Search

Citrobacter rodentium, a natural mouse pathogen which colonises the colon of immuno-competent mice, provides a robust model for interrogating host-pathogen-microbiota interactions in vivo. This model has been key to providing new insights into local host responses to enteric infection, including changes in intestinal epithelial cell immunometabolism and mucosal immunity. C. rodentium injects 31 bacterial effectors into epithelial cells via a type III secretion system (T3SS). Recently, these effectors were shown to be able to form multiple intracellular subnetworks which can withstand significant contractions whilst maintaining virulence. Here we highlight recent advances in understanding gut mucosal responses to infection and effector biology, as well as potential uses for artificial intelligence (AI) in understanding infectious disease and speculate on the role of T3SS effector networks in host adaption.

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae , modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. An analysis of large K. pneumoniae genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine-to-thymine transition at position 25 (25c > t) in ompK36. We show that the 25c > t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates K. pneumoniae in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c > t transition tips the balance toward treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c > t transition mediates an intramolecular messenger RNA (mRNA) interaction between a uracil encoded by 25t and the first adenine within the Shine–Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

Nutrients not only act as building blocks but also as signaling molecules. Nutrient-availability promotes cell growth and proliferation and suppresses catabolic processes, such as macroautophagy/autophagy. These effects are mediated by checkpoint kinases such as MTOR (mechanistic target of rapamycin kinase), which is activated by amino acids and growth factors, and AMP-activated protein kinase (AMPK), which is activated by low levels of glucose or ATP. These kinases have wide-ranging activities that can be co-opted by immune cells upon exposure to danger signals, cytokines or pathogens. Here, we discuss recent insight into the regulation and repurposing of nutrient-sensing responses by the innate immune system during infection. Moreover, we examine how natural mutations and pathogen-mediated interventions can alter the balance between anabolic and autophagic pathways leading to a breakdown in tissue homeostasis and/or host defense. Abbreviations: AKT1/PKB: AKT serine/threonine kinase 1; ATG: autophagy related; BECN1: beclin 1; CGAS: cyclic GMP-AMP synthase; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; FFAR: free fatty acid receptor; GABARAP: GABA type A receptor-associated protein; IFN: interferon; IL: interleukin; LAP: LC3-associated phagocytosis; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; NLR: NOD (nucleotide-binding oligomerization domain) and leucine-rich repeat containing proteins; PI3K, phosphoinositide 3-kinase; PRR: pattern-recognition receptor; PtdIns3K: phosphatidylinositol 3-kinase; RALB: RAS like proto-oncogene B; RHEB: Ras homolog, MTORC1 binding; RIPK1: receptor interacting serine/threonine kinase 1; RRAG: Ras related GTP binding; SQSTM1/p62: sequestosome 1; STING1/TMEM173: stimulator of interferon response cGAMP interactor 1; STK11/LKB1: serine/threonine kinase 11; TBK1: TANK binding kinase 1; TLR: toll like receptor; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; TRIM: tripartite motif protein; ULK1: unc-51 like autophagy activating kinase 1; V-ATPase: vacuolar-type H+-proton-translocating ATPase.

Mycobacterium tuberculosis infection causes high rates of morbidity and mortality. Host-directed therapy may enhance the immune response, reduce tissue damage and shorten treatment duration. The inflammasome is integral to innate immune responses but over-activation has been described in tuberculosis (TB) pathology and TB-immune reconstitution syndrome. Here we explore how clinical isolates differentially activate the inflammasome and how inflammasome inhibition can lead to enhanced bacterial clearance. Wild-type, Nlrp3−/−/Aim2−/−, Casp1/11−/− and Asc−/− murine bone-marrow derived macrophages (BMDMs) were infected with laboratory strain M. tuberculosis H37Rv or clinical isolates from various lineages. Inflammasome activation and bacterial numbers were measured, and pharmacological inhibition of NLRP3 was achieved using MCC950. Clinical isolates of M. tuberculosis differed in their ability to activate inflammasomes. Beijing isolates had contrasting effects on IL-1β and caspase-1 activation, but all clinical isolates induced lower IL-1β release than H37Rv. Our studies suggest the involvement of NLRP3, AIM2 and an additional unknown sensor in IL-1β maturation. Pharmacological blockade of NLRP3 with MCC950 reduced bacterial survival, and combined treatment with the antimycobacterial drug rifampicin enhanced the effect. Modulating the inflammasome is an attractive adjunct to current anti-mycobacterial therapy that warrants further investigation.

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

Type III secretion system (T3SS) effectors are key virulence factors that underpin the infection strategy of many clinically important Gram-negative pathogens, including Salmonella enterica, Shigella spp., enteropathogenic and enterohemorrhagic Escherichia coli and their murine equivalent, Citrobacter rodentium. The cellular processes or proteins targeted by the effectors can be common to multiple pathogens or pathogen-specific. The main approach to understanding T3SS-mediated pathogenesis has been to determine the contribution of one effector at a time, with the aim of piecing together individual functions and unveiling infection mechanisms. However, in contrast to this prevailing approach, simultaneous deletion of multiple effectors revealed that they function as an interconnected network in vivo, uncovering effector codependency and context-dependent effector essentiality. This paradigm shift in T3SS biology is at the heart of this opinion article.

INTRODUCTION Infections with many Gram-negative pathogens, including Escherichia coli, Salmonella, Shigella, and Yersinia, rely on the injection of effectors via type III secretion systems (T3SSs). The effectors hijack cellular processes through multiple mechanisms, including molecular mimicry and diverse enzymatic activities. Although in vitro analyses have shown that individual effectors can exhibit complementary, interdependent, or antagonistic relationships, most in vivo studies have focused on the contribution of single effectors to pathogenesis. Citrobacter rodentium is a natural mouse pathogen that shares infection strategies and virulence factors with the human pathogens enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). The ability of these pathogens to colonize the gastrointestinal tract is mediated by the injection of effectors via a T3SS. Although C. rodentium infects 31 effectors, the prototype EPEC strain E2348/69 translocates 21 effectors. RATIONALE The aim of this study was to test the hypotheses that, rather than operating individually, the T3SS effectors form robust intracellular networks that can sustain large contractions and that expanded effector repertoires play a role in distinct disease phenotypes and host adaption. RESULTS We tested the effector-network paradigm by infecting mice with >100 C. rodentium effector mutant combinations. First, using machine learning prediction algorithms, we discovered additional effectors, NleN and NleO. We then sequentially deleted effector genes from two distinct starting points to reach sustainable endpoints, which resulted in strains missing 19 unrelated effectors (CR14) or 10 effectors involved in the modulation of innate immune responses in intestinal epithelial cells (IECs) (CRi9). Moreover, we deleted Map and EspF, which target the mitochondria and disrupt tight junctions. Unexpectedly, all strains colonized the colon and activated conserved metabolic and antimicrobial processes in the IECs while eliciting distinct cytokine and immune cell infiltration responses. In particular, although infection with C. rodentium Δmap/ΔespF failed to induce secretion of interleukin-22 (IL-22), CR14 and CRi9 triggered heightened secretion of IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and of IL-22, interferon-γ (IFN-γ), and IL-17 from colonic explants, respectively. Nonetheless, infection with CR14 or CRi9 induced protective immunity against secondary infections. Although Tir, EspZ, and NleA are essential, other effectors exhibit context-dependent essentiality in vivo. Moreover, C. rodentium expressing the effector repertoire of EPEC E2348/69 failed to efficiently colonize mice. We used curated functional information and our in vivo data to train a machine learning model that predicted values for colonization efficiency of previously uncharacterized mutant combinations. Notably, a mutant with a low predicted value, lacking only nleF, nleG8, nleG1, nleB, and espL, failed to colonize. CONCLUSION Our analysis revealed that T3SS effectors form robust networks, which can sustain substantial contractions while maintaining virulence, and that the composition of the effector network contributes to host adaptation. Alternative effector networks within a single pathogen triggered markedly different immune responses yet induced protective immunity. CR14 did not tolerate any further contraction, which suggests that this network reached its robustness limit with only 12 effectors. As the robustness limits of other effector networks depend on the contraction starting point and the order of the deletions, machine learning models could transform our ability to predict alternative network functions. Together, this study demonstrates the robustness of T3SS effector networks and the ability of IECs to withstand drastic perturbations while maintaining antibacterial functions. The work of T.I.R. and J.S.C. was funded by the CRUK Centre grant with reference no. C309/A25144. N.W. was supported by PhD fellowships from the Manna Center Program for Food Safety and Security at Tel Aviv University and the Edmond J. Safra Center for Bioinformatics at Tel-Aviv University. The work of E. N.-B. was funded by a PhD FPU grant from the Spanish government (FPU2017/04179). A. R.-P. was partially funded by project PID2019-106960GB-I00 (AEI/FEDER, Spain) and by InGEMICS-CM, FSE/FEDER, Comunidad de Madrid Project B2017/BMD-3691. L.A.F. is supported by grant BIO2017-89081-R (AEI/MICIU/FEDER, European Union). G.F. is supported by an MRC program grant (MR/R02671/), a Royal Society grant (IC160080), and a Wellcome Investigator Award (107057/Z/15/Z). Peerreview

Salmonella Paratyphi A (SPtA) remains one of the leading causes of enteric (typhoid) fever. Yet, despite the recent increased rate of isolation from patients in Asia, our understanding of its pathogenesis is incomplete. Here we investigated inflammasome activation in human macrophages infected with SPtA. We found that SPtA induces GSDMD‐mediated pyroptosis via activation of caspase‐1, caspase‐4 and caspase‐8. Although we observed no cell death in the absence of a functional Salmonella pathogenicity island‐1 (SPI‐1) injectisome, HilA‐mediated overexpression of the SPI‐1 regulon enhances pyroptosis. SPtA expresses FepE, an LPS O‐antigen length regulator, which induces the production of very long O‐antigen chains. Using a ΔfepE mutant we established that the very long O‐antigen chains interfere with bacterial interactions with epithelial cells and impair inflammasome‐mediated macrophage cell death. Salmonella Typhimurium (STm) serovar has a lower FepE expression than SPtA, and triggers higher pyroptosis, conversely, increasing FepE expression in STm reduced pyroptosis. These results suggest that differential expression of FepE results in serovar‐specific inflammasome modulation, which mirrors the pro‐ and anti‐inflammatory strategies employed by STm and SPtA, respectively. Our studies point towards distinct mechanisms of virulence of SPtA, whereby it attenuates inflammasome‐mediated detection through the elaboration of very long LPS O‐polysaccharides.

Enteric pathogen–host interactions occur at multiple interfaces, including the intestinal epithelium and deeper organs of the immune system. Microbial ligands and activities are detected by host sensors that elicit a range of immune responses. Membrane‐bound toll‐like receptors and cytosolic inflammasome pathways are key signal transducers that trigger the production of pro‐inflammatory molecules, such as cytokines and chemokines, and regulate cell death in response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tussle between bacterial pathogens and the host. Inflammasomes are complexes that activate caspase‐1 and are regulated by related caspases, such as caspase‐11, ‐4, ‐5 and ‐8. Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasome activation (e.g. Listeria monocytogenes, Helicobacter pylori), others (e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasome pathways and their immune functions, and discuss how enteric bacterial pathogens interact with them. These studies have not only shed light on inflammasome‐mediated immunity, but also the exciting area of mammalian cytosolic immune surveillance.

Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection., Two Shigella species, Shigella flexneri and Shigella sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle- and high-income countries. S. flexneri is a prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species.

Lentiviral vectors are gene delivery vehicles that integrate into the host genome of dividing and non-dividing mammalian cells facilitating long-term transgene expression. Lentiviral vector versatility is greatly increased by incorporating heterologous viral envelope proteins onto the vector particles instead of the native envelope, conferring on these pseudotyped vectors a modified tropism and host range specificity. We investigated the pseudotyping efficiency of HIV-1 based lentiviral vectors with alphaviral envelope proteins from the Chikungunya Virus (CHIKV-G) and Sindbis Virus (SINV-G). Following vector production optimisation, titres for the CHIKV-G pseudotype were comparable to the VSV-G pseudotype but those for the SINV-G pseudotype were significantly lower. High titre CHIKV-G pseudotyped vector efficiently transduced various human and mouse neural cell lines and normal human astrocytes (NHA) in vitro. Although transduction was broad, tropism for NHAs was observed. In vivo stereotaxic delivery in striatum, thalamus and hippocampus respectively in the adult rat brain revealed localised transduction restricted to striatal astrocytes and hippocampal dentate granule neurons. Transduction of different subtypes of granule neurons from precursor to post-mitotic stages of differentiation was evident in the sub-granular zone and dentate granule cell layer. No significant inflammatory response was observed, but comparable to that of VSV-G pseudotyped lentiviral vectors. Robust long-term expression followed for three months post-transduction along with absence of neuroinflammation, coupled to the selective and unique neuron/glial tropism indicates that these vectors could be useful for modelling and gene therapy studies in the CNS.

TwoShigellaspecies,flexneriandsonnei, cause approximately 90% of bacterial dysentery worldwide. WhileS. flexneriis the dominant species in low-income countries,S. sonneicauses the majority of infections in middle and high-income countries.S. flexneriis a prototypic cytosolic bacterium; once intracellular it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. By contrast little is known about the invasion, vacuole escape and induction of pyroptosis duringS. sonneiinfection of macrophages. We demonstrate thatS. sonneicauses substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolicS. sonneiand hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen, which inS. sonneiis contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the T3SS was required for vacuole escape. Our findings suggest thatS. sonneihas adapted to an extracellular lifestyle by incorporating additional O-antigen into its surface structures compared to otherShigellaspecies.

Citrobacter rodentium is an extracellular enteric mouse-specific pathogen used to model infections with human pathogenic Escherichia coli and inflammatory bowel disease. C. rodentium injects type III secretion system effectors into intestinal epithelial cells (IECs) to target inflammatory, metabolic and cell survival pathways and establish infection. While the host responds to infection by activating innate and adaptive immune signalling, required for clearance, the IECs respond by rapidly shifting bioenergetics to aerobic glycolysis, which leads to oxygenation of the epithelium, an instant expansion of mucosal-associated commensal Enterobacteriaceae and a decline of obligate anaerobes. Moreover, infected IECs reprogramme intracellular metabolic pathways, characterized by simultaneous activation of cholesterol biogenesis, import and efflux, leading to increased serum and faecal cholesterol levels. In this Review we summarize recent advances highlighting the intimate relationship between C. rodentium pathogenesis, metabolism and the gut microbiota. Citrobacter rodentium, an extracellular mouse-specific enteric pathogen, provides a robust model for the study of physiological host–pathogen–microbiota interactions. In this Review, Frankel and colleagues highlight how the C. rodentium model has advanced our understanding of enteric infections and inflammatory bowel disease, in particular changes to host metabolism and inflammation.

The multidomain scaffold protein p62 (also called sequestosome-1) is involved in autophagy, antimicrobial immunity, and oncogenesis. Mutations in SQSTM1, which encodes p62, are linked to hereditary inflammatory conditions such as Paget’s disease of the bone, frontotemporal dementia (FTD), amyotrophic lateral sclerosis, and distal myopathy with rimmed vacuoles. Here, we report that p62 was proteolytically trimmed by the protease caspase-8 into a stable protein, which we called p62TRM. We found that p62TRM, but not full-length p62, was involved in nutrient sensing and homeostasis through the mechanistic target of rapamycin complex 1 (mTORC1). The kinase RIPK1 and caspase-8 controlled p62TRM production and thus promoted mTORC1 signaling. An FTD-linked p62 D329G polymorphism and a rare D329H variant could not be proteolyzed by caspase-8, and these noncleavable variants failed to activate mTORC1, thereby revealing the detrimental effect of these mutations. These findings on the role of p62TRM provide new insights into SQSTM1-linked diseases and mTORC1 signaling.

Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.