Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections.

Neutrophils have an important role in the antimicrobial defense and resolution of urinary tract infections (UTIs). Our research suggests that a mechanism known as neutrophil extracellular trap (NET) formation is a defense strategy to combat pathogens that have invaded the urinary tract. A set of human urine specimens with very high neutrophil counts had microscopic evidence of cellular aggregation and lysis. Deoxyribonuclease I (DNase) treatment resulted in disaggregation of such structures, release of DNA fragments and a proteome enriched in histones and azurophilic granule effectors whose quantitative composition was similar to that of previously described in vitro-formed NETs. The effector proteins were further enriched in DNA-protein complexes isolated in native PAGE gels. Immunofluorescence microscopy revealed a flattened morphology of neutrophils associated with decondensed chromatin, remnants of granules in the cell periphery, and myeloperoxidase co-localized with extracellular DNA, features consistent with early-phase NETs. Nuclear staining revealed that a considerable fraction of bacterial cells in these structures were dead. The proteomes of two pathogens, Staphylococcus aureus and Escherichia coli, were indicative of adaptive responses to early-phase NETs, specifically the release of virulence factors and arrest of ribosomal protein synthesis. Finally, we discovered patterns of proteolysis consistent with widespread cleavage of proteins by neutrophil elastase, proteinase 3 and cathepsin G and evidence of citrullination in many nuclear proteins.

A second-generation protein-protein interaction network of Helicobacter pylori.

Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein-protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.

Diagnosing inflammation and infection in the urinary system via proteomics.

BACKGROUND: Current methodology for the diagnosis of diseases in the urinary system includes patient symptomology, urine analysis and urine culture. Asymptomatic bacteriuria from urethral colonization or indwelling catheters, sample contamination from perineal or vaginal sources, and non-infectious inflammatory conditions can mimic UTIs, leading to uncertainty on medical treatment decisions. METHODS: Innovative shotgun metaproteomic methods were used to analyze urine sediments from 120 patients also subjected to conventional urinalysis for various clinical reasons including suspected UTIs. The proteomic data were simultaneously searched for the presence of microbial agents, inflammation, immune responses against pathogens, and evidence of urothelial tissue injury. Hierarchical clustering analysis was performed to identify host protein patterns discerning UTI from urethral colonization and vaginal contamination of urine samples. RESULTS: Organisms causing more than 98% of all UTIs and commensal microbes of the urogenital and perineal area were identified from 76 urine sediments with detection sensitivities estimated to be similar to urine culture. Proteomic data permitted a thorough evaluation of inflammatory and antimicrobial immune responses. Hierarchical clustering of the data revealed that high abundances of proteins from activated neutrophils were associated with pathogens in most cases, and correlated well with leukocyte esterase activities and leukocyte counts via microscopy. Proteomic data also allowed assessments of urothelial injury, by quantifying proteins highly expressed in red blood cells and contributing to the acute phase response. Lactobacillus and Gardnerella vaginalis were frequently identified suggesting urethral colonization and/or vaginal contamination of urine. CONCLUSIONS: A metaproteomic approach of interest for routine urine clinical diagnostics is presented. As compared to urinalysis and urine culture methods, the data are derived from a single experiment for a given sample and provide additional insights into presence or absence of inflammatory responses and vaginal contamination of urine specimens.

The Rbf1, Hfl1 and Dbp4 of Candida albicans regulate common as well as transcription factor-specific mitochondrial and other cell activities.

BACKGROUND: Our interest in Candida albicans mitochondria began with the identification of GOA1. We demonstrated its role in cell energy production, cross-talk among mitochondria and peroxisomes, non-glucose energy metabolism, maintenance of stationary phase growth, and prevention of premature apoptosis. Its absence results in avirulence. However, what regulated transcription of GOA1 was unknown. RESULTS: To identify transcriptional regulators (TRs) of GOA1, we screened a C. albicans TF knockout library (TRKO) and identified Rbf1p, Hfl1p, and Dpb4p as positive TRs of GOA1. The phenotypes of each mutant (reduced respiration, inability to grow on glycerol, reduced ETC CI and CIV activities) are reasonable evidence for their required roles especially in mitochondrial functions. While the integration of mitochondria with cell metabolic activities is presumed to occur, there is minimal information on this subject at the genome level. Therefore, microarray analysis was used to provide this information for each TR mutant. Transcriptional profiles of Rbf1p and Hfl1p are more similar than that of Dpn4p. Our data demonstrate common and also gene-specific regulatory functions for each TR. We establish their roles in carbon metabolism, stress adaptation, cell wall synthesis, transporter efflux, peroxisomal metabolism, phospholipid synthesis, rRNA processing, and nuclear/mtDNA replication. CONCLUSIONS: The TRs regulate a number of common genes but each also regulates specific gene transcription. These data for the first time create a genome roadmap that can be used to integrate mitochondria with other cell processes. Of interest, the TRs are fungal-specific, warranting consideration as antifungal drug targets.

Urine sample preparation in 96-well filter plates for quantitative clinical proteomics.

Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ~10 µg of total protein were processed by 96FASP and LC-MS resulting in 700-900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics.

Studying protein complexes by the yeast two-hybrid system.

Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

Fueling the fire of B cell activation.

Antigen activated naïve B cells undergoing germinal center responses have distinct metabolic requirements.

Serum metabolomics of treatment response in myasthenia gravis.

OBJECTIVE: High-dose prednisone use, lasting several months or longer, is the primary initial therapy for myasthenia gravis (MG). Upwards of a third of patients do not respond to treatment. Currently no biomarkers can predict clinical responsiveness to corticosteroid treatment. We conducted a discovery-based study to identify treatment responsive biomarkers in MG using sera obtained at study entry to the thymectomy clinical trial (MGTX), an NIH-sponsored randomized, controlled study of thymectomy plus prednisone versus prednisone alone. METHODS: We applied ultra-performance liquid chromatography coupled with electro-spray quadrupole time of flight mass spectrometry to obtain comparative serum metabolomic and lipidomic profiles at study entry to correlate with treatment response at 6 months. Treatment response was assessed using validated outcome measures of minimal manifestation status (MMS), MG-Activities of Daily Living (MG-ADL), Quantitative MG (QMG) score, or a strictly defined composite measure of response. RESULTS: Increased serum levels of phospholipids were associated with treatment response as assessed by QMG, MMS, and the Responders classification, but all measures showed limited overlap in metabolomic profiles, in particular the MG-ADL. A panel including histidine, free fatty acid (13:0), γ-cholestenol and guanosine was highly predictive of the strictly defined treatment response measure. The AUC in Responders' prediction for these markers was 0.90 irrespective of gender, age, thymectomy or baseline prednisone use. Pathway analysis suggests that xenobiotic metabolism could play a major role in treatment resistance. There was no association with outcome and gender, age, thymectomy or baseline prednisone use. INTERPRETATION: We have defined a metabolomic and lipidomic profile that can now undergo validation as a treatment predictive marker for MG patients undergoing corticosteroid therapy. Metabolomic profiles of outcome measures had limited overlap consistent with their assessing distinct aspects of treatment response and supporting unique biological underpinning for each outcome measure. Interindividual variation in prednisone metabolism may be a determinate of how well patients respond to treatment.

Enzymatic dysfunction of mitochondrial complex I of the Candida albicans goa1 mutant is associated with increased reactive oxidants and cell death.

We have previously shown that deletion of GOA1 (growth and oxidant adaptation) of Candida albicans results in a loss of mitochondrial membrane potential, ATP synthesis, increased sensitivity to oxidants and killing by human neutrophils, and avirulence in a systemic model of candidiasis. We established that translocation of Goa1p to mitochondria occurred during peroxide stress. In this report, we show that the goa1Δ (GOA31), compared to the wild type (WT) and a gene-reconstituted (GOA32) strain, exhibits sensitivity to inhibitors of the classical respiratory chain (CRC), including especially rotenone (complex I [CI]) and salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway (AOX), while potassium cyanide (KCN; CIV) causes a partial inhibition of respiration. In the presence of SHAM, however, GOA31 has an enhanced respiration, which we attribute to the parallel respiratory (PAR) pathway and alternative NADH dehydrogenases. Interestingly, deletion of GOA1 also results in a decrease in transcription of the alternative oxidase gene AOX1 in untreated cells as well as negligible AOX1 and AOX2 transcription in peroxide-treated cells. To explain the rotenone sensitivity, we measured enzyme activities of complexes I to IV (CI to CIV) and observed a major loss of CI activity in GOA31 but not in control strains. Enzymatic data of CI were supported by blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments which demonstrated less CI protein and reduced enzyme activity. The consequence of a defective CI in GOA31 is an increase in reactive oxidant species (ROS), loss of chronological aging, and programmed cell death ([PCD] apoptosis) in vitro compared to control strains. The increase in PCD was indicated by an increase in caspase activity and DNA fragmentation in GOA31. Thus, GOA1 is required for a functional CI and partially for the AOX pathway; loss of GOA1 compromises cell survival. Further, the loss of chronological aging is new to studies of Candida species and may offer an insight into therapies to control these pathogens. Our observation of increased ROS production associated with a defective CI and PCD is reminiscent of mitochondrial studies of patients with some types of neurodegenerative diseases where CI and/or CIII dysfunctions lead to increased ROS and apoptosis.

A Flow Cytometry-Based Assay to Measure C3b Deposition on Protozoan Parasites Such as Toxoplasma gondii.

Flow cytometry is a powerful tool that can be used to study protozoan parasite interactions with the complement system. We developed a flow cytometric assay to measure the deposition of complement activation product C3b and to assess resistance to complement-mediated lysis. This assay involves exposing cultured parasites to human serum (the source of human complement) and staining parasites with antibodies against complement proteins to detect and quantify complement components on the parasite surface by flow cytometry. The assay can be used to compare complement activation across a variety of different species of protozoan parasites. As a proof of concept, we describe protocols to study C3 deposition on the single-cell protist Toxoplasma gondii. This parasite actively regulates C3 deposition and proteolytic inactivation to eliminate the formation of lytic pores targeted to the parasite surface coat, which is the end-product of the complement cascade. The antibodies used in this assay recognize both active and inactive forms of C3 deposited on parasite surfaces. Hence, the assay facilitates the identification and characterization of parasite resistance factors that regulate complement deposition and catabolic inactivation. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Culturing human foreskin fibroblasts and Toxoplasma gondii strains Basic Protocol 2: In vitro complement activation assay Support Protocol: Screening of normal human serum Basic Protocol 3: Flow cytometric analysis of C3b deposition.

A Protective and Pathogenic Role for Complement During Acute Toxoplasma gondii Infection.

The infection competence of the protozoan pathogen Toxoplasma gondii is critically dependent on the parasite's ability to inactivate the host complement system. Toxoplasma actively resists complement-mediated killing in non-immune serum by recruiting host-derived complement regulatory proteins C4BP and Factor H (FH) to the parasite surface to inactivate surface-bound C3 and limit formation of the C5b-9 membrane attack complex (MAC). While decreased complement activation on the parasite surface certainly protects Toxoplasma from immediate lysis, the biological effector functions of C3 split products C3b and C3a are maintained, which includes opsonization of the parasite for phagocytosis and potent immunomodulatory effects that promote pro-inflammatory responses and alters mucosal defenses during infection, respectively. In this review, we discuss how complement regulation by Toxoplasma controls parasite burden systemically but drives exacerbated immune responses locally in the gut of genetically susceptible C57BL/6J mice. In effect, Toxoplasma has evolved to strike a balance with the complement system, by inactivating complement to protect the parasite from immediate serum killing, it generates sufficient C3 catabolites that signal through their cognate receptors to stimulate protective immunity. This regulation ultimately controls tachyzoite proliferation and promotes host survival, parasite persistence, and transmissibility to new hosts.

Urethral Catheter Biofilms Reveal Plasticity in Bacterial Composition and Metabolism and Withstand Host Immune Defenses in Hypoxic Environment.

Biofilms composed of multiple microorganisms colonize the surfaces of indwelling urethral catheters that are used serially by neurogenic bladder patients and cause chronic infections. Well-adapted pathogens in this niche are Escherichia coli, Proteus, and Enterococcus spp., species that cycle through adhesion and multilayered cell growth, trigger host immune responses, are starved off nutrients, and then disperse. Viable microbial foci retained in the urinary tract recolonize catheter surfaces. The molecular adaptations of bacteria in catheter biofilms (CBs) are not well-understood, promising new insights into this pathology based on host and microbial meta-omics analyses from clinical specimens. We examined catheters from nine neurogenic bladder patients longitudinally over up to 6 months. Taxonomic analyses from 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed that 95% of all catheter and corresponding urinary pellet (UP) samples contained bacteria. CB biomasses were dominated by Enterobacteriaceae spp. and often accompanied by lactic acid and anaerobic bacteria. Systemic antibiotic drug treatments of patients resulted in either transient or lasting microbial community perturbations. Neutrophil effector proteins were abundant not only in UP but also CB samples, indicating their penetration of biofilm surfaces. In the context of one patient who advanced to a kidney infection, Proteus mirabilis proteomic data suggested a combination of factors associated with this disease complication: CB biomasses were high; the bacteria produced urease alkalinizing the pH and triggering urinary salt deposition on luminal catheter surfaces; P. mirabilis utilized energy-producing respiratory systems more than in CBs from other patients. The NADH:quinone oxidoreductase II (Nqr), a Na+ translocating enzyme not operating as a proton pump, and the nitrate reductase A (Nar) equipped the pathogen with electron transport chains promoting growth under hypoxic conditions. Both P. mirabilis and E. coli featured repertoires of transition metal ion acquisition systems in response to human host-mediated iron and zinc sequestration. We discovered a new drug target, the Nqr respiratory system, whose deactivation may compromise P. mirabilis growth in a basic pH milieu. Animal models would not allow such molecular-level insights into polymicrobial biofilm metabolism and interactions because the complexity cannot be replicated.

Monoclonal Antibody-Based Therapies for Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune, neuromuscular disorder that produces disabling weakness through a compromise of neuromuscular transmission. The disease fulfills strict criteria of an antibody-mediated disease. Close to 90% of patients have antibodies directed towards the nicotinic acetylcholine receptor (AChR) on the post-synaptic surface of skeletal muscle and another 5% to the muscle-specific kinase, which is involved in concentrating the AChR to the muscle surface of the neuromuscular junction. Conventional treatments of intravenous immunoglobulin and plasma exchange reduce autoantibody levels to produce their therapeutic effect, while prednisone and immunosuppressives do so by moderating autoantibody production. None of these treatments were specifically developed for MG and have a range of adverse effects. The extensive advances in monoclonal antibody technology allowing specific modulation of biological pathways has led to a tremendous increase in the potential treatment options. For MG, monoclonal antibody therapeutics target the effector mechanism of complement inhibition and the reduction of antibody levels by FcRn inhibition. Antibodies directed against CD20 and signaling pathways, which support lymphocyte activity, have been used to reduce autoantibody production. Thus far, only eculizumab, an antibody against C5, has reached the clinic. We review the present status of monoclonal antibody-based treatments for MG that have entered human testing and offer the promise to transform treatment of MG.

Toxoplasma gondii Recruits Factor H and C4b-Binding Protein to Mediate Resistance to Serum Killing and Promote Parasite Persistence in vivo.

Regulating complement is an important step in the establishment of infection by microbial pathogens. Toxoplasma gondii actively resists complement-mediated killing in non-immune human serum (NHS) by inactivating C3b, however the precise molecular basis is unknown. Here, a flow cytometry-based C3b binding assay demonstrated that Type II strains had significantly higher levels of surface-bound C3b than Type I strains. However, both strains efficiently inactivated C3b and were equally resistant to serum killing, suggesting that resistance is not strain-dependent. Toxoplasma activated both the lectin (LP) and alternative (AP) pathways, and the deposition of C3b was both strain and lectin-dependent. A flow cytometry-based lectin binding assay identified strain-specific differences in the level and heterogeneity of surface glycans detected. Specifically, increased lectin-binding by Type II strains correlated with higher levels of the LP recognition receptor mannose binding lectin (MBL). Western blot analyses demonstrated that Toxoplasma recruits both classical pathway (CP) and LP regulator C4b-binding proteins (C4BP) and AP regulator Factor H (FH) to the parasite surface to inactivate bound C3b-iC3b and C3dg and limit formation of the C5b-9 attack complex. Blocking FH and C4BP contributed to increased C5b-9 formation in vitro. However, parasite susceptibility in vitro was only impacted when FH was blocked, indicating that down regulation of the alternative pathway by FH may be more critical for parasite resistance. Infection of C3 deficient mice led to uncontrolled parasite growth, acute mortality, and reduced antibody production, indicating that both the presence of C3, and the ability of the parasite to inactivate C3, was protective. Taken together, our results establish that Toxoplasma regulation of the complement system renders mice resistant to acute infection by limiting parasite proliferation in vivo, but susceptible to chronic infection, with all mice developing transmissible cysts to maintain its life cycle.

Comprehensive Metaproteomic Analyses of Urine in the Presence and Absence of Neutrophil-Associated Inflammation in the Urinary Tract.

Inflammation in the urinary tract results in a urinary proteome characterized by a high dynamic range of protein concentrations and high variability in protein content. This proteome encompasses plasma proteins not resorbed by renal tubular uptake, renal secretion products, proteins of immune cells and erythrocytes derived from trans-urothelial migration and vascular leakage, respectively, and exfoliating urothelial and squamous epithelial cells. We examined how such proteins partition into soluble urine (SU) and urinary pellet (UP) fractions by analyzing 33 urine specimens 12 of which were associated with a urinary tract infection (UTI). Using mass spectrometry-based metaproteomic approaches, we identified 5,327 non-redundant human proteins, 2,638 and 4,379 of which were associated with SU and UP fractions, respectively, and 1,206 non-redundant protein orthology groups derived from pathogenic and commensal organisms of the urogenital tract. Differences between the SU and UP proteomes were influenced by local inflammation, supported by respective comparisons with 12 healthy control urine proteomes. Clustering analyses showed that SU and UP fractions had proteomic signatures discerning UTIs, vascular injury, and epithelial cell exfoliation from the control group to varying degrees. Cases of UTI revealed clusters of proteins produced by activated neutrophils. Network analysis supported the central role of neutrophil effector proteins in the defense against invading pathogens associated with subsequent coagulation and wound repair processes. Our study expands the existing knowledge of the urinary proteome under perturbed conditions, and should be useful as reference dataset in the search of biomarkers.

The binary protein-protein interaction landscape of Escherichia coli.

Efforts to map the Escherichia coli interactome have identified several hundred macromolecular complexes, but direct binary protein-protein interactions (PPIs) have not been surveyed on a large scale. Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys (∼70% of the E. coli proteome) in duplicate to generate a map of 2,234 interactions, which approximately doubles the number of known binary PPIs in E. coli. Integration of binary PPI and genetic-interaction data revealed functional dependencies among components involved in cellular processes, including envelope integrity, flagellum assembly and protein quality control. Many of the binary interactions that we could map in multiprotein complexes were informative regarding internal topology of complexes and indicated that interactions in complexes are substantially more conserved than those interactions connecting different complexes. This resource will be useful for inferring bacterial gene function and provides a draft reference of the basic physical wiring network of this evolutionarily important model microbe.