The great divide: rhamnolipids mediate separation between P. aeruginosa and S. aureus.

The interactions between bacterial species during infection can have significant impacts on pathogenesis. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that can co-infect hosts and cause serious illness. The factors that dictate whether one species outcompetes the other or whether the two species coexist are not fully understood. We investigated the role of surfactants in the interactions between these two species on a surface that enables P. aeruginosa to swarm. We found that P. aeruginosa swarms are repelled by colonies of clinical S. aureus isolates, creating physical separation between the two strains. This effect was abolished in mutants of S. aureus that were defective in the production of phenol-soluble modulins (PSMs), which form amyloid fibrils around wild-type S. aureus colonies. We investigated the mechanism that establishes physical separation between the two species using Imaging of Reflected Illuminated Structures (IRIS), which is a non-invasive imaging method that tracks the flow of surfactants produced by P. aeruginosa. We found that PSMs produced by S. aureus deflected the surfactant flow, which in turn, altered the direction of P. aeruginosa swarms. These findings show that rhamnolipids mediate physical separation between P. aeruginosa and S. aureus, which could facilitate coexistence between these species. Additionally, we found that a number of molecules repelled P. aeruginosa swarms, consistent with a surfactant deflection mechanism. These include Bacillus subtilis surfactant, the fatty acids oleic acid and linoleic acid, and the synthetic lubricant polydimethylsiloxane. Lung surfactant repelled P. aeruginosa swarms and inhibited swarm expansion altogether at higher concentration. Our results suggest that surfactant interactions could have major impacts on bacteria-bacteria and bacteria-host relationships. In addition, our findings uncover a mechanism responsible for P. aeruginosa swarm development that does not rely solely on sensing but instead is based on the flow of surfactant.

Swarming of P. aeruginosa: Through the lens of biophysics.

Swarming is a collective flagella-dependent movement of bacteria across a surface that is observed across many species of bacteria. Due to the prevalence and diversity of this motility modality, multiple models of swarming have been proposed, but a consensus on a general mechanism for swarming is still lacking. Here, we focus on swarming by Pseudomonas aeruginosa due to the abundance of experimental data and multiple models for this species, including interpretations that are rooted in biology and biophysics. In this review, we address three outstanding questions about P. aeruginosa swarming: what drives the outward expansion of a swarm, what causes the formation of dendritic patterns (tendrils), and what are the roles of flagella? We review models that propose biologically active mechanisms including surfactant sensing as well as fluid mechanics-based models that consider swarms as thin liquid films. Finally, we reconcile recent observations of P. aeruginosa swarms with early definitions of swarming. This analysis suggests that mechanisms associated with sliding motility have a critical role in P. aeruginosa swarm formation.

Understanding how bacterial collectives organize on surfaces by tracking surfactant flow.

Swarming is a collective bacterial behavior in which a dense population of bacterial cells moves over a porous surface, resulting in the expansion of the population. This collective behavior can guide bacteria away from potential stressors such as antibiotics and bacterial viruses. However, the mechanisms responsible for the organization of swarms are not understood. Here, we briefly review models that are based on bacterial sensing and fluid mechanics that are proposed to guide swarming in the pathogenic bacterium Pseudomonas aeruginosa. To provide further insight into the role of fluid mechanics in P. aeruginosa swarms, we track the movement of tendrils and the flow of surfactant using a novel technique that we have developed, Imaging of Reflected Illuminated Structures (IRIS). Our measurements show that tendrils and surfactants form distinct layers that grow in lockstep with each other. The results raise new questions about existing swarming models and the possibility that the flow of surfactants impacts tendril development. These findings emphasize that swarm organization involves an interplay between biological processes and fluid mechanics.

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.

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response.

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.

PQS Produced by the Pseudomonas aeruginosa Stress Response Repels Swarms Away from Bacteriophage and Antibiotics.

We investigate the effect of bacteriophage infection and antibiotic treatment on the coordination of swarming, a collective form of flagellum- and pilus-mediated motility in bacteria. We show that phage infection of the opportunistic bacterial pathogen Pseudomonas aeruginosa abolishes swarming motility in the infected subpopulation and induces the release of the Pseudomonas quinolone signaling molecule PQS, which repulses uninfected subpopulations from approaching the infected area. These mechanisms have the overall effect of limiting the infection to a subpopulation, which promotes the survival of the overall population. Antibiotic treatment of P. aeruginosa elicits the same response, abolishing swarming motility and repulsing approaching swarms away from the antibiotic-treated area through a PQS-dependent mechanism. Swarms are entirely repelled from the zone of antibiotic-treated P. aeruginosa, consistent with a form of antibiotic evasion, and are not repelled by antibiotics alone. PQS has multiple functions, including serving as a quorum-sensing molecule, activating an oxidative stress response, and regulating the release of virulence and host-modifying factors. We show that PQS serves additionally as a stress warning signal that causes the greater population to physically avoid cell stress. The stress response at the collective level observed here in P. aeruginosa is consistent with a mechanism that promotes the survival of bacterial populations.IMPORTANCE We uncover a phage- and antibiotic-induced stress response in the clinically important opportunistic pathogen Pseudomonas aeruginosa Phage-infected P. aeruginosa subpopulations are isolated from uninfected subpopulations by the production of a stress-induced signal. Activation of the stress response by antibiotics causes P. aeruginosa to physically be repelled from the area containing antibiotics altogether, consistent with a mechanism of antibiotic evasion. The stress response observed here could increase P. aeruginosa resilience against antibiotic treatment and phage therapy in health care settings, as well as provide a simple evolutionary strategy to avoid areas containing stress.

New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation.

Protein L-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the pcmt1 gene, catalyzes repair of abnormal L-isoaspartyl linkages in age-damaged proteins. Pcmt1 knockout mice exhibit a profound neuropathology and die 30-60 days postnatal from an epileptic seizure. Here we characterize four new SNP variants of human PIMT with respect to enzymatic activity, thermal stability, and propensity to aggregation. Under standard assay conditions, L191S, A150V, P174H and A65V showed activity losses of 72%, 64%, 61%, and 11% respectively. By differential scanning fluorimetry, melting temperature deviations were -5.2, -4.5, +0.5, and -3.4°C. SDS-PAGE of purified protein reveal significant aggregation of L191S, A150V, and P174H, but not A65V. We also report new data on three unusual PIMT variants among the 13 recently characterized by our laboratory. A7P and I58V were previously found to have 1.8-2.0 times the activity of WT PIMT in the standard assay; however, upon kinetic analysis, we find both variants exhibit reduced catalytic efficiency (Vmax/Km) due to weak isoaspartyl substrate binding. The near complete loss of activity (<1%) seen in R36C was investigated by comparing activity of two artificial variants. R36K shows 4.6X the activity of R36C, while R36A shows no improvement, suggesting the guanidino nitrogens of the R36 play a key role in binding the methyl donor S-adenosyl-L-methionine (AdoMet). The new findings reported here extend the list of human PIMT variants that may contribute to neurological diseases in the young and the decline of CNS function in the aged.

Polymorphic Variants of Human Protein l-Isoaspartyl Methyltransferase Affect Catalytic Activity, Aggregation, and Thermal Stability: IMPLICATIONS FOR THE ETIOLOGY OF NEUROLOGICAL DISORDERS AND COGNITIVE AGING.

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the human pcmt1 gene, catalyzes repair of abnormal l-isoaspartyl linkages in age-damaged proteins. Pcmt1 knock-out mice exhibit a profound neuropathology and die 30-60 days postnatal from an epileptic seizure. Here we express 15 reported variants of human PIMT and characterize them with regard to their enzymatic activity, thermal stability, and propensity to aggregation. One mutation, R36C, renders PIMT completely inactive, whereas two others, A7P and I58V, exhibit activity that is 80-100% higher than wild type. G175R is highly prone to aggregation and has greatly reduced activity. R17S and R17H show markedly enhanced sensitivity to thermal denaturation. Based on previous studies of moderate PIMT variation in humans and mice, we predict that heterozygosity for R36C, G175R, R17S, and R17H will prove detrimental to cognitive function and successful aging, whereas homozygosity (if it ever occurs) will lead to severe neurological problems in the young.