Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn's disease associated adherent-invasive Escherichia coli infection.

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohn's disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNFα), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model.

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

Salmonella enterica Serovar Typhimurium Travels to Mesenteric Lymph Nodes Both with Host Cells and Autonomously.

Salmonella infection is a globally important cause of gastroenteritis and systemic disease and is a useful tool to study immune responses in the intestine. Although mechanisms leading to immune responses against Salmonella have been extensively studied, questions remain about how bacteria travel from the intestinal mucosa to the mesenteric lymph nodes (MLN), a key site for Ag presentation. In this study, we used a mouse model of infection with Salmonella enterica serovar Typhimurium (STM) to identify changes in intestinal immune cells induced during early infection. We then used fluorescently labeled STM to identify interactions with immune cells from the site of infection through migration in lymph to the MLN. We show that viable STM can be carried in the lymph by any subset of migrating dendritic cells but not by macrophages. Moreover, approximately half of the STM in lymph are not associated with cells at all and travel autonomously. Within the MLN, STM associates with dendritic cells and B cells but predominantly with MLN-resident macrophages. In conclusion, we describe the routes used by STM to spread systemically in the period immediately postinfection. This deeper understanding of the infection process could open new avenues for controlling it.

SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection.

Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors.

Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment.

Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

Bacterial secreted effectors and caspase-3 interactions.

Apoptosis is a critical process that intrinsically links organism survival to its ability to induce controlled death. Thus, functional apoptosis allows organisms to remove perceived threats to their survival by targeting those cells that it determines pose a direct risk. Central to this process are apoptotic caspases, enzymes that form a signalling cascade, converting danger signals via initiator caspases into activation of the executioner caspase, caspase-3. This enzyme begins disassembly of the cell by activating DNA degrading enzymes and degrading the cellular architecture. Interaction of pathogenic bacteria with caspases, and in particular, caspase-3, can therefore impact both host cell and bacterial survival. With roles outside cell death such as cell differentiation, control of signalling pathways and immunomodulation also being described for caspase-3, bacterial interactions with caspase-3 may be of far more significance in infection than previously recognized. In this review, we highlight the ways in which bacterial pathogens have evolved to subvert caspase-3 both through effector proteins that directly interact with the enzyme or by modulating pathways that influence its activation and activity.

Mapping the Influence of the Gut Microbiota on Small Molecules across the Microbiome Gut Brain Axis.

Microbes exert influence across the microbiome-gut-brain axis through neurotransmitter production, induction of host immunomodulators, or the release or induction of other microbial or host molecules. Here, we used mass spectrometry imaging (MSI), a label-free imaging tool, to map molecular changes in the gut and brain in germ-free, antibiotic-treated and control mice. We determined spatial distribution and relative quantification of neurotransmitters and their precursors in response to the microbiome. Using untargeted MSI, we detected a significant change in the levels of four identified small molecules in the brains of germ-free animals compared to controls. However, antibiotic treatment induced no significant changes in these same metabolites in the brain after 1 week of treatment. This work exemplifies the utility of MSI as a tool for the study of known and discovery of novel, mediators of microbiome-gut-brain axis communication.

Regulatory T cells control the dynamic and site-specific polarization of total CD4 T cells following Salmonella infection.

FoxP3+ regulatory T cells (Tregs) control inflammation and maintain mucosal homeostasis, but their functions during infection are poorly understood. Th1, Th2, and Th17 cells can be identified by master transcription factors (TFs) T-bet, GATA3, and RORγT; Tregs also express these TFs. While T-bet+ Tregs can selectively suppress Th1 cells, it is unclear whether distinct Treg populations can alter Th bias. To address this, we used Salmonella enterica serotype Typhimurium to induce nonlethal colitis. Following infection, we observed an early colonic Th17 response within total CD4 T cells, followed by a Th1 bias. The early Th17 response, which contains both Salmonella-specific and non-Salmonella-specific cells, parallels an increase in T-bet+ Tregs. Later, Th1 cells and RORγT+ Tregs dominate. This reciprocal dynamic may indicate that Tregs selectively suppress Th cells, shaping the immune response. Treg depletion 1-2 days post-infection shifted the early Th17 response to a Th1 bias; however, Treg depletion 6-7 days post-infection abrogated the Th1 bias. Thus, Tregs are necessary for the early Th17 response, and for a maximal Th1 response later. These data show that Tregs shape the overall tissue CD4 T cell response and highlight the potential for subpopulations of Tregs to be used in targeted therapeutic approaches.

Author Correction: Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

This Article was originally published under Nature Research's License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly.

Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence.

Microbiome-derived carnitine mimics as previously unknown mediators of gut-brain axis communication.

Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen-free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function.

Caspase-3 cleavage of Salmonella type III secreted effector protein SifA is required for localization of functional domains and bacterial dissemination.

SifA is a bi-functional Type III Secretion System (T3SS) effector protein that plays an important role in Salmonella virulence. The N-terminal domain of SifA binds SifA-Kinesin-Interacting-Protein (SKIP), and via an interaction with kinesin, forms tubular membrane extensions called Sif filaments (Sifs) that emanate from the Salmonella Containing Vacuole (SCV). The C-terminal domain of SifA harbors a WxxxE motif that functions to mimic active host cell GTPases. Taken together, SifA functions in inducing endosomal tubulation in order to maintain the integrity of the SCV and promote bacterial dissemination. Since SifA performs multiple, unrelated functions, the objective of this study was to determine how each functional domain of SifA becomes processed. Our work demonstrates that a linker region containing a caspase-3 cleavage motif separates the two functional domains of SifA. To test the hypothesis that processing of SifA by caspase-3 at this particular site is required for function and proper localization of the effector protein domains, we developed two tracking methods to analyze the intracellular localization of SifA. We first adapted a fluorescent tag called phiLOV that allowed for type-III secretion system (T3SS) mediated delivery of SifA and observation of its intracellular colocalization with caspase-3. Additionally, we created a dual-tagging strategy that permitted tracking of each of the SifA functional domains following caspase-3 cleavage to different subcellular locations. The results of this study reveal that caspase-3 cleavage of SifA is required for the proper localization of functional domains and bacterial dissemination. Considering the importance of these events in Salmonella pathogenesis, we conclude that caspase-3 cleavage of effector proteins is a more broadly applicable effector processing mechanism utilized by Salmonella to invade and persist during infection.

Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

Bacterial-mediated cancer therapy has shown great promise in in vivo tumour models with increased survival rates post-bacterial treatment. Improving efficiency of bacterial-mediated tumour regression has focused on controlling and exacerbating bacterial cytotoxicity towards tumours. One mechanism that has been used to carry this out is the process of bactofection where post-invasion, bacteria deliver plasmid-borne mammalian genes into target cells for expression. Here we utilised the cancer-targeting Salmonella Typhimurium strain, SL7207, to carry out bactofection into triple negative breast cancer MDA-MB-231 cells. However, we noted that post-transformation with the commonly used mammalian expression vector pEGFP, S. Typhimurium became filamentous, attenuated and unable to invade target cells efficiently. Filamentation did not occur in Escherichia coli-transformed with the same plasmid. Further investigation identified the region inducing S. Typhimurium filamentation as being the f1 origin of replication (f1 ori), an artefact of historic use of mammalian plasmids for single stranded DNA production. Other f1 ori-containing plasmids also induced the attenuated phenotype, while removal of the f1 ori from pEGFP restored S. Typhimurium virulence and increased the bactofection capacity. This work has implications for interpretation of prior bactofection studies employing f1 ori-containing plasmids in S. Typhimurium, while also indicating that future use of S. Typhimurium in targeting tumours should avoid the use of these plasmids.

Inflammation associated ethanolamine facilitates infection by Crohn's disease-linked adherent-invasive Escherichia coli.

BACKGROUND: The predominance of specific bacteria such as adherent-invasive Escherichia coli (AIEC) within the Crohn's disease (CD) intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is however readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. METHODS: Quantitative RT-PCR (qRT-PCR) to determine expression of genes central to ethanolamine metabolism; transmission electron microscopy to detect presence of bacterial microcompartments (MCPs); in vitro infections of both murine and human macrophage cell lines examining intracellular replication of the AIEC-type strain LF82 and clinical E. coli isolates in the presence of ethanolamine; determination of E. coli ethanolamine utilization (eut) operon transcription in faecal samples from healthy patients, patients with active CD and the same patients in remission following treatment. RESULTS: Growth on the intestinal short chain fatty acid propionic acid (PA) stimulates significantly increased transcription of the eut operon (fold change relative to glucose: >16.9; p-value <.01). Additionally ethanolamine was accessible to intra-macrophage AIEC and stimulated significant increases in growth intracellularly when it was added extracellularly at concentrations comparable to those in the human intestine. Finally, qRT-PCR indicated that expression of the E. coli eut operon was increased in children with active CD compared to healthy controls (fold change increase: >4.72; P < .02). After clinical remission post-exclusive enteral nutrition treatment, the same CD patients exhibited significantly reduced eut expression (Pre vs Post fold change decrease: >15.64; P < .01). INTERPRETATION: Our data indicates a role for ethanolamine metabolism in selecting for AIEC that are consistently overrepresented in the CD intestine. The increased E. coli metabolism of ethanolamine seen in the intestine during active CD, and its decrease during remission, indicates ethanolamine use may be a key factor in shaping the intestinal microbiome in CD patients, particularly during times of inflammation. FUND: This work was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/K008005/1 & BB/P003281/1 to DMW; by a Tenovus Scotland grant to MJO; by Glasgow Children's Hospital Charity, Nestle Health Sciences, Engineering and Physical Sciences Research Council (EPSRC) and Catherine McEwan Foundation grants awarded to KG; and by a Natural Environment Research Council (NERC) fellowship (NE/L011956/1) to UZI. The IBD team at the Royal Hospital for Children, Glasgow are supported by the Catherine McEwan Foundation and Yorkhill IBD fund. RKR and RH are supported by NHS Research Scotland Senior fellowship awards.

Correction: Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

This Article was originally published with one of the panels in Figure 5A inserted twice (SL-pEGFP). In Figure 5B there was also a typo. SL-LacZ should have read SL-pEGFP(-f1). Both 5A and 5B are corrected in both the PDF and HTML versions of the Article.

Draft Genome Sequence of the Tumor-Targeting Salmonella enterica Serovar Typhimurium Strain SL7207.

Salmonella enterica serovar Typhimurium strain SL7207 is a genetically modified derivative of strain SL1344, which preferentially accumulates in tumors and can be used as a vehicle for tissue-specific gene delivery in vivo Here, we report the draft genome sequence of SL7207, confirming a purported aroA deletion and four single-nucleotide polymorphisms compared to SL1344.

Mass spectrometry imaging identifies palmitoylcarnitine as an immunological mediator during Salmonella Typhimurium infection.

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome.

Draft Genome Sequence of the Commensal Escherichia coli Strain F-18.

Here, we report the draft genome sequence of Escherichia coli strain F-18, originally isolated from the feces of a healthy individual in 1977. The draft genome is 5,246,829 bp, with a G+C content of 50.50%, and it encodes 4,933 predicted coding sequences (CDSs), 10 rRNAs, and 84 tRNAs.

An Unbalanced Rearrangement of Chromosomes 4:20 is Associated with Childhood Osteoporosis and Reduced Caspase-3 Levels.

The purpose of this study was to investigate the association of a chromosome 4:20 imbalance with osteoporosis in three related children. Bone biochemistry, bone turnover markers, and dual-energy X-ray absorptiometry (DXA) scanning were performed in all three cases and bone biopsy and histomorphometry in one. The chromosome imbalance was delineated by array comparative genomic hybridization (aCGH) and analyzed for candidate genes. A potential candidate gene within the deleted region is caspase-3, previously linked to low bone mineral density (BMD) in heterozygous mice thus caspase-3 activity was measured in cases and controls. Routine bone biochemistry and markers of bone turnover did not reveal any abnormality. DXA showed reduced total and lumbar spine bone mineral content. aCGH showed an 8 megabase (Mb) deletion of terminal chromosome 4q incorporating a region previously linked to low BMD and a 15 Mb duplication of terminal chromosome 20p. Bone biopsy showed a high bone turnover state, trabecularisation of cortical bone and numerous small osteoclasts coupled with normal bone formation. Basal serum caspase-3 activity was lower in cases compared with controls. We conclude that the early-onset osteoporosis with low basal levels of caspase-3 and abnormal osteoclasts is a feature of this chromosomal translocation. Further investigation of the role of the deleted and duplicated genes and especially caspase-3 is required.