Emergence of mcr-4.3 genes in a novel Shewanella specie isolated from the Arctic environment.

Mobile colistin resistance (mcr) genes are pivotal contributors to last-line of antimicrobial resistance in human infections. Shewanella, historically recognized as a natural environmental bacterium with metal reduction capabilities, recently has been observed in clinical settings. However, limited knowledge has been explored on genetic differences between strains from non-clinical and clinical strains. In this study, we conducted the whole genome sequencing on six Arctic strains, illustrated the phylogenetic relationships on published 393 Shewanella strains that categorized the genus into four lineages (L1 to L4). Over 86.4% of clinical strain group (CG) strains belonged to L1 and L4, carrying mcr-4 genes and a complete metal-reduction pathways gene cluster. Remarkably, a novel Arctic Shewanella strain in L3, exhibits similar genetic characteristics with CG strains that carried both mcr-4 genes and a complete metal reduction pathway gene cluster. It raised concerns about the transmission ability from environment to clinic setting causing in the potential infections, and emphasized the need for monitoring the emerging strains with human infections.

Detecting horizontal gene transfer with metagenomics co-barcoding sequencing.

Horizontal gene transfer (HGT) is the process through which genetic information is transferred between different genomes and that played a crucial role in bacterial evolution. HGT can enable bacteria to rapidly acquire antibiotic resistance and bacteria that have acquired resistance is spreading within the microbiome. Conventional methods of characterizing HGT patterns include short-read metagenomic sequencing (short-reads mNGS), long-read sequencing, and single-cell sequencing. These approaches present several limitations, such as short-read fragments, high amounts of input DNA, and sequencing costs, respectively. Here, we attempt to circumvent present limitations to detect HGT by developing a metagenomics co-barcode sequencing workflow (MECOS) and applying it to the human and mouse gut microbiomes. In addition to that, we have over 10-fold increased contig length compared to short-reads mNGS; we also obtained exceeding 30 million paired reads with co-barcode information. Applying the novel bioinformatic pipeline, we integrated this co-barcoding information and the context information from long reads, and observed over 50-fold HGT events after we corrected the potential wrong HGT events. Specifically, we detected approximately 3,000 HGT blocks in individual samples, encompassing ~6,000 genes and ~100 taxonomic groups, including loci conferring tetracycline resistance through ribosomal protection. MECOS provides a valuable tool for investigating HGT and advance our understanding on the evolution of natural microbial communities within hosts.IMPORTANCEIn this study, to better identify horizontal gene transfer (HGT) in individual samples, we introduce a new co-barcoding sequencing system called metagenomics co-barcoding sequencing (MECOS), which has three significant improvements: (i) long DNA fragment extraction, (ii) a special transposome insertion, (iii) hybridization of DNA to barcode beads, and (4) an integrated bioinformatic pipeline. Using our approach, we have over 10-fold increased contig length compared to short-reads mNGS, and observed over 50-fold HGT events after we corrected the potential wrong HGT events. Our results indicate the presence of approximately 3,000 HGT blocks, involving roughly 6,000 genes and 100 taxonomic groups in individual samples. Notably, these HGT events are predominantly enriched in genes that confer tetracycline resistance via ribosomal protection. MECOS is a useful tool for investigating HGT and the evolution of natural microbial communities within hosts, thereby advancing our understanding of microbial ecology and evolution.

Co-expression IL-15 receptor alpha with IL-15 reduces toxicity via limiting IL-15 systemic exposure during CAR-T immunotherapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapy is a powerful adoptive immunotherapy against both B-cell malignancies and some types of solid tumors. Interleukin (IL) -15 is an important immune stimulator that may provide ideal long-term persistent CAR-T cells. However, higher base line or peak serum IL-15 levels are also related to severe toxicity, such as cytokine release syndrome (CRS), graft-versus-host disease (GVHD), and neurotoxicity. METHODS: We successfully constructed CD19 specific armored CAR-T cells overexpressing IL-I5 and IL-15 receptor alpha (IL-15Ra). In vitro cell differentiation and viability were monitored by flow cytometry, and an in vivo xenograft mouse models was used to evaluate the anti-tumor efficiency and liver damage of CAR-T cells. RESULTS: CAR-T cells overexpressing IL-15 alone demonstrated enhanced viability, retarded exhaustion in vitro and superior tumor-inhibitory effects in vivo. However, these tumor-free mice had lower survival rates, with serious liver injuries, as a possible result of toxicity. As expected, CAR-T cells overexpressing IL-15 combined with IL-15Ra had reduced CD132 expression and released fewer cytokines (IFNγ, IL-2 and IL-15) in vitro, as well as had the tendency to improve mouse survival via repressing the growth of tumor cells and keeping livers healthier compared to CAR-IL-15 T cells. CONCLUSIONS: These results indicated the importance of IL-15 in enhancing T cells persistence and IL-15Ra in reducing the adverse effects of IL-15, with superior tumor retardation during CAR-T therapy. This study paves the way for the rapid exploitation of IL-15 in adoptive cell therapy in the future.

Bone erosion in inflammatory arthritis is attenuated by Trichinella spiralis through inhibiting M1 monocyte/macrophage polarization.

Helminths and helminth-derived products hold promise for treating joint bone erosion in rheumatoid arthritis (RA). However, the mechanisms of helminths ameliorating the osteoclastic bone destruction are incompletely understood. Here, we report that Trichinella spiralis infection or treatment with the excreted/secreted products of T. spiralis muscle larvae (MES) attenuated bone erosion and osteoclastogenesis in mice with collage-induced arthritis (CIA) through inhibiting M1 monocyte/macrophage polarization and the production of M1-related proinflammatory cytokines. In vitro, MES inhibited LPS-induced M1 macrophage activation while promoting IL-4-induced M2 macrophage polarization. Same effects of MES were also observed in monocytes derived from RA patients, wherein MES treatment suppressed LPS-induced M1 cytokine production. Moreover, MES treatment attenuated LPS and RANKL co-stimulated osteoclast differentiation from the RAW264.7 macrophages through inhibiting activation of the NF-κB rather than MAPK pathway. This study provides insight into the M1 subset as a potential target for helminths to alleviate osteoclastic bone destruction in RA.

Trichinella spiralis Calreticulin S-Domain Binds to Human Complement C1q to Interfere With C1q-Mediated Immune Functions.

Helminths develop strategies to escape host immune responses that facilitate their survival in the hostile host immune environment. Trichinella spiralis, a tissue-dwelling nematode, has developed a sophisticated strategy to escape complement attack. Our previous study demonstrated that T. spiralis secretes calreticulin (TsCRT) to inhibit host classical complement activation through binding to C1q; however, the C1q binding site in TsCRT and the specific mechanism involved with complement-related immune evasion remains unknown. Using molecular docking modeling and fragment expression, we determined that TsCRT-S, a 153-aa domain of TsCRT, is responsible for C1q binding. Recombinant TsCRT-S protein expressed in Escherichia coli had the same capacity to bind and inhibit human C1q-induced complement and neutrophil activation, as full-length TsCRT. TsCRT-S inhibited neutrophil reactive oxygen species and elastase release by binding to C1q and reduced neutrophil killing of newborn T. spiralis larvae. Binding of TsCRT-S to C1q also inhibited formation of neutrophil extracellular traps (NETs), which are involved in autoimmune pathologies and have yet to be therapeutically targeted. These findings provide evidence that the TsCRT-S fragment, rather than the full-length TsCRT, is a potential target for vaccine or therapeutic development for trichinellosis, as well as for complement-related autoimmune disease therapies.

Excretory/Secretory Products From Trichinella spiralis Adult Worms Attenuated DSS-Induced Colitis in Mice by Driving PD-1-Mediated M2 Macrophage Polarization.

Helminth-modulated macrophages contribute to attenuating inflammation in inflammatory bowel diseases. The programmed death 1 (PD-1) plays an important role in macrophage polarization and is essential in the maintenance of immune system homeostasis. Here, we investigate the role of PD-1-mediated polarization of M2 macrophages and the protective effects of excretory/secretory products from Trichinella spiralis adult worms (AES) on DSS-induced colitis in mice. Colitis in mice was induced by oral administration of dextran sodium sulfate (DSS) daily. Mice with DSS-induced colitis were treated with T. spiralis AES intraperitoneally, and pathological manifestations were evaluated. Macrophages in mice were depleted with liposomal clodronate. Markers for M1-type (iNOS, TNF-α) and M2-type (CD206, Arg-1) macrophages were detected by qRT-PCR and flow cytometry. Macrophage expression of PD-1 was quantified by flow cytometry; RAW 264.7 cells and peritoneal macrophages were used for in vitro tests, and PD-1 gene knockout mice were used for in vivo investigation of the role of PD-1 in AES-induced M2 macrophage polarization. Macrophage depletion was found to reduce DSS-induced colitis in mice. Treatment with T. spiralis AES significantly increased macrophage expression of CD206 and Arg-1 and simultaneously attenuated colitis severity. We found T. spiralis AES to enhance M2 macrophage polarization; these findings were confirmed studying in vitro cultures of RAW264.7 cells and peritoneal macrophages from mice. Further experimentation revealed that AES upregulated PD-1 expression, primarily on M2 macrophages expressing CD206. The AES-induced M2 polarization was found to be decreased in PD-1 deficient macrophages, and the therapeutic effects of AES on colitis was reduced in PD-1 knockout mice. In conclusion, the protective effects of T. spiralis AES on DSS-induced colitis were found to associate with PD-1 upregulation and M2 macrophage polarization. Thus, PD-1-mediated M2 macrophage polarization is a key mechanism of helminth-induced modulation of the host immune system.

Mapping of the complement C1q binding site on Trichinella spiralis paramyosin.

BACKGROUND: Trichinella spiralis is a tissue-dwelling parasite has developed the ability to evade the host immune attack to establish parasitism in a host. One of the strategies evolved by the nematode is to produce proteins that immunomodulate the host immune system. TsPmy is a paramyosin secreted by T. spiralis on the surface of larvae and adult worms that can interact with complement components C1q and C8/C9 to compromise their activation and functions. To better understand the mechanism of TsPmy involved in the C1q inactivation and immune evasion, the C1q-binding site on TsPmy was investigated. METHODS: The TsPmy C1q-binding site was investigated by sequential narrow-down fragment expression in bacteria and peptide binding screening. C1q binding activity was identified by Far-Western blotting and ELISA assays. RESULTS: After several runs of sequential fragment expression, the C1q binding site was narrowed down to fragments of N-terminal TsPmy226-280aa and TsPmy231-315aa, suggesting the final C1q binding site is probably located to TsPmy231-280aa. A total of nine peptides covering different amino acid sequences within TsPmy231-280aa were synthesized. The binding assay to C1q determined that only P2 peptide covering TsPmy241-280aa binds to C1q, indicating that the C1q binding domain may need both the linearized sequence and conformational structure required for binding to C1q. The binding of peptide P2 to C1q significantly inhibited both C1q-initiated complement classical activation and C1q-induced macrophage chemotaxis. CONCLUSIONS: This study identifies the C1q binding site within TsPmy which provides helpful information for developing a vaccine against trichinellosis by targeting the C1q-binding activity of TsPmy.

Trichinella spiralis Infection Mitigates Collagen-Induced Arthritis via Programmed Death 1-Mediated Immunomodulation.

Helminth infection induces Th2-biased immune responses and inhibitory/regulatory pathways that minimize excessive inflammation to facilitate the chronic infection of helminth in the host and in the meantime, prevent host hypersensitivity from autoimmune or atopic diseases. However, the detailed molecular mechanisms behind modulation on inflammatory diseases are yet to be clarified. Programmed death 1 (PD-1) is one of the important inhibitory receptors involved in the balance of host immune responses during chronic infection. Here, we used the murine model to examine the role of PD-1 in CD4+ T cells in the effects of Trichinella spiralis infection on collagen-induced arthritis (CIA). Mice infected with T. spiralis demonstrated higher expression of PD-1 in the spleen CD4+ T cells than those without infection. Mice infected with T. spiralis 2 weeks prior to being immunized with type II collagen displayed lower arthritis incidence and significantly attenuated pathology of CIA compared with those of uninfected mice. The therapeutic effect of T. spiralis infection on CIA was reversed by blocking PD-1 with anti-PD-1 antibody, associated with enhanced Th1/Th17 pro-inflammatory responses and reduced Th2 responses. The role of PD-1 in regulating CD4+ T cell differentiation and proliferation during T. spiralis infection was further examined in PD-1 knockout (PD-1-/-) C57BL/6 J mice. Interestingly, T. spiralis-induced alteration of attenuated Th1 and enhanced Th2/regulatory T cell differentiation in wild-type (WT) mice was effectively diminished in PD-1-/- mice characterized by recovered Th1 cytokine levels, reduced levels of Th2 and regulatory cytokines and CD4+CD25+Foxp3+ cells. Moreover, T. spiralis-induced CD4+ T cell proliferation suppression in WT mice was partially restored in PD-1-/- mice. This study introduces the first evidence that PD-1 plays a critical role in helminth infection-attenuated CIA in a mouse model by regulating the CD4+ T cell function, which may provide the new insights into the mechanisms of helminth-induced immunomodulation of host autoimmunity.