MALDI-TOF MS and 16S RNA Identification of Culturable Gastric Microbiota: Variability Associated with the Presence of Helicobacter pylori

Helicobacter pylori is the main bacteria associated with gastroduodenal diseases. Recent studies have reported that gastric microbiota might be modified by the H. pylori colonization, favoring gastric lesions′ development. In Chile, the region of La Araucanía concentrates a high risk of gastric cancer associated with Helicobacter pylori colonization, rurality, poverty, and Mapuche ethnicity. Hence, we aimed to identify the culturable gastric microbiota and characterize its variability at different stages of epithelial injury, based on its H. pylori colonization in dyspeptic patients from this Chilean region. Microaerophilic bacteria strains were isolated from antrum biopsies of 155 dyspeptic patients′ biopsies and identified using MALDI-TOF MS or 16sRNA gene sequencing for non-pylori species identification, and UreC gene amplification for H. pylori confirmation. We found 48 species from 18 families, mainly belonging to Neisseriaceae (21.3%), Streptococcaceae (20.0%), Actynomicetaceae (9.0%), Enterobacteriaceae, and Lactobacillaceae (4.5%); however, Streptococcaceae and Actinomycetaceae families showed a significant reduction in samples infected with H. pylori, along with a considerably lower diversity of species. Our results revealed a microbiota modification due to H. pylori colonization associated with the gastric epithelial state, suggesting a potential microbiota role for developing and progressing gastric diseases.

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A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1

10.1101/081331 ◽

2016 ◽

Author(s):

Rahul Mahadev Shelake ◽

Yuki Ito ◽

Junya Masumoto ◽

Eugene Hayato Morita ◽

Hidenori Hayashi

Keyword(s):

Helicobacter Pylori ◽

Metal Binding ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Metal Species ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Sds Page ◽

Gel Shift ◽

Tof Ms

AbstractSodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed “gel shifting” appears to be common for histidine-rich proteins but not yet studied in detail. We investigated “gel shifting” in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining “gel shifting” of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3-4 kDa) than apo-Hpn, phenomenon termed “metal gel-shift” demonstrating an intimate link between Ni2+ binding and “gel shifting”. To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66±0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact "metal-gel shift" band on SDS-PAGE. In view of presented data metal-binding and “metal-gel shift” models are discussed.

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Differentiation of Gastric Helicobacter Species Using MALDI-TOF Mass Spectrometry

Pathogens ◽

10.3390/pathogens10030366 ◽

2021 ◽

Vol 10 (3) ◽

pp. 366

Author(s):

Helena Berlamont ◽

Chloë De Witte ◽

Sofie De Bruyckere ◽

James G. Fox ◽

Steffen Backert ◽

...

Keyword(s):

Species Identification ◽

Growth Conditions ◽

Reference Database ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Animal Pathogens ◽

H Pylori ◽

Specific Species ◽

Individual Mass ◽

Tof Ms

Gastric helicobacters (Helicobacter (H.) pylori and non-H. pylori Helicobacter species (NHPHs)) colonize the stomach of humans and/or animals. Helicobacter species identification is essential since many of them are recognized as human and/or animal pathogens. Currently, Helicobacter species can only be differentiated using molecular methods. Differentiation between NHPHs using MALDI-TOF MS has not been described before, probably because these species are poorly represented in current MALDI-TOF MS databases. Therefore, we identified 93 gastric Helicobacter isolates of 10 different Helicobacter species using MALDI-TOF MS in order to establish a more elaborate Helicobacter reference database. While the MALDI Biotyper database was not able to correctly identify any of the isolates, the in-house database correctly identified all individual mass spectra and resulted in 82% correct species identification based on the two highest log score matches (with log scores ≥2). In addition, a dendrogram was constructed using all newly created main spectrum profiles. Nine main clusters were formed, with some phylogenetically closely related Helicobacter species clustering closely together and well-defined subclusters being observed in specific species. Current results suggest that MALDI-TOF MS allows rapid differentiation between gastric Helicobacter species, provided that an extensive database is at hand and variation due to growth conditions and agar-medium-related peaks are taken into account.

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