Manipulation of microbial gene essentiality in Pseudomonas aeruginosa as the new reaffirmation for novel therapeutic development.
Declaration
The work presented in this thesis is entirely my own, except where I have either acknowledged help from a known person or given a publication reference.
Abstract
Originally the genus Pseudomonas comprised of non-fermentative bacteria that were classified by their morphological similarity. The species was described as pseudomonas due to common cell pair arrangement resembling a singular cell (appendix Figure 1). However due to the controversy surrounding this genera, a vast amount of changes have been done in recent years. Regardless of the changes to the genera currently the most clinically significant isolate is Pseudomonas aeruginosa (PA).
PA contains broad environmental distribution and is efficient in adapting and regulating phenotype based on the site of infection. This adaptability has facilitated an emergence of multi-drug resistant strains creating a dire necessity for new treatments to combat the continuous rise of antimicrobial resistance. However, currently, there is a significant advancement gap with few novel antimicrobial frameworks introduced in the current clinical practice.
To address the necessity, this thesis is focused on exploiting essential genes such as biotin protein ligase (BPL) for gram-negative bacterium Pseudomonas aeruginosa. Manipulation of essential biotin genes could lead to new reaffirmation methods for therapeutic scaffold development and plausible clinical effectiveness. BPL universally regulates lipid biosynthesis and incorporated an essential metabolic enzyme – biotin. Exploiting mutant strains indicated evident growth inhibition and impaired virulence. Genomic manipulation on gene essentiality produced data that is indicative of no other potential pathway for biotinylation of biotin, reaffirming it as a very effective and lucrative target. Allowing the user to focus on biotin inhibitor development with an efficient vector. Opening new effective therapeutics via inhibition.
1. Introduction
1.1 Pseudomonas aeruginosa
Pseudomonas sensu stricto genus consists of many species that are grouped collectively by their metabolic heterogeneity and the ability to colonise a vast range of surfaces. Pseudomonas aeruginosa (PA) a ubiquitously dispersed opportunistic gram-negative, aerobic non-sporulating rod and motile pathogen. That has the capacity to populate soil and water reservoirs, colonising nearly any surface (biotic or abiotic)1. The genus strains are particularly troublesome in terms of medical implications. As in some cases, bacteria are able to utilise nitrate compounds for alternative means when are in anaerobic conditions as well as grow with sucrose exploiting both constituent monosaccharides, while other species exploit glucose moiety, accruing fructose in the arrangement of polysaccaharide2. In microbiological terms, PA is categorised as one of the major threats and classified as one of the markers for hygiene attributes for human consumption. Thus, PA has been subjected to vast amounts of research and publications exploiting its virulence and pathogenicity towards patients with reduced immunity as well as the components that can be developed from the nutritional and physiological resourcefulness. Don't use plagiarised sources.Get your custom essay just from $11/page
1.2 Colonisation, infection and disease
The effective colonisation attributes of PA are associated with the increased adaptability.
1.3 Biofilm formation
The innate ability of PA to form biofilms, “a functional consortium of microorganisms organized within an extensive exopolysaccharide matrix”6, is associated with profound resistance to antimicrobials. The cause of biofilm formation by Pseudomonas spp. is generated by the key concentration of N-acyl homoserine lactone (AHL). Due to quorum sensing and AHL production, sufficient numbers of the pathogen are present, enabling a coordinated gene expression, promoting the colonial survival of the pathogen. This intrinsic ability of biofilm posses’ tremendous negative impact on the host prospects of recovery. PA is able to habituate within any environment and populate as a biofilm, resulting in chronic infection. Most common incidences of chronic lung infections from PA are from patients with Cystic Fibrosis (CF), whom due to impaired lung function has a high frequency of developing a Pseudomonas lung infection at an early age. However, the isolates from such infections at the initial acute stage of the infection do not exhibit the same genomic virulence factors as the one from the chronic stage. Consistently chronic phase isolates display differences such as reduced inflammation and cytotoxicity and other down-regulated pathogenicity factors. The difference is the disappearance of the flagellum and pili, that are required for attachment, mobility and type III secretion system (T3SS) .
Chronic phase isolates of PA is able to form biofilms more readily and possess the ability to overexpress exopolysaccharide alginate. Exopolysaccharides with the extracellular DNA and polypeptides confer to the structural and conformational design of the biofilm. The innate biofilm formation is also dependant on the strain and/or nutritional conditions. As such, we hypothesised that the mutant strains in the presence of a standard antimicrobial front-line agent (macrolide class erythromycin and tetracycline) would inhibit and/or delay the formation a biofilm.
T3SS is one of the main virulence factors that equally traverses the internal and external membranes and is frequently associated with increased mortality in acute PA infections. The system is comprised of more than 20 protein units that constitute a complex resembling a syringe-like conformation, enabling administration of toxins to the surrounding host’s cell membranes. T3SS is shared among many gram-negative pathogenic bacteria and is evolutionarily related to flagella. Thus, the expression of T3SS in PA’s is a major component that is determinant of the pathogen’s virulence abilities7. The PA is contributing to epithelial cell and macrophage damage through four known bacterial effectors8: Exoenzyme (Exo) S and T – inhibits with cell division, cell migration/junction, and prompts apoptosis, Exo Y – is a host-dependent factor, which interrupts actin structural conformation via adenylate cyclase, Exo U – hold phospholipase that leads to brisk cell death. Majority of the PA strains can be classed into two sections based on the bacterial effector production (Exo S/T and Exo U/T). The genotypes are mutually correlated to acute/ chronic infection in man. However, the PA sp. contains a significantly lower variety of bacterial effectors compared to other well-categorized T3SS for example; Salmonella enterica exerts 13 effectors whereas Shigella spp. encode 25 effectors.
1.4 Toxins of PA
Toxins are microbial products that have the capability to directly cause tissue injuries or inflict destructive biological behaviours. In several instances, the toxin is entirety accountable for inflicting the characteristic disease symptoms. The negative toxin-like actions are caused by the multitude of degradative enzymes that are able to cause cell lysis or the individual receptor binding proteins that induct noxious reactions into the focal tissue.
PA toxigenesis consists of numerous noxious compounds, that appear to be produced based on the disease stage: adhesion, surface colonisation, bacterial dissemination and disease.
Adhesins are crucial for establishing infection9. To facilitate adherence PA exploits at least four surface factors: (1) lipopolysaccharide (LPS), (2) flagella (central motility element), (3) alginate, and (4) pili (delicate hair-like structures). The motility elements (flagella and pili) and the lipid A factor of LPS are accountable for the endotoxin function. Alginate, a mucoidal exopolysaccharide, enables capsule formation, protecting PA from hosts defence mechanisms or antibiotic mediated killing. Once the infection is established endotoxin production
ExoU is a phospholipase and is estimated to be 100 times more potent a cytotoxin than ExoS and capable of causing rapid death of host eukaryotic cells due to loss of plasma membrane integrity consistent with necrosis (Kipnis et al., 2006; Hauser, 2009). The exact contribution of each of the toxins to pathogenesis is unclear
PA is an excellent source of siderophore compounds such as the major fluorescent siderophore pyoverdine.
Exotoxin
Pyocyanin
Pyoverdine
1.5 Multidrug resistance
1.6 Biotin gene essentiality
Biotin is a crucial part of bacterial amino acid metabolism, gluconeogenesis and Fatty acid synthesis (FAS)10. Recent studies have proposed that the essential cofactor biotin may also takeover FAS biosynthetic enzymes, as the fatty acids are utilised in two central metabolic cofactors lipoate and biotin. Lipoate is a central cofactor in alfa-ketoacid dehydrogenases incorporating pyruvate dehydrogenase. Biotin is a crucial cofactor in Biotin Protein Ligase (BPL). BLP is a sole bifunctional pivotal enzyme that employs an ordered ligand-binding mechanism for the biotinylation and activation of biotin-dependent enzymes11. In many bacterial pathogens, BPL is encoded and regulated in BirA gene cluster12. BPL is crucial for the activation of biotin-dependant enzymes such as acetyl CoA carboxylase (ACC) and pyruvate carboxylase (PC) that catalyse central reactions. Moreover, BPL inhibition assay demonstrated that growth in the absence of the enzyme, the bacterial growth is abolished, making it a novel drug target, and mechanism of action (MOA).
BLP is a pivotal enzyme that can be found in all classes of biotic organisms. Which is further classified into two groups of BPL’s: mono-functional enzymes defined by the Pyrococcus horikoshii BPL (PhBPL), which exclusively catalyse post-translational biotinylation, and the bifunctional enzymes defined by the quintessential Escherichia coli BPL (EcBPL), which are able to act as transcriptional repressors as well.13
BPL catalyses protein biotinylation via 2 step process. During the initial step, condensation is carried out: biotin with ATP producing the biotinyl-5’-AMP intermediate and pyrophosphate (PPi). During the second step the intermediate forms protein-protein interaction with the unaligned apoenzyme to facilitate biotin transfer for the holoenzyme with the release of the AMP.
The pivotal cofactor is functional when it is covalently linked to the designated cognate protein. Structural confirmation of biotin consists of heterocyclic rings with a valeric acid chain. Biosynthetic pathway grouped into early and late phases; late phase biotin enzyme is needed for the assembly of two heterocyclic rings12, which are fairly structurally conserved and associated with the bifunctionality of BPL. Early phase in distinction to the late phase is quite varied; it is subjected to the biosynthesis of pimelate thioester, contributing to the valeric side chain and initial ring carbons. However, this pathway in biotin synthesis is still largely unknown. Only recently it has been demonstrated in E. coli, consisting of catalysts that are encoded in BioH and BioC gene clusters, which in part allow FAS pathway to intake pimelate.
1.6 Metformin applications for bacterial infections
Metformin is not a conventional antibiotic even though it does posse’s antimicrobial activity against gram-positive, gram-negative pathogens including M. Tuberculosis. Previous studies have shown that diabetic patients that have been receiving metformin therapy had a significantly decreased risk of tuberculosis14. Other in vitro investigations indicated that pathogen growth in co-cultures increases when basolateral glucose concentrations are elevated.14
Quorum Sensing (QS) controls the virulence factors of PA such as elastase, hemolysin, protease, pyocyanin excretion, pathogenic mobility, biofilm genesis and resistance to oxidative stress.15
Regardless of the advancing endeavours to control PA infections, the underlying virulence mechanisms are elusive. Many models, including mammalian, have not been able to replicate the vast pathogenicity factors relevant to human disease. In this study, the chosen model is Caenorhabditis elegans (CE) as the host. The selected nematode can provide a favourable host-infection high-throughput model. The rapid generation times and susceptibility for human pathogens are especially advantageous for the PA-CE model, as the majority of virulence-associated elements are preserved throughout differing taxa (biotic and abiotic). Besides CE model can mimic a relatively simplistic innate human immune system response due to shared characteristics.
Further expansion of investigations on BPL of gram-negative pathogens such as P. Aeruginosa could lead to novel treatment possibilities (stand-alone or combinatorial). As the current low infection ( gram-negative) recovery rates are dependant on the expedient response from the innate host immunity and pathogenic elements that mainly act to counteract the host ability for recovery. Combined with the seriousness of PA infections and the constrained antimicrobial resource for treatments, discovering other possible prevention and treatment approaches such a biotin and BPL inhibitors is an imperative urgency.4
2. Aims and objectives
Exploiting gene essentiality of biotin, this study has been developed to assess the probability of an effective biotin inhibitor treatment for PA infections. To reaffirm the prospects of biotin inhibitor as the novel antimicrobial treatment, the genomic sequence encoding biotin metabolism was identified and manipulated. Mutant strains produced to mimic plausible therapeutic effects for multi-combinatorial virulence mechanisms such as:
3. Materials and Methods
3.1.Reagents
Metformin hydrochloride, biotin, avidin and all other chemicals and reagents were purchased from Sigma Aldrich otherwise stated.
3.2. Bacterial strains
Pseudomonas aeruginosa wild type (WT) (PA14) strain, auxotroph mutants of P. aeruginosa (PA∆bioC, PA∆bioA, PA∆bioD, PA∆bioF) and Escherichia coli (OP50) from frozen glycerol stock (− 80 °C)
3.3 Media
3.3.1 Bacterial growth media
Luria-Bertani rich medium (LB) broth powder (Miller’s)
3.3.2 Nematode growth media
Nematode growth medium (NGM), Minimal salt media (M9W) and Caenorhabditis elegans nematodes were prepared following springer protocols, Pseudomonas methods and protocols16.
3.4 Methods
3.4.2 In vitro growth assay of PA mutants.
Biotin deficient strains PA∆BioA, ∆BioC, ∆BioD, ∆BioF and wild type PA14 were grown on LB agar medium overnight at 37°C from frozen stock. 5 ml of LB media inoculated with a single colony of the strain, grown in aeration in a tube rotator at 37°C for 12-16h. 10 μL of each saturated culture was seeded on LB agar (metformin – 0 mM, 25 mM, 50 mM, 100 mM). Plates were incubated at 37°C overnight.
Results obtained from the plates indicated successful growth inhibition of all mutant strains in a similar fashion. The second set of plates were prepared to observe if the mutant strain is able to grow with Biotin supplementation. The range for supplementation was from 0.5 ng/ml to 2.5 ng/ml of biotin.
3.4.3. Bacterial growth curve analysis.
PA growth curve of PA14 strain and PaBioA mutant cultured in LB broth and M9 media, in the presence and absence of metformin, biotin and avidin was analysed. An overnight culture of PA was inoculated separately in 100 ml of LB and M9 broth, incubated at 37°C, and the optical density (OD) was measured 1-hour intervals (up to 24h) at 600 nm
3.4.4 Microtiter Biofilm assay.
Wild type and mutant strain of PA (PA14 and BioA) cultured in LB and M9 broth overnight. Prepared cultures were diluted 1:10(3) and 1:10(6) into fresh medium. 100 μL aliquots distributed in a 96-well plate in triplicates. Microtiter plate incubated at 37°C for 18 h. Once incubation is complete, discard the contents and wash plate (three times) in water, 150 μL of 0.1% crystal violet solution was added to the wells, incubated at room temp. for 15 min. Stain rinsed out plate air-dried for 2 h.
Contents in the wells solubilised in 150 μL of 30% acetic acid. Contents transferred into plate reader and OD is taken at 550 nm.
3.4.5.C. elegans (N2) maintenance.
N2 Bristol nematodes were employed fro the host-pathogen screening methods. Egg prep and hatching completed at 20°C in M9 broth media overnight. Starved worms were transferred onto Nematode growth media (NGM) plates that previously have been seeded with OP50 E. coli. Nematodes were grown at 20°C for 24 h.
3.4.6 In vivo, C. elegans killing assay.
An overnight saturated bacterial broth culture (10 μl) was distributed onto a 6cm plate NGM (slow killing assay) and PGS (fast killing assay), incubated overnight at 37°C. Late NGM plates were left at room temperature (20- 25°C) for 8-24 hours, PGS- 8-12 hours. NGM plates were seeded with 40-50, PGS – seeded with 30-40 hermaphrodite L4/adult worms. Both plates incubated at 25°C, assessment for live worms carried out every 4-6 hours. For statistical significance 3 replicate experiments were conducted. E. coli OP50 was used as a negative control measure. A nematode was considered dead when it seized to respond to external stimuli. For accuracy, any nematodes that have died from being stuck on the wall of the plate were not incorporated in the data.
3.4.7.Liquid killing assay.
4. Results and discussion
Current computational methods for developing novel antimicrobials or other metabolic manipulations have led to vast biochemical libraries and bacterial strains. Relying on substitute biological intentions such as exploiting growth rate to reduce metabolic intrusions without incorporating flux dimensions that are requirements for wild type strains17. By encompassing the genomic integrity, that is a vital factor of any inhabiting organism, has a potential reaffirmation application for further development. There is substantial resource exploitation for predetermined proteins to certify the genome.
Biofilm formation and essential gene mutant.
Compounds that affect PvrR function could have an important role in the treatment
5. Conclusion and future research
6. Appendix
7. References
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641844/
Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death
Microtiter Dish Biofilm Formation Assay
doi: 10.3791/2437