Trained immunity (TI) is the process wherein innate immune cells gain functional memory upon exposure to specific ligands or pathogens, leading to augmented inflammatory responses and pathogen clearance upon secondary exposure. While the differentiation of hematopoietic stem cells (HSCs) and reprogramming of bone marrow (BM) progenitors are well-established mechanisms underpinning durable TI protection, remodeling of the cellular architecture within the tissue during TI remains underexplored. Here, we study the effects of peritoneal Bacillus Calmette–Guérin (BCG) administration to find TI-mediated protection in the spleen against a subsequent heterologous infection by the Gram-negative pathogen Salmonella Typhimurium ( S .Tm). Utilizing single cell RNA-sequencing and flow cytometry, we discerned STAT1-regulated genes in TI-associated resident and recruited splenic myeloid populations. The temporal dynamics of TI were further elucidated, revealing both early and delayed myeloid subsets with time-dependent, cell-type-specific STAT1 signatures. Using lineage tracing, we find that tissue-resident red pulp macrophages (RPM), initially depleted by BCG exposure, are restored from both tissue-trained, self-renewing macrophages and from bone marrow-derived progenitors, fostering long lasting local defense. Early inhibition of STAT1 activation, using specific JAK-STAT inhibitors, reduces both RPM loss and recruitment of trained monocytes. Our study suggests a temporal window soon after BCG vaccination, in which STAT1-dependent activation of long-lived resident cells in the tissue mediates localized protection.

Encounters between host cells and intracellular bacterial pathogens lead to complex phenotypes that determine the outcome of infection. Single-cell RNA sequencing (scRNA-seq) is increasingly used to study the host factors underlying diverse cellular phenotypes but has limited capacity to analyze the role of bacterial factors. Here, we developed scPAIR-seq, a single-cell approach to analyze infection with a pooled library of multiplex-tagged, barcoded bacterial mutants. Infected host cells and barcodes of intracellular bacterial mutants are both captured by scRNA-seq to functionally analyze mutant-dependent changes in host transcriptomes. We applied scPAIR-seq to macrophages infected with a library of Salmonella Typhimurium secretion system effector mutants. We analyzed redundancy between effectors and mutant-specific unique fingerprints and mapped the global virulence network of each individual effector by its impact on host immune pathways. ScPAIR-seq is a powerful tool to untangle bacterial virulence strategies and their complex interplay with host defense strategies that drive infection outcome.

Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1+ monocytes into lymphoid organs during Salmonella Typhimurium (STm) infection disrupted pre-existing germinal center (GC) reactions. GC responses induced by influenza, plasmodium, or commensals deteriorated following STm infection. GC disruption was independent of the direct bacterial interactions with B cells and instead was induced through recruitment of CCR2-dependent Sca-1+ monocytes into the lymphoid organs. GC collapse was associated with impaired cellular respiration and was dependent on TNFα and IFNγ, the latter of which was essential for Sca-1+ monocyte differentiation. Monocyte recruitment and GC disruption also occurred during LPS-supplemented vaccination and Listeria monocytogenes infection. Thus, systemic activation of the innate immune response upon severe bacterial infection is induced at the expense of antibody-mediated immunity.

Key to the success of intracellular pathogens is the ability to sense and respond to a changing host cell environment. Macrophages exposed to microbial products undergo metabolic changes that drive inflammatory responses. However, the role of macrophage metabolic reprogramming in bacterial adaptation to the intracellular environment has not been explored. Here, using metabolic profiling and dual RNA sequencing, we show that succinate accumulation in macrophages is sensed by intracellular Salmonella Typhimurium (S. Tm) to promote antimicrobial resistance and type III secretion. S. Tm lacking the succinate uptake transporter DcuB displays impaired survival in macrophages and in mice. Thus, S. Tm co-opts the metabolic reprogramming of infected macrophages as a signal that induces its own virulence and survival, providing an additional perspective on metabolic host-pathogen cross-talk.

Interactions between intracellular bacteria and mononuclear phagocytes give rise to diverse cellular phenotypes that may determine the outcome of infection. Recent advances in single cell RNA-seq (scRNA-seq) have identified multiple subsets within the mononuclear population defined by unique molecular features, but the implications to their function during infection is unknown. Here, we applied high resolution kinetic analysis using microscopy, flow cytometry and scRNA-seq to survey the mononuclear niche of intracellular Salmonella Typhimurium ( S .Tm) during early systemic infection in mice. We describe an eclipse like growth kinetics in the spleen, with a first phase of bacterial control mediated by tissue resident red pulp macrophages. A second phase involved bacterial growth mediated by intracellular replication within a macrophage population we termed CD9 macrophages, that originate from non-classical monocytes. Nr4a1 e2−/− mice, specifically depleted of non-classical monocytes but not other mononuclear cells, are more resistant to S .Tm infection. Our study underscores a cell-type specific host-pathogen interaction that determines early infection growth dynamics and has implications to the infection outcome of the entire organism.

Interactions between intracellular bacteria and mononuclear phagocytes give rise to diverse cellular phenotypes that may determine the outcome of infection. Recent advances in single-cell RNA sequencing (scRNA-seq) have identified multiple subsets within the mononuclear population, but implications to their function during infection are limited. Here, we surveyed the mononuclear niche of intracellular Salmonella Typhimurium (S.Tm) during early systemic infection in mice. We described eclipse-like growth kinetics in the spleen, with a first phase of bacterial control mediated by tissue-resident red-pulp macrophages. A second phase involved extensive bacterial replication within a macrophage population characterized by CD9 expression. We demonstrated that CD9+ macrophages induced pathways for detoxificating oxidized lipids, that may be utilized by intracellular S.Tm. We established that CD9+ macrophages originated from non-classical monocytes (NCM), and NCM-depleted mice were more resistant to S.Tm infection. Our study defines macrophage subset-specific host-pathogen interactions that determine early infection dynamics and infection outcome of the entire organism.

Methionine is a nutritionally essential sulfur-containing amino acid found at low levels in plant tissues. Yet, the factors that regulate its synthesis and accumulation in seeds are not fully known. Recent genetic studies demonstrate that Arabidopsis seeds are able to synthesize methionine de novo through the aspartate family pathway similarly to vegetative tissues; however, additional biochemical studies suggest that the S-methylmethionine (SMM) cycle also plays a major role in methionine synthesis in seeds. To better understand the contribution of these two pathways to methionine synthesis, we have sampled various vegetative and reproductive tissues during the Arabidopsis life cycle and determined the contents of soluble and protein-incorporated methionine, SMM, as well as the expression levels of the key genes involved in these two pathways. Our results strengthen the hypothesis that SMM that is produced in the rosette leaves from methionine contributes to methionine accumulation in seeds. However, the SMM cycle may have additional functions in plant tissues since its key genes were expressed in all of the examined tissues, although at different rates. The accumulation patterns of soluble and protein-incorporated methionine during the Arabidopsis life cycle were found to be similar to most of the other amino acids, especially to those belonging to the branched-chain and aromatic amino acids that are produced in chloroplasts together with methionine. This indicates that similar factors regulate the levels of amino acids during development.