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 ...

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 ...

University of California

University of California, Irvine

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or . Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system.

sideSPIM - selective plane illumination based on a conventional inverted microscope.

Bacteria colonize environments that contain networks of moving fluids, including digestive pathways, blood vasculature in animals, and the xylem and phloem networks in plants. In these flow networks, bacteria form distinct biofilm structures that have an important role in pathogenesis. The physical mechanisms that determine the spatial organization of bacteria in flow are not understood. Here, we show that the bacterium  colonizes flow networks using a cyclical process that consists of surface attachment, upstream movement, detachment, movement with the bulk flow, and surface reattachment. This process, which we have termed dynamic switching, distributes bacterial subpopulations upstream and downstream in flow through two phases: movement on surfaces and cellular movement via the bulk. The model equations that describe dynamic switching are identical to those that describe dynamic instability, a process that enables microtubules in eukaryotic cells to search space efficiently to capture chromosomes. Our results show that dynamic switching enables bacteria to explore flow networks efficiently, which maximizes dispersal and colonization and establishes the organizational structure of biofilms. A number of eukaryotic and mammalian cells also exhibit movement in two phases in flow, which suggests that dynamic switching is a modality that enables efficient dispersal for a broad range of cell types.

Dynamic switching enables efficient bacterial colonization in flow.

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  abolishes swarming motility in the infected subpopulation and induces the release of the  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  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 , 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  is consistent with a mechanism that promotes the survival of bacterial populations. We uncover a phage- and antibiotic-induced stress response in the clinically important opportunistic pathogen  Phage-infected  subpopulations are isolated from uninfected subpopulations by the production of a stress-induced signal. Activation of the stress response by antibiotics causes  to physically be repelled from the area containing antibiotics altogether, consistent with a mechanism of antibiotic evasion. The stress response observed here could increase  resilience against antibiotic treatment and phage therapy in health care settings, as well as provide a simple evolutionary strategy to avoid areas containing stress.

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

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  We established a metabolic profile for  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. 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  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.

Albert Siryaporn

Department of Physics and Astronomy,
Department of Molecular Biology and Biochemistry

University of California

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

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

Albert Siryaporn

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physics and Astronomy,Department of Molecular Biology and Biochemistry

University of California

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

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

Department of Physics and Astronomy,
Department of Molecular Biology and Biochemistry