A Shift in Central Metabolism Accompanies Virulence Activation in Pseudomonas aeruginosa.

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach

Surface Attachment Induces Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa infects every type of host that has been examined by deploying multiple virulence factors. Previous studies of virulence regulation have largely focused on chemical cues, but P. aeruginosa may also respond to mechanical cues. Using a rapid imaging-based virulence assay, we demonstrate that P. aeruginosa activates virulence in response to attachment to a range of chemically distinct surfaces, suggesting that this bacterial species responds to mechanical properties of its substrates. Surface-activated virulence requires quorum sensing, but activating quorum sensing does not induce virulence without surface attachment. The activation of virulence by surfaces also requires the surface-exposed protein PilY1, which has a domain homologous to a eukaryotic mechanosensor. Specific mutation of the putative PilY1 mechanosensory domain is sufficient to induce virulence in non-surface-attached cells, suggesting that PilY1 mediates surface mechanotransduction. Triggering virulence only when cells are both at high density and attached to a surface—two host-nonspecific cues—explains how P. aeruginosa precisely regulates virulence while maintaining broad host specificity

Roadmap on emerging concepts in the physical biology of bacterial biofilms: from surface sensing to community formation

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor

Cross-talk between bacterial two-component circuits

Bacteria sense and respond to a variety of environmental stimuli through two-component systems, which consist of an extensive network of histidine kinases and response regulators. Many bacteria possess a large number of two-component systems. Given the high level of sequence and structural similarity between different systems, the existence of cross-talk or interference between systems is possible. This work explores cross-talk between the CpxA-CpxR and EnvZ-OmpR two-component systems in E. coli and describes mechanisms that provide insulation against such interference. In particular, two mechanisms appear to suppress cross-talk between these systems, which depend on their cognate partners and on the bifunctional nature of the histidine kinases. This also gives rise to mutational robustness, i.e. it masks the effects of mutations that would otherwise lead to increased cross-talk. While the molecular specificity associated with interacting domains of histidine kinases and response regulators is likely to be the greatest contributor to the high level of signaling specificity, the mechanisms described here likely enhance this effect. In addition, this work explores the molecular determinants of specificity using results from a co-evolution analysis of histidine kinase and response regulator residues and also through an extensive saturation mutagenesis screen. Substitutions at key residues along the lower portion of the DHp domain alter the specificity of EnvZ and substantially increase cross-talk between non-cognate pairs. In contrast, random mutation at other sites, most notably those in close proximity to the conserved histidine involved in auto-phosphorylation, impact negatively on cross-talk. While this work is chiefly focused on characterizing cross-talk from kinase activity, it also lays the foundation for future work exploring the possibility of phosphatase crosstalk

Cross-talk between bacterial two-component circuits

Bacteria sense and respond to a variety of environmental stimuli through two-component systems, which consist of an extensive network of histidine kinases and response regulators. Many bacteria possess a large number of two-component systems. Given the high level of sequence and structural similarity between different systems, the existence of cross-talk or interference between systems is possible. This work explores cross-talk between the CpxA-CpxR and EnvZ-OmpR two-component systems in E. coli and describes mechanisms that provide insulation against such interference. In particular, two mechanisms appear to suppress cross-talk between these systems, which depend on their cognate partners and on the bifunctional nature of the histidine kinases. This also gives rise to mutational robustness, i.e. it masks the effects of mutations that would otherwise lead to increased cross-talk. While the molecular specificity associated with interacting domains of histidine kinases and response regulators is likely to be the greatest contributor to the high level of signaling specificity, the mechanisms described here likely enhance this effect. In addition, this work explores the molecular determinants of specificity using results from a co-evolution analysis of histidine kinase and response regulator residues and also through an extensive saturation mutagenesis screen. Substitutions at key residues along the lower portion of the DHp domain alter the specificity of EnvZ and substantially increase cross-talk between non-cognate pairs. In contrast, random mutation at other sites, most notably those in close proximity to the conserved histidine involved in auto-phosphorylation, impact negatively on cross-talk. While this work is chiefly focused on characterizing cross-talk from kinase activity, it also lays the foundation for future work exploring the possibility of phosphatase crosstalk

A Rapid Image-based Bacterial Virulence Assay Using Amoeba.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa