Summary Bacterial lineages that chronically infect cystic fibrosis (CF) patients genetically diversify during infection. However, the mechanisms driving diversification are unknown. By dissecting 10 CF lung pairs and studying ~12,000 regional isolates, we were able to investigate whether clonally-related Pseudomonas aeruginosa inhabiting different lung regions evolve independently and differ functionally. Phylogenetic analysis of genome sequences showed that regional isolation of P. aeruginosa drives divergent evolution. We investigated the consequences of regional evolution by studying isolates from mildly and severely-diseased lung regions and found evolved differences in bacterial nutritional requirements, host-defense and antibiotic resistance, and virulence due to hyperactivity of type 3 secretion systems. These findings suggest that bacterial intermixing is limited in CF lungs, and that regional selective pressures may markedly differ. The findings also may explain how specialized bacterial variants arise during infection, and raise the possibility that pathogen diversification occurs in other chronic infections characterized by spatially heterogeneous conditions.

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Protein interaction topologies are critical determinants of biological function. Large-scale or proteome-wide measurements of protein interaction topologies in cells currently pose an unmet challenge that could dramatically improve understanding of complex biological systems. A primary impediment includes direct protein topology and interaction measurements from living systems since interactions that lack biological significance may be introduced during cell lysis. Furthermore, many biologically relevant protein interactions will likely not survive the lysis/sample preparation and may only be measured with in vivo methods. As a step toward meeting this challenge, a new mass spectrometry method called Real-time Analysis for Cross-linked peptide Technology (ReACT) has been developed that enables assignment of cross-linked peptides “on-the-fly”. Using ReACT, 708 unique cross-linked (<5% FDR) peptide pairs were identified from cross-linked E. coli cells. These data allow assembly of the first protein interaction network that also contains topological features of every interaction, as it existed in cells during cross-linker application. Of the identified interprotein cross-linked peptide pairs, 40% are derived from known interactions and provide new topological data that can help visualize how these interactions exist in cells. Other identified cross-linked peptide pairs are from proteins known to be involved within the same complex, but yield newly discovered direct physical interactors. ReACT enables the first view of these interactions inside cells, and the results acquired with this method suggest cross-linking can play a major role in future efforts to map the interactome in cells.

The unique and remarkable physicochemical properties of protein surface topologies give rise to highly specific biomolecular interactions, which form the framework through which living systems are able to carry out their vast array of functions. Technological limitations undermine efforts to probe protein structures and interactions within unperturbed living systems on a large scale. Rapid chemical stabilization of proteins and protein complexes through chemical cross-linking offers the alluring possibility to study details of the protein structure to function relationships as they exist within living cells. Here we apply the latest technological advances in chemical cross-linking combined with mass spectrometry to study protein topologies and interactions from living human cells identifying a total of 368 cross-links. These include cross-links from all major cellular compartments including membrane, cytosolic and nuclear proteins. Intraprotein and interprotein cross-links were also observed for core histone proteins, including several cross-links containing post-translational modifications which are known histone marks conferring distinct epigenetic functions. Proteins are the principal operatives within cells, involved in carrying out essentially all biological functions. A complex network of intra-and intermolecular interactions, post-translational modifications and abundance levels is required to maintain the delicate balance of function essential for life. Subtle changes within this network can give rise to specific biological responses to environmental factors, onset of disease, normal aging, and other biological processes. Therefore, direct experimental observation of protein structures and interactions in relation to biological function is paramount to improved understanding of living systems.Chemical cross-linking has long been used as a method of fixation to preserve biological samples in the fields of histology and pathology (1). Protein interactions and topologies have also been studied with chemical cross-linking methods for many years (2-4). Chemical cross-linking with mass spectrometry (XL-MS) 1 is emerging as a powerful technology to study protein structures and interactions in complex biological systems (5). Technological advances in chemistry, analytical instrumentation, and informatics are beginning to allow the successful application of XL-MS to study protein topologies and interactions on a large scale in complex biological systems. These methods are able to provide low resolution spatial information on protein topologies through distance constraints imposed by the chemical linker arm distance. The resultant distance constraints are often used to refine crystal structure measurements and to assist de novo structure prediction with molecular modeling techniques (6, 7). Structural information derived through cross-linking experiments is largely complementary to structural information obtained through other techniques including hydrogen-deuterium exchange mass spectrometry, NMR, and x-ray cryst...

Protein interactions and topologies are key features that enable specificity, function and the evolution of highly integrated, regulated networks in biological systems. Primary challenges associated with the study of biological systems include identification of protein interactions and measurement of topological features of proteins and their interactions in vivo. Advancements such as the Yeast Two-Hybrid (1), coimmunoprecipitation (2), and Tandem Affinity Purification tags (3) have greatly increased the ability to identify hundreds or even thousands of interactions from complex biological samples (2, 4 -6). Despite the many thousands of protein interactions that are now known (7) however, for only a tiny fraction is there any knowledge of their in vivo topology. On the other hand, if topologies of interactions were more widely known, this information could improve understanding of underlying fundamental factors that drive interactions, improve development of highly specific modulators of protein interactions, improve interaction prediction capabilities, and improve comprehension on biological systems. Unfortunately, exceedingly few methods exist to allow unbiased measurement of proteinprotein interaction topological features in cells.Chemical cross-linking has great potential for in vivo interaction topological studies (8 -10). Cross-linked peptides contain information about interacting protein identities and can uniquely define regions of protein sequences that are near one another when proteins are present within the native cellular environment. Challenges associated with in vivo crosslinking analysis that have precluded this achievement include the difficulty in identification of cross-linked peptides and the severe dynamic range constraints resultant from the overwhelming majority of noncross-linked peptides. Our efforts to overcome these challenges resulted in development of Protein Interaction Reporter (PIR) 1 technology (11) that uses a novel type of cross-linker and mass spectrometry to identify peptides that are close to one another within protein complexes in cells. These efforts resulted in the first reported identification of cross-linked peptides from live cells (9) including the first in vivo identification of an interaction among two outer membrane cytochrome c proteins, an interaction that appears to be critical to electron transport properties of Shewanella oneidensis (12).Here we present the first application of PIR technology to the study of interactions in E. coli cells where 65 cross-linked peptide pairs were unambiguously identified. To date, this constitutes the largest in vivo cross-linked peptide data set ever produced. In this system, we are also able to compare many of our results with known protein and protein complex crystal structures that demonstrate excellent agreement with our in vivo data. Importantly, this comparative analysis was also used to define distance constraints that enable refinements of structural prediction of in vivo protein complexes never before possible. Furthe...