Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping

Abstract

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases. Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases. Respiratory tract infections (RTIs) 1The abbreviations used are:RTIrespiratory tract infectionAMRantimicrobial resistanceTCUPtyping and characterization of bacteria using bottom-up tandem MS proteomicsHRAMhigh-resolution accurate-massLPIlipid-based protein immobilizationANIaverage nucleotide identityBLASTBasic Local Alignment Search ToolSDCsodium deoxycholateNCBINational Center for Biotechnology InformationPRMparallel reaction monitoringSRMselected reaction monitoringMRMmultiple reaction monitoring. 1The abbreviations used are:RTIrespiratory tract infectionAMRantimicrobial resistanceTCUPtyping and characterization of bacteria using bottom-up tandem MS proteomicsHRAMhigh-resolution accurate-massLPIlipid-based protein immobilizationANIaverage nucleotide identityBLASTBasic Local Alignment Search ToolSDCsodium deoxycholateNCBINational Center for Biotechnology InformationPRMparallel reaction monitoringSRMselected reaction monitoringMRMmultiple reaction monitoring. are a major reason for hospital admissions and are often treated with antibiotics (1Kronman M.P. Zhou C. Mangione-Smith R. Bacterial prevalence and antimicrobial prescribing trends for acute respiratory tract infections.Pediatrics. 2014; 134: e956-e965Crossref PubMed Scopus (102) Google Scholar). Today, a clinical assessment performed by the physician, is mainly based on symptoms, together with supporting clinical laboratory microbiological confirmation (2van Houten C.B. de Groot J.A.H. Klein A. Srugo I. Chistyakov I. de Waal W. Meijssen C.B. Avis W. Wolfs T.F.W. Shachor-Meyouhas Y. Stein M. Sanders E.A.M. Bont L.J. A host-protein based assay to differentiate between bacterial and viral infections in preschool children (OPPORTUNITY): a double-blind, multicentre, validation study.Lancet Infect. Dis. 2017; 17: 431-440Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Microbiological characterization of a clinical sample traditionally relies on cultivation of bacteria, which not only takes precious time, but in many cases is inconclusive because of the difficulty to recover viable bacteria. For example, in only ∼50% of the cases, are Streptococcus pneumoniae, a responsible agent for pneumococcal infections, recovered by culturing (3Song J.Y. Eun B.W. Nahm M.H. Diagnosis of pneumococcal pneumonia: current pitfalls and the way forward.Infect. Chemother. 2013; 45: 351-366Crossref PubMed Scopus (52) Google Scholar). Because bacterial infection can lead rapidly to invasive life-threatening situations, physicians may prescribe broad-spectrum antibiotics before knowing whether the infection is caused by bacteria or virus. Overuse of broad-spectrum antibiotics is a significant contributor to the emergence of anti-microbial resistance (AMR). One of the key counter-measures in the battle against AMR will be the development of improved, rapid, accurate and comprehensive diagnostic methods. respiratory tract infection antimicrobial resistance typing and characterization of bacteria using bottom-up tandem MS proteomics high-resolution accurate-mass lipid-based protein immobilization average nucleotide identity Basic Local Alignment Search Tool sodium deoxycholate National Center for Biotechnology Information parallel reaction monitoring selected reaction monitoring multiple reaction monitoring. respiratory tract infection antimicrobial resistance typing and characterization of bacteria using bottom-up tandem MS proteomics high-resolution accurate-mass lipid-based protein immobilization average nucleotide identity Basic Local Alignment Search Tool sodium deoxycholate National Center for Biotechnology Information parallel reaction monitoring selected reaction monitoring multiple reaction monitoring. DNA-based diagnostic approaches, such as real-time polymerase chain reaction (RT-PCR) is currently implemented in the routine protocols of the clinical microbiology laboratory and whole-genome sequencing is increasingly applied. However, PCR is a targeted approach and, thus, detects and identifies only the known and selected targets, which can lead to biased results and insufficient species resolution and characterization. One example is in the differentiation of closely related species within the Mitis Group of the genus Streptococcus, using PCR-based analyses of house-keeping genes or virulence factors (4Johnston C. Hinds J. Smith A. van der Linden M. Van Eldere J. Mitchell T.J. Detection of large numbers of pneumococcal virulence genes in streptococci of the mitis group.J. Clin. Microbiol. 2010; 48: 2762-2769Crossref PubMed Scopus (58) Google Scholar, 5Rolo D.A.S.S Domenech A. Fenoll A. Linares J. de Lencastre H. Ardanuy C. Sa-Leao R. Disease isolates of Streptococcus pseudopneumoniae and non-typeable S. pneumoniae presumptively identified as atypical S. pneumoniae in Spain.PloS One. 2013; 8: e57047Crossref PubMed Scopus (35) Google Scholar, 6Simoes A.S. Sa-Leao R. Eleveld M.J. Tavares D.A. Carrico J.A. Bootsma H.J. Hermans P.W. Highly penicillin-resistant multidrug-resistant pneumococcus-like strains colonizing children in Oeiras, Portugal: genomic characteristics and implications for surveillance.J. Clin. Microbiol. 2010; 48: 238-246Crossref PubMed Scopus (30) Google Scholar). Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) MS-based microbial species identification has emerged as an alternative to traditional phenotypic- or genotypic-based methods (7Erhard M. von Dohren H. Jungblut P. Rapid typing and elucidation of new secondary metabolites of intact cyanobacteria using MALDI-TOF mass spectrometry.Nat. 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