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The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1β production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to E. coli, and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with E. coli, this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome., Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) engages interleukin-10 (IL-10) as an early innate immune response to regulate inflammation and promote the control of bladder infection. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. Here, we identify the major UPEC flagellar filament, FliC, as a key bacterial component sensed by the bladder innate immune system responsible for the induction of IL-10 synthesis. IL-10 responses of human as well as mouse bladder epithelial cell-monocyte cocultures were triggered by flagella of three major UPEC representative strains, CFT073, UTI89, and EC958. FliC purified to homogeneity induced IL-10 in vitro and in vivo as well as other functionally related cytokines, including IL-6. The genome-wide innate immunological context of FliC-induced IL-10 in the bladder was defined using RNA sequencing that revealed a network of transcriptional and antibacterial defenses comprising 1,400 genes that were induced by FliC. Of the FliC-responsive bladder transcriptome, altered expression of il10 and 808 additional genes were dependent on Toll-like receptor 5 (TLR5), according to analysis of TLR5-deficient mice. Examination of the potential of FliC and associated innate immune signature in the bladder to boost host defense, based on prophylactic or therapeutic administration to mice, revealed significant benefits for the control of UPEC. We conclude that detection of FliC through TLR5 triggers rapid IL-10 synthesis in the bladder, and FliC represents a potential immune modulator that might offer benefit for the treatment or prevention of UPEC UTI. IMPORTANCE Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to E. coli, and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with E. coli, this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome.

Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) engages interleukin-10 (IL-10) as an early innate immune response to regulate inflammation and promote the control of bladder infection. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. Here, we identify the major UPEC flagellar filament, FliC, as a key bacterial component sensed by the bladder innate immune system responsible for the induction of IL-10 synthesis. IL-10 responses of human as well as mouse bladder epithelial cell-monocyte cocultures were triggered by flagella of three major UPEC representative strains, CFT073, UTI89, and EC958. FliC purified to homogeneity induced IL-10 in vitro and in vivo as well as other functionally related cytokines, including IL-6. The genome-wide innate immunological context of FliC-induced IL-10 in the bladder was defined using RNA sequencing that revealed a network of transcriptional and antibacterial defenses comprising 1,400 genes that were induced by FliC. Of the FliC-responsive bladder transcriptome, altered expression of il10 and 808 additional genes were dependent on Toll-like receptor 5 (TLR5), according to analysis of TLR5-deficient mice. Examination of the potential of FliC and associated innate immune signature in the bladder to boost host defense, based on prophylactic or therapeutic administration to mice, revealed significant benefits for the control of UPEC. We conclude that detection of FliC through TLR5 triggers rapid IL-10 synthesis in the bladder, and FliC represents a potential immune modulator that might offer benefit for the treatment or prevention of UPEC UTI. IMPORTANCE Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to E. coli, and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with E. coli, this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome.

Summary Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic E scherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional programme associated with intramacrophage survival, we performed host–pathogen co‐transcriptome analyses using RNA sequencing. Mouse bone marrow‐derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated and quantified the mammalian and bacterial transcriptomes. Bone marrow‐derived macrophages responded to the two UPEC strains with a broadly similar gene expression programme. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes up‐regulated at 24 h post‐infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co‐cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.

Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug resistant clone associated with urinary tract and bloodstream infections. Most ST131 strains exhibit resistance to multiple antibiotics and cause infections associated with limited treatment options. The largest sub-clonal ST131 lineage is resistant to fluoroquinolones, contains the type 1 fimbriae fimH30 allele and expresses an H4 flagella antigen. Flagella are motility organelles that contribute to UPEC colonisation of the upper urinary tract. In this study, we examined the specific role of H4 flagella in ST131 motility and interaction with host epithelial and immune cells. We show that the majority of H4-positive ST131 strains are motile and are enriched for flagella expression during static pellicle growth. We also tested the role of H4 flagella in ST131 through the construction of specific mutants, over-expression strains and isogenic mutants that expressed alternative H1 and H7 flagellar subtypes. Overall, our results revealed that H4, H1 and H7 flagella possess conserved phenotypes with regards to motility, epithelial cell adhesion, invasion and uptake by macrophages. In contrast, H4 flagella trigger enhanced induction of the anti-inflammatory cytokine IL-10 compared to H1 and H7 flagella, a property that may contribute to ST131 fitness in the urinary tract.

Uropathogenic Escherichia coli (UPEC) is the cause of ~75% of all urinary tract infections (UTIs) and is increasingly associated with multidrug resistance. This includes UPEC strains from the recently emerged and globally disseminated sequence type 131 (ST131), which is now the dominant fluoroquinolone-resistant UPEC clone worldwide. Most ST131 strains are motile and produce H4-type flagella. Here, we applied a combination of saturated Tn5 mutagenesis and transposon directed insertion site sequencing (TraDIS) as a high throughput genetic screen and identified 30 genes associated with enhanced motility of the reference ST131 strain EC958. This included 12 genes that repress motility of E. coli K-12, four of which (lrhA, ihfA, ydiV, lrp) were confirmed in EC958. Other genes represented novel factors that impact motility, and we focused our investigation on characterisation of the mprA, hemK and yjeA genes. Mutation of each of these genes in EC958 led to increased transcription of flagellar genes (flhD and fliC), increased expression of the FliC flagellin, enhanced flagella synthesis and a hyper-motile phenotype. Complementation restored all of these properties to wild-type level. We also identified Tn5 insertions in several intergenic regions (IGRs) on the EC958 chromosome that were associated with enhanced motility; this included flhDC and EC958_1546. In both of these cases, the Tn5 insertions were associated with increased transcription of the downstream gene(s), which resulted in enhanced motility. The EC958_1546 gene encodes a phage protein with similarity to esterase/deacetylase enzymes involved in the hydrolysis of sialic acid derivatives found in human mucus. We showed that over-expression of EC958_1546 led to enhanced motility of EC958 as well as the UPEC strains CFT073 and UTI89, demonstrating its activity affects the motility of different UPEC strains. Overall, this study has identified and characterised a number of novel factors associated with enhanced UPEC motility.