Cystic fibrosis lung infections are polymicrobial, complex, and challenging to treat.

<p>The airways of patients with CF are colonized from various host (as indicated) and environmental (not depicted) sources. Patients subsequently develop chronic, polymicrobial infections composed of diverse bacterial, fungal, and viral organisms. These polymicrobial communities both influence and are impacted by their human host through complex, multifactorial interactions. As highlighted in the figure above and throughout this review, CF lung microbial communities encounter frequent antibiotic therapy, host immune factors, and an altered lung environment (including the presence of hypoxic [low oxygen] and anoxic [no oxygen] regions) throughout disease progression, all of which contribute to the development of chronic communities that often have decreased microbial diversity and are populated by organisms that have become highly adapted and resilient to treatment. The combination of diverse colonization sources, dynamic inter-domain interactions, microbial adaptation, environmental factors, and patient therapy mediates patient outcome. Eventually, chronic pulmonary infection culminates in a decline in lung function, which becomes most severe during pulmonary exacerbations and late stage disease progression. This steep decline in lung function ultimately leads to respiratory failure, the primary cause of morbidity and mortality in CF patients today [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005258#ppat.1005258.ref006" target="_blank">6</a>]. Figure illustration and design copyright 2015 William Scavone and used with permission.</p

Single-Cell and Single-Molecule Analysis Deciphers the Localization, Adhesion, and Mechanics of the Biofilm Adhesin LapA

The large adhesin protein LapA mediates adhesion and biofilm formation by <i>Pseudomonas fluorescens</i>. Although adhesion is thought to involve the long multiple repeats of LapA, very little is known about the molecular mechanism by which this protein mediates attachment. Here we use atomic force microscopy to unravel the biophysical properties driving LapA-mediated adhesion. Single-cell force spectroscopy shows that expression of LapA on the cell surface <i>via</i> biofilm-inducing conditions (<i>i.e.</i>, phosphate-rich medium) or deletion of the gene encoding the LapG protease (LapA+ mutant) increases the adhesion strength of <i>P. fluorescens</i> toward hydrophobic and hydrophilic substrates, consistent with the adherent phenotypes observed in these conditions. Substrate chemistry plays an unexpected role in modulating the mechanical response of LapA, with sequential unfolding of the multiple repeats occurring only on hydrophilic substrates. Biofilm induction also leads to shortening of the protein extensions, reflecting stiffening of their conformational properties. Using single-molecule force spectroscopy, we next demonstrate that the adhesin is randomly distributed on the surface of wild-type cells and can be released into the solution. For LapA+ mutant cells, we found that the adhesin massively accumulates on the cell surface without being released and that individual LapA repeats unfold when subjected to force. The remarkable adhesive and mechanical properties of LapA provide a molecular basis for the “multi-purpose” adhesion function of LapA, thereby making <i>P. fluorescens</i> capable of colonizing diverse environments

Strains used in this study.

<p>^a Minimum inhibitory concentration of tobramycin for <i>P</i>. <i>aeruginosa</i> strains as measured by Biomerieux E-test strips according to manufacture’s instructions.</p><p>Strains used in this study.</p

Mannitol does not sensitize non-mucoid, laboratory strain <i>P</i>. <i>aeruginosa</i> PA14 to tobramycin.

<p>A. Mannitol is minimally cytotoxic to CFBE cells. Normalized cytotoxicity as measured by fraction of LDH release. Cytotoxicity was measured after 24 hours of treatment with 0, 40 or 60 mM mannitol as indicated. Cells lysed with Triton X-100 served as a control to determine total lysis. Columns indicate mean of at least three biological replicates, error bars indicate standard deviation (S.D.). **, P<0.01, comparison of indicated sample to total lysis control by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons. B. Viability of <i>P</i>. <i>aeruginosa</i> PA14 grown as a biofilm on CFBE cells after treatment with 0 μg/mL tobramycin (open bars), 8 μg/mL tobramycin (hatched bars), 0 mM mannitol (white bars), 60 mM mannitol (gray bars) or co-treatment with 8 μg/mL tobramycin and 60 mM mannitol, as indicated. Columns indicate mean of at least three biological replicates, error bars indicate S.D. ***, P<0.001 by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons. There is no significant difference between <i>P</i>. <i>aeruginosa</i> PA14 treated with tobramycin +/- mannitol.</p

Mannitol does not sensitize <i>P</i>. <i>aeruginosa</i> clinical isolates grown as biofilms on CF airway cells to tobramycin.

<p>A. Viability of <i>P</i>. <i>aeruginosa</i> clinical isolates grown as biofilms on CFBE cells and treated with 0 μg/mL tobramycin (open bars), 8 μg/mL tobramycin (hatched bars), 0 mM mannitol (white bars), 60 mM mannitol (gray bars) or co-treatment with 8 μg/mL tobramycin and 60 mM mannitol, as indicated. Columns indicate mean of at least three biological replicates, error bars indicate S.D. **, P<0.01 by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons. B. The viability of strains <i>P</i>. <i>aeruginosa</i> PAO1 (left) and FRD1 (right) as biofilms on CFBE cells and treated with 0 μg/mL tobramycin (open bars), 8 μg/mL tobramycin (hatched bars), 0 mM mannitol (white bars), 60 mM mannitol (gray bars) or co-treatment with 8 μg/mL tobramycin and 60 mM mannitol, as indicated. **, P<0.01 or ***, P<0.001 by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons. ns, not significant compared to tobramycin treatment in the absence of mannitol. C. The viability of strain <i>P</i>. <i>aeruginosa</i> PAO1 as a biofilm on plastic and treated with 0 μg/mL tobramycin (open bars), 80 μg/mL tobramycin (hatched bars), 0 mM mannitol (white bars), 60 mM mannitol (gray bars) or co-treatment with 80 μg/mL tobramycin and 60 mM mannitol, as indicated. *, P<0.05 compared to treatment with 80 μg/mL tobramycin with no mannitol. **, P<0.01 or ***, P<0.001 by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons.</p

High-Speed “4D” Computational Microscopy of Bacterial Surface Motility

Bacteria exhibit surface motility modes that play pivotal roles in early-stage biofilm community development, such as type IV pili-driven “twitching” motility and flagellum-driven “spinning” and “swarming” motility. Appendage-driven motility is controlled by molecular motors, and analysis of surface motility behavior is complicated by its inherently 3D nature, the speed of which is too fast for confocal microscopy to capture. Here, we combine electromagnetic field computation and statistical image analysis to generate 3D movies close to a surface at 5 ms time resolution using conventional inverted microscopes. We treat each bacterial cell as a spherocylindrical lens and use finite element modeling to solve Maxwell’s equations and compute the diffracted light intensities associated with different angular orientations of the bacterium relative to the surface. By performing cross-correlation calculations between measured 2D microscopy images and a library of computed light intensities, we demonstrate that near-surface 3D movies of <i>Pseudomonas aeruginosa</i> translational and rotational motion are possible at high temporal resolution. Comparison between computational reconstructions and detailed hydrodynamic calculations reveals that <i>P. aeruginosa</i> act like low Reynolds number spinning tops with unstable orbits, driven by a flagellum motor with a torque output of ∼2 pN μm. Interestingly, our analysis reveals that <i>P. aeruginosa</i> can undergo complex flagellum-driven dynamical behavior, including precession, nutation, and an unexpected taxonomy of surface motility mechanisms, including upright-spinning bacteria that diffuse laterally across the surface, and horizontal bacteria that follow helicoidal trajectories and exhibit superdiffusive movements parallel to the surface