Biology subjects

Shuppara, A. M.

Publications and source records attributed to Shuppara, A. M..

6 recordsLinked to original sources

bioRxiv2026

Orthogonal microfluidic approaches reveal force-enhanced migration of bacterial populations on surfaces

Host-generated flow is expected to oppose bacterial migration by sweeping cells downstream, yet some bacteria can migrate upstream against the direction of flow. However, it is unclear how the magnitude of shear force influences upstream bacterial migration. Here, we use microfluidics to examine upstream migration of the human pathogen Pseudomonas aeruginosa under host-relevant shear forces (0.1-10 pN). By independently varying flow rate and solution viscosity, we discover that increasing shear force counterintuitively enhances population-level upstream migration. Single-cell tracking reveals that force-enhanced migration is driven by an increase in twitching motility speed. Using a microfluidic-based trigonometry approach, we link the increase in speed to the cell-surface angle. At low shear forces, type IV pilus retraction generates a torque that tilts cells toward a vertical orientation, geometrically constraining forward movement and decreasing twitching speed. In contrast, higher shear forces push cells toward a horizontal orientation, increasing twitching speed. Collectively, our results reveal how shear force can enhance bacterial motility and upstream migration, providing a framework to understand how host shear forces may promote the spread of bacterial infections.

microbiology↗

bioRxiv2026

Detoxification of host-relevant H2O2 shapes Pseudomonas aeruginosa population gradients in flow

During infection, bacterial populations need to overcome low levels of host-generated H2O2. However, experiments almost always use high H2O2 levels that are rarely found in hosts. Here, we use microfluidics to investigate how host-relevant H2O2 impacts populations of the human pathogen Pseudomonas aeruginosa in flow. Using long-channel microfluidic devices, we establish that cells at the front of a population remove H2O2 and protect their downstream neighbors. Population-level protection is mediated by three OxyR-regulated scavenging systems (KatA, KatB, AhpCF), which are each sufficient to detoxify host-relevant H2O2. Mutants lacking all three systems are sensitive to H2O2 but can be cross-protected by resistant cells when co-cultured. Cross-protection is abolished in higher flow regimes, where H2O2 is delivered faster than cells can remove it. Our results demonstrate how local detoxification provides global protection, which results in spatial gradients across bacterial populations in flow. Together, our findings highlight how biological, chemical, and physical factors collectively determine the fate of bacterial populations in host-relevant environments.

microbiology↗

bioRxiv2026

Flow-driven delivery boosts antibiotic effectiveness by overwhelming bacterial defenses

Physical, chemical, and biological factors collectively determine antibiotic effectiveness. However, laboratory studies often consider only one or two of these factors and fail to capture key interaction effects. Here, we use microfluidics to study the interplay of host-relevant shear flow, the antibiotic gentamicin, and the human pathogen Pseudomonas aeruginosa. We discover bacterial populations can defend against gentamicin by chemical inactivation or physical sequestration. In low flow regimes, bacterial defenses succeed by eliminating gentamicin faster than it is delivered. As flow increases, delivery overwhelms bacterial defenses, allowing gentamicin to penetrate populations and inhibit cells. Combining biophysical simulations and long-channel microfluidics, we demonstrate that antibiotic susceptibility depends on spatial context, as cells at the beginning of the channel shield cells at the end. Surprisingly, we show that populations of sensitive cells can successfully defend against gentamicin, resulting in spatial gradients that are shaped by flow and cell density. Collectively, our interdisciplinary experiments reveal how flow overwhelms bacterial defenses, providing a framework to better understand antibiotic effectiveness in dynamic environments.

microbiology↗

bioRxiv2024

Combining multiple stressors unexpectedly blocks bacterial migration and growth

In nature, organisms experience combinations of stressors. However, laboratory studies typically simplify reality and focus on the effects of an individual stressor. Here, we use a microfluidic approach to simultaneously provide a physical stressor (shear flow) and a chemical stressor (H2O2) to the human pathogen Pseudomonas aeruginosa. By treating cells with levels of flow and H2O2 that commonly co-occur in nature, we discover that previous reports significantly overestimate the H2O2 levels required to block bacterial growth. Specifically, we establish that flow increases H2O2 effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H2O2 levels, we identify the core H2O2 regulon, characterize OxyR-mediated dynamic regulation, and dissect the redundant roles of multiple H2O2 scavenging systems. By examining single-cell behavior, we serendipitously discover that the combined effects of H2O2 and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H2O2 and flow synergize to restrict bacterial colonization and survival. By studying two stressors at once, our research highlights the limitations of oversimplifying nature and demonstrates that physical and chemical stress can combine to yield unpredictable effects.

microbiology↗

bioRxiv2024

Fluid flow overcomes antimicrobial resistance by boosting delivery

Antimicrobial resistance is an emerging global threat to humanity. As resistance outpaces development, new perspectives are required. For decades, scientists have prioritized chemical optimization, while largely ignoring the physical process of delivery. Here, we used biophysical simulations and microfluidic experiments to explore how fluid flow delivers antimicrobials into communities of the highly resistant pathogen Pseudomonas aeruginosa. We discover that increasing flow overcomes bacterial resistance towards three chemically distinct antimicrobials: hydrogen peroxide, gentamicin, and carbenicillin. Without flow, resistant P. aeruginosa cells generate local zones of depletion by neutralizing all three antimicrobials through degradation or chemical modification. As flow increases, delivery overwhelms neutralization, allowing antimicrobials to regain effectiveness against resistant bacteria. Additionally, we discover that cells on the edge of a community shield internal cells, and cell-cell shielding is abolished in higher flow regimes. Collectively, our quantitative experiments reveal the unexpected result that physical flow and chemical dosage are equally important to antimicrobial effectiveness. Thus, our results should inspire the incorporation of flow into the discovery, development, and implementation of antimicrobials, and could represent a new strategy to combat antimicrobial resistance.

microbiology↗

bioRxiv2022

Fluid flow sensitizes bacterial pathogens to chemical stress

Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa. In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H2O2) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H2O2. High shear rates replenish H2O2, abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that cells in flow are sensitive to a H2O2 concentration that is 100-1000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H2O2 concentration required to trigger a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H2O2 levels in experimental and natural systems. Finally, we demonstrate that the shear rate and H2O2 concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus, suggesting that flow sensitizes bacteria to chemical stress in natural environments.

microbiology↗