Identifying components of the Shewanella phage LambdaSo lysis system.

Phage-induced lysis of Gram-negative bacterial hosts usually requires a set of phage lysis proteins, a holin, an endopeptidase, and a spanin system, to disrupt each of the three cell envelope layers. Genome annotations and previous studies identified a gene region in the Shewanella oneidensis prophage LambdaSo, which comprises potential holin- and endolysin-encoding genes but lacks an obvious spanin system. By a combination of candidate approaches, mutant screening, characterization, and microscopy, we found that LambdaSo uses a pinholin/signal-anchor-release (SAR) endolysin system to induce proton leakage and degradation of the cell wall. Between the corresponding genes, we found that two extensively nested open-reading frames encode a two-component spanin module Rz/Rz1. Unexpectedly, we identified another factor strictly required for LambdaSo-induced cell lysis, the phage protein Lcc6. Lcc6 is a transmembrane protein of 65 amino acid residues with hitherto unknown function, which acts at the level of holin in the cytoplasmic membrane to allow endolysin release. Thus, LambdaSo-mediated cell lysis requires at least four protein factors (pinholin, SAR endolysin, spanin, and Lcc6). The findings further extend the known repertoire of phage proteins involved in host lysis and phage egress. IMPORTANCE: Lysis of bacteria can have multiple consequences, such as the release of host DNA to foster robust biofilm. Phage-induced lysis of Gram-negative cells requires the disruption of three layers, the outer and inner membranes and the cell wall. In most cases, the lysis systems of phages infecting Gram-negative cells comprise holins to disrupt or depolarize the membrane, thereby releasing or activating endolysins, which then degrade the cell wall. This, in turn, allows the spanins to become active and fuse outer and inner membranes, completing cell envelope disruption and allowing phage egress. Here, we show that the presence of these three components may not be sufficient to allow cell lysis, implicating that also in known phages, further factors may be required.

Resident regulatory T cells reflect the immune history of individual lymph nodes.

Regulatory T cells (Tregs) are present in lymphoid and nonlymphoid tissues where they restrict immune activation, prevent autoimmunity, and regulate inflammation. Tregs in nonlymphoid tissues are typically resident, whereas those in lymph nodes (LNs) are considered to recirculate. However, Tregs in LNs are not a homogenous population, and circulation kinetics of different Treg subsets are poorly characterized. Furthermore, whether Tregs can acquire memory T cell properties and persist for extended periods after their activation in LNs is unclear. Here, we used in situ labeling with a stabilized photoconvertible protein to uncover turnover rates of Tregs in LNs in vivo. We found that, whereas most Tregs in LNs recirculate, 10 to 20% are memory-like resident cells that remain in their respective LNs for weeks to months. Single-cell RNA sequencing revealed that LN-resident cells are a functionally and ontogenetically heterogeneous population and share the same core residency gene signature with conventional CD4+ and CD8+ T cells. Resident cells in LNs did not actively proliferate and did not require continuous T cell receptor (TCR) signaling for their residency. However, resident and circulating Tregs had distinct TCR repertoires, and each LN contained exclusive clonal subpopulations of resident Tregs. Our results demonstrate that, similar to conventional T cells, Tregs can form resident memory-like populations in LNs after adaptive immune responses. Specific and local suppression of immune responses by resident Tregs in draining LNs might provide previously unidentified therapeutic opportunities for the treatment of local chronic inflammatory conditions.

MHCII expression on gut macrophages supports T cell homeostasis and is regulated by microbiota and ontogeny.

Macrophages are traditionally considered antigen-presenting cells. However, their ability to present antigen and the factors regulating macrophage MHCII expression are poorly understood. Here, we demonstrate that MHCII expression on murine intestinal macrophages is differentially controlled by their residence in the small intestine (SI) or the colon, their ontogeny and the gut microbiota. Monocyte-derived macrophages are uniformly MHCIIhi, independently of the tissue of residence, microbial status or the age of the mouse, suggesting a common monocyte differentiation pathway. In contrast, MHCII expression on long-lived, prenatally-derived Tim4+ macrophages is low after birth but significantly increases at weaning in both SI and colon. Furthermore, MHCII expression on colonic Tim4+, but not monocyte-derived macrophages, is dependent on recognition of microbial stimuli, as MHCII expression is significantly downregulated in germ-free, antibiotic-treated and MyD88 deficient mice. To address the function of MHCII presentation by intestinal macrophages we established two models of macrophage-specific MHCII deficiency. We observed a significant reduction in the overall frequency and number of tissue-resident, but not newly arrived, SI CD4+ T cells in the absence of macrophage-expressed MHCII. Our data suggest that macrophage MHCII provides signals regulating gut CD4+ T cell maintenance with different requirements in the SI and colon.

Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities.

Microbiome research needs comprehensive repositories of cultured bacteria from the intestine of mammalian hosts. We expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, all publicly available and taxonomically described. This includes strain-level diversity, small-sized bacteria, and previously undescribed taxa (one family, 10 genera, and 39 species). This collection enabled metagenome-educated prediction of synthetic communities (SYNs) that capture key functional differences between microbiomes, notably identifying communities associated with either resistance or susceptibility to DSS-induced colitis. Additionally, nine species were used to amend the Oligo-Mouse Microbiota (OMM)12 model, yielding the OMM19.1 model. The added strains compensated for phenotype differences between OMM12 and specific pathogen-free mice, including body composition and immune cells in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available for future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.

Dendritic cell functions in the inductive and effector sites of intestinal immunity.

The intestine is constantly exposed to foreign antigens, which are mostly innocuous but can sometimes be harmful. Therefore, the intestinal immune system has the delicate task of maintaining immune tolerance to harmless food antigens while inducing tailored immune responses to pathogens and regulating but tolerating the microbiota. Intestinal dendritic cells (DCs) play a central role in these functions as sentinel cells able to prime and polarize the T cell responses. DCs are deployed throughout the intestinal mucosa but with local specializations along the gut length and between the diffuse effector sites of the gut lamina propria (LP) and the well-organized immune inductive sites comprising isolated lymphoid follicles (ILFs), Peyer's patches (PPs), and other species-specific gut-associated lymphoid tissues (GALTs). Understanding the specificities of each intestinal DC subset, how environmental factors influence DC functions, and how these can be modulated is key to harnessing the therapeutic potential of mucosal adaptive immune responses, whether by enhancing the efficacy of mucosal vaccines or by increasing tolerogenic responses in inflammatory disorders. In this review, we summarize recent findings related to intestinal DCs in steady state and upon inflammation, with a special focus on their functional specializations, highly dependent on their microenvironment.

Isolation and Characterization of Shewanella Phage Thanatos Infecting and Lysing Shewanella oneidensis and Promoting Nascent Biofilm Formation.

Species of the genus Shewanella are widespread in nature in various habitats, however, little is known about phages affecting Shewanella sp. Here, we report the isolation of phages from diverse freshwater environments that infect and lyse strains of Shewanella oneidensis and other Shewanella sp. Sequence analysis and microscopic imaging strongly indicate that these phages form a so far unclassified genus, now named Shewanella phage Thanatos, which can be positioned within the subfamily of Tevenvirinae (Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Caudovirales; Myoviridae; Tevenvirinae). We characterized one member of this group in more detail using S. oneidensis MR-1 as a host. Shewanella phage Thanatos-1 possesses a prolate icosahedral capsule of about 110 nm in height and 70 nm in width and a tail of about 95 nm in length. The dsDNA genome exhibits a GC content of about 34.5%, has a size of 160.6 kbp and encodes about 206 proteins (92 with an annotated putative function) and two tRNAs. Out of those 206, MS analyses identified about 155 phage proteins in PEG-precipitated samples of infected cells. Phage attachment likely requires the outer lipopolysaccharide of S. oneidensis, narrowing the phage's host range. Under the applied conditions, about 20 novel phage particles per cell were produced after a latent period of approximately 40 min, which are stable at a pH range from 4 to 12 and resist temperatures up to 55°C for at least 24 h. Addition of Thanatos to S. oneidensis results in partial dissolution of established biofilms, however, early exposure of planktonic cells to Thanatos significantly enhances biofilm formation. Taken together, we identified a novel genus of Myophages affecting S. oneidensis communities in different ways.