Host-Microbe Protein Interactions during Bacterial Infection

Schweppe, Devin K.; Harding, Christopher L.; Chavez, Juan D.; Wu, Xia; Ramage, Elizabeth; Singh, Pradeep Kumar; Manoil, Colin; Bruce, James E.

doi:10.1016/j.chembiol.2015.09.015

Cited by 110 publications

(102 citation statements)

References 85 publications

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“…Chemical cross-linking provides evidence of proximal solventaccessible sites on proteins in vivo and is a means to predict and assemble models of protein structures (34,35,39). It is important to note that the chemical cross-linking workflow in this study was performed on whole, functional mitochondria.…”

Section: Resultsmentioning

confidence: 99%

“…Large-scale XL-MS offers two key benefits. First, in conjunction with in silico modeling of structures, the identification of cross-linked interactions enables the observation of the physical features of proteins, including membrane proteins (33,34). These capabilities can uncover novel functions derived from protein-protein interactions, and crosslinks have provided data complementary to cryo-EM and crystallography to enhance the understanding of the structure and function of complex systems (27).…”

mentioning

confidence: 99%

“…Second, large-scale chemical cross-linking analyses applied to living systems can enable the determination of protein interactions and conformational changes in complex, dynamic native environments, including the quantification of changes in response to pharmacological intervention (35). In the present study protein interaction reporter (PIR)-based XL-MS was performed using a peptide-based chemical crosslinking molecule for unambiguous identification of cross-linked peptides (33,34,(36)(37)(38). PIR workflows leverage tandem mass spectrometry (MS n , where n = stages of MS) methods based on the expected PIR mass relationships observed between MS and Significance Mitochondria meet the majority of living cells' demand for ATP and, as important regulators of redox homeostasis, metabolite levels, and calcium buffering, are a critical link between cell energetics and signaling.…”

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confidence: 99%

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Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

Schweppe

Chavez

Lee

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

182

206

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Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly. mitochondria | mass spectrometry | interactome | cross-linking | protein interaction reporter M itochondrial proteins play a diverse role in cellular biology and disease. Mitochondrial dysfunction directly causes multiple inherited diseases (1) and is implicated in common diseases, including neurological developmental disorders (2, 3), neurodegenerative and cardiovascular diseases (4-6), diabetes (7), asthma (8), cancer (9), and age-related disease (10). In mammals, these organelles have evolved to retain more than 1,000 proteins that interact within a complex, i.e., dual membrane architecture (11,12). Within the mitochondrial proteome, the "powerhouse" functions are carried out by the core constituents of the oxidative phosphorylation (OXPHOS) system [complexes I-IV of the electron transport chain (ETC) and ATP synthase (complex V)]. These proteins are necessary for creation of the mitochondrial electrochemical gradient that powers synthesis of ATP. This system includes critical protein-protein interactions within individual OXPHOS complexes as well as "supercomplex" interactions between ETC complexes I, III, and IV in the respirasome (13). Deficient supercomplex formation has been proposed as a critical mitochondrial defect in failing hearts (5,6,14,15), and dynamic rearrangement of supercomplexes has been implicated in noncanonical mitochondrial functions such as antibacterial innate immune responses (16). Assessing these interactions is further complicated by regulatory posttranslational modification and conformational changes of mitochondrial proteins (17)(18)(19)(20). Advances in this area have been impeded, in part, by the lack of large-scale detection of dynamic, sometimes transient, interactions between membrane proteins. Thus, large-scale determination of the protein interactome within mitochondria would provide a valuable tool to adv...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

Schweppe

Chavez

Lee

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

182

206

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“…Of particular mention is outer membrane protein A (OmpA) which has been suggested to have a variety of functions, including adhesion to host epithelial cells, biofilm function, and complement resistance (100). In a recent lethal model of A. baumannii pneumonia, transposon-mediated disruption of OmpA reduced mortality in small numbers of mice, suggesting a virulence function of the OmpA (100).…”

Section: Acinetobacter Virulence Factorsmentioning

confidence: 99%

Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges

et al. 2017

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SUMMARY Acinetobacter is a complex genus, and historically, there has been confusion about the existence of multiple species. The species commonly cause nosocomial infections, predominantly aspiration pneumonia and catheter-associated bacteremia, but can also cause soft tissue and urinary tract infections. Community-acquired infections by Acinetobacter spp. are increasingly reported. Transmission of Acinetobacter and subsequent disease is facilitated by the organism's environmental tenacity, resistance to desiccation, and evasion of host immunity. The virulence properties demonstrated by Acinetobacter spp. primarily stem from evasion of rapid clearance by the innate immune system, effectively enabling high bacterial density that triggers lipopolysaccharide (LPS)–Toll-like receptor 4 (TLR4)-mediated sepsis. Capsular polysaccharide is a critical virulence factor that enables immune evasion, while LPS triggers septic shock. However, the primary driver of clinical outcome is antibiotic resistance. Administration of initially effective therapy is key to improving survival, reducing 30-day mortality threefold. Regrettably, due to the high frequency of this organism having an extreme drug resistance (XDR) phenotype, early initiation of effective therapy is a major clinical challenge. Given its high rate of antibiotic resistance and abysmal outcomes (up to 70% mortality rate from infections caused by XDR strains in some case series), new preventative and therapeutic options for Acinetobacter spp. are desperately needed.

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“…PIR-derived cross-linked sites identified between or within proteins are then used as distance constraints to develop structural models that enable visualization of proteins and protein complexes, as they exist in cells during cross-linker application (19,23,24). A recent study using in vivo PIR application confirmed OmpA homodimer existence and interfacial regions that were originally identified in bacterial cells by traditional MS methods (25), demonstrating the ability to derive meaningful structural information from this experimental approach.…”

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confidence: 80%

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

DeBlasio

Chavez

Alexander

et al. 2016

J Virol

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Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection-hallmarks of host-pathogen interactions-were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCEThe exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy. P hloem-limited viruses, such as poleroviruses, are among the most devastating, but least understood pathogens infecting plants due to the technical challenges encountered when studying pathogens that naturally replicate and move within intractable cell types (1). Mutagenesis studies performed with infectious clones have greatly enhanced our understanding of the domains within viral proteins that are re...

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Host-Microbe Protein Interactions during Bacterial Infection

Cited by 110 publications

References 85 publications

Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

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