Real-Time Electrochemical Detection of Pseudomonas aeruginosa Phenazine Metabolites Using Transparent Carbon Ultramicroelectrode Arrays.

Here, we use a recently developed electrochemical sensing platform of transparent carbon ultramicroelectrode arrays (T-CUAs) for the in vitro detection of phenazine metabolites from the opportunistic human pathogen Pseudomonas aeruginosa. Specifically, redox-active metabolites pyocyanin (PYO), 5-methylphenazine-1-carboxylic acid (5-MCA), and 1-hydroxyphenazine (OHPHZ) are produced by P. aeruginosa, which is commonly found in chronic wound infections and in the lungs of cystic fibrosis patients. As highly diffusible chemicals, PYO and other metabolites are extremely toxic to surrounding host cells and other competing microorganisms, thus their detection is of great importance as it could provide insights regarding P. aeruginosa virulence mechanisms. Phenazine metabolites are known to play important roles in cellular functions; however, very little is known about how their concentrations fluctuate and influence cellular behaviors over the course of infection and growth. Herein we report the use of easily assembled, low-cost electrochemical sensors that provide rapid response times, enhanced sensitivity, and high reproducibility. As such, these T-CUAs enable real-time electrochemical monitoring of PYO and another extremely reactive and distinct redox-active phenazine metabolite, 5-methylphenazine-1-carboxylic acid (5-MCA), from a highly virulent laboratory P. aeruginosa strain, PA14. In addition to quantifying phenazine metabolite concentrations, changes in phenazine dynamics are observed in the biosynthetic route for the production of PYO. Our quantitative results, over a 48-h period, show increasing PYO concentrations during the first 21 h of bacterial growth, after which PYO levels plateau and then slightly decrease. Additionally, we explore environmental effects on phenazine dynamics and PYO concentrations in two growth media, tryptic soy broth (TSB) and lysogeny broth (LB). The maximum concentrations of cellular PYO were determined to be 190 ± 5 µM and 150 ± 1 µM in TSB and LB, respectively. Finally, using desorption electrospray ionization (DESI) and nanoelectrospray ionization (nano-ESI) mass spectrometry we confirm the detection and identification of reactive phenazine metabolites.

Towards mapping electrostatic interactions between Kdo2-lipid A and cationic antimicrobial peptides via ultraviolet photodissociation mass spectrometry.

Cationic antimicrobial peptides (CAMPs) have been known to act as multi-modal weapons against Gram-negative bacteria. As a new approach to investigate the nature of the interactions between CAMPs and the surfaces of bacteria, native mass spectrometry and two MS/MS strategies (ultraviolet photodissociation (UVPD) and higher energy collisional activation (HCD)) are used to examine formation and disassembly of saccharolipid·peptide complexes. Kdo2-lipid A (KLA) is used as a model saccharolipid to evaluate complexation with a series of cationic peptides (melittin and three analogs). Collisional activation of the KLA·peptide complexes results in the disruption of electrostatic interactions, resulting in apo-sequence ions with shifts in the distribution of ions compared to the fragmentation patterns of the apo-peptides. UVPD of the KLA·peptide complexes results in both apo- and holo-sequence ions of the peptides, the latter in which the KLA remains bound to the truncated peptide fragment despite cleavage of a covalent bond of the peptide backbone. Mapping both the N- and C-terminal holo-product ions gives insight into the peptide motifs (specifically an electropositive KRKR segment and a proline residue) that are responsible for mediating the electrostatic interactions between the cationic peptides and saccharolipid.

Spatial determinants of quorum signaling in a Pseudomonas aeruginosa infection model.

Quorum sensing (QS) is a bacterial communication system that involves production and sensing of extracellular signals. In laboratory models, QS allows bacteria to monitor and respond to their own cell density and is critical for fitness. However, how QS proceeds in natural, spatially structured bacterial communities is not well understood, which significantly hampers our understanding of the emergent properties of natural communities. To address this gap, we assessed QS signaling in the opportunistic pathogen Pseudomonas aeruginosa in a cystic fibrosis (CF) lung infection model that recapitulates the biogeographical aspects of the natural human infection. In this model, P. aeruginosa grows as spatially organized, highly dense aggregates similar to those observed in the human CF lung. By combining this natural aggregate system with a micro-3D-printing platform that allows for confinement and precise spatial positioning of P. aeruginosa aggregates, we assessed the impact of aggregate size and spatial positioning on both intra- and interaggregate signaling. We discovered that aggregates containing ∼2,000 signal-producing P. aeruginosa were unable to signal neighboring aggregates, while those containing ≥5,000 cells signaled aggregates as far away as 176 µm. Not all aggregates within this "calling distance" responded, indicating that aggregates have differential sensitivities to signal. Overexpression of the signal receptor increased aggregate sensitivity to signal, suggesting that the ability of aggregates to respond is defined in part by receptor levels. These studies provide quantitative benchmark data for the impact of spatial arrangement and phenotypic heterogeneity on P. aeruginosa signaling in vivo.

Oxygen limitation within a bacterial aggregate.

ABSTRACT Cells within biofilms exhibit physiological heterogeneity, in part because of chemical gradients existing within these spatially structured communities. Previous work has examined how chemical gradients develop in large biofilms containing >10(8) cells. However, many bacterial communities in nature are composed of small, densely packed aggregates of cells (≤ 10(5) bacteria). Using a gelatin-based three-dimensional (3D) printing strategy, we confined the bacterium Pseudomonas aeruginosa within picoliter-sized 3D "microtraps" that are permeable to nutrients, waste products, and other bioactive small molecules. We show that as a single bacterium grows into a maximally dense (10(12) cells ml(-1)) clonal population, a localized depletion of oxygen develops when it reaches a critical aggregate size of ~55 pl. Collectively, these data demonstrate that chemical and phenotypic heterogeneity exists on the micrometer scale within small aggregate populations. IMPORTANCE Before developing into large, complex communities, microbes initially cluster into aggregates, and it is unclear if chemical heterogeneity exists in these ubiquitous micrometer-scale aggregates. We chose to examine oxygen availability within an aggregate since oxygen concentration impacts a number of important bacterial processes, including metabolism, social behaviors, virulence, and antibiotic resistance. By determining that oxygen availability can vary within aggregates containing ≤ 10(5) bacteria, we establish that physiological heterogeneity exists within P. aeruginosa aggregates, suggesting that such heterogeneity frequently exists in many naturally occurring small populations.