-based deep learning were employed for identifying potential key interactions and dynamic correlations between each subtype. Two dynamic combinations regions were revealed (α1 helix/N-terminal, β9-α15 loop, β7-β8 loop, hinge region, and C terminal, β1-β2 loop, β8-β9 loop) which exist in all four L2 β-lactamases. Stabilising these two combinations could possibly help inhibit the function of L2 β-lactamases. Besides, several potential key residues which result in high dynamic regions were also identified. Since very few research targeted on L2 β-lactamases, this work could be a starting point for the following-up work. The improved understanding of the dynamics of L1 and L2 β-lactamases will help in the design of their inhibitors and discovery of novel resistance breakers

A LysR-family transcriptional regulator is involved in the selenium-dependent transcriptional regpression of selenium-free hydrogenase gene groups in Methanococcus voltae

Methanococcus voltae besitzt zwei Coenzym F420 reduzierende und zwei Coenzym 420 nicht reduzierende Hydrogenasen. Zwei davon, Fru und Vhu, sind Selenoproteine. Eine F420 reduzierende Hydrogenase (Frc) und eine F420 nicht reduzierende Hydrogenase (Vhc) enthalten kein Selenocystein. Die Gengruppen, die für diese Enzyme codieren, sind durch eine gemeinsame intergene Region verbunden. Es wurde beschrieben, dass die Transkription der vhc-frc-Cluster durch die Anwesenheit von Selen im Wachstumsmedium inhibiert wird, obwohl die Anwesenheit von Spuren von Selen die Voraussetzung dafür ist, dass unter Laborbedingungen maximale Wachstumsraten erreicht werden. Ortsspezifische Mutagenese in der intergenen Region führte zur Identifizierung von Bindungsstellen für positiv und negativ regulatorischen Proteine geführt. In den hier beschriebenen Untersuchungen wurde das Gen für ß-Glucuronidase (uidA) benutzt, um das Transkriptionsniveau der frc- und vhc-Gengruppen in M. voltae-Stämmen (F3 und V1) in vivo indirekt zu verfolgen. Insertionsvektoren wurden konstruiert, um Zufalls-insertionen zu erzeugen. Mit diesem Ansatz wurden keine deregulierten Mutanten gefunden. Jedoch führte die Transformation mit einem Integrationsvektor, der die frc-vhc-intergene Region als Promotorregion für den Selektionsmarker trug, zur Derepression eines Hydrogenasepromotors unter gleichzeitiger Amplifikation des Vektors im Chromosom. Dies wurde als weiterer Anhaltspunkt dafür angesehen, dass es eine Bindungsstelle für den negativen Regulator in der intergenen Region gibt. Daraufhin wurde DNA-Affinitiätschromatogrphie eingesetzt, um zu versuchen, (das) negative Regulatorprotein(e) zu reinigen. An biotinmarkierter DNA, die die hypothetische Bindungsstelle für den negativen Regulator in der intergenen Region enthielt, wurde ein Protein teilweise gereinigt. Die N-terminale Sequenz des Proteins wurde bestimmt. BLAST-Analysen ergaben, dass es zur LysR-Familie prokaryotischer Regulationsproteine gehört. Nach der Erstellung der kompletten Nukelotidsequenz, wurde ein Knockout des Gens im M. voltae-Stamm V1 durchgeführt. Die Zerstörung des Gens im Stamm V1 führte zur Transkription des Reportergens und auch der Hydrogenasegene in Gegenwart von Selen. Dieser Regulator erhielt daher den Namen HrsM (selenabhängiger Repressor von Hydrogenasen in Methanococcus voltae). HrsM ist der erste beschriebene zur bakteriellen LysR-Familie gehörige Regulator in Archaea

Oxidative stress response in pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative environmental and human opportunistic pathogen highly adapted to many different environmental conditions. It can cause a wide range of serious infections, including wounds, lungs, the urinary tract, and systemic infections. The high versatility and pathogenicity of this bacterium is attributed to its genomic complexity, the expression of several virulence factors, and its intrinsic resistance to various antimicrobials. However, to thrive and establish infection, P. aeruginosa must overcome several barriers. One of these barriers is the presence of oxidizing agents (e.g., hydrogen peroxide, superoxide, and hypochlorous acid) produced by the host immune system or that are commonly used as disinfectants in a variety of different environments including hospitals. These agents damage several cellular molecules and can cause cell death. Therefore, bacteria adapt to these harsh conditions by altering gene expression and eliciting several stress responses to survive under oxidative stress. Here, we used PubMed to evaluate the current knowledge on the oxidative stress responses adopted by P. aeruginosa. We will describe the genes that are often differently expressed under oxidative stress conditions, the pathways and proteins employed to sense and respond to oxidative stress, and how these changes in gene expression influence pathogenicity and the virulence of P. aeruginosa. Understanding these responses and changes in gene expression is critical to controlling bacterial pathogenicity and developing new therapeutic agents

The role of DNA gyrase in illegitimate recombination

DNA, due to its double-helical structure, is subject to changes in topology due to the nature of transcription and replication. To overcome this, cells have processes and enzymes that ameliorate these changes. One such group of enzymes are the DNA topoisomerases, which are responsible for the maintenance of DNA topology. Despite this important role, these enzymes participate in illegitimate recombination (IR), which is genetic recombination between regions of DNA that share little or no homology. This can result in chromosomal rearrangements and is often a consequence of DNA-damaging agents. A consequence of topoisomerase-induced IR is thought to be therapy-related acute myeloid leukaemia (tAML). Analogously, there is evidence that exposure to sublethal concentrations of ciprofloxacin, a topoisomerase inhibitor, can cause resistance to non-quinolone antibiotics. This may work by a similar mechanism as that proposed for t-AML. This project centres around the examination of DNA gyrase-mediated IR focussing on the proposed subunit-exchange model. Using Blue-Native PAGE, I set up an assay to examine subunit exchange in topoisomerases. I have also characterised previously identified gyrase hyper-recombination mutations, known to increase the frequency of IR. Furthermore, I have investigated quinolone-induced antibiotic resistance and what the mechanism is. Here, I show that DNA gyrase can undergo subunit exchange, and that this seems to occur within higherorder oligomers of the enzyme, which have not been investigated before. Biochemical characterisation of the hyper-recombination mutations shows that they impair DNA gyrase activity which, in vivo, may have downstream consequences that may lead to IR. Using an in vivo assay where E. coli is treated with subinhibitory levels of quinolones, I have seen resistance to other non-quinolone antibiotics. This is not seen when other antibiotics, including other topoisomerase inhibitors, are tested. Whole genome sequencing has revealed point mutations that explain the resistances seen, however other larger chromosomal modifications have been observed as well

A genomic approach to understanding the molecular epidemiology and clinical burden of multi-drug resistant Enterobacterale Infections in Bangladesh

This PhD was the first comprehensive study in South-Asia, investigating epidemiology of AMR in a Bangladeshi health setting by aligning demographic, clinical, and genomic data. Carbapenem-resistant Enterobacterales (CRE) from clinical specimens were 11.1% (210/1893) and carbapenem-sensitive Enterobacterales (CSE) were 22.8% (433/1893). CRE was associated with age (6-25 years), gender, burn unit and ICU patients. Additionally, with patients given levofloxacin, amikacin, clindamycin, and meropenem during hospital stay (p<0.05). CRE cases were associated with allcause in-hospital 30-days mortality (27.8%) than CSE (13.5%) (p<0.05). Clinical CRE clustered in particular clonal types compared to CSE e.g. ST167, ST448, ST8346, ST405, and ST648 in E. coli, ST16; and ST231, ST11, ST515, and ST23 in K. pneumoniae (p<0.05). CRE clades were associated with direct clonal transmission in putative outbreak clusters (contained isolates of 0-2 SNPs differences), designated as KP5 (K. pneumoniae ST23), KP1 (K. pneumoniae ST15), EC9 (E. coli ST648), and Eco1 (E. cloacae ST113). Plasmid-mediated horizontal transfer of CRE was linked mostly linked to IncFII and IncX3. CRE faecal carriage was 34.8% (244/700) and significantly higher among inpatients (53.8%) than the outpatients (12%) (p<0.05). The clinical and colonisation studies were undertaken about a year apart; however, clusters were found across clinical and faecal isolates (≤20 SNP); these were, EC4 (E. coli ST8346), EC6 (E. coli ST405), EC7 (E. coli ST5954), KP1 (K. pneumoniae ST15), and Eco1 (E. cloacae ST113). Additionally, this PhD describes outbreaks at Dhaka Medical College Hospital e.g. an MDR Klebsiella variicola clone (ST771) in neonatal unit from October 2016 to January 2017, associated with high mortality (54.5%), and by Burkholderia cepacia ST1578 from burn sepsis cases. This study reported the first human-associated mobile colistin resistance in Bangladesh (mcr-1 in faecal colonisation and mcr-8 in clinical infections). Data derived from this study indicate an urgent need of antibiotic stewardship program and standard infection control policy in Bangladeshi hospitals