Phenotypic and Molecular Characterization of The Emerging Pathogen Campylobacter concisus
Creators: name: Wahid, Syeda Umme Habiba
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310704
subject: Microbial genetics
classificationCode: 310206
subject: Sequence analysis
classificationCode: 310701
subject: Bacteriology

description: Campylobacter concisus is an emerging bacterial pathogen linked with a wide range of human diseases including gum diseases, gastroenteritis and inflammatory bowel disease. C. concisus is classified into two genotypically divergent groups: genomospecies 1 (GS1) and genomospecies 2 (GS2). However, very little is known about the mode of transmission, pathogenic potential, disease propensities and severity linked with particular genomospecies. Bacteria utilize various strategies to survive environmental, nutritional, and host defences, including inducing a state known as viable but non-culturable (VBNC), employed by over 100 pathogenic bacteria. In this state, bacteria remain metabolically active but cannot be detected by conventional culture method, posing potential health risks that may be underestimated. Information on the VBNC state and resuscitation of VBNC cells in C. concisus is very limited. This study sought to explore, quantify, and characterize the VBNC state of the bacterium. It demonstrated that C. concisus cells entered the VBNC state when subjected to starvation under low temperature (4°C) for more than three weeks, marking the first observation of this phenomenon. The newly developed and optimized propidium monoazide coupled with qPCR (PMAxx-qPCR) assay effectively inhibited qPCR signals from dead cells at a final concentration of 20 µM. In this study, C. concisus entered the VBNC state within 15 days for ATCC 51562 and within 9 days for ATCC 51561. Viable cell counts, determined by PMAxx-qPCR, remained around to the initial level of 107 CFU/mL, indicating a significant portion of the cell population had entered the VBNC state. Electron microscopy revealed that the VBNC cells became coccoid and significantly smaller. The VBNC cells could be resuscitated through a temperature increase in the presence of a highly nutritious growth medium. The morphology of resuscitated cells was similar to C. concisus cells in exponential growth but larger than the VBNC cells. In conclusion, under environmental stress, most C. concisus cells were transitioned to the VBNC state. The PMAxx-qPCR method reliably detects and quantifies both viable and VBNC cells of C. concisus, promising accurate bacterial load assessment. These findings underscore the potential significance of the VBNC state in C. concisus survival and transmission.
Bacterial flagella play an important role in cellular motility, chemotaxis and disease development in the host. Bioinformatic analysis has revealed that, unlike the well-characterized human pathogen, Campylobacter jejuni, C. concisus harbors a single flagellin gene, encoding the flagellar filament protein, upstream of the flagellin glycosylation island (FGI), as do other hydrogen-requiring Campylobacter species. The FGI harbor genes for the biosynthesis of either pseudaminic (Pse) acid or legionaminic (Leg) acid. This study employed swarming motility assay and differential dynamic microscopy to assess C. concisus motility, revealing strain-dependent variability. Motile strains exhibited higher expression of the flagellin gene compared to non-motile strains. Electron microscopy (EM) revealed the presence of polar flagella in some strains that showed higher expression of the flagellin gene. EM also revealed the presence of spherical-shaped outer membrane vesicles similar to that of other Gram-negative bacteria budding out from the outer membrane. A 3D homology model of C. concisus flagellin (CcFla) was generated using bio-computational tools. CcFla harbors four domains (D0-D3) and shares a fold similar to other Gram-negative bacterial flagellins. The inner domains (D0 and D1) that form the core of the flagellar filament are common to all the available flagellin structures whereas the outer domains (D2 and D3) varied among bacterial species in terms of their sequence, packing, and oligomeric state within the filament. Comparative analysis with the DALI server identified conserved hydrophobic and β-hairpin motifs. Modpred server predicted multiple O-glycosylation sites on CcFla. These findings unveil structural features potentially relevant for host immune response and cross-reactivity with other Campylobacter species.
The thesis further explores the role of outer membrane proteins including flagella and post-translational modification, and their role in host cell adhesion and invasion. OMPs play an important role in Gram-negative bacteria for growth, survival, and pathogenesis. Flagellin O-glycosylation is essential for proper flagellar formation, motility, and virulence. Profiling the OMP repertoire of C. concisus has revealed distinct variations in banding patterns across different strains within each genomospecies. Predominant OMPs were identified using Nano liquid chromatography-tandem mass spectrometry (nano-LC-ESI MS/MS). These include major outer membrane protein, flagellin, motility protein B, Elongation factor-Tu (Ef-Tu) and phase proteins. The flagellin protein (53 kDa in size) was present in some strains coinciding with their higher motility. Notably, flagellated GS2 strains exhibited greater diversity in OMP profiles compared to their non-flagellated counterparts. Furthermore, flagellated GS2 strains harboured Ef-Tu and phage proteins, which were absent in GS1 strains. Whole genomic analysis confirmed the incorporation of lysogenic phage genomes only into the genomes of tested GS2 strains. Genomic analysis has also corroborated that flagellin glycosylation islands within C. concisus genomes harbor a single flagellin, alongside genes responsible for the biosynthesis of either pseudaminic acid (Pse) or legionaminic acid (Leg). The nano-LC-ESI MS/MS unveiled strain-specific variations in flagellin O-glycosylation patterns. Interestingly, flagellin was found to be modified with either Pse or Leg and their derivatives, but not both simultaneously. Notably, Leg acid modification was found to be only in GS2 strains. Through 3D mapping, it was revealed that the majority of glycosylation sites were situated within the outer domains (D2-D3) of the flagellin structure. These findings underscore the diverse distribution of glycosylation within C. concisus flagellin.
The virulence potential of the characterized strains was assessed through examination of their adherence and invasion capacity. All strains of C. concisus, spanning both genomospecies, displayed adherence to human enterocyte-like Caco-2 cells. However, notable differences in invasion proportions were observed among the tested strains. Specifically, the flagellated strains exhibited higher adherence and invasion potential as compared with the non-flagellated strains. Notably, strains with a higher number of modifications on their flagellin demonstrated significantly higher adhesion and invasion potential compared to those with fewer modifications. Given the outer domain's recognized antigenic nature, this suggests a pivotal role in host-pathogen interactions. These findings have provided important insights into the role of flagella in C. concisus virulence and host colonization.