Nikolay Stoynov

Immunoproteomic Discovery of Novel T Cell Antigens from the Obligate Intracellular Pathogen Chlamydia

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-Ab) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.

Quantitative Proteomic Analysis of Type III Secretome of Enteropathogenic Escherichia coli Reveals an Expanded Effector Repertoire for Attaching/Effacing Bacterial Pathogens

Type III secretion systems are central to the pathogenesis and virulence of many important Gram-negative bacterial pathogens, and elucidation of the secretion mechanism and identification of the secreted substrates are critical to our understanding of their pathogenic mechanisms and developing potential therapeutics. Stable isotope labeling with amino acids in cell culture-based mass spectrometry is a quantitative and highly sensitive proteomics tool that we have previously used to successfully analyze the type III secretomes of Citrobacter rodentium and Salmonella enterica serovar Typhimurium. In this report, stable isotope labeling with amino acids in cell culture was used to analyze the type III secretome of enteropathogenic Escherichia coli (EPEC), an important human pathogen, which, together with enterohemorrhagic E. coli and C. rodentium, represents the family of attaching and effacing bacterial pathogens. We not only confirmed all 25 known EPEC type III-secreted proteins and effectors previously identified by conventional molecular and bioinformatical techniques but also identified several new type III-secreted proteins, including two novel effectors, C_0814/NleJ and LifA, that were shown to be translocated into host cells. LifA is a known virulence factor believed to act as a toxin as well as an adhesin, but its mechanism of secretion and function is not understood. With a predicted molecular mass of 366 kDa, LifA is the largest type III effector identified thus far in any pathogen. We further demonstrated that Efa1, ToxB, and Z4332 (homologs of LifA in enterohemorrhagic E. coli) are also type III effectors. This study has comprehensively characterized the type III secretome of EPEC, expanded the repertoire of type III-secreted effectors for the attaching and effacing pathogens, and provided new insights into the mode of function for LifA/Efa1/ToxB/Z4332, an important family of virulence factors.

Mapping the protein interaction network in methicillin-resistant Staphylococcus aureus.

Mortality attributable to infection with methicillin-resistant Staphylococcus aureus (MRSA) has now overtaken the death rate for AIDS in the United States, and advances in research are urgently needed to address this challenge. We report the results of the systematic identification of protein-protein interactions for the hospital-acquired strain MRSA-252. Using a high-throughput pull-down strategy combined with quantitative proteomics to distinguish specific from nonspecific interactors, we identified 13,219 interactions involving 608 MRSA proteins. Consecutive analyses revealed that this protein interaction network (PIN) exhibits scale-free organization with the characteristic presence of highly connected hub proteins. When clinical and experimental antimicrobial targets were queried in the network, they were generally found to occupy peripheral positions in the PIN with relatively few interacting partners. In contrast, the hub proteins identified in this MRSA PIN that are essential for network integrity and stability have largely been overlooked as drug targets. Thus, this empirical MRSA-252 PIN provides a rich source for identifying critical proteins essential for network stability, many of which can be considered as prospective antimicrobial drug targets.

Biomarkers for Severity of Spinal Cord Injury in the Cerebrospinal Fluid of Rats

One of the major challenges in management of spinal cord injury (SCI) is that the assessment of injury severity is often imprecise. Identification of reliable, easily quantifiable biomarkers that delineate the severity of the initial injury and that have prognostic value for the degree of functional recovery would significantly aid the clinician in the choice of potential treatments. To find such biomarkers we performed quantitative liquid chromatography-mass spectrometry (LC-MS/MS) analyses of cerebrospinal fluid (CSF) collected from rats 24 h after either a moderate or severe SCI. We identified a panel of 42 putative biomarkers of SCI, 10 of which represent potential biomarkers of SCI severity. Three of the candidate biomarkers, Ywhaz, Itih4, and Gpx3 were also validated by Western blot in a biological replicate of the injury. The putative biomarkers identified in this study may potentially be a valuable tool in the assessment of the extent of spinal cord damage.

Quantitative Mass Spectrometry Catalogues Salmonella Pathogenicity Island-2 Effectors and Identifies Their Cognate Host Binding Partners

Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.

Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click–SILAC approach

Messenger RNA-binding translational regulatory proteins determine in large part the spectrum of transcripts that are translated under specific cellular contexts. Y-box binding protein-1 (YB-1) is a conserved eukaryotic translational regulator that is implicated in cancer progression. To identify specific proteins that are translationally regulated by YB-1, we established a pulse-labelling approach combining Click chemistry and stable isotope labelling by amino acids in cell culture (SILAC). The proteome of TC32 human Ewing sarcoma cells, which robustly express YB-1, was compared with or without YB-1 siRNA knockdown. Cells labelled with light or heavy isotopologs of Arg and Lys were then cotranslationally pulsed with the methionine derivative, azidohomoalanine (AHA). Cells were lysed and newly synthesized proteins were selectively derivatized via a Click (3+2 cycloaddition) reaction to add an alkyne biotin tag. They were then affinity purified and subjected to liquid chromatography-tandem mass spectrometry. This combined Click-SILAC approach enabled us to catalog and quantify newly synthesized proteins regulated by YB-1 after only 45 min of labelling. Bioinformatic analysis revealed that YB-1 regulated proteins are involved in diverse biological pathways. We anticipate that this Click-SILAC strategy will be useful for studying short-term protein synthesis in different cell culture systems and under diverse biological contexts.

A search for protein biomarkers links olfactory signal transduction to social immunity

BackgroundThe Western honey bee (Apis mellifera L.) is a critical component of human agriculture through its pollination activities. For years, beekeepers have controlled deadly pathogens such as Paenibacillus larvae, Nosema spp. and Varroa destructor with antibiotics and pesticides but widespread chemical resistance is appearing and most beekeepers would prefer to eliminate or reduce the use of in-hive chemicals. While such treatments are likely to still be needed, an alternate management strategy is to identify and select bees with heritable traits that allow them to resist mites and diseases. Breeding such bees is difficult as the tests involved to identify disease-resistance are complicated, time-consuming, expensive and can misidentify desirable genotypes. Additionally, we do not yet fully understand the mechanisms behind social immunity. Here we have set out to discover the molecular mechanism behind hygienic behavior (HB), a trait known to confer disease resistance in bees.ResultsAfter confirming that HB could be selectively bred for, we correlated measurements of this behavior with protein expression over a period of three years, at two geographically distinct sites, using several hundred bee colonies. By correlating the expression patterns of individual proteins with HB scores, we identified seven putative biomarkers of HB that survived stringent control for multiple hypothesis testing. Intriguingly, these proteins were all involved in semiochemical sensing (odorant binding proteins), nerve signal transmission or signal decay, indicative of the series of events required to respond to an olfactory signal from dead or diseased larvae. We then used recombinant versions of two odorant-binding proteins to identify the classes of ligands that these proteins might be helping bees detect.ConclusionsOur data suggest that neurosensory detection of odors emitted by dead or diseased larvae is the likely mechanism behind a complex and important social immunity behavior that allows bees to co-exist with pathogens.

Studies with plant cell cultures of Podophyllum peltatum L. II: Biotransformation of dibenzylbutanolides to lignans. Development of a biological factory' for lignan synthesis

An efficient and versatile synthetic route to appropriate dibenzylbutanolides suitable for biotransformation to lignans as potential intermediates for the syntheses of the clinical anti-cancer drug etoposide has been developed. Biotransformation of such substrates, for example 10, with cell cultures of Podophyllum peltatum affords a potentially exciting route to this drug. Of particular significance is the development of a semi-continuous fermentation process with these plant cells wherein successive additions of substrate and isolation of end products can be pursued with a given batch of cells and over a period of several months

Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A

BackgroundThe pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in individuals suffering from HIV/AIDS. The cyclic-AMP/protein kinase A (PKA) signal transduction pathway regulates the production of extracellular virulence factors in C. neoformans, but the influence of the pathway on the secretome has not been investigated. In this study, we performed quantitative proteomics using galactose-inducible and glucose-repressible expression of the PKA1 gene encoding the catalytic subunit of PKA to identify regulated proteins in the secretome.MethodsThe proteins in the supernatants of cultures of C. neoformans were precipitated and identified using liquid chromatography-coupled tandem mass spectrometry. We also employed multiple reaction monitoring in a targeted approach to identify fungal proteins in samples from macrophages after phagocytosis of C. neoformans cells, as well as from the blood and bronchoalveolar fluid of infected mice.ResultsWe identified 61 secreted proteins and found that changes in PKA1 expression influenced the extracellular abundance of five proteins, including the Cig1 and Aph1 proteins with known roles in virulence. We also observed a change in the secretome profile upon induction of Pka1 from proteins primarily involved in catabolic and metabolic processes to an expanded set that included proteins for translational regulation and the response to stress. We further characterized the secretome data using enrichment analysis and by predicting conventional versus non-conventional secretion. Targeted proteomics of the Pka1-regulated proteins allowed us to identify the secreted proteins in lysates of phagocytic cells containing C. neoformans, and in samples from infected mice. This analysis also revealed that modulation of PKA1 expression influences the intracellular survival of cryptococcal cells upon phagocytosis.ConclusionsOverall, we found that the cAMP/PKA pathway regulates specific components of the secretome including proteins that affect the virulence of C. neoformans. The detection of secreted cryptococcal proteins from infected phagocytic cells and tissue samples suggests their potential utility as biomarkers of infection. The proteomics data are available via ProteomeXchange with identifiers PXD002731 and PASS00736.

Establishment of Camellia sinensis cell culture with high peroxidase activity and oxidative coupling reaction of dibenzylbutanolides

Abstract We succeeded in establishment of Camellia sinensis cell culture with high peroxidase activity. When dibenzylbutanolides ( 1a – c ) were reacted with C. sinensis cell culture, peroxidase-catalyzed oxidative coupling of 1a – c to cyclic products ( 2a – c ) proceeded quantitatively in the absence of foreign hydrogen peroxide as cofactor.