SummaryBackgroundDysbiosis is associated with many diseases, including irritable bowel syndrome (IBS), inflammatory bowel diseases (IBD), obesity and diabetes. Potential clinical impact of imbalance in the intestinal microbiota suggests need for new standardised diagnostic methods to facilitate microbiome profiling.AimTo develop and validate a novel diagnostic test using faecal samples to profile the intestinal microbiota and identify and characterise dysbiosis.MethodsFifty‐four DNA probes targeting ≥300 bacteria on different taxonomic levels were selected based on ability to distinguish between healthy controls and IBS patients in faecal samples. Overall, 165 healthy controls (normobiotic reference collection) were used to develop a dysbiosis model with a bacterial profile and Dysbiosis Index score output. The model algorithmically assesses faecal bacterial abundance and profile, and potential clinically relevant deviation in the microbiome from normobiosis. This model was tested in different samples from healthy volunteers and IBS and IBD patients (n = 330) to determine the ability to detect dysbiosis.ResultsValidation confirms dysbiosis was detected in 73% of IBS patients, 70% of treatment‐naïve IBD patients and 80% of IBD patients in remission, vs. 16% of healthy individuals. Comparison of deep sequencing and the GA‐map Dysbiosis Test, (Genetic Analysis AS, Oslo, Norway) illustrated good agreement in bacterial capture; the latter showing higher resolution by targeting pre‐determined highly relevant bacteria.ConclusionsThe GA‐map Dysbiosis Test identifies and characterises dysbiosis in IBS and IBD patients, and provides insight into a patient's intestinal microbiota. Evaluating microbiota as a diagnostic strategy may allow monitoring of prescribed treatment regimens and improvement in new therapeutic approaches.

Several major bacterial pathogens and related commensal species colonizing the human mucosa express phosphocholine (PC) at their cell surfaces. PC appears to impact host-microbe biology by serving as a ligand for both C-reactive protein and the receptor for platelet-activating factor. Type IV pili of Neisseria gonorrhoeae (Ng) and Neisseria meningitidis, filamentous protein structures critical to the colonization of their human hosts, are known to react variably with monoclonal antibodies recognizing a PC epitope. However, the structural basis for this reactivity has remained elusive. To address this matter, we exploited the finding that the PilE pilin subunit in Ng mutants lacking the PilV protein acquired the PC epitope independent of changes in pilin primary structure. Specifically, we show by using mass spectrometry that PilE derived from the pilV background is composed of a mixture of subunits bearing O-linked forms of either phosphoethanolamine (PE) or PC at the same residue, whereas the wild-type background carries only PE at that same site. Therefore, PilV can influence pilin structure and antigenicity by modulating the incorporation of these alternative modifications. The disaccharide covalently linked to Ng pilin was also characterized because it is present on the same peptides bearing the PE and PC modifications and, contrary to previous reports, was found to be linked by means of 2,4-diacetamido-2,4,6-trideoxyhexose. Taken together, these findings provide new insights into Ng type IV pilus structure and antigenicity and resolve long-standing issues regarding the nature of both the PC epitope and the pilin glycan.

Type IV pili (Tfp) of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Tfp are assumed to play a key role in the initial adherence to human epithelial cells by virtue of the associated adhesin protein PilC. To examine the structural and functional basis for adherence in more detail, we identified potential genes encoding polypeptides sharing structural similarities to PilE (the Tfp subunit) within the N. gonorrhoeae genome sequence database. We show here that a fiber subunit-like protein, termed PilV, is essential to organelle-associated adherence but dispensable for Tfp biogenesis and other pilus-related phenotypes, including autoagglutination, competence for natural transformation, and twitching motility. The adherence defect in pilV mutants cannot be attributed to reduced levels of piliation, defects in fiber anchoring to the bacterial cell surface, or to unstable pilus expression related to organelle retraction. PilV is expressed at low levels relative to PilE and copurifies with Tfp fibers in a PilC-dependent fashion. Purified Tfp from pilV mutants contain PilC adhesin at reduced levels. Taken together, these data support a model in which PilV functions in adherence by promoting the functional display of PilC in the context of the pilus fiber.