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BackgroundAccumulation of lipid in the liver is the first hallmark of both alcohol-related liver disease (ALD) and non-alcohol-related fatty liver disease (NAFLD). Recent studies indicate that specific mutations in lipid genes confer risk and might influence disease progression to irreversible liver cirrhosis. This study aimed to understand the function/s of lipid risk genes driving disease development in zebrafish genetic models of alcohol- and non-alcohol related fatty liver.MethodsWe used zebrafish larvae to investigate the effect of alcohol and high fat to model fatty liver and tested the utility of this model to study lipid risk gene functions. CRISPR/Cas9 gene editing was used to create knockdowns in 5 days post-fertilization zebrafish larvae for the available orthologs of human cirrhosis risk genes (pnpla3, faf2, tm6sf2). To establish fatty liver models, larvae were exposed to ethanol and a high fat diet (HFD) consisting of chicken egg yolk. Changes in morphology (imaging), survival, liver injury (biochemical tests, histopathology), gene expression (qPCR) and lipid accumulation (dye specific live imaging) were analysed across treatment groups to test the functions of these genes.ResultsExposure of 5-day post-fertilization (dpf) WT larvae to 2% ethanol or HFD for 48 hours developed measurable hepatic steatosis. CRISPR-Cas9 genome editing depletedpnpla3, faf2andtm6sf2gene expression in these CRISPR knockdown larvae (crispants). Knockdown significantly increased effects of ethanol and HFD toxicity by increasing hepatic steatosis and hepatic neutrophil recruitment ≥2-fold in all three crispants. Furthermore, ethanol or HFD exposure significantly altered the expression of genes associated with ethanol metabolism (cyp2y3) and lipid metabolism-related gene expression, includingatgl(triglyceride hydrolysis),axox1, echs1(fatty acid β-oxidation),fabp10a(transport),hmgcra(metabolism),notch1(signaling) andsrebp1(lipid synthesis), in all threepnpla3, faf2andtm6sf2crispants. Nile Red staining in all three crispants revealed significantly increased lipid droplet size and triglyceride accumulation in the livers following exposure to ethanol or HFD.ConclusionsWe identified roles forpnpla3, faf2andtm6sf2genes in triglyceride accumulation and fatty acid oxidation pathways in a zebrafish larvae model of fatty liver.

Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.

Mycobacterial granuloma formation involves significant stromal remodeling including the growth of leaky, granuloma-associated vasculature. These permeable blood vessels aid mycobacterial growth, as antiangiogenic or vascular normalizing therapies are beneficial host-directed therapies in preclinical models of tuberculosis across host-mycobacterial pairings. Using the zebrafish–Mycobacterium marinum infection model, we demonstrate that vascular normalization by inhibition of vascular endothelial protein tyrosine phosphatase (VE-PTP) decreases granuloma hypoxia, the opposite effect of hypoxia-inducing antiangiogenic therapy. Inhibition of VE-PTP decreased neutrophil recruitment to granulomas in adult and larval zebrafish, and decreased the proportion of neutrophils that extravasated distal to granulomas. Furthermore, VE-PTP inhibition increased the accumulation of T cells at M. marinum granulomas. Our study provides evidence that, similar to the effect in solid tumors, vascular normalization during mycobacterial infection increases the T cell:neutrophil ratio in lesions which may be correlates of protective immunity.

Pathogenic mycobacteria inhibit inflammasome activation to establish infection. Although it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux, and inflammasome activation has not been investigated. Here, we use Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis infection of THP-1 cells to demonstrate that pathogenic mycobacteria up-regulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are at least partially mediated by NLRP3 inflammasome activation, caspase-mediated release of IL-1β, and downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections.

Ohmyungsamycin A and ecumicin are structurally related cyclic depsipeptide natural products that possess activity against

Heme oxygenase function is highly conserved between vertebrates where it plays important roles in normal embryonic development and controls oxidative stress. Expression of the zebrafish heme oxygenase 1 genes is known to be responsive to oxidative stress suggesting a conserved physiological function. In this study, we generate a knockout allele of zebrafish

Herein, we report the design and synthesis of inhibitors of

Mycobacterium abscessus infections are of increasing global prevalence and are often difficult to treat due to complex antibiotic resistance profiles. While there are similarities between the pathogenesis of M. abscessus and tuberculous mycobacteria, including granuloma formation and stromal remodeling, there are distinct molecular differences at the host-pathogen interface. Here we have used a zebrafish-M. abscessus model and host-directed therapies that were previously identified in the zebrafish-M. marinum model to identify potential host-directed therapies against M. abscessus infection. We find efficacy of anti-angiogenic and vascular normalizing therapies against rough M. abscessus infection, but no effect of anti-platelet drugs.

Uropathogenic Escherichia coli (UPEC) causes urinary tract infections that can result in sepsis. The haemostatic system is protective in the pyelonephritis stage of ascending UPEC infection, but the role of the haemostatic system has not been investigated during sepsis. Here we utilize a zebrafish-UPEC systemic infection model to visualize infection-induced coagulation and examine the effects of commonly prescribed anti-haemostatic medications on the infection severity. Treatment of systemically infected zebrafish with warfarin, aspirin, or ticagrelor reduced host survival, while stabilization of clots with aminocaproic acid increased host survival. Anti-haemostatic drug treatment increased UPEC burden. Our findings provide evidence that commonly prescribed anti-haemostatic medications may worsen the outcome of severe UPEC infection.

Pidotimod (PDT) is a synthetic dipeptide molecule which can improve immune responses in mice and humans, protecting hosts from infection. However, the exact mechanism of protection remains ill-defined. The effect of pidotimod has not yet been investigated in the inflammatory response of zebrafish. In this study, we used tail wound and infection models of zebrafish to study the effect of PDT on inflammation. We found that zebrafish larvae were sensitive to PDT immersion causing toxicity at doses above 50 μg/mL. The tail wound assay showed that PDT increased the recruitment of neutrophils and macrophages to the wound site and promoted the transcription of the pro-inflammatory cytokine il1b. However, we did not observe protection of uropathogenic Escherichia coli or Mycobacterium marinum infected zebrafish larvae following PDT treatment. This study provides a new platform for PDT research, which is worthy of further research to identify further effects of PDT therapy.

Regulation of host microRNA (miRNA) expression is a contested node that controls the host immune response to mycobacterial infection. The host must overcome concerted subversive efforts of pathogenic mycobacteria to launch and maintain a protective immune response. Here we examine the role of miR-126 in the zebrafish model of Mycobacterium marinum infection and identify a protective role for this infection-induced miRNA through multiple effector pathways. Specifically, we analyse the impact of the miR-126 knockdown-induced tsc1a and cxcl12a/ccl2/ccr2 signalling axes during early host-M. marinum interactions. We find a strong detrimental effect of tsc1a upregulation that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death despite dramatically higher recruitment of macrophages to the site of infection. We demonstrate that infection-induced miR-126 suppresses tsc1 and cxcl12a expression thus improving macrophage function early in infection, partially through activation of mTOR signalling and strongly through preventing the recruitment of Ccr2+ permissive macrophages, resulting in the recruitment of protective tnfa-expressing macrophages. Together our results demonstrate an important role for infection-induced miR-126 in shaping an effective immune response to M. marinum infection in zebrafish embryos.

Pathogenic mycobacteria induce the formation of hypoxic granulomas during latent tuberculosis (TB) infection, in which the immune system contains, but fails to eliminate the mycobacteria. Fatty acid metabolism-related genes are relatively overrepresented in the mycobacterial genome and mycobacteria favor host-derived fatty acids as nutrient sources. However, whether and how mycobacteria modulate host fatty acid metabolism to drive granuloma progression remains unknown. Here, we report that mycobacteria under hypoxia markedly secrete the protein Rv0859/MMAR_4677 (Fatty-acid degradation A, FadA), which is also enriched in tuberculous granulomas. FadA acts as an acetyltransferase that converts host acetyl-CoA to acetoacetyl-CoA. The reduced acetyl-CoA level suppresses H3K9Ac-mediated expression of the host proinflammatory cytokine Il6, thus promoting granuloma progression. Moreover, supplementation of acetate increases the level of acetyl-CoA and inhibits the formation of granulomas. Our findings suggest an unexpected mechanism of a hypoxia-induced mycobacterial protein suppressing host immunity via modulation of host fatty acid metabolism and raise the possibility of a novel therapeutic strategy for TB infection.

Gut dysbiosis is an important modifier of pathologies including cardiovascular disease but our understanding of the role of individual microbes is limited. Here, we have used transplantation of mouse microbiota into microbiota-deficient zebrafish larvae to study the interaction between members of a mammalian high fat diet-associated gut microbiota with a lipid rich diet challenge in a tractable model species. We find zebrafish larvae are more susceptible to hyperlipidaemia when exposed to the mouse high fat-diet-associated microbiota and that this effect can be driven by two individual bacterial species fractionated from the mouse high fat-diet-associated microbiota. We find Stenotrophomonas maltophilia increases the hyperlipidaemic potential of chicken egg yolk to zebrafish larvae independent of direct interaction between S. maltophilia and the zebrafish host. Colonisation by live, or exposure to heat-killed, Enterococcus faecalis accelerates hyperlipidaemia via host Myd88 signalling. The hyperlipidaemic effect is replicated by exposure to the Gram positive Toll-like receptor agonists peptidoglycan and lipoteichoic acid in a MyD88-dependent manner. In this work, we demonstrate the applicability of zebrafish as a tractable host for the identification of gut microbes that can induce conditional host phenotypes via microbiota transplantation and subsequent challenge with a high fat diet.

Cigarette smoke (CS)-induced inflammation leads to a range of diseases including chronic obstructive pulmonary disease and cancer. The gut microbiota is a major modifying environmental factor that determine the severity of cigarette smoke-induced pathology. Microbiomes and metabolites from CS-exposed mice exacerbate lung inflammation via the gut-lung axis of shared mucosal immunity in mice but these systems are expensive to establish and analyse. Zebrafish embryos and larvae have been used to model the effects of cigarette smoking on a range of physiological processes and offer an amenable platform for screening modifiers of cigarette smoke-induced pathologies with key features of low cost and rapid visual readouts. Here we exposed zebrafish larvae to cigarette smoke extract (CSE) and characterised a CSE-induced leukocytic inflammatory phenotype with increased neutrophilic and macrophage inflammation in the gut. The CSE-induced phenotype was exacerbated by co-exposure to microbiota from the faeces of CS-exposed mice, but not control mice. Microbiota could be recovered from the gut of zebrafish and studied in isolation in a screening setting. This demonstrates the utility of the zebrafish-CSE exposure platform for identifying environmental modifiers of cigarette smoking-associated pathology and demonstrates that the CS-exposed mouse gut microbiota potentiates the inflammatory effects of CSE across host species.

Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of exogenous glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals.

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary co-factor for host immunity. Heme oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of heme. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here, we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish heme oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from M. marinum infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against M. marinum infection by reducing infection-induced iron accumulation and ferrostatin-sensitive cell death.

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin‐related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F‐actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3‐knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon‐like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three‐dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3‐dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities., Quantification of cytotoxic effector T lymphocytes (CTLs) morphology using standard biomedical assays and developed image analysis algorithms showed that the reduction of actin‐related protein (Arp)3 levels impaired F‐actin content maintenance, leading to defects in CTL functionality. The knockdown CTL motility mode, instead of the actin‐rich lamellipodia‐based migratory strategy, displays blebbing‐like migration at the cost of reduced migration speed. Thus, the role of the Arp2/3 complex is not redundant among the various mechanochemical coordinators involved in the leading‐edge formation in CTLs.

The spectre of the coming post-antibiotic age demands novel therapies for infectious diseases. Tuberculosis, caused by Mycobacterium tuberculosis, is the single deadliest infection throughout human history. M. tuberculosis has acquired antibiotic resistance at an alarming rate with some strains reported as being totally drug resistant. Host-directed therapies attempt to overcome the evolution of antibiotic resistance by targeting relatively immutable host processes. Here I hypothesise the induction of hypoxia via anti-angiogenic therapy will be an efficacious host-directed therapy against tuberculosis. I argue that anti-angiogenic therapy is a modernisation of industrial revolution era sanatoria treatment for tuberculosis, and present a view of the tuberculosis granuloma as a “bacterial tumour” that can be treated with anti-angiogenic therapies to reduce bacterial burden and spare host immunopathology. I suggest two complementary modes of action, induction of bacterial dormancy and activation of host Hypoxia Induced Factor-mediated immunity, and define the experimental tools necessary to test this hypothesis. This work was supported by an Australian National Health and Medical Research Council CJ Martin Early Career Fellowship APP1053407 and Project Grant APP1099912; The University of Sydney Fellowship G197581; NSW Ministry of Health under the NSW Health Early-Mid Career Fellowships Scheme H18/31086; and the Kenyon Family Foundation Inflammation Award (S.H.O.).

Tuberculosis is a chronic inflammatory disease caused by persistent infection with Mycobacterium tuberculosis. The rise of antibiotic resistant strains necessitates the design of novel treatments. Recent evidence shows that not only is M. tuberculosis highly resistant to oxidative killing, it also co-opts host oxidant production to induce phagocyte death facilitating bacterial dissemination. We have targeted this redox environment with the cyclic nitroxide derivative 4-methoxy-TEMPO (MetT) in the zebrafish-M. marinum infection model. MetT inhibited the production of mitochondrial ROS and decreased infection-induced cell death to aid containment of infection. We identify a second mechanism of action whereby stress conditions, including hypoxia, found in the infection microenvironment appear to sensitise M. marinum to killing by MetT both in vitro and in vivo. Together, our study demonstrates MetT inhibited the growth and dissemination of M. marinum through host and bacterial targets. This work was supported by the Australian National Health and Medical Research Council grants APP1099912 and APP1053407; The University of Sydney, Australia Fellowship G197581; NSW Ministry of Health, Australia under the NSW Health Early-Mid Career Fellowships Scheme H18/31086; and the Kenyon Family Inflammation Award (S.H.O.).

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary co-factor for host immunity. Heme oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of heme. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish heme oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from mycobacterial infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against mycobacterial infection by reducing infection-induced iron accumulation and ferroptosis.

SummaryPathogenic mycobacteria inhibit inflammasome activation as part of their pathogenesis. While it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux and inflammasome activation has not been investigated. Here we use Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis of human THP-1 cells to demonstrate that pathogenic mycobacteria upregulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are mediated by NLRP3 inflammasome activation and are dependent on caspase-mediated release of IL-1β and the downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections.

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas., Author summary Mycobacterial infections cause significant disease burden to humans and animals, the most widely known example being tuberculosis which has killed more humans than any other infectious disease throughout history. Infectious mycobacteria are highly evolved to hijack host processes, including the very immune cells tasked with destroying them. MicroRNAs are host molecules that control wide-ranging programs of host gene expression and are important in the immune response to infections. Here we use the zebrafish model of mycobacterial infection to determine the role of the infection-induced microRNA miR-206 in the host response to infection. We found pathogenic mycobacteria trigger the host to produce more miR-206 in order to suppress the otherwise protective recruitment of neutrophils to sites of infection via the host Cxcl12/Cxcr4 signalling pathway. Our study provides new insight into the role of mycobacterial infection-induced miR-206 function in the context of a whole host.

Predation by heterotrophic protists drives the emergence of adaptive traits in bacteria, and often these traits lead to altered interactions with hosts and persistence in the environment. Here we studied adaptation of the cholera pathogen, Vibrio cholerae during long-term co-incubation with the protist host, Acanthamoeba castellanii. We determined phenotypic and genotypic changes associated with long-term intra-amoebal host adaptation and how this impacts pathogen survival and fitness. We showed that adaptation to the amoeba host leads to temporal changes in multiple phenotypic traits in V. cholerae that facilitate increased survival and competitive fitness in amoeba. Genome sequencing and mutational analysis revealed that these altered lifestyles were linked to non-synonymous mutations in conserved regions of the flagellar transcriptional regulator, flrA. Additionally, the mutations resulted in enhanced colonisation in zebrafish, establishing a link between adaptation of V. cholerae to amoeba predation and enhanced environmental persistence. Our results show that pressure imposed by amoeba on V. cholerae selects for flrA mutations that serves as a key driver for adaptation. Importantly, this study provides evidence that adaptive traits that evolve in pathogens in response to environmental predatory pressure impact the colonisation of eukaryotic organisms by these pathogens.

UropathogenicEscherichia coli(UPEC) causes urinary tract infections that can result in sepsis. The haemostatic system is protective in the pyelonephritis stage of ascending UPEC infection, but the role of the haemostatic system has not been investigated during sepsis. Here we utilize a zebrafish-UPEC systemic infection model to visualize infection-induced coagulation and examine the effects of commonly prescribed anti-haemostatic medications on the infection severity. Treatment of systemically infected zebrafish with warfarin, aspirin, or ticagrelor reduced host survival, while stabilization of clots with aminocaproic acid increased host survival. Anti-haemostatic drug treatment increased UPEC burden. Our findings provide evidence that commonly prescribed anti-haemostatic medications may worsen the outcome of severe UPEC infection.

Cigarette smoke (CS)-induced inflammation leads to a range of diseases including chronic obstructive pulmonary disease and cancer. The gut microbiota is a major modifying environmental factor that determine the severity of cigarette smoke-induced pathology. Microbiomes and metabolites from CS-exposed mice exacerbate lung inflammation via the gut-lung axis of shared mucosal immunity in mice but these systems are expensive to establish and analyse. Zebrafish embryos and larvae have been used to model the effects of cigarette smoking on a range of physiological processes and offer an amenable platform for screening modifiers of cigarette smoke-induced pathologies with key features of low cost and rapid visual readouts. Here we exposed zebrafish larvae to cigarette smoke extract (CSE) and characterised a CSE-induced leukocytic inflammatory phenotype with increased neutrophilic and macrophage inflammation in the gut. The CSE-induced phenotype was exacerbated by co-exposure to microbiota from the faeces of CS-exposed mice, but not control mice. Microbiota could be recovered from the gut of zebrafish and studied in isolation in a screening setting. This demonstrates the utility of the zebrafish-CSE exposure platform for identifying environmental modifiers of cigarette smoking-associated pathology and demonstrates that the CS-exposed mouse gut microbiota potentiates the inflammatory effects of CSE across host species.

Short-chain fatty acids (SCFAs) are produced by microbial fermentation of dietary fiber in the gut. Butyrate is a particularly important SCFA with anti-inflammatory properties and is generally present at lower levels in inflammatory diseases associated with gut microbiota dysbiosis in mammals. We aimed to determine if SCFAs are produced by the zebrafish microbiome and if SCFAs exert conserved effects on zebrafish immunity as an example of the non-mammalian vertebrate immune system. We demonstrate that bacterial communities from adult zebrafish intestines synthesize all three main SCFA in vitro, although SCFA were below our detectable limits in zebrafish intestines in vivo. Immersion in butyrate, but not acetate or propionate, reduced the recruitment of neutrophils and M1-type pro-inflammatory macrophages to wounds. We found conservation of butyrate sensing by neutrophils via orthologs of the hydroxycarboxylic acid receptor 1 (hcar1) gene. Neutrophils from Hcar1-depleted embryos were no longer responsive to the anti-inflammatory effects of butyrate, while macrophage sensitivity to butyrate was independent of Hcar1. Our data demonstrate conservation of anti-inflammatory butyrate effects and identify the presence of a conserved molecular receptor in fish.

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection.

Mycobacteria have co-evolved with their hosts resulting in pathogens adept at intracellular survival. Pathogenic mycobacteria actively manipulate infected macrophages to drive granuloma formation while subverting host cell processes to create a permissive niche. Granuloma residency confers phenotypic antimicrobial resistance by physically excluding or neutralising antibiotics. Host-directed therapies (HDTs) combat infection by restoring protective immunity and reducing immunopathology independent of pathogen antimicrobial resistance status. This review covers innovative research that has discovered ‘secondary’ symptoms of infection in the granuloma stroma are actually primary drivers of infection and that relieving these stromal pathologies with HDTs benefits the host. Advances in our understanding of the relationship between tuberculosis and the host vasculature, haemostatic system and extracellular matrix reorganisation are discussed. Preclinical and clinical use of HDTs against these stromal targets are summarised.

Comorbidities are an important factor in tuberculosis pathophysiology and treatment but are understudied in animal models. Schild et al. present a zebrafish model of Mycobacterium marinum infection and wound comorbidity that retains responsiveness to protective hypoxia-inducible factor-1α activation as an example of a host-directed therapy. This platform is a new paradigm for the zebrafish-M. marinum infection model and provides a blueprint to test therapeutic interventions on infection and comorbid pathologies. Comment on: https://doi.org/10.1111/febs.15433.

We recently reported the discovery of non-toxic cyclam-derived compounds that are active against drug-resistant Mycobacterium tuberculosis. In this paper we report exploration of the structure-activity relationship for this class of compounds, identifying several simpler compounds with comparable activity. The most promising compound identified, possessing significantly improved water solubility, displayed high levels of bacterial clearance in an in vivo zebrafish embryo model, suggesting this compound series has promise for in vivo treatment of tuberculosis. This work was supported by the National Health and Medical Research Council (NHMRC) Project APP1084266, the NHMRC Center of Research Excellence in Tuberculosis Control (APP1043225), Project Grant APP1099912, NHMRC CJ Martin Early Career Fellowship to S.O., APP1053407 and the University of Sydney Sydnovate Fund.

Investigation into the macromolecular structure–activity relationship of synthetic antimicrobial polymers has been gaining scientific interest due to the possibility of discovering new alternatives for combating the increase of multidrug resistance in bacteria. Recently, we reported the development of new antimicrobial polymers in the form of amphiphilic ternary copolymers that consist of low-fouling (oligoethylene glycol), cationic and hydrophobic side chains. The combination of these three main functional groups is crucial in endowing the polymers with high antimicrobial potency against Gram-negative pathogens and low cytotoxicity. Following on from our previous study, we herein present a systematic assessment on the effects of the polymer chain length and architecture (i.e., random vs. block copolymers and linear vs. hyperbranched) on the antimicrobial activity and hemocompatibility of antimicrobial ternary copolymers. The polymer chain length in random copolymers slightly affects the antimicrobial activity where longer chains are marginally more bacteriostatic against Pseudomonas aeruginosa and Escherichia coli. In terms of hemocompatibility, polymers with shorter chains are more prone to hemagglutination. Interestingly, when the hydrophilic and hydrophobic segments are separated into diblock copolymers, the antimicrobial activity is lost, possibly due to the stable core–shell architecture. The hyperbranched structure which consists of 2-ethylhexyl groups as hydrophobic side-chains yields the best overall biological properties, having similar antimicrobial activity (MIC = 64 μg mL−1) and >4-fold increase in HC50 compared to the linear random copolymers (HC50 > 10 000 μg mL−1) with no hemagglutination. The hyperbranched polymers are also bactericidal and kill ≥99% and 90% of planktonic and biofilm Pseudomonas aeruginosa, respectively. This study thus highlights the importance of determining macromolecular structural aspects that govern the biological activity of antimicrobial polymers.

Infections caused by Mycobacterium abscessus are increasing in prevalence within patient groups with respiratory comorbidities. Initial colonisation by the smooth colony M. abscessus (S) can be followed by an irreversible genetic switch into a highly inflammatory rough colony M. abscessus (R), often associated with a decline in pulmonary function. Our understanding of the role of adaptive immunity in M. abscessus pathogenesis is largely unknown. Here, we have used intraperitoneal infection of adult zebrafish to model M. abscessus pathogenesis in the context of fully functioning host immunity. We find infection with the R variant penetrates host organs causing an inflammatory immune response leading to necrotic granuloma formation within 2 weeks. The R bacilli are targeted by T cell-mediated immunity and burden is constrained. Strikingly, the S variant colonises host internal surfaces at high loads and is met with a robust innate immune response but little T cell-mediated immunity. Invasive granuloma formation is delayed in S variant infection compared to R variant infection upon which T cell-mediated immunity is required to control infection. In mixed infections, the S variant outcompetes the R variant. We also find the R variant activates host immunity to the detriment of S variant M. abscessus in mixed infections. These findings demonstrate the applicability of the adult zebrafish to model persistent M. abscessus infection and provide insight into the immunopathogenesis of chronic M. abscessus infection.

Infection of zebrafish with natural pathogen Mycobacterium marinum is a useful surrogate for studying the human granulomatous inflammatory response to infection by Mycobacterium tuberculosis. The adaptive immune system of the adult stage zebrafish offers an advance on the commonly used embryo infection model as adult zebrafish form granulomas with striking similarities to human-M. tuberculosis granulomas. Here, we present workflows to perform high content analyses of granulomas in adult zebrafish infected with M. marinum by cryosectioning to take advantage of strong endogenous transgenic fluorescence adapted from common zebrafish embryo infection tools. Specific guides to classifying granuloma necrosis and organisation, quantifying bacterial burden and leukocyte infiltration of granulomas, visualizing foam cell formation, analysing extracellular matrix remodelling and granuloma fibrosis are also provided. We use these methods to characterize neutrophil recruitment to M. marinum granulomas across time and find an inverse relation to granuloma necrosis suggesting granuloma necrosis is not a marker of immunopathology in the natural infection system of the adult zebrafish-M. marinum pairing. The methods can be easily translated to studying the zebrafish adaptive immune response to other chronic and granuloma-forming pathogens. This work was supported by an Australian National Health and Medical Research Council CJ Martin Early Career Fellowship APP1053407 and Project Grant APP1099912; The University of Sydney Fellowship G197581; NSW Ministry of Health under the NSW Health Early-Mid Career Fellowships Scheme H18/31086; and the Kenyon Family Foundation Inflammation Award (S.H.O.).

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-seq analysis of zebrafish granulomas as well as analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell-autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

Mycobacterium tuberculosis infection in humans triggers formation of granulomas, which are tightly organized immune cell aggregates that are the central structure of tuberculosis. Infected and uninfected macrophages interdigitate, assuming an altered, flattened appearance. Although pathologists have described these changes for over a century, the molecular and cellular programs underlying this transition are unclear. Here, using the zebrafish-Mycobacterium marinum model, we found that mycobacterial granuloma formation is accompanied by macrophage induction of canonical epithelial molecules and structures. We identified fundamental macrophage reprogramming events that parallel E-cadherin-dependent mesenchymal-epithelial transitions. Macrophage-specific disruption of E-cadherin function resulted in disordered granuloma formation, enhanced immune cell access, decreased bacterial burden, and increased host survival, suggesting that the granuloma can also serve a bacteria-protective role. Granuloma macrophages in humans with tuberculosis were similarly transformed. Thus, during mycobacterial infection, granuloma macrophages are broadly reprogrammed by epithelial modules, and this reprogramming alters the trajectory of infection and the associated immune response.

5 pages, 4 figures, Changes to lipid metabolism are well-characterised consequences of human tuberculosis infection but their functional relevance are not clearly elucidated in these or other host-mycobacterial systems. The zebrafish-Mycobacterium marinum infection model is used extensively to model many aspects of human-M. tuberculosis pathogenesis but has not been widely used to study the role of infection-induced lipid metabolism. We find mammalian mycobacterial infection-induced alterations in host Low Density Lipoprotein metabolism are conserved in the zebrafish model of mycobacterial pathogenesis. Depletion of LDLR, a key lipid metabolism node, decreased M. marinum burden, and corrected infection-induced altered lipid metabolism resulting in decreased LDL and reduced the rate of macrophage transformation into foam cells. Our results demonstrate a conserved role for infection-induced alterations to host lipid metabolism, and specifically the LDL-LDLR axis, across host-mycobacterial species pairings, This work was supported by the Australian National Health and Medical Research Council (APP1099912 and APP1053407 to S.H.O.); Meat and Livestock Australia (P.PSH. 0813 to A.C.P. and K. dS); the Marie Bashir Institute for Infectious Diseases and Biosecurity (grant to S.H.O., A.C.P. and K. dS); the Kenyon Family Foundation Inflammation Award (grant to S.H.O.); the University of Sydney (fellowship to S.H.O.); Consellería de Economía, Emprego e Industria (GAIN), Xunta de Galicia (grant IN607B 2016/12 to Institute of Marine Research (IIM-CSIC))

Infection-induced thrombocytosis is a clinically important complication of tuberculosis (TB). Recent studies have separately highlighted a correlation of platelet activation with TB severity and utility of aspirin as a host-directed therapy for TB that modulates the inflammatory response. Here we investigate the possibility that the beneficial effects of aspirin are related to an anti-platelet mode of action. We utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin or platelet-specific glycoprotein IIb/IIIa inhibitors reduced mycobacterial burden demonstrating a detrimental role for infection-induced thrombocyte activation. We found platelet inhibition reduced thrombocyte-macrophage interactions and restored indices of macrophage-mediated immunity to mycobacterial infection. Pathological thrombocyte activation and granuloma formation were found to be intrinsically linked illustrating a bidirectional relationship between host hemostasis and TB pathogenesis. Our study illuminates platelet activation as an efficacious target of anti-platelets drugs including aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.Key PointsInhibition of thrombocyte activation improves control of mycobacterial infection.Inhibition of thrombocyte activation reduces thrombocyte-macrophage interactions and improves indices of macrophage immune function against mycobacterial infection.

Host lipid metabolism is an important target for subversion by pathogenic mycobacteria such as Mycobacterium tuberculosis. The appearance of foam cells within the granuloma are well-characterised effects of chronic tuberculosis. The zebrafish-Mycobacterium marinum infection model recapitulates many aspects of human-M. tuberculosis infection and is used as a model to investigate the structural components of the mycobacterial granuloma. Here, we demonstrate that the zebrafish-M. marinum granuloma contains foam cells and that the transdifferentiation of macrophages into foam cells is driven by the mycobacterial ESX1 pathogenicity locus. This report demonstrates conservation of an important aspect of mycobacterial infection across species.

Host lipid metabolism is an important target for subversion by pathogenic mycobacteria such as Mycobacterium tuberculosis. The appearance of foam cells within the granuloma are well-characterised effects of chronic tuberculosis. The zebrafish-Mycobacterium marinum infection model recapitulates many aspects of human-M. tuberculosis infection and is used as a model to investigate the structural components of the mycobacterial granuloma. Here, we demonstrate that the zebrafish-M. marinum granuloma contains foam cells and that the transdifferentiation of macrophages into foam cells is driven by the mycobacterial ESX1 pathogenicity locus. This report demonstrates conservation of an important aspect of mycobacterial infection across species. This work was supported by the Australian National Health and Medical Research Council (APP1099912 and APP1053407 to S.H.O.); Meat and Livestock Australia (P.PSH. 0813 to A.C.P.); the Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney (grant to S.H.O. and A.C.P.); University of Sydney Fellowship (grant to S.H.O.); and the Kenyon Family Inflammation Award (grant to S.H.O.).

Significance Salmonella enterica serovar Typhi ( S . Typhi) causes ∼20 million cases of typhoid fever every year. We carried out a genome-wide association study to identify genetic differences that correlate with the susceptibility of cells from hundreds of individuals to S . Typhi invasion. A SNP in VAC14 was associated with susceptibility to S . Typhi invasion and VAC14 expression. Cells mutated for VAC14 displayed increased S . Typhi docking due to increased plasma membrane cholesterol levels. The same SNP was associated with risk of typhoid fever in a Vietnamese population. Furthermore, treating zebrafish with a cholesterol-lowering drug reduced their susceptibility to S . Typhi infection. Therefore, this work demonstrates the power of coupling multiple genetic association studies with mechanistic dissection for understanding infectious disease susceptibility.