Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were ...abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.
Graphical abstract Highlights ► Communities of microbes exhibit increased production rates and metabolic efficiency. ► Synthetic microbial consortia can perform complex functions by distributing ...tasks. ► Cell–cell communication and synthetic biology can program ecosystem behavior. ► Metabolic models can predict community behavior and synergy between organisms.
Pattern formation is a hallmark of coordinated cell behaviour in both single and multicellular organisms. It typically involves cell-cell communication and intracellular signal processing. Here we ...show a synthetic multicellular system in which genetically engineered 'receiver' cells are programmed to form ring-like patterns of differentiation based on chemical gradients of an acyl-homoserine lactone (AHL) signal that is synthesized by 'sender' cells. In receiver cells, 'band-detect' gene networks respond to user-defined ranges of AHL concentrations. By fusing different fluorescent proteins as outputs of network variants, an initially undifferentiated 'lawn' of receivers is engineered to form a bullseye pattern around a sender colony. Other patterns, such as ellipses and clovers, are achieved by placing senders in different configurations. Experimental and theoretical analyses reveal which kinetic parameters most significantly affect ring development over time. Construction and study of such synthetic multicellular systems can improve our quantitative understanding of naturally occurring developmental processes and may foster applications in tissue engineering, biomaterial fabrication and biosensing.
Microbial fatty acid-derived fuels have emerged as promising alternatives to petroleum-based transportation fuels. Here we report a modular engineering approach that systematically removed metabolic ...pathway bottlenecks and led to significant titre improvements in a multi-gene fatty acid metabolic pathway. On the basis of central pathway architecture, E. coli fatty acid biosynthesis was re-cast into three modules: the upstream acetyl coenzyme A formation module; the intermediary acetyl-CoA activation module; and the downstream fatty acid synthase module. Combinatorial optimization of transcriptional levels of these three modules led to the identification of conditions that balance the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. Refining protein translation efficiency by customizing ribosome binding sites for both the upstream acetyl coenzyme A formation and fatty acid synthase modules enabled further production improvement. Fed-batch cultivation of the engineered strain resulted in a final fatty acid production of 8.6 g l(-1). The modular engineering strategies demonstrate a generalized approach to engineering cell factories for valuable metabolites production.
In light of emerging antibiotic resistance, bacterial cell wall lytic enzymes are promising antimicrobial agents that degrade bacterial peptidoglycan while specifically recognizing the target ...bacterium. The efficacy of lytic enzymes against several multi-drug-resistant pathogens infecting humans has led to many efforts focused on in vivo therapeutic applications. However, the potential for lytic enzymes to combat bacterial contamination in environments outside the human body is underexplored. The persistence of pathogenic bacteria, in either planktonic or biofilm states and on various surfaces, has facilitated the spread of bacterial infections, necessitating the development of robust strategies for detecting and killing resistant bacteria in diverse environments. Here, we present an overview of the current state-of-the-art of exploiting lytic enzymes for non-therapeutic applications including pathogen decontamination in social infrastructures and food decontamination, as well as pathogen detection.
Key points
• Lytic enzymes are effective antimicrobial, antibiofilm, and sporicidal agents.
• Pathogen detection using lytic enzyme–binding domains is rapid and highly sensitive.
• Domain engineering is required for enhanced enzyme activity in complex environments.
The role that the complex microbial communities play in human and environmental health cannot be understated. The increased information about community complexity, as well as the overuse of ...broad-spectrum antibiotics, suggest that new approaches to target specific organisms within a community context are essential towards new antimicrobial therapies. Here, we have assessed the activity and selectivity of two cell wall lytic enzymes, lysostaphin (Lst) and PlyPH, in the presence of multiple bacteria and under varied media conditions. Lst and PlyPH target the clinically relevant pathogens
Staphylococcus aureus
and
Bacillus cereus
, respectively. Lst was effective under all conditions resulting in ~ 4-log and ~ 3-log reduction at 100 μg/mL in actively growing monoculture and co-culture, respectively. PlyPH was also selective but less active and more susceptible to media and cell population changes. Lst and PlyPH activities could be increased in supernatants from actively growing cultures in the presence of a protease inhibitor cocktail, suggesting a possible role played by proteases secreted during cell growth in reducing lytic enzyme activity. This work demonstrates the utility of cell wall lytic enzymes for targeted pathogen killing or microbial community remodeling.
•Cell wall binding of lysostaphin and PlyPH is impacted by complex growth media.•Differences in binding properties exist between a bacteriolysin and an endolysin.•Off rate of PlyPH binding is 20-fold ...lower than lysostaphin binding in growth media.•Endolysin PlyPH binding is strongly sensitive to medium composition.•Results suggest evolutionary differences between a bacteriolysin and an endolysin.
The bacteriolysin lysostaphin (Lst) and endolysin PlyPH are potent modular lytic enzymes with activity against clinically-relevant Gram-positive Staphylococcus aureus and Bacillus cereus, respectively. Both enzymes possess an N-terminal catalytic domain and C-terminal binding domain, with the latter conferring significant enzyme specificity. Lst and PlyPH show reduced activity in the presence of bacterial growth-supporting conditions, such as complex media. Here, we hypothesize that Lst and PlyPH bind poorly to their targets in growth media, which may influence their use in antimicrobial applications in the food industry, as therapeutics, and for control of microbial communities. To this end, binding of isolated Lst and PlyPH binding domains to target bacteria was quantified in the presence of three increasingly complex media – phosphate buffered saline (PBS), defined growth medium (AAM) and undefined complex medium (TSB) by surface plasmon resonance (SPR) and flow cytometry. Evaluation of binding kinetics by SPR demonstrated that PlyPH binding was particularly sensitive to medium composition, with 8-fold lower association and 3.4-fold lower dissociation rate constants to B. cereus in TSB compared to PBS. Flow cytometry studies indicated a decrease in the binding-dependent fluorescent populations of S. aureus and B. cereus, for lysostaphin binding domain and PlyPH binding domain, respectively, in TSB compared to PBS. Enzyme binding behavior was consistent with the enzymes’ catalytic activity in the three media, thereby suggesting that compromised enzyme binding could be responsible for poor activity in more complex growth media.
Background & Aims The role of interleukin (IL)-5 in the pathogenesis of eosinophilic esophagitis (EoE) has been established in animal models; anti–IL-5 therapy has been reported to be effective in ...adults. We investigated whether IL-5 has a role in accumulation of esophageal eosinophils in children with EoE and whether therapy with mepolizumab, an antibody against IL-5, reduces the number of esophageal intraepithelial eosinophils in children with EoE. Methods We performed an international, multicenter, double-blind, randomized, prospective study of 59 children with EoE, defined as baseline peak count of esophageal intraepithelial eosinophils of ≥20 in at least 1 high-power field (hpf). Patients received an infusion every 4 weeks (a total of 3 infusions) of 0.55, 2.5, or 10 mg/kg mepolizumab. No placebo group was used. Results Baseline peak and mean esophageal intraepithelial eosinophil counts were (mean ± SE) 122.5 ± 8.78 and 39.1 ± 3.63 per hpf, respectively. Four weeks after the third infusion, peak eosinophil counts were <5 per hpf in 5 of 57 children (8.8%); we did not observe differences among groups given different doses of mepolizumab. Reduced peak and mean eosinophil counts, to <20 per hpf, were observed in 18 of 57 (31.6%) and 51 of 57 (89.5%) children, respectively. Peak and mean esophageal intraepithelial eosinophil counts decreased significantly to 40.2 ± 5.17 and 9.3 ± 1.25 per hpf, respectively ( P < .0001). An analysis to evaluate predictors of response associated a higher mean baseline esophageal intraepithelial eosinophil count with a greater reduction in mean count ( P < .0001). Conclusions IL-5 is involved in the pathogenesis of EoE in children. Mepolizumab, an antibody against IL-5, reduces esophageal eosinophilic inflammation in these patients.
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