Biology subjects

Koch, M. D.

Publications and source records attributed to Koch, M. D..

13 recordsLinked to original sources

bioRxiv2026

The construction and operation of type IV pili impose a variable energetic burden across phylogenetically distant bacteria

Type IV pili (T4P) are dynamic surface appendages that mediate essential biological functions and virulence traits, yet their energetic burden on cellular budgets in light of fluctuating host environments remains unexplored. Here, we present a comprehensive economic analysis of T4P construction and operation in ATP equivalents, following established frameworks of flagella analyses. Using Pseudomonas aeruginosa as a model system, we quantify the total cellular burden to synthesize the T4P machinery, maintain the inner-membrane pool of major pilin (PilA), and drive repeated cycles of pilus extension and retraction over a generation. We estimate that the T4P system consumes ~0.7% of the total cellular energy budget, dominated by PilA monomer production. Conversely, the operational cost of dynamic T4P fibers is negligible due to their intermittent activity - contrasting sharply with the high continuous cost of rotating a polar flagellum. Extending this framework across five phylogenetically diverse species (P. aeruginosa, Vibrio cholerae, Caulobacter crescentus, Neisseria spp., and Myxococcus xanthus) reveals that T4P investment varies tenfold (0.2 - 1.8% of the cellular budget), driven by differences in pilin size, machine number, pilus extension rates, and cell volume. Neisseria is a distinct outlier whose high extension rate makes operational costs approach construction costs, while in all other species construction dominates. These findings indicate that changes in nutrient availability or surface association may modulate pilus number and length as a strategy to optimize energetic burdens during host-pathogen interaction.

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↗

bioRxiv2026

Rapid activation of dormant type IV pili enables a dispersal-infection tradeoff in environments with fluctuating nutrients

Bacteria in fluctuating environments must balance the high metabolic costs of motility against risks of bacteriophage predation and immune clearance. While flagellar trade-off mechanisms are well-documented, regulation of type IV pilus (T4P) activity during environmental transitions remains unclear. We show that Pseudomonas aeruginosa uses an energy-dependent idling strategy to synchronize T4P-mediated surface motility with nutrient availability. In nutrient-depleted stationary phase, T4P transcription and protein levels remain constant, pre-assembled machines persist at the cell pole, yet cells produce only sparse, truncated pili that extend and retract slowly. Using a single-cell ATP biosensor, we show that T4P dynamics respond directly to cellular adenylate energy charge. Carbon source addition rapidly elevates intracellular ATP, reactivating pre-assembled T4P within minutes. This bypasses de novo protein synthesis, restoring pilus number, length, and extension/retraction rates. This rapid response drives opportunistic biofilm dispersal but, at the same time, creates an immediate tradeoff: reactivated T4P restore susceptibility to pilus-specific phages upon nutrient upshift. Thus, energetic gating of T4P enables P. aeruginosa to minimize exposure to phages during starvation while remaining poised for rapid reactivation. Importantly, T4P promote resistance to opsonization and phagocytosis by macrophages and neutrophils. Upon nutrient upshift, full T4P activity therefore supports dispersal and host colonization while conferring immune protection, revealing a fundamental dispersal-infection tradeoff at the host- microbe interface in fluctuating environments such as the lung and gut.

microbiology↗

bioRxiv2026

Type IV pilus length determines virulence by regulating a hidden subpopulation of non-contributing filaments

Many clinically important bacterial pathogens, including Pseudomonas, Vibrio, Neisseria, and Acinetobacter species, employ dynamic extracellular appendages called type IV pili (T4P) to facilitate virulence through cyclical extension and retraction of pilus filaments. To dissect how T4P dynamics govern pathogenesis, we engineered a genetic system to precisely tune pilus length across a continuum. We demonstrate that pilus length critically determines four major T4P-dependent virulence traits in Pseudomonas aeruginosa (motility, surface sensing, biofilm formation, and phage infection) and reveal a hidden subpopulation of pili that are unable to interact with environmental substrates or host cells, rendering them non-contributing to any T4P-mediated function. Integrating molecular dynamics simulations, we show that low inner-membrane abundance of the major pilin forces the extension mechanism into transient idle states, restricting both velocity and final length. Molecularly, this finding reveals how two key biophysical parameters, pilin abundance and diffusion, impose a fundamental physical constraint on T4P assembly, and that regulating pilin abundance presents a strong lever over regulating pilus count for controlling the amount of functionally contributing filaments. Contrary to the prevailing view that retraction force generation primarily dictates T4P-mediated behaviors, our results establish extension dynamics as the overlooked bottleneck constraining all retraction-enabled virulence traits, with population heterogeneity in length enabling adaptive bet-hedging.

microbiology↗

bioRxiv2024

Flow rapidly replenishes scarce nutrients to promote bacterial growth

In laboratory settings, bacteria grow in static culture with more nutrients than they require. However, bacteria in nature experience flowing environments that are nutrient limited. Using microfluidics and single cell imaging, we discover that flow promotes growth of the human pathogen Pseudomonas aeruginosa at surprisingly low nutrient concentrations. In static environments, cells require high nutrient concentrations as they steadily consume non-renewable resources. In flowing environments, cells grow robustly at very low nutrient concentrations that are constantly replenished. Our simulation-guided experiments show how stopping flow halts growth within minutes and varying flow impacts growth gradients across bacterial populations. By precisely delivering nutrients using microfluidics, we learned that cells in flow can grow on glucose concentrations 1,000 times lower than those observed in typical laboratory experiments. The ultralow glucose concentrations sufficient for growth in flow closely align with the affinity of bacterial glucose transporters, suggesting that bacteria have evolved in flowing environments with scarce nutrients. Collectively, our results emphasize the limits of traditional culturing approaches and highlight how depleted environments can unexpectedly support bacterial growth.

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↗

bioRxiv2024

Prophages block cell surface receptors to ensure survival of their viral progeny

In microbial communities, viruses compete for host cells to infect, and thus evolved diverse ways to inhibit their competitors. One mechanism is Superinfection exclusion (Sie), whereby a virus that has established an infection prevents a secondary infection. We identified a Pseudomonas prophage Sie protein that alters pilus dynamics through the pilus assembly chaperone, PilZ. This protein, known as Zip for PilZ interacting protein, does not abrogate pilus activity, but fine tunes it, providing strong phage resistance without a fitness cost. This tuning is modulated through quorum sensing, which coordinates Zip production in concert with bacterial cell density to ensure maximal protection when bacterial populations are at the highest risk of phage infection. Most notably, Zip activity prevents internalization and destruction of phage progeny. We refer to this as the "anti-Kronos effect" after the Greek god who devoured his own children and show that it is a conserved feature of diverse prophage-encoded Sie systems.

microbiology↗

bioRxiv2023

Type-IV pili tune an adhesion-migration trade-off during surface colonization of Pseudomonas aeruginosa

Bacterial pathogenicity relies on both firm surface adhesion and cell dissemination. How twitching bacteria resolve the fundamental contradiction between adhesion and migration is unknown. To address this question, we employ live-cell imaging of type-IV pili (T4P) and therewith construct a comprehensive mathematical model of Pseudomonas aeruginosa migration. The data show that only 10% to 50% of T4P bind to substrates and contribute to migration through random extension and retraction. Individual T4P do not display a measurable sensory response to surfaces, but their number increases on cellular surface contact. Attachment to surfaces is mediated, besides T4P, by passive adhesive forces acting on the cell body. Passive adhesions slow down cell migration and result in local random motion on short time scales, which is followed by directionally persistent, superdiffusive motion on longer time scales. Moreover, passive adhesions strongly enhance surface attachment under shear flow. {Delta}pilA mutants, which produce no T4P, robustly stick to surfaces under shear flow. In contrast, rapidly migrating {Delta}pilH cells, which produce an excessive number of T4P, are easily detached by shear. Wild-type cells sacrifice migration speed for robust surface attachment by maintaining a low number of active pili. The different cell strains pertain to disjunct regimes in a generic adhesion-migration trait space. Depending on the nature of the adhesion structures, adhesion and migration are either compatible or a trade-off is required for efficient bacterial surface colonization under different conditions.

biophysics↗

bioRxiv2022

Massively-parallel Microbial mRNA Sequencing (M3-Seq) reveals heterogenous behaviors in bacteria at single-cell resolution

Bacterial populations are highly adaptive. They can respond to stress and survive in shifting environments. How the behaviors of individual bacteria vary during stress, however, is poorly understood. To identify and characterize rare bacterial subpopulations, technologies for single-cell transcriptional profiling have been developed. Existing approaches, though, are all limited in some technical capacity (e.g., number of cells or transcripts that can be profiled). Due in part to these limitations, few conditions have yet been studied with these tools. Here, we develop Massively-parallel Microbial mRNA sequencing (M3-Seq), a single-cell RNA-sequencing platform for bacteria that pairs combinatorial cell indexing with post hoc rRNA depletion. We show that M3-Seq can profile hundreds of thousands of bacterial cells from different species under a range of conditions in single experiments. We then apply M3-Seq to reveal rare populations, insights into bet hedging strategies during stress responses, and host responses to phage infection.

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↗

bioRxiv2021

Pseudomonas aeruginosa differentiates substrate rigidity using retraction of type IV pili

The ability of eukaryotic cells to differentiate substrate stiffness is fundamental for many processes such as the development of stem cells into mature tissue. Here, we establish that bacteria feel their microenvironment in a similar manner. We show that Pseudomonas aeruginosa actively probes and measures substrate stiffness using type IV pili (TFP). The activity of the major virulence factor regulator Vfr is peaked with stiffness in a physiologically important range between 0.1 kPa (mucus) and 1000 kPa (cartilage). The local concentration of PilA at the base of dynamic TFP changes during extension and retraction in a surface dependent manner due to slow PilA diffusion in the cell membrane. Traction force measurements reveal that TFP retraction deforms even stiff substrates. Modeling of the measured substrate deformation and optical tweezers experiments suggest that TFP adhere at the tip only. Informed by these experimental results, we developed a model that describes substrate stiffness dependent dynamics of the polar PilA concentration which are quantitatively consistent with the transcriptional response to stiffness. Manipulating the ATPase activity of the TFP motors changes the TFP extension and retraction velocities and consequently the PilA concentration dynamics in a manner that is predictive of the experimental stiffness response. This work points to the use of a competition between PilA diffusion and TFP extension-retraction as a molecular shear rheometer. Our results highlight that stiffness sensing is a conserved property between the kingdoms of life.

microbiology↗

bioRxiv2020

Pseudomonas aeruginosa detachment from surfaces via a self-made small molecule

Pseudomonas aeruginosa is a significant threat in both healthcare and industrial biofouling. Surface attachment of P. aeruginosa is particularly problematic as surface association induces virulence and biofilm formation, which hamper later antibiotic treatments. Previous efforts have searched for biofilm dispersal agents, but there are no known factors that specifically disperse surface-attached P. aeruginosa. In this study we develop a quantitative surface-dispersal assay and use it to show that P. aeruginosa itself produces factors that can stimulate its dispersal. Through bioactivity-guided fractionation, Mass Spectrometry, and Nuclear Magnetic Resonance, we elucidated the structure of one such factor, 2-methyl-4-hydroxyquinoline (MHQ). MHQ is an alkyl-quinolone with a previously unknown activity and is synthesized by the PqsABC enzymes. Pure MHQ is sufficient to disperse P. aeruginosa, but the dispersal activity of natural P. aeruginosa conditioned media requires additional factors. Whereas other alkyl quinolones have been shown to act as antibiotics or membrane depolarizers, MHQ lacks these activities and known antibiotics do not induce dispersal. In contrast, we show that MHQ inhibits the activity of Type IV Pili (TFP) and that TFP targeting can explain its dispersal activity. Our work thus identifies surface dispersal as a new activity of P. aeruginosa-produced small molecules, characterizes MHQ as a promising dispersal agent, and establishes TFP inhibition as a viable mechanism for P. aeruginosa dispersal. Significance StatementWe discovered that the clinically relevant human bacterial pathogen P. aeruginosa, typically associated with surface-based infections, is dispersed by a small molecule that the bacteria themselves produce. We elucidate the chemical structure of this molecule and find that mechanistically it functions to inhibit the activity of the P. aeruginosa extra cellular surface motility appendage, the type IV pilus.

microbiology↗

bioRxiv2020

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

The functions of type IV pili (TFP) are mediated by cycles of extension and retraction. The coordination of these cycles remains mysterious due to poor quantification of TFP dynamics. Here we fluorescently label the TFP in the opportunistic pathogen Pseudomonas aeruginosa and track the full extension and retraction cycles of individual TFP to quantify their dynamics. We test several models for the switch between extension and retraction using quantitative experiments, biophysical modeling and genetics. We invalidate the prominent hypothesis that this switch is triggered by surface contact. Instead, we show that the entire repetitive cycle of extension and retraction of individual TFP is governed by the stochastic binding of antagonistic extension and retraction motors and explain how this mechanism quantitatively defines physiologically-important features like TFP length and their production rate. Interestingly, our results suggest that the major throttle of TFP production is the unbinding of the retraction motor.

microbiology↗