paper to read for exam Flashcards
the key findings and conclusions from the 2019 study by Lloyd et al. on Pseudomonas aeruginosa and the RpoN* molecular roadblock include:
Virulence Phenotypes in CF Patient Isolates:
Clinical isolates of P. aeruginosa from cystic fibrosis (CF) patients showed diverse virulence phenotypes, including variability in motility, biofilm formation, and pathogenicity, with SCH0057-7 being notably pathogenic.
RpoN Protein Levels Variability:
Levels of the sigma factor RpoN, which regulates virulence and antibiotic resistance, varied among clinical isolates. While some strains showed high levels, others had low or undetectable levels.
Impact of RpoN on Virulence*:
Expression of RpoN*, a cis-acting peptide designed to block RpoN’s transcriptional activity, reduced virulence-associated traits, like motility and biofilm formation, in specific isolates.
Increased Antibiotic Susceptibility:
In laboratory strain PA19660 Xen5, RpoN* expression increased sensitivity to multiple antibiotics, particularly beta-lactams like cefepime and piperacillin. This demonstrates RpoN*’s potential for combating antibiotic resistance.
Enhanced Survival in Infection Model:
In a P. aeruginosa-C. elegans infection model, RpoN* expression in the highly pathogenic SCH0057-7 strain significantly increased worm survival, suggesting reduced virulence.
Clinical Potential:
The findings indicate that RpoN* has therapeutic potential as an alternative to traditional antibiotics by reducing virulence and enhancing antibiotic susceptibility without directly targeting bacterial survival pathways, which could limit the development of resistance.
Introduction
. Pseudomonas aeruginosa is an opportunistic pathogen
in burn patients and individuals with cystic fbrosis (CF), and a leading cause of nosocomial infections- Nosocomial infections are infections that develop while a person is receiving medical attention
- P. aeruginosa is inherently resistant to many antibiotics and can develop resistance to others, limiting
treatment options. P. aeruginosa has multiple sigma factors to regulate transcription. The alternative
sigma factor, RpoN (σ54), regulates many virulence genes and is linked to antibiotic resistance.
Recently, we described a cis-acting peptide, RpoN, which is a “molecular roadblock”, binding
consensus promoters at the -24 site, blocking transcription. RpoN reduces virulence of P. aeruginosa
laboratory strains, but its efects in clinical isolates was unknown. We investigated the efects of
RpoN* on phenotypically varied P. aeruginosa strains isolated from CF patients. RpoN* expression
reduced motility, bioflm formation, and pathogenesis in a P. aeruginosa-C. elegans infection model.
Furthermore, we investigated RpoN* efects on antibiotic susceptibility in a laboratory strain. RpoN*
expression increased susceptibility to several beta-lactam-based antibiotics in strain P. aeruginosa
PA19660 Xen5. We show that using a cis-acting peptide to block RpoN consensus promoters has
potential clinical implications in reducing virulence and improving antibiotic susceptibility
- A cis-acting peptide is a peptide that negatively regulates translation by interfering with the functions of a ribosome
A cis-acting peptide is a molecule that influences gene expression by interacting with DNA or other elements in close proximity on the same molecule of DNA. This type of peptide does not diffuse through the cell to affect distant genes but instead acts locally, usually by binding directly to specific DNA promoter sequences or regulatory sites to control the expression of nearby genes.
In the context of the Lloyd et al. study, RpoN* is a cis-acting peptide designed to bind to RpoN (σ54) promoter sites at the conserved -24 position in the genome of Pseudomonas aeruginosa. This blocks RpoN from initiating transcription of virulence and resistance genes, effectively reducing their expression. Thus, the peptide acts directly at the target site (i.e., at the location of the RpoN-controlled promoter on the DNA) to modulate gene expression without impacting other distant regulatory sequences or genes
Context and Problem: The paper addresses the challenge of treating Pseudomonas aeruginosa infections, which are increasingly multidrug-resistant (MDR) and problematic in healthcare, especially among burn and cystic fibrosis (CF) patients. The bacteria’s resistance to many antibiotics and its capacity to gain additional resistance complicate treatment.
RpoN (σ54) Role: RpoN is a key sigma factor in P. aeruginosa, regulating virulence factors like motility, biofilm formation, and antibiotic resistance. Due to its central role, targeting RpoN could theoretically reduce bacterial virulence and resistance without affecting bacterial survival, potentially slowing resistance development.
RpoN (Molecular Roadblock)**: RpoN is a synthetic peptide designed to block RpoN activity by binding to its consensus promoter sites, reducing transcription of virulence genes. While RpoN* had shown effectiveness in lab strains, its impact on clinical isolates from CF patients was unknown before this study.
Pseudomonas aeruginosa, a Gram-negative, opportunistic pathogen
is a leading cause of nosocomial infections and is associated with infections in burn patients
. P. aeruginosa is
also responsible for colonizing the respiratory tract and causing chronic infections in individuals with cystic
fbrosis (CF)
. It is the most common pathogen isolated from individuals with CF and is a major source of morbidity and mortality
In CF patients, P. aeruginosa undergoes a transformation from a non-mucoid form upon initial colonization
of the lungs to a mucoid form as the disease progresses. Tis results in a chronic debilitating pulmonary infection
characterized by the overexpression of alginate. Mucoid strains synthesize large quantities of alginate, enhancing bioflm formation and protecting P. aeruginosa from antibiotics or the immune possibly through
formation of microcolonies
-, multidrug resistant strains are still prevalent and occurred in 19.4% of CF infections in
2015
P. aeruginosa is inherently resistant to a number of antibiotics. It can also acquire resistance through exogenous resistance genes via horizontal gene transfer or mutations, limiting treatment options.
current Promising strategies include enhancing
activity of currently available antibiotics and decreasing virulence of the bacteria once an infection occurs.
P. aeruginosa virulence is caused by many factors, including toxins, proteases, phospholipases, the presence
of pili and fagella, and bioflm formation. Virulence is regulated by a network of transcription factors, such as
sigma factors RpoS and RpoN, and quorum sensing regulators. Te alternative sigma factor, σ54 or RpoN, regulates nitrogen assimilation, quorum sensing, motility, bioflm formation and other virulence factors. RpoN
regulation was recently linked to P. aeruginosa tolerance to several antibiotics. RpoN binds to specifc promoters with conserved −24, −12 sequences upstream of RpoN-regulated genes throughout the genome and is a
key virulence regulator. Te specifc and conserved nature through which RpoN controls its regulon led us to develop the RpoN molecular roadblock
- the specifc and conserved nature through which RpoN controls its regulon led us to
develop the RpoN molecular roadblock, RpoN* - RpoN* is a cis-acting peptide that specifcally binds the −24 site
of RpoN consensus promoters, blocking transcription by RpoN and other factors - When RpoN* is expressed in P. aeruginosa laboratory strains, transcription is afected
globally and virulence is attenuated - We showed that more than 700 genes are afected, either directly regulated
by RpoN or indirectly by other transcription factors under RpoN control
-Furthermore, in P. aeruginosa, some
genes may have promoter binding sites for multiple sigma factors31. Tus, loss of RpoN does not always equate to
loss of transcription and gene expression. We showed that RpoN* afects virulence in a RpoN-deletion strain of P.
aeruginosa PAO1. Tis demonstrates the roadblock’s ability to attenuate gene expression by blocking transcription
of genes under dual-regulation with RpoN and other sigma factors. Tis strategy of blocking multiple promoters
throughout the P. aeruginosa genome may be an efective method to combat virulence and evade development of resistance.
-We also describe RpoN* efects on antibiotic
resistance in a laboratory strain. Expression of RpoN* reduced virulence-associated phenotypes in clinical isolates and improved P. aeruginosa susceptibility to multiple antibiotics. Tis study demonstrates that RpoN* has
potential clinical applications and represents an efecti
Te specifc and conserved nature through which RpoN controls its regulon led us to
develop the RpoN molecular roadblock, RpoN. RpoN is a cis-acting peptide that specifcally binds the −24 site
of RpoN consensus promoters, blocking transcription by RpoN and other factors
Methods
Clinical Isolate Characterization: Patient isolates of P. aeruginosa from CF patients were characterized for motility, biofilm formation, and virulence. RpoN protein levels were measured using Western blot to understand the baseline expression across strains.
Transformation with RpoN: Select CF isolates were transformed with RpoN to test its effects on virulence-related traits (motility and biofilm formation).
Infection Model: A P. aeruginosa-C. elegans infection model was used to assess the pathogenicity of the isolates, with and without RpoN* expression, via survival assays.
Antibiotic Susceptibility Testing: Using MicroScan panels, the effects of RpoN* on antibiotic susceptibility in a laboratory strain of P. aeruginosa were evaluated.
The methods and materials used in the Lloyd et al. study were carefully chosen to address key questions about RpoN*’s potential to reduce virulence and improve antibiotic sensitivity in Pseudomonas aeruginosa, especially in strains isolated from cystic fibrosis (CF) patients. Here’s why each major method and material was essential to the study:
- Clinical Isolates of Pseudomonas aeruginosa from CF Patients
Reason: Clinical isolates from CF patients exhibit a range of phenotypes that are distinct from lab strains, particularly in motility, biofilm formation, and resistance patterns. Using these isolates enabled the researchers to test RpoN’s effects in a realistic, heterogeneous population that mirrors clinical challenges.
Objective: To evaluate the impact of RpoN on virulence-related traits and antibiotic sensitivity in diverse, clinically relevant strains rather than standardized lab strains alone. - Plasmid-Based Delivery of RpoN*
Reason: The plasmid-based approach provided a controlled way to express RpoN* in transformed P. aeruginosa strains, allowing the researchers to selectively induce RpoN* expression in specific isolates.
Objective: To verify whether RpoN* could reduce virulence and resistance traits when expressed in P. aeruginosa. The plasmid (pBBR1MCS-5) with the inducible promoter enabled researchers to manage RpoN* expression experimentally and assess its effects. - Phenotyping Assays for Motility and Biofilm Formation
Reason: Motility and biofilm formation are critical virulence factors for P. aeruginosa, particularly in chronic lung infections in CF patients. Motility enables colonization and spread, while biofilm formation protects bacteria from antibiotics and immune responses.
Objective: To determine if RpoN* expression could effectively reduce these virulence traits, potentially making P. aeruginosa less pathogenic and easier to treat with antibiotics. - Western Blot Analysis for RpoN Protein Levels
Reason: Variability in RpoN levels among clinical isolates could influence RpoN’s effectiveness, as some isolates may naturally express lower RpoN levels due to mutations. Western blots allowed the researchers to quantify RpoN protein levels in each isolate, providing context for interpreting RpoN’s impact.
Objective: To assess the relationship between baseline RpoN levels and the efficacy of RpoN* in reducing virulence. This information helped determine if RpoN* would be effective across strains with varying RpoN expression. - P. aeruginosa – C. elegans Infection Model
Reason: C. elegans infection models allow for in vivo testing of pathogenicity, providing insights into how RpoN* affects virulence in a living organism. This model is frequently used to study bacterial virulence and host-pathogen interactions.
Objective: To test if RpoN* expression could reduce pathogenicity in a biologically relevant infection model, supporting the hypothesis that RpoN* can reduce virulence in an in vivo context. - Antibiotic Susceptibility Testing Using MicroScan Panels
Reason: MicroScan panels are a standardized clinical tool for determining antibiotic minimum inhibitory concentrations (MICs) and susceptibility profiles, making results comparable to clinical standards.
Objective: To test whether RpoN* could increase sensitivity to certain antibiotics, especially beta-lactams, as this would demonstrate RpoN’s potential to counteract resistance mechanisms. This testing focused on measuring how RpoN expression altered antibiotic MICs in P. aeruginosa, providing a clinically relevant perspective on RpoN*’s utility. - Gentamicin and IPTG Induction for Plasmid Maintenance and RpoN Expression*
Reason: Gentamicin was used to select for transformed cells carrying the plasmid, while IPTG (isopropyl β-D-1-thiogalactopyranoside) induced the expression of RpoN.
Objective: To maintain plasmid stability and control RpoN expression in assays, ensuring consistent and reliable results across experiments.
Each method and material contributed to testing the central hypothesis: that RpoN* could serve as a “molecular roadblock” to reduce virulence and improve antibiotic susceptibility in P. aeruginosa. Together, they allowed the researchers to assess RpoN*’s effects in both lab-based and clinically relevant conditions, supporting a comprehensive evaluation of its therapeutic potential.
Results
Virulence phenotypes were variable in P. aeruginosa isolates from CF patients. P. aeruginosa
isolated from diferent CF patients or within the same CF patient have varied phenotypes and genotypes36,37. P.
aeruginosa adapts over time, leading to mutations and changes in expression of genes related to motility, quorum
sensing, and overall virulence38,40. To determine the virulence-related phenotypic profles of the strains used in
this study (Table 1), each P. aeruginosa patient isolate was evaluated for motility and bioflm formation, compared
to the virulent positive control strain P. aeruginosa PA19660 Xen5.
we evaluated relative protein levels of RpoN in these patient isolates by western blot. RpoN levels were moderately high in the
positive control P. aeruginosa PAO1-S, while low or minimal protein levels were detected in the isogenic ΔrpoN
mutant negative control (Fig. 3). Te faint bands in the ∆rpoN strain and several CF patient isolates are background signals due to non-specifc antibody binding to another protein or sigma factor with a similar apparent
molecular weight
Diverse Virulence Profiles: CF isolates showed variability in motility, biofilm formation, and virulence. Strains SCH0057-7, SCH0256-1, and UUH0201 displayed high motility, while SCH0254-118 exhibited strong biofilm formation, suggesting adaptations for persistence in CF lung environments.
Variable RpoN Protein Levels: RpoN levels were inconsistent across isolates. High levels in some isolates correlated with greater motility and biofilm production, but even low-RpoN isolates displayed virulence traits, indicating redundancy or alternate pathways in virulence regulation
RpoN Reduces Virulence Traits:
Motility and Biofilm Reduction: RpoN significantly reduced motility and biofilm formation in the CF isolates that could be transformed. This shows RpoN*’s ability to reduce traits that are vital for colonization and persistence.
C. elegans Model: In the paralytic killing assay, RpoN* increased C. elegans survival, especially in the SCH0057-7 isolate. This confirms that RpoN* attenuates pathogenicity in a host-mimicking environment.
Enhanced Antibiotic Sensitivity: In a lab strain, RpoN* increased sensitivity to beta-lactam antibiotics, including imipenem, cefepime, and piperacillin. Importantly, RpoN* lowered the minimum inhibitory concentration (MIC) for these antibiotics, which suggests clinical relevance, as it could shift some resistant strains to a susceptible category.
RpoN* increased susceptibility to select antibiotics in vitro-The antibiotics with improved susceptibility were cefotaxime, cefepime, and cefazidime (three cephalosporins),
piperacillin (a ureidopenicillin), and imipenem (a carbapenem). Susceptibility to some antibiotics was unchanged
(data not shown). For piperacillin, there was at least a 2-fold increase in susceptibility. Tis improvement is clinically relevant as it changed the status from resistant to sensitive (Table 2). For the other drugs, which there was
no change in clinical susceptibility status, presence of RpoN* increased the therapeutic potency of the drugs. Te
results demonstrate that RpoN* expression increased P. aeruginosa susceptibility to several antibiotics
Discussion
Here, we confrm and expand results of previous studies by showing the ability of RpoN* to abrogate virulence
phenotypes in P. aeruginosa isolates from CF patients and to improve susceptibility to several antibiotics. Our
working model of the mechanism of action of RpoN* is that it binds the -24 promoter consensus sites, blocking
transactivation by RpoN and other sigma factors. By altering the transcriptome, RpoN* reduced virulence in
well-characterized laboratory strains32. Tus, the motivation for this study was to understand the clinical relevance of RpoN. We demonstrated that RpoN expressed in CF patient isolates reduced motility and bioflm
formation in vitro, independently of RpoN protein levels. Te RpoN* molecular roadblock protected C. elegans
from a highly virulent P. aeruginosa
Mechanism of RpoN Effectiveness: The authors suggest that RpoN functions as a “molecular roadblock” by binding to RpoN’s conserved -24, -12 consensus promoters, preventing transcription of virulence and resistance genes.
Potential for Multi-Pathogen Application: RpoN*’s mechanism could theoretically apply to other Gram-negative pathogens since the RpoN binding sites are conserved.
Impact on Antibiotic Resistance: RpoN* could complement antibiotics by making bacteria more susceptible, without applying direct pressure that selects for resistance. This is advantageous, as it could prolong the effectiveness of existing antibiotics.
Limitations: Only a subset of patient isolates could be transformed, limiting in vivo validation across diverse isolates. The need for plasmid transformation limits the current clinical feasibility of RpoN*, but it points toward future potential, such as developing small molecules mimicking RpoN
Conclusion
Conclusion
The study provides a promising look at RpoN* as a therapeutic agent that targets virulence rather than viability, a shift from traditional antibiotics. The findings suggest that RpoN* could be an innovative component in managing MDR infections, particularly those affecting CF patients. This work encourages the development of drugs mimicking RpoN*’s mechanism, potentially as adjuncts to enhance existing antibiotic therapies.
limitations of the study
limitations of the study
the study by Lloyd et al. has several limitations, which are important to consider for assessing the clinical applicability and scalability of RpoN* as a therapeutic tool. Key limitations include:
Transformation Challenges with Clinical Isolates:
Only a subset of the P. aeruginosa clinical isolates could be successfully transformed with the RpoN* plasmid, which restricted the study’s ability to generalize findings across diverse CF isolates. This transformation challenge limits the validation of RpoN* effects across the full spectrum of virulence phenotypes found in CF patients.
Plasmid-Based Delivery:
RpoN* was delivered via a plasmid, a method not feasible for direct therapeutic application in clinical settings. The use of plasmids means that the delivery system is unsuitable for routine use in human infections, pointing to the need for alternative delivery systems, such as small molecules or peptide mimics of RpoN*.
Inability to Maintain RpoN Expression in Some Models*:
The study encountered difficulties in maintaining RpoN* expression in long-term infection assays, like the slow-killing assay in C. elegans. The plasmid and its expression required specific conditions, such as gentamicin selection and IPTG induction, which were not durable in the model. This suggests challenges in ensuring sustained RpoN* activity in vivo.
Limited Testing on Antibiotic-Resistant Clinical Strains:
The study focused on a laboratory strain for antibiotic susceptibility testing and did not include a range of antibiotic-resistant clinical isolates. This leaves an open question about RpoN*’s effectiveness in actual multidrug-resistant (MDR) P. aeruginosa strains from patients, particularly those resistant to antibiotics like quinolones, carbapenems, and tobramycin.
Scope of Antibiotic Susceptibility Testing:
Antibiotic testing was limited to a specific set of drugs on the MicroScan panel, and the study did not test whether RpoN* affects susceptibility to other antibiotics, such as tobramycin and quinolones, which are often critical in CF infections. Expanding testing to a wider array of antibiotics would provide a more comprehensive view of RpoN*’s impact on resistance.
Potential Redundancy Among Sigma Factors:
P. aeruginosa has multiple sigma factors that can regulate overlapping sets of genes. The redundancy among these factors may mean that blocking RpoN with RpoN* could have variable effects across different isolates, depending on other compensatory regulatory mechanisms. This could limit RpoN*’s effectiveness if other sigma factors offset the loss of RpoN activity.
Laboratory vs. Clinical Relevance:
The findings are promising in controlled laboratory settings, but the clinical relevance remains untested. For example, it is unclear whether RpoN* or similar peptides can penetrate infected tissues or biofilms in the human body or maintain sufficient activity in the complex environment of a CF patient’s lungs.
These limitations indicate the need for further studies to refine RpoN* delivery mechanisms, expand testing on MDR clinical isolates, and explore alternatives to ensure consistent expression and activity in realistic infection models.
