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 (H 2 O 2 ) to the human pathogen Pseudomonas aeruginosa . By treating cells with levels of flow and H 2 O 2 that commonly co-occur in nature, we discover that previous reports significantly overestimate the H 2 O 2 levels required to block bacterial growth. Specifically, we establish that flow increases H 2 O 2 effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H 2 O 2 levels, we identify the core H 2 O 2 regulon, characterize OxyR-mediated dynamic regulation, and dissect the redundant roles of multiple H 2 O 2 scavenging systems. By examining single-cell behavior, we serendipitously discover that the combined effects of H 2 O 2 and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H 2 O 2 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.

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.

Shear rate sensitizes bacterial pathogens to H2O2 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 flow triggers an effect like "wind-chill" that sensitizes cells to H2O2 concentrations 100 to 1,000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H2O2 concentration required to generate 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 host environments. 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.

Bacteria in Fluid Flow.

Bacteria thrive in environments rich in fluid flow, such as the gastrointestinal tract, bloodstream, aquatic systems, and the urinary tract. Despite the importance of flow, how flow affects bacterial life is underappreciated. In recent years, the combination of approaches from biology, physics, and engineering has led to a deeper understanding of how bacteria interact with flow. Here, we highlight the wide range of bacterial responses to flow, including changes in surface adhesion, motility, surface colonization, quorum sensing, virulence factor production, and gene expression. To emphasize the diversity of flow responses, we focus our review on how flow affects four ecologically distinct bacterial species: Escherichia coli, Staphylococcus aureus, Caulobacter crescentus, and Pseudomonas aeruginosa. Additionally, we present experimental approaches to precisely study bacteria in flow, discuss how only some flow responses are triggered by shear force, and provide perspective on flow-sensitive bacterial signaling.

Sequence signatures of two public antibody clonotypes that bind SARS-CoV-2 receptor binding domain.

Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short complementarity-determining region (CDR) H3. Germline-encoded sequence motifs in heavy chain CDRs H1 and H2 have a major function, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, is not clear. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that seem to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. These results advance understanding of the antibody response to SARS-CoV-2.

Sequence signatures of two IGHV3-53/3-66 public clonotypes to SARS-CoV-2 receptor binding domain.

Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short CDR H3. Germline-encoded sequence motifs in CDRs H1 and H2 play a major role, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, have not been elucidated. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that appear to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. Overall, our results advance fundamental understanding of the antibody response to SARS-CoV-2.