Correction: Quinn et al. GlnH, a Novel Antigen That Offers Partial Protection against Verocytotoxigenic Escherichia coli Infection. Vaccines 2023, 11, 175.

In the original publication [...].

GlnH, a Novel Antigen That Offers Partial Protection against Verocytotoxigenic Escherichia coli Infection.

Verotoxin-producing Escherichia coli (VTEC) causes zoonotic infections, with potentially devastating complications, and children under 5 years old are particularly susceptible. Antibiotic treatment is contraindicated, and due to the high proportion of infected children that suffer from severe and life-changing complications, there is an unmet need for a vaccine to prevent VTEC infections. Bacterial adhesins represent promising candidates for the successful development of a vaccine against VTEC. Using a proteomic approach to identify bacterial proteins interacting with human gastrointestinal epithelial Caco-2 and HT-29 cells, we identified eleven proteins by mass spectrometry. These included a glutamine-binding periplasmic protein, GlnH, a member of the ABC transporter family. The glnH gene was identified in 13 of the 15 bovine and all 5 human patient samples tested, suggesting that it is prevalent. We confirmed that GlnH is involved in the host cell attachment of an O157:H7 prototype E. coli strain to gastrointestinal cells in vitro. Recombinant GlnH was expressed and purified prior to the immunisation of mice. When alum was used as an adjuvant, GlnH was highly immunogenic, stimulating strong serological responses in immunised mice, and it resulted in a modest reduction in faecal shedding but did not reduce colonisation. GlnH immunisation with a T-cell-inducing adjuvant (SAS) also showed comparable antibody responses and an IgG1/IgG2a ratio suggestive of a mixed Th1/Th2 response but was partially protective, with a 1.5-log reduction in colonisation of the colon and caecum at 7 days relative to the adjuvant only (p = 0.0280). It is clear that future VTEC vaccine developments should consider the contribution of adjuvants in addition to antigens. Moreover, it is likely that a combined cellular and humoral response may prove more beneficial in providing protective interventions against VTEC.

Structural Analysis of the Black-Legged Tick Saliva Protein Salp15.

Salp15 is one of the proteins in the saliva of the tick Ixodes scapularis. Together with other biomolecules injected into the mammalian host at the biting site, it helps the tick to sustain its blood meal for days. Salp15 interferes with the cellular immune response of the mammalian host by inhibiting the activation of CD4+ T-lymphocytes. This function is co-opted by pathogens that use the tick as a vector and invade the host when the tick bites, such as Borrelia burgdorferi, the causative agent of Lyme borreliosis. Because of the immunity-suppressing role of Salp15, it has been proposed as a candidate for therapeutic applications in disorders of the immune system. The protein is produced as a 135-residue long polypeptide and secreted without its N-terminal signal 1-21 sequence. Detailed structural studies on Salp15 are lacking because of the difficulty in producing large amounts of the folded protein. We report the production of Salp15 and its structural analysis by NMR. The protein is monomeric and contains a flexible N-terminal region followed by a folded domain with mixed α + ß secondary structures. Our results are consistent with a three-dimensional structural model derived from AlphaFold, which predicts the formation of three disulfide bridges and a free C-terminal cysteine.

A structurally unique Fusobacterium nucleatum tannase provides detoxicant activity against gallotannins and pathogen resistance.

Colorectal cancer pathogenesis and progression is associated with the presence of Fusobacterium nucleatum and the reduction of acetylated derivatives of spermidine, as well as dietary components such as tannin-rich foods. We show that a new tannase orthologue of F. nucleatum (TanBFnn ) has significant structural differences with its Lactobacillus plantarum counterpart affecting the flap covering the active site and the accessibility of substrates. Crystallographic and molecular dynamics analysis revealed binding of polyamines to a small cavity that connects the active site with the bulk solvent which interact with catalytically indispensable residues. As a result, spermidine and its derivatives, particularly N8 -acetylated spermidine, inhibit the hydrolytic activity of TanBFnn and increase the toxicity of gallotannins to F. nucleatum. Our results support a model in which the balance between the detoxicant activity of TanBFnn and the presence of metabolic inhibitors can dictate either conducive or unfavourable conditions for the survival of F. nucleatum.

The mitochondrial negative regulator MCJ modulates the interplay between microbiota and the host during ulcerative colitis.

Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.

A multi-omic analysis reveals the regulatory role of CD180 during the response of macrophages to Borrelia burgdorferi.

Macrophages are cells of the innate immune system with the ability to phagocytose and induce a global pattern of responses that depend on several signaling pathways. We have determined the biosignature of murine bone marrow-derived macrophages and human blood monocytes using transcriptomic and proteomic approaches. We identified a common pattern of genes that are transcriptionally regulated and overall indicate that the response to B. burgdorferi involves the interaction of spirochetal antigens with several inflammatory pathways corresponding to primary (triggered by pattern-recognition receptors) and secondary (induced by proinflammatory cytokines) responses. We also show that the Toll-like receptor family member CD180 is downregulated by the stimulation of macrophages, but not monocytes, with the spirochete. Silencing Cd180 results in increased phagocytosis while tempering the production of the proinflammatory cytokine TNF. Cd180-silenced cells produce increased levels of Itgam and surface CD11b, suggesting that the regulation of CD180 by the spirochete initiates a cascade that increases CR3-mediated phagocytosis of the bacterium while repressing the consequent inflammatory response.

Corrigendum: mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

This corrects the article DOI: 10.1038/nature22964.

The immunosuppressive effect of the tick protein, Salp15, is long-lasting and persists in a murine model of hematopoietic transplant.

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73.

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

Fine-Tuning of CD8(+) T Cell Mitochondrial Metabolism by the Respiratory Chain Repressor MCJ Dictates Protection to Influenza Virus.

Mitochondrial respiration is regulated in CD8(+) T cells during the transition from naive to effector and memory cells, but mechanisms controlling this process have not been defined. Here we show that MCJ (methylation-controlled J protein) acted as an endogenous break for mitochondrial respiration in CD8(+) T cells by interfering with the formation of electron transport chain respiratory supercomplexes. Metabolic profiling revealed enhanced mitochondrial metabolism in MCJ-deficient CD8(+) T cells. Increased oxidative phosphorylation and subcellular ATP accumulation caused by MCJ deficiency selectively increased the secretion, but not expression, of interferon-γ. MCJ also adapted effector CD8(+) T cell metabolism during the contraction phase. Consequently, memory CD8(+) T cells lacking MCJ provided superior protection against influenza virus infection. Thus, MCJ offers a mechanism for fine-tuning CD8(+) T cell mitochondrial metabolism as an alternative to modulating mitochondrial mass, an energetically expensive process. MCJ could be a therapeutic target to enhance CD8(+) T cell responses.

Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.