MALDI-TOF MS Profiling-Advances in Species Identification of Pests, Parasites, and Vectors

Invertebrate pests and parasites of humans, animals, and plants continue to cause serious diseases and remain as a high treat to agricultural productivity and storage. The rapid and accurate species identification of the pests and parasites are needed for understanding epidemiology, monitoring outbreaks, and designing control measures. Matrix-assisted laser desorption ionization time- of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as a rapid, cost effective, and high throughput technique of microbial species identification in modern diagnostic laboratories. The development of soft ionization techniques and the release of commercial pattern matching software platforms has resulted in the exponential growth of applications in higher organisms including parasitology. The present review discusses the proof-of- principle experiments and various methods of MALDI MS profiling in rapid species identification of both laboratory and field isolates of pests, parasites and vectors

Isolate Specific Cold Response of Yersinia enterocolitica in Transcriptional, Proteomic, and Membrane Physiological Changes

Yersinia enterocolitica, a zoonotic foodborne pathogen, is able to withstand low temperatures. This psychrotrophic ability allows it to multiply in food stored in refrigerators. However, little is known about the Y. enterocolitica cold response. In this study, isolate-specific behavior at 4°C was demonstrated and the cold response was investigated by examining changes in phenotype, gene expression, and the proteome. Altered expression of cold-responsive genes showed that the ability to survive at low temperature depends on the capacity to acclimate and adapt to cold stress. This cold acclimation at the transcriptional level involves the transient induction and effective repression of cold-shock protein (Csp) genes. Moreover, the resumption of expression of genes encoding other non-Csp is essential during prolonged adaptation. Based on proteomic analyses, the predominant functional categories of cold-responsive proteins are associated with protein synthesis, cell membrane structure, and cell motility. In addition, changes in membrane fluidity and motility were shown to be important in the cold response of Y. enterocolitica. Isolate-specific differences in the transcription of membrane fluidity- and motility-related genes provided evidence to classify strains within a spectrum of cold response. The combination of different approaches has permitted the systematic description of the Y. enterocolitica cold response and gives a better understanding of the physiological processes underlying this phenomenon

Mass spectrometry data from label-free quantitative proteomic analysis of harmless and pathogenic strains of infectious microalgae, Prototheca spp

Here, we provide the dataset associated with our research article ‘label-free quantitative proteomic analysis of harmless and pathogenic strains of infectious microalgae, Prototheca spp.’ (Murugaiyan et al., 2017) [1]. This dataset describes liquid chromatography–mass spectrometry (LC–MS)-based protein identification and quantification of a non-infectious strain, Prototheca zopfii genotype 1 and two strains associated with severe and mild infections, respectively, P. zopfii genotype 2 and Prototheca blaschkeae. Protein identification and label-free quantification was carried out by analysing MS raw data using the MaxQuant-Andromeda software suit. The expressional level differences of the identified proteins among the strains were computed using Perseus software and the results were presented in [1]. This DiB provides the MaxQuant output file and raw data deposited in the PRIDE repository with the dataset identifier PXD005305

Proteomics of Brucella: Technologies and Their Applications for Basic Research and Medical Microbiology

Brucellosis is a global zoonosis caused by Gram-negative, facultative intracellular bacteria of the genus Brucella (B.). Proteomics has been used to investigate a few B. melitensis and B. abortus strains, but data for other species and biovars are limited. Hence, a comprehensive analysis of proteomes will significantly contribute to understanding the enigmatic biology of brucellae. For direct identification and typing of Brucella, matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI—TOF MS) has become a reliable tool for routine diagnosis due to its ease of handling, price and sensitivity highlighting the potential of proteome-based techniques. Proteome analysis will also help to overcome the historic but still notorious Brucella obstacles of infection medicine, the lack of safe and protective vaccines and sensitive serologic diagnostic tools by identifying the most efficient protein antigens. This perspective summarizes past and recent developments in Brucella proteomics with a focus on species identification and serodiagnosis. Future applications of proteomics in these fields are discussed

Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross- reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI—TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae

Subclinical pulmonary pathogenic infection in camels slaughtered in Cairo, Egypt

Introduction: Camels migrate between the open boundaries of Sudan and Egypt either for grazing or for slaughtering. Bad hygiene and stress is often related to pulmonary diseases in camels. This study investigated whether camels slaughtered in Cairo carried pulmonary infections. Methodology: Five hundred lung tissues of slaughtered camels were examined and 100 samples suspected for pulmonary infection were subjected to microbial identification and histopathology. Results: A total of 70 lung tissues revealed 97 bacterial isolates of 8 species, including Staphylococcus aureus (37.14%), Escherichia coli (27.14%), Klebsiella pneumoniae (26.71%), Bacillus spp. (25.72%), Streptococcus pyogenes (10%), Corynebacterium spp. (8.85 %), Pasteurella spp. (2.85%), and Arcanobacterium pyogenes (1.4%). Some of these species were earlier reported to be associated with pulmonary infection. Histopathology revealed different types of pneumonia in 50% of the investigated lungs. Conclusions: A considerable number of apparently healthy camels carry pathogenic agents in their lower respiratory tracts. Immunosuppression and stressful conditions might influence these pathogens to induce respiratory diseases in camels. Thus, the infected camels might act as reservoir of these infections agents. If adequate care is not taken, this might be a threat to abattoir workers and may spread infections to humans

Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix- Assisted Laser Desorption/Ionization Mass Spectrometry

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology

Nematode species identification – Current status, challenges and future perspectives for cyathostomin

Human and animal health is globally affected by a variety of parasitic helminths. The impact of co-infections and development of anthelmintic resistance requires improved diagnostic tools, especially for parasitic nematodes e.g., to identify resistant species or attribute pathological effects to individual species or particular species combinations. In horses, co-infection with cyathostomins is rather a rule than an exception with typically 5 to 15 species (out of more than 40 described) per individual host. In cyathostomins, reliable morphological species differentiation is currently limited to adults and requires highly specialized expertize while precise morphological identification of eggs and early stage larvae is impossible. The situation is further complicated by a questionable validity of some cyathostomins while others might actually represent cryptic species complexes. Several molecular methods using different target sequences were established to overcome these limitations. For adult worms, PCR followed by sequencing of mitochondrial genes or external or internal ribosomal RNA spacers is suitable to genetically confirm morphological identifications. The most commonly used method to differentiate eggs or larvae is the reverse-line-blot hybridization assay. However, both methods suffer from the fact that target sequences are not available for many species or even that GenBank® entries are unreliable regarding the cyathostomin species. Recent advances in proteomic tools for identification of metazoans including insects and nematodes of the genus Trichinella will be evaluated for suitability to diagnose cyathostomins. Future research should focus on the comparative analysis of morphological, molecular and proteomic data from the same cyathostomin specimen to optimize tools for species-specific identification

Pan-Proteomic Analysis and Elucidation of Protein Abundance among the Closely Related Brucella Species, Brucella abortus and Brucella melitensis

Brucellosis is a zoonotic infection caused by bacteria of the genus Brucella. The species, B. abortus and B. melitensis, major causative agents of human brucellosis, share remarkably similar genomes, but they differ in their natural hosts, phenotype, antigenic, immunogenic, proteomic and metabolomic properties. In the present study, label-free quantitative proteomic analysis was applied to investigate protein expression level differences. Type strains and field strains were each cultured six times, cells were harvested at a midlogarithmic growth phase and proteins were extracted. Following trypsin digestion, the peptides were desalted, separated by reverse-phase nanoLC, ionized using electrospray ionization and transferred into an linear trap quadrapole (LTQ) Orbitrap Velos mass spectrometer to record full scan MS spectra (m/z 300–1700) and tandem mass spectrometry (MS/MS) spectra of the 20 most intense ions. Database matching with the reference proteomes resulted in the identification of 826 proteins. The Cluster of Gene Ontologies of the identified proteins revealed differences in bimolecular transport and protein synthesis mechanisms between these two strains. Among several other proteins, antifreeze proteins, Omp10, superoxide dismutase and 30S ribosomal protein S14 were predicted as potential virulence factors among the proteins differentially expressed. All mass spectrometry data are available via ProteomeXchange with identifier PXD006348

Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two- dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan- Brucella, B. abortus- and B. melitensis-specific antibodies