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1. Cephalosporins Interfere With Quorum Sensing and Improve the Ability of Caenorhabditis elegans to Survive Pseudomonas aeruginosa Infection

   https://doi.org/10.3389/fmicb.2021.598498  

   Kumar, Lokender ; Brenner, Nathanael ; Brice, John ; Klein-Seetharaman, Judith ; Sarkar, Susanta K. ( January 2021 , Frontiers in Microbiology)

   null (Ed.)

   Pseudomonas aeruginosa utilizes the quorum sensing (QS) system to strategically coordinate virulence and biofilm formation. Targeting QS pathways may be a potential anti-infective approach to treat P. aeruginosa infections. In the present study, we define cephalosporins’ anti-QS activity using Chromobacterium violaceum CV026 for screening and QS-regulated mutants of P. aeruginosa for validation. We quantified the effects of three cephalosporins, cefepime, ceftazidime, and ceftriaxone, on (1) pyocyanin production using spectrophotometric assay, (2) bacterial motility using agar plate assay, and (3) biofilm formation using scanning electron microscopy. We also studied isogenic QS mutant strains of PAO1 (Δ lasR ,Δ rhlR ,Δ pqsA , and Δ pqsR) to compare and distinguish QS-mediated effects on the motility phenotypes and bacterial growth with and without sub-MIC concentrations of antibiotics. Results showed that cephalosporins have anti-QS activity and reduce bacterial motility, pyocyanin production, and biofilm formation for CV026 and PAO1. Also, sub-MICs of cefepime increased aminoglycosides’ antimicrobial activity against P. aeruginosa PAO1, suggesting the advantage of combined anti-QS and antibacterial treatment. To correlate experimentally observed anti-QS effects with the interactions between cephalosporins and QS receptors, we performed molecular docking with ligand binding sites of quorum sensing receptors using Autodock Vina. Molecular docking predicted cephalosporins’ binding affinities to the ligand-binding pocket of QS receptors (CviR, LasR, and PqsR). To validate our results using an infection model, we quantified the survival rate of C aenorhabditis elegans following P. aeruginosa PAO1 challenge at concentrations less than the minimum inhibitory concentration (MIC) of antibiotics. C. elegans infected with PAO1 without antibiotics showed 0% survivability after 72 h. In contrast, PAO1-infected C. elegans showed 65 ± 5%, 58 ± 4%, and 49 ± 8% survivability after treatment with cefepime, ceftazidime, and ceftriaxone, respectively. We determined the survival rates of C. elegans infected by QS mutant strains Δ lasR (32 ± 11%), Δ rhlR (27 ± 8%), Δ pqsA (27 ± 10%), and Δ pqsR (37 ± 6%), which suggest essential role of QS system in virulence. In summary, cephalosporins at sub-MIC concentrations show anti-QS activity and enhance the antibacterial efficacy of aminoglycosides, a different class of antibiotics. Thus, cephalosporins at sub-MIC concentrations in combination with other antibiotics are potential candidates for developing therapies to combat infections caused by P. aeruginosa. 

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2. The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa

   https://doi.org/10.1128/spectrum.02108-21  

   Simanek, Kayla A. ; Taylor, Isabelle R. ; Richael, Erica K. ; Lasek-Nesselquist, Erica ; Bassler, Bonnie L. ; Paczkowski, Jon E. ( February 2022 , Microbiology Spectrum)

   LaRock, Christopher N. (Ed.)

   ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals and individuals with underlying pulmonary disorders. P. aeruginosa virulence is controlled by quorum sensing (QS), a bacterial cell-cell communication mechanism that underpins transitions between individual and group behaviors. In P. aeruginosa , the PqsE enzyme and the QS receptor RhlR directly interact to control the expression of genes involved in virulence. Here, we show that three surface-exposed arginine residues on PqsE comprise the site required for interaction with RhlR. We show that a noninteracting PqsE variant [PqsE(NI)] possesses catalytic activity, but is incapable of promoting virulence phenotypes, indicating that interaction with RhlR, and not catalysis, drives these PqsE-dependent behaviors. Biochemical characterization of the PqsE-RhlR interaction coupled with RNA-seq analyses demonstrates that the PqsE-RhlR complex increases the affinity of RhlR for DNA, enabling enhanced expression of genes encoding key virulence factors. These findings provide the mechanism for PqsE-dependent regulation of RhlR and identify a unique regulatory feature of P. aeruginosa QS and its connection to virulence. IMPORTANCE Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI). QS is required for virulence in the human pathogen Pseudomonas aeruginosa , which can cause fatal infections in patients with underlying pulmonary disorders. In this study, we determine the molecular basis for the physical interaction between two virulence-driving QS components, PqsE and RhlR. We find that the ability of PqsE to bind RhlR correlates with virulence factor production. Since current antimicrobial therapies exacerbate the growing antibiotic resistance problem because they target bacterial growth, we suggest that the PqsE-RhlR interface discovered here represents a new candidate for targeting with small molecule inhibition. Therapeutics that disrupt the PqsE-RhlR interaction should suppress virulence. Targeting bacterial behaviors such as QS, rather than bacterial growth, represents an attractive alternative for exploration because such therapies could potentially minimize the development of resistance. 

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3. Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

   https://doi.org/10.1186/s12934-021-01703-2  

   Chun, Kayla ; Stephens, Kristina ; Wang, Sally ; Tsao, Chen-Yu ; Payne, Gregory F. ; Bentley, William E. ( November 2021 , Microbial Cell Factories)

   Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication.

   We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with thesoxRSoxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulonsoxRS,was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture.

   Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

    

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4. Phage Infection Restores PQS Signaling and Enhances Growth of a Pseudomonas aeruginosa lasI Quorum-Sensing Mutant

   https://doi.org/10.1128/jb.00557-21  

   Høyland-Kroghsbo, Nina Molin ; Bassler, Bonnie L. ( January 2022 , Journal of Bacteriology)

   Bondy-Denomy, Joseph (Ed.)

   ABSTRACT Chemical communication between bacteria and between bacteria and the bacteriophage (phage) viruses that prey on them can shape the outcomes of phage-bacterial encounters. Quorum sensing (QS) is a bacterial cell-to-cell communication process that promotes collective undertaking of group behaviors. QS relies on the production, release, accumulation, and detection of signal molecules called autoinducers. Phages can exploit QS-mediated communication to manipulate their hosts and maximize their own survival. In the opportunistic pathogen Pseudomonas aeruginosa , the LasI/R QS system induces the RhlI/R QS system, and in opposing manners, these two systems control the QS system that relies on the autoinducer called PQS. A P. aeruginosa Δ lasI mutant is impaired in PQS synthesis, leading to accumulation of the precursor molecule HHQ, and HHQ suppresses growth of the P. aeruginosa Δ lasI strain. We show that, in response to a phage infection, the P. aeruginosa Δ lasI mutant reactivates QS, which, in turn, restores pqsH expression, enabling conversion of HHQ into PQS. Moreover, downstream QS target genes encoding virulence factors are induced. Additionally, phage-infected P. aeruginosa Δ lasI cells transiently exhibit superior growth compared to uninfected cells. IMPORTANCE Clinical isolates of P. aeruginosa frequently harbor mutations in particular QS genes. Here, we show that infection by select temperate phages restores QS, a cell-to-cell communication mechanism in a P. aeruginosa QS mutant. Restoration of QS increases expression of genes encoding virulence factors. Thus, phage infection of select P. aeruginosa strains may increase bacterial pathogenicity, underscoring the importance of characterizing phage-host interactions in the context of bacterial mutants that are relevant in clinical settings. 

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5. The Antimicrobial Peptide Human Beta-Defensin 2 Inhibits Biofilm Production of Pseudomonas aeruginosa Without Compromising Metabolic Activity

   https://doi.org/doi.org/10.3389/fimmu.2020.00805  

   Parducho, Kevin R ; Beadell, Brent ; Ybarra, Tiffany ; Bush, Mabel ; Escalera, Erick ; Mendez, Marlon ; Anderson, Chance ; Trejos, Aldo T ; Chieng, Andy ; Park, Hyunsook ; et al ( May 2020 , Frontiers in immunology)

   Biofilm production is a key virulence factor that facilitates bacterial colonization on host surfaces and is regulated by complex pathways, including quorum sensing, that also control pigment production, among others. To limit colonization, epithelial cells, as part of the first line of defense, utilize a variety of antimicrobial peptides (AMPs) including defensins. Pore formation is the best investigated mechanism for the bactericidal activity of AMPs. Considering the induction of human beta-defensin 2 (HBD2) secretion to the epithelial surface in response to bacteria and the importance of biofilm in microbial infection, we hypothesized that HBD2 has biofilm inhibitory activity. We assessed the viability and biofilm formation of a pyorubin-producing Pseudomonas aeruginosa strain in the presence and absence of HBD2 in comparison to the highly bactericidal HBD3. At nanomolar concentrations, HBD2 – independent of its chiral state – significantly reduced biofilm formation but not metabolic activity, unlike HBD3, which reduced biofilm and metabolic activity to the same degree. A similar discrepancy between biofilm inhibition and maintenance of metabolic activity was also observed in HBD2 treated Acinetobacter baumannii, another Gram-negative bacterium. There was no evidence for HBD2 interference with the regulation of biofilm production. The expression of biofilm-related genes and the extracellular accumulation of pyorubin pigment, another quorum sensing controlled product, did not differ significantly between HBD2 treated and control bacteria, and in silico modeling did not support direct binding of HBD2 to quorum sensing molecules. However, alterations in the outer membrane protein profile accompanied by surface topology changes, documented by atomic force microscopy, was observed after HBD2 treatment. This suggests that HBD2 induces structural changes that interfere with the transport of biofilm precursors into the extracellular space. Taken together, these data support a novel mechanism of biofilm inhibition by nanomolar concentrations of HBD2 that is independent of biofilm regulatory pathways. 

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