The Natural History and Composition of Urinary Catheter Biofilms: Early Uropathogen Colonization with Intraluminal and Distal Predominance.

PURPOSE: We sought to determine the composition and initiation site of bacterial biofilm on indwelling urinary catheters and to track biofilm progression with time. MATERIALS AND METHODS: Indwelling urinary catheters were collected from 2 tertiary care centers following removal from patients. Indwelling time was noted and catheters were de-identified. Catheters were sectioned, stained for biofilms and analyzed by spectrophotometry and visualization. Biofilm colonization patterns were analyzed using descriptive statistical analysis and bacterial composition was determined using next generation sequencing. RESULTS: We collected and analyzed a total of 33 catheters from 26 males and 7 females with indwelling time ranging from 15 minutes to 43 days. Biofilm colonization was consistently high on the region of the balloon for all indwelling times. After week 1 the distal third of the catheter had higher biofilm colonization than the proximal third (week 2 p=0.034). At all indwelling times the intraluminal surface of the catheter had greater biofilm colonization than the outer surface. Next generation sequencing detected potential uropathogenic bacteria in all 10 analyzed samples. CONCLUSIONS: The catheter balloon, its distal aspect and its lumen were the predominant locations of biofilm comprising uropathogenic bacteria. Strategies to prevent or treat biofilm should be targeted to these areas.

Heterogeneous and Highly Dynamic Interface in Plastocyanin-Cytochrome f Complex Revealed by Site-Specific 2D-IR Spectroscopy.

Transient protein complexes are crucial for sustaining dynamic cellular processes. The complexes of electron-transfer proteins are a notable example, such as those formed by plastocyanin (Pc) and cytochrome f (cyt f) in the photosynthetic apparatus. The dynamic and heterogeneous nature of these complexes, however, makes their study challenging. To better elucidate the complex of Nostoc Pc and cyt f, 2D-IR spectroscopy coupled to site-specific labeling with cyanophenylalanine infrared (IR) probes was employed to characterize how the local environments at sites along the surface of Pc were impacted by cyt f binding. The results indicate that Pc most substantially engages with cyt f via the hydrophobic patch around the copper redox site. Complexation with cyt f led to an increase in inhomogeneous broadening of the probe absorptions, reflective of increased heterogeneity of interactions with their environment. Notably, most of the underlying states interconverted very rapidly (1 to 2 ps), suggesting a complex with a highly mobile interface. The data support a model of the complex consisting of a large population of an encounter complex. Additionally, the study demonstrates the application of 2D-IR spectroscopy with site-specifically introduced probes to reveal new quantitative insight about dynamic biochemical systems.

Evaluation of p-(13C,15N-Cyano)phenylalanine as an Extended Time Scale 2D IR Probe of Proteins.

Two-dimensional infrared (2D IR) spectroscopy provides a powerful approach for the direct study of molecular dynamics with high spatial and temporal resolution. Its application for investigating specific locations in proteins requires the incorporation of IR probe groups with spectrally isolated absorptions to avoid the congestion inherent to protein spectra. This has motivated extensive efforts toward the development of new IR probes, but there remains a need for those that can extend the experimental time range, which is limited by their vibrational lifetimes. Toward this goal, isotopically labeled p-(13C15N-cyano)phenylalanine was synthesized, site-selectively incorporated into the protein plastocyanin, and evaluated for its potential as a 2D IR probe. The isotopic labeling increases the vibrational lifetime about 2-fold, which results in larger signals at longer time scales. However, isotopic labeling simultaneously shifts the absorption to a spectral region with greater water absorbance, which results in greater heating-induced signals in the background that overlap those of the nitrile probe. The study demonstrates the use of a new 2D IR probe to measure the side chain dynamics in a protein and also illustrates the multiple factors to consider in development of 2D IR probes for studying proteins.

Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy.

The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes.

Applications of two-dimensional infrared spectroscopy.

Two-dimensional infrared (2D IR) spectroscopy has recently emerged as a powerful tool with applications in many areas of scientific research. The inherent high time resolution coupled with bond-specific spatial resolution of IR spectroscopy enable direct characterization of rapidly interconverting species and fast processes, even in complex systems found in chemistry and biology. In this minireview, we briefly outline the fundamental principles and experimental procedures of 2D IR spectroscopy. Using illustrative example studies, we explain the important features of 2D IR spectra and their capability to elucidate molecular structure and dynamics. Primarily, this minireview aims to convey the scope and potential of 2D IR spectroscopy by highlighting select examples of recent applications including the use of innate or introduced vibrational probes for the study of nucleic acids, peptides/proteins, and materials.