Establishing Personalized Blood Protein Reference Ranges Using Noninvasive Microsampling and Targeted Proteomics: Implications for Antidoping Strategies.

To prevent doping practices in sports, the World Anti-Doping Agency implemented the Athlete Biological Passport (ABP) program, monitoring biological variables over time to indirectly reveal the effects of doping rather than detect the doping substance or the method itself. In the context of this program, a highly multiplexed mass spectrometry-based proteomics assay for 319 peptides corresponding to 250 proteins was developed, including proteins associated with blood-doping practices. "Baseline" expression profiles of these potential biomarkers in capillary blood (dried blood spots (DBS)) were established using multiple reaction monitoring (MRM). Combining DBS microsampling with highly multiplexed MRM assays is the best-suited technology to enhance the effectiveness of the ABP program, as it represents a cost-effective and robust alternative analytical method with high specificity and selectivity of targets in the attomole range. DBS data were collected from 10 healthy athlete volunteers over a period of 140 days (28 time points per participant). These comprehensive findings provide a personalized targeted blood proteome "fingerprint" showcasing that the targeted proteome is unique to an individual and likely comparable to a DNA fingerprint. The results can serve as a baseline for future studies investigating doping-related perturbations.

Syphilis and the host: multi-omic analysis of host cellular responses to Treponema pallidum provides novel insight into syphilis pathogenesis.

Introduction: Syphilis is a chronic, multi-stage infection caused by the extracellular bacterium Treponema pallidum ssp. pallidum. Treponema pallidum widely disseminates through the vasculature, crosses endothelial, blood-brain and placental barriers, and establishes systemic infection. Although the capacity of T. pallidum to traverse the endothelium is well-described, the response of endothelial cells to T. pallidum exposure, and the contribution of this response to treponemal traversal, is poorly understood. Methods: To address this knowledge gap, we used quantitative proteomics and cytokine profiling to characterize endothelial responses to T. pallidum. Results: Proteomic analyses detected altered host pathways controlling extracellular matrix organization, necroptosis and cell death, and innate immune signaling. Cytokine analyses of endothelial cells exposed to T. pallidum revealed increased secretion of interleukin (IL)-6, IL-8, and vascular endothelial growth factor (VEGF), and decreased secretion of monocyte chemoattractant protein-1 (MCP-1). Discussion: This study provides insight into the molecular basis of syphilis disease symptoms and the enhanced susceptibility of individuals infected with syphilis to HIV co-infection. These investigations also enhance understanding of the host response to T. pallidum exposure and the pathogenic strategies used by T. pallidum to disseminate and persist within the host. Furthermore, our findings highlight the critical need for inclusion of appropriate controls when conducting T. pallidum-host cell interactions using in vitro- and in vivo-grown T. pallidum.

Deep proteome coverage advances knowledge of Treponema pallidum protein expression profiles during infection.

Comprehensive proteome-wide analysis of the syphilis spirochete, Treponema pallidum ssp. pallidum, is technically challenging due to high sample complexity, difficulties with obtaining sufficient quantities of bacteria for analysis, and the inherent fragility of the T. pallidum cell envelope which further complicates proteomic identification of rare T. pallidum outer membrane proteins (OMPs). The main aim of the present study was to gain a deeper understanding of the T. pallidum global proteome expression profile under infection conditions. This will corroborate and extend genome annotations, identify protein modifications that are unable to be predicted at the genomic or transcriptomic levels, and provide a foundational knowledge of the T. pallidum protein expression repertoire. Here we describe the optimization of a T. pallidum-specific sample preparation workflow and mass spectrometry-based proteomics pipeline which allowed for the detection of 77% of the T. pallidum protein repertoire under infection conditions. When combined with prior studies, this brings the overall coverage of the T. pallidum proteome to almost 90%. These investigations identified 27 known/predicted OMPs, including potential vaccine candidates, and detected expression of 11 potential OMPs under infection conditions for the first time. The optimized pipeline provides a robust and reproducible workflow for investigating T. pallidum protein expression during infection. Importantly, the combined results provide the deepest coverage of the T. pallidum proteome to date.

Sample Preparation Methods for Targeted Single-Cell Proteomics.

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540-660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390-540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5-25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation.

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

An extracellular Leptospira interrogans leucine-rich repeat protein binds human E- and VE-cadherins.

Pathogenic Leptospira bacteria are the causative agents of leptospirosis, a zoonotic disease affecting animals and humans worldwide. These pathogenic species have the ability to rapidly cross host tissue barriers by a yet unknown mechanism. A comparative analysis of pathogens and saprophytes revealed a higher abundance of genes encoding proteins with leucine-rich repeat (LRR) domains in the genomes of pathogens. In other bacterial pathogens, proteins with LRR domains have been shown to be involved in mediating host cell attachment and invasion. One protein from the pathogenic species Leptospira interrogans, LIC10831, has been previously analysed via X-ray crystallography, with findings suggesting it may be an important bacterial adhesin. Herein we show that LIC10831 elicits an antibody response in infected animals, is actively secreted by the bacterium, and binds human E- and VE-cadherins. These results provide biochemical and cellular evidences of LRR protein-mediated host-pathogen interactions and identify a new multireceptor binding protein from this infectious Leptospira species.

Molecular phenotyping of laboratory mouse strains using 500 multiple reaction monitoring mass spectrometry plasma assays.

Mouse is the predominant experimental model for the study of human disease due, in part, to phylogenetic relationship, ease of breeding, and the availability of molecular tools for genetic manipulation. Advances in genome-editing methodologies, such as CRISPR-Cas9, enable the rapid production of new transgenic mouse strains, necessitating complementary high-throughput and systematic phenotyping technologies. In contrast to traditional protein phenotyping techniques, multiple reaction monitoring (MRM) mass spectrometry can be highly multiplexed without forgoing specificity or quantitative precision. Here we present MRM assays for the quantitation of 500 proteins and subsequently determine reference concentration values for plasma proteins across five laboratory mouse strains that are typically used in biomedical research, revealing inter-strain and intra-strain phenotypic differences. These 500 MRM assays will have a broad range of research applications including high-throughput phenotypic validation of novel transgenic mice, identification of candidate biomarkers, and general research applications requiring multiplexed and precise protein quantification.

Multiple Reaction Monitoring Using Double Isotopologue Peptide Standards for Protein Quantification.

Multiple reaction monitoring (MRM) is a technique used in tandem mass spectrometry where the first mass analyzer preselects parent ions for fragmentation and the second mass analyzer transmits selected product ions to the detector. This targeted technique has found widespread application in bottom-up proteomics for monitoring target peptides in a complex enzymatic digest. Quantitative MRM can be performed on enzymatically digested samples using spiked-in synthetic peptide standards, providing unsurpassed quantitative accuracy and a dynamic range of four orders of magnitude, often eliminating the need for prior depletion of high-abundance proteins. The development of MRM assays requires technical rigor, and this chapter details a methodology for sample preparation, data acquisition, and analyses to successfully perform quantitative MRM assays using two distinct isotopologue peptide standards to quantify proteins in mouse plasma and heart tissue.

Respiratory Microbiome of Endangered Southern Resident Killer Whales and Microbiota of Surrounding Sea Surface Microlayer in the Eastern North Pacific.

In the Salish Sea, the endangered Southern Resident Killer Whale (SRKW) is a high trophic indicator of ecosystem health. Three major threats have been identified for this population: reduced prey availability, anthropogenic contaminants, and marine vessel disturbances. These perturbations can culminate in significant morbidity and mortality, usually associated with secondary infections that have a predilection to the respiratory system. To characterize the composition of the respiratory microbiota and identify recognized pathogens of SRKW, exhaled breath samples were collected between 2006-2009 and analyzed for bacteria, fungi and viruses using (1) culture-dependent, targeted PCR-based methodologies and (2) taxonomically broad, non-culture dependent PCR-based methodologies. Results were compared with sea surface microlayer (SML) samples to characterize the respective microbial constituents. An array of bacteria and fungi in breath and SML samples were identified, as well as microorganisms that exhibited resistance to multiple antimicrobial agents. The SML microbes and respiratory microbiota carry a pathogenic risk which we propose as an additional, fourth putative stressor (pathogens), which may adversely impact the endangered SRKW population.

Diet-Induced Obesity Does Not Alter Tigecycline Treatment Efficacy in Murine Lyme Disease.

Obese individuals more frequently suffer from infections, as a result of increased susceptibility to a number of bacterial pathogens. Furthermore, obesity can alter antibiotic treatment efficacy due to changes in drug pharmacokinetics which can result in under-dosing. However, studies on the treatment of bacterial infections in the context of obesity are scarce. To address this research gap, we assessed efficacy of antibiotic treatment in diet-induced obese mice infected with the Lyme disease pathogen, Borrelia burgdorferi. Diet-induced obese C3H/HeN mice and normal-weight controls were infected with B. burgdorferi, and treated during the acute phase of infection with two doses of tigecycline, adjusted to the weights of diet-induced obese and normal-weight mice. Antibiotic treatment efficacy was assessed 1 month after the treatment by cultivating bacteria from tissues, measuring severity of Lyme carditis, and quantifying bacterial DNA clearance in ten tissues. In addition, B. burgdorferi-specific IgG production was monitored throughout the experiment. Tigecycline treatment was ineffective in reducing B. burgdorferi DNA copies in brain. However, diet-induced obesity did not affect antibiotic-dependent bacterial DNA clearance in any tissues, regardless of the tigecycline dose used for treatment. Production of B. burgdorferi-specific IgGs was delayed and attenuated in mock-treated diet-induced obese mice compared to mock-treated normal-weight animals, but did not differ among experimental groups following antibiotic treatment. No carditis or cultivatable B. burgdorferi were detected in any antibiotic-treated group. In conclusion, obesity was associated with attenuated and delayed humoral immune responses to B. burgdorferi, but did not affect efficacy of antibiotic treatment.

Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans.

Leptospira are emerging zoonotic pathogens transmitted from animals to humans typically through contaminated environmental sources of water and soil. Regulatory pathways of pathogenic Leptospira spp. underlying the adaptive response to different hosts and environmental conditions remains elusive. In this study, we provide the first global Transcriptional Start Site (TSS) map of a Leptospira species. RNA was obtained from the pathogen Leptospira interrogans grown at 30°C (optimal in vitro temperature) and 37°C (host temperature) and selectively enriched for 5' ends of native transcripts. A total of 2865 and 2866 primary TSS (pTSS) were predicted in the genome of L. interrogans at 30 and 37°C, respectively. The majority of the pTSSs were located between 0 and 10 nucleotides from the translational start site, suggesting that leaderless transcripts are a common feature of the leptospiral translational landscape. Comparative differential RNA-sequencing (dRNA-seq) analysis revealed conservation of most pTSS at 30 and 37°C. Promoter prediction algorithms allow the identification of the binding sites of the alternative sigma factor sigma 54. However, other motifs were not identified indicating that Leptospira consensus promoter sequences are inherently different from the Escherichia coli model. RNA sequencing also identified 277 and 226 putative small regulatory RNAs (sRNAs) at 30 and 37°C, respectively, including eight validated sRNAs by Northern blots. These results provide the first global view of TSS and the repertoire of sRNAs in L. interrogans. These data will establish a foundation for future experimental work on gene regulation under various environmental conditions including those in the host.

Infection with the Lyme disease pathogen suppresses innate immunity in mice with diet-induced obesity.

Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high-fat diet-induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil- and macrophage-based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi-infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high-fat diet, toll-like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow-derived macrophages from obese, B. burgdorferi-infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence.

Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model.

Pathogenic Leptospira interrogans exoproteins are primarily involved in heterotrophic processes.

Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.

Post-translational modification of LipL32 during Leptospira interrogans infection.

BACKGROUND: Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin. METHODOLOGY/PRINCIPAL FINDINGS: Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32. CONCLUSIONS/SIGNIFICANCE: The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira.

A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

High-temperature protein G is an essential virulence factor of Leptospira interrogans.

Leptospira interrogans is a global zoonotic pathogen and is the causative agent of leptospirosis, an endemic disease of humans and animals worldwide. There is limited understanding of leptospiral pathogenesis; therefore, further elucidation of the mechanisms involved would aid in vaccine development and the prevention of infection. HtpG (high-temperature protein G) is the bacterial homolog to the highly conserved molecular chaperone Hsp90 and is important in the stress responses of many bacteria. The specific role of HtpG, especially in bacterial pathogenesis, remains largely unknown. Through the use of an L. interrogans htpG transposon insertion mutant, this study demonstrates that L. interrogans HtpG is essential for virulence in the hamster model of acute leptospirosis. Complementation of the htpG mutant completely restored virulence. Surprisingly, the htpG mutant did not appear to show sensitivity to heat or oxidative stress, phenotypes common in htpG mutants in other bacterial species. Furthermore, the mutant did not show increased sensitivity to serum complement, reduced survival within macrophages, or altered protein or lipopolysaccharide expression. The underlying cause for attenuation thus remains unknown, but HtpG is a novel leptospiral virulence factor and one of only a very small number identified to date.