Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis.

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase-α, ß, ß'. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the ß or ß' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.

The human symbiont Mucispirillum schaedleri: causality in health and disease.

Trillions of bacteria inhabit the mammalian gastrointestinal tract. In the majority of hosts, these symbionts contribute largely to beneficial functions promoting microbe-host homeostasis. However, an increasing number of human diseases is associated with altered microbiota composition and enrichment of certain bacterial species. A well-known example of this is Mucispirillum schaedleri, which has been associated with inflammatory conditions in the intestine. Mucispirillum spp. belong to the phylum Deferribacteres and are prevalent but low abundant members of the rodent, pig and human microbiota. Recently, M. schaedleri was causally linked to the development of Crohn's disease-like colitis in immunodeficient mice. While this study certifies a considerable pathogenic potential, the same organism can also promote health in the immunocompetent host: M. schaedleri protects from Salmonella enterica serovar Typhimurium (S. Tm)-induced colitis by interfering with the expression of the pathogen´s invasion machinery. In this review, we summarize the current knowledge on the mammalian gut symbiont M. schaedleri and its role in intestinal homeostasis and discuss open questions and perspectives for future research.

Complete genome sequence of Helicobacter pylori B128 7.13 and a single-step method for the generation of unmarked mutations.

BACKGROUND: Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction-modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori, there are relatively few genetic tools for the mutagenesis of this bacterium. MATERIALS AND METHODS: Here, we use PacBio and Illumina sequencing to reveal the complete genome sequence of H pylori B128 7.13. Furthermore, we describe a system to generate markerless and scarless mutations on the H pylori chromosome using the counter-selection marker, galactokinase from Escherichia coli. RESULTS: We show that this mutagenesis strategy can be used to generate in-frame insertions, gene deletions, and multiple independent mutations in B128 7.13. Using the closed genome as a reference, we also report the absence of second site chromosomal mutations and/or rearrangements in our mutagenized strains. We compare the genome sequence of H pylori B128 7.13 with a closely related strain, H pylori B8, and reveal one notable region of difference, which is a 1430 bp insertion encoding a H pylori-specific DUF874 family protein of unknown function. CONCLUSIONS: This article reports the closed genome of the important H pylori B128 7.13 strain and a mutagenesis method that can be adopted by researchers as an alternative strategy to generate isogenic mutants of H pylori in order to further our understanding of this bacterium.

Intestinal colonization resistance in the context of environmental, host, and microbial determinants.

Microbial communities that colonize the human gastrointestinal (GI) tract defend against pathogens through a mechanism known as colonization resistance (CR). Advances in technologies such as next-generation sequencing, gnotobiotic mouse models, and bacterial cultivation have enhanced our understanding of the underlying mechanisms and the intricate microbial interactions involved in CR. Rather than being attributed to specific microbial clades, CR is now understood to arise from a dynamic interplay between microbes and the host and is shaped by metabolic, immune, and environmental factors. This evolving perspective underscores the significance of contextual factors, encompassing microbiome composition and host conditions, in determining CR. This review highlights recent research that has shifted its focus toward elucidating how these factors interact to either promote or impede enteric infections. It further discusses future research directions to unravel the complex relationship between host, microbiota, and environmental determinants in safeguarding against GI infections to promote human health.

Systemic and Mucosal Immunogenicity of Monovalent XBB.1.5-Adapted COVID-19 mRNA Vaccines in Patients with Inflammatory Bowel Disease.

Recently updated COVID-19 mRNA vaccines encode the spike protein of the omicron subvariant XBB.1.5 and are recommended for patients with inflammatory bowel disease (IBD) on immunosuppressive treatment. Nonetheless, their immunogenicity in patients with IBD against rapidly expanding virus variants remains unknown. This prospective multicenter cohort study is the first study to investigate the immunogenicity of XBB.1.5-adapted vaccines in patients with IBD. Systemic and mucosal antibodies targeting the receptor-binding domains (RBDs) of the omicron subvariants XBB.1.5, EG.5.1, and BA.2.86, as well as their neutralization were quantified before and two to four weeks after vaccination with monovalent XBB.1.5-adapted mRNA vaccines. Vaccination increased levels of serum anti-RBD IgG targeting XBB.1.5, EG.5.1, and BA.2.86 (1.9-fold, 1.8-fold, and 2.6-fold, respectively) and enhanced corresponding neutralization responses (2.3-fold, 3.1-fold, and 3.5-fold, respectively). Following vaccination, anti-TNF-treated patients had reduced virus neutralization compared to patients on treatments with other cellular targets. 11.1% and 16.7% of patients lacked EG.5.1 and BA.2.86 neutralization, respectively; all these patients received anti-TNF treatment. At mucosal sites, vaccination induced variant-specific anti-RBD IgG but failed to induce RBD-targeting IgA. Our findings provide a basis for future vaccine recommendations while highlighting the importance of frequent booster vaccine adaptation and the need for mucosal vaccination strategies in patients with IBD.

XBB.1.5-Adapted COVID-19 mRNA Vaccines but Not Infections With Previous Omicron Variants Boost Neutralisation Against the SARS-CoV-2 JN.1 Variant in Patients With Inflammatory Bowel Disease.

BACKGROUND: Variant-adapted COVID-19 vaccines are recommended for patients with inflammatory bowel disease (IBD). However, many patients rely on pre-existing immunity by original vaccines or prior infections. AIM: To assess whether such immunity sufficiently combats the highly immune-evasive SARS-CoV-2 JN.1 variant. METHODS: Utilising two longitudinal cohorts, we evaluated immunity against JN.1 induced by original vaccines (IBD: n = 98; healthy: n = 48), omicron breakthrough infection (IBD: n = 55; healthy: n = 57) or XBB.1.5-adapted vaccines (IBD: n = 18). Neutralisation and anti-receptor-binding domain (RBD) IgG levels against wild-type SARS-CoV-2 and JN.1 were assessed using multiplex immunoassays. Study outcomes were wild-type and JN.1 neutralisation following three doses of original mRNA vaccines, stratified by immunosuppressive therapy (primary outcome), and JN.1 neutralisation following third-dose breakthrough infection or a fourth dose of XBB.1.5-adapted mRNA vaccines (secondary outcomes). RESULTS: Following original vaccines, JN.1 neutralisation was lower than wild-type neutralisation in all study groups (healthy, anti-TNF and non-anti-TNF; each p < 0.001); most individuals lacked JN.1 neutralisation (healthy: 97.9%; anti-TNF: 98.3% and non-anti-TNF: 92.3%). Confounder-adjusted multivariable modelling strongly associated anti-TNF therapy with low levels of anti-JN.1-RBD IgG (fold-change 0.48 [95% CI 0.39-0.59]). JN.1 neutralisation was similar in patients with or without breakthrough infection (anti-TNF, non-anti-TNF; each p > 0.05); neutralisation failure was 100% despite breakthrough infection. XBB.1.5-adapted vaccines enhanced JN.1 neutralisation (p < 0.001) and reduced neutralisation failure rates in patients with IBD (94.4% pre-vaccination vs. 44.4% post-vaccination; p = 0.003). CONCLUSIONS: Only variant-adapted vaccines protect against emerging SARS-CoV-2 variants. Patients with IBD and healthy individuals without recent vaccination may lack protection against the JN.1 subvariant KP.3 which causes current COVID-19 surges.

Pulsed antibiotic treatments of gnotobiotic mice manifest in complex bacterial community dynamics and resistance effects.

Bacteria can evolve to withstand a wide range of antibiotics (ABs) by using various resistance mechanisms. How ABs affect the ecology of the gut microbiome is still poorly understood. We investigated strain-specific responses and evolution during repeated AB perturbations by three clinically relevant ABs, using gnotobiotic mice colonized with a synthetic bacterial community (oligo-mouse-microbiota). Over 80 days, we observed resilience effects at the strain and community levels, and we found that they were correlated with modulations of the estimated growth rate and levels of prophage induction as determined from metagenomics data. Moreover, we tracked mutational changes in the bacterial populations, and this uncovered clonal expansion and contraction of haplotypes and selection of putative AB resistance-conferring SNPs. We functionally verified these mutations via reisolation of clones with increased minimum inhibitory concentration (MIC) of ciprofloxacin and tetracycline from evolved communities. This demonstrates that host-associated microbial communities employ various mechanisms to respond to selective pressures that maintain community stability.

Systemic and T cell-associated responses to SARS-CoV-2 immunisation in gut inflammation (STAR SIGN study): effects of biologics on vaccination efficacy of the third dose of mRNA vaccines against SARS-CoV-2.

BACKGROUND: Immunosuppressed patients with inflammatory bowel disease (IBD) experience increased risk of vaccine-preventable diseases such as COVID-19. AIMS: To assess humoral and cellular immune responses following SARS-CoV-2 booster vaccination in immunosuppressed IBD patients and healthy controls. METHODS: In this prospective, multicentre, case-control study, 139 IBD patients treated with biologics and 110 healthy controls were recruited. Serum anti-SARS-CoV-2 spike IgG concentrations were measured 2-16 weeks after receiving a third mRNA vaccine dose. The primary outcome was to determine if humoral immune responses towards booster vaccines differ in IBD patients under anti-TNF versus non-anti-TNF therapy and healthy controls. Secondary outcomes were antibody decline, impact of previous infection and SARS-CoV-2-targeted T cell responses. RESULTS: Anti-TNF-treated IBD patients showed reduced anti-spike IgG concentrations (geometric mean 2357.4 BAU/ml [geometric SD 3.3]) when compared to non-anti-TNF-treated patients (5935.7 BAU/ml [3.9]; p < 0.0001) and healthy controls (5481.7 BAU/ml [2.4]; p < 0.0001), respectively. In multivariable modelling, prior infection (geometric mean ratio 2.00 [95% CI 1.34-2.90]) and vaccination with mRNA-1273 (1.53 [1.01-2.27]) increased antibody concentrations, while anti-TNF treatment (0.39 [0.28-0.54]) and prolonged time between vaccination and antibody measurement (0.72 [0.58-0.90]) decreased anti-SARS-CoV-2 spike antibodies. Antibody decline was comparable in IBD patients independent of anti-TNF treatment and antibody concentrations could not predict breakthrough infections. Cellular and humoral immune responses were uncoupled, and more anti-TNF-treated patients than healthy controls developed inadequate T cell responses (15/73 [20.5%] vs 2/100 [2.0%]; p = 0.00031). CONCLUSIONS: Anti-TNF-treated IBD patients have impaired humoral and cellular immunogenicity following SARS-CoV-2 booster vaccination. Fourth dose administration may be beneficial for these patients.

STAR SIGN study: Evaluation of COVID-19 vaccine efficacy against the SARS-CoV-2 variants BQ.1.1 and XBB.1.5 in patients with inflammatory bowel disease.

BACKGROUND: Vaccine-elicited immune responses are impaired in patients with inflammatory bowel disease (IBD) treated with anti-TNF biologics. AIMS: To assess vaccination efficacy against the novel omicron sublineages BQ.1.1 and XBB.1.5 in immunosuppressed patients with IBD. METHODS: This prospective multicentre case-control study included 98 biologic-treated patients with IBD and 48 healthy controls. Anti-spike IgG concentrations and surrogate neutralisation against SARS-CoV-2 wild-type, BA.1, BA.5, BQ.1.1, and XBB.1.5 were measured at two different time points (2-16 weeks and 22-40 weeks) following third dose vaccination. Surrogate neutralisation was based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Primary outcome was surrogate neutralisation against tested SARS-CoV-2 sublineages. Secondary outcomes were proportions of participants with insufficient surrogate neutralisation, impact of breakthrough infection, and correlation of surrogate neutralisation with anti-spike IgG concentration. RESULTS: Surrogate neutralisation against all tested sublineages was reduced in patients with IBD who were treated with anti-TNF biologics compared to patients treated with non-anti-TNF biologics and healthy controls (each p ≤ 0.001) at visit 1. Anti-TNF therapy (odds ratio 0.29 [95% CI 0.19-0.46]) and time since vaccination (0.85 [0.72-1.00]) were associated with low, and mRNA-1273 vaccination (1.86 [1.12-3.08]) with high wild-type surrogate neutralisation in a ß-regression model. Accordingly, higher proportions of patients treated with anti-TNF biologics had insufficient surrogate neutralisation against omicron sublineages at visit 1 compared to patients treated with non-anti-TNF biologics and healthy controls (each p ≤ 0.015). Surrogate neutralisation against all tested sublineages decreased over time but was increased by breakthrough infection. Anti-spike IgG concentrations correlated with surrogate neutralisation. CONCLUSIONS: Patients with IBD who are treated with anti-TNF biologics show impaired neutralisation against novel omicron sublineages BQ.1.1 and XBB.1.5 and may benefit from prioritisation for future variant-adapted vaccines.