Adaptation of Prokaryotic Toxins for Negative Selection and Cloning-Independent Markerless Mutagenesis in Streptococcus Species.

The Streptococcus mutans genetic system offers a variety of strategies to rapidly engineer targeted chromosomal mutations. Previously, we reported the first S. mutans negative selection system that functions in a wild-type background. This system utilizes induced sensitivity to the toxic amino acid analog p-chlorophenylalanine (4-CP) as a negative selection mechanism and was developed for counterselection-based cloning-independent markerless mutagenesis (CIMM). While we have employed this system extensively for our ongoing genetic studies, we have encountered a couple limitations with the system, mainly its narrow host range and the requirement for selection on a toxic substrate. Here, we report the development of a new negative selection system that addresses both limitations, while still retaining the utility of the previous 4-CP-based markerless mutagenesis system. We placed a variety of toxin-encoding genes under the control of the xylose-inducible gene expression cassette (Xyl-S) and found the Fst-sm and ParE toxins to be suitable candidates for inducible negative selection. We combined the inducible toxins with an antibiotic resistance gene to create several different counterselection cassettes. The most broadly useful of these contained a wild-type fst-sm open reading frame transcriptionally fused to a point mutant form of the Xyl-S expression system, which we subsequently named IFDC4. IFDC4 was shown to exhibit exceptionally low background resistance, with 3- to 4-log reductions in cell number observed when plating on xylose-supplemented medium. IFDC4 also functioned similarly in multiple strains of S. mutans as well as with Streptococcus gordonii and Streptococcus sanguinis. We performed CIMM with IFDC4 and successfully engineered a variety of different types of markerless mutations in all three species. The counterselection strategy described here provides a template approach that should be adaptable for the creation of similar counterselection systems in many other bacteria. IMPORTANCE Multiple medically significant Streptococcus species, such as S. mutans, have highly sophisticated genetic systems available, largely as a consequence of their amenability to genetic manipulation via natural competence. Despite this, few options are available for the creation of markerless mutations in streptococci, especially within wild-type strains. Markerless mutagenesis is a critical tool for genetic studies, as it allows the user to explore many fundamental questions that are not easily addressable using marked mutagenesis. Here, we describe a new approach for streptococcal markerless mutagenesis that offers a variety of advantages over the current approach, which employs induced sensitivity to the toxic substrate 4-CP. The approach employed here should be readily adaptable for the creation of similar markerless mutagenesis systems in other organisms.

Homeostatic interferon-lambda response to bacterial microbiota stimulates preemptive antiviral defense within discrete pockets of intestinal epithelium.

Interferon-lambda (IFN-λ) protects intestinal epithelial cells (IECs) from enteric viruses by inducing expression of antiviral IFN-stimulated genes (ISGs). Here, we find that bacterial microbiota stimulate a homeostatic ISG signature in the intestine of specific pathogen-free mice. This homeostatic ISG expression is restricted to IECs, depends on IEC-intrinsic expression of IFN-λ receptor (Ifnlr1), and is associated with IFN-λ production by leukocytes. Strikingly, imaging of these homeostatic ISGs reveals localization to pockets of the epithelium and concentration in mature IECs. Correspondingly, a minority of mature IECs express these ISGs in public single-cell RNA sequencing datasets from mice and humans. Furthermore, we assessed the ability of orally administered bacterial components to restore localized ISGs in mice lacking bacterial microbiota. Lastly, we find that IECs lacking Ifnlr1 are hyper-susceptible to initiation of murine rotavirus infection. These observations indicate that bacterial microbiota stimulate ISGs in localized regions of the intestinal epithelium at homeostasis, thereby preemptively activating antiviral defenses in vulnerable IECs to improve host defense against enteric viruses.

Selective Interferon Responses of Intestinal Epithelial Cells Minimize Tumor Necrosis Factor Alpha Cytotoxicity.

Interferon (IFN) family cytokines stimulate genes (interferon-stimulated genes [ISGs]) that are integral to antiviral host defense. Type I IFNs act systemically, whereas type III IFNs act preferentially at epithelial barriers. Among barrier cells, intestinal epithelial cells (IECs) are particularly dependent on type III IFN for the control and clearance of virus infection, but the physiological basis of this selective IFN response is not well understood. Here, we confirm that type III IFN treatment elicits robust and uniform ISG expression in neonatal mouse IECs and inhibits the replication of IEC-tropic rotavirus. In contrast, type I IFN elicits a marginal ISG response in neonatal mouse IECs and does not inhibit rotavirus replication. In vitro treatment of IEC organoids with type III IFN results in ISG expression that mirrors the in vivo type III IFN response. However, IEC organoids have increased expression of the type I IFN receptor relative to neonate IECs, and the response of IEC organoids to type I IFN is strikingly increased in magnitude and scope relative to type III IFN. The expanded type I IFN-specific response includes proapoptotic genes and potentiates toxicity triggered by tumor necrosis factor alpha (TNF-α). The ISGs stimulated in common by type I and III IFNs have strong interferon-stimulated response element (ISRE) promoter motifs, whereas the expanded set of type I IFN-specific ISGs, including proapoptotic genes, have weak ISRE motifs. Thus, the preferential responsiveness of IECs to type III IFN in vivo enables selective ISG expression during infection that confers antiviral protection but minimizes disruption of intestinal homeostasis.IMPORTANCE Enteric viral infections are a major cause of gastroenteritis worldwide and have the potential to trigger or exacerbate intestinal inflammatory diseases. Prior studies have identified specialized innate immune responses that are active in the intestinal epithelium following viral infection, but our understanding of the benefits of such an epithelium-specific response is incomplete. Here, we show that the intestinal epithelial antiviral response is programmed to enable protection while minimizing epithelial cytotoxicity that can often accompany an inflammatory response. Our findings offer new insight into the benefits of a tailored innate immune response at the intestinal barrier and suggest how dysregulation of this response could promote inflammatory disease.

Persistence of Systemic Murine Norovirus Is Maintained by Inflammatory Recruitment of Susceptible Myeloid Cells.

Viral persistence can contribute to chronic disease and promote virus dissemination. Prior work demonstrated that timely clearance of systemic murine norovirus (MNV) infection depends on cell-intrinsic type I interferon responses and adaptive immunity. We now find that the capsid of the systemically replicating MNV strain CW3 promotes lytic cell death, release of interleukin-1α, and increased inflammatory cytokine release. Correspondingly, inflammatory monocytes and neutrophils are recruited to sites of infection in a CW3-capsid-dependent manner. Recruited monocytes and neutrophils are subsequently infected, representing a majority of infected cells in vivo. Systemic depletion of inflammatory monocytes or neutrophils from persistently infected Rag1-/- mice reduces viral titers in a tissue-specific manner. These data indicate that the CW3 capsid facilitates lytic cell death, inflammation, and recruitment of susceptible cells to promote persistence. Infection of continuously recruited inflammatory cells may be a mechanism of persistence broadly utilized by lytic viruses incapable of establishing latency.

Genetic variation in human carboxylesterase CES1 confers resistance to hepatic steatosis.

Obesity often leads non-alcoholic fatty liver disease, insulin resistance and hyperlipidemia. Expression of carboxylesterase CES1 is positively correlated with increased lipid storage and plasma lipid concentration. Here we investigated structural and metabolic consequences of a single nucleotide polymorphism in CES1 gene that results in p.Gly143Glu amino acid substitution. We generated a humanized mouse model expressing CES1WT (control), CES1G143E and catalytically dead CES1S221A (negative control) in the liver in the absence of endogenous expression of the mouse orthologous gene. We show that the CES1G143E variant exhibits only 20% of the wild-type lipolytic activity. High-fat diet fed mice expressing CES1G143E had reduced liver and plasma triacylglycerol levels. The mechanism by which decreased CES1 activity exerts this hypolipidemic phenotype was determined to include decreased very-low density lipoprotein secretion, decreased expression of hepatic lipogenic genes and increased fatty acid oxidation as determined by increased plasma ketone bodies and hepatic mitochondrial electron transport chain protein abundance. We conclude that attenuation of human CES1 activity provides a beneficial effect on hepatic lipid metabolism. These studies also suggest that CES1 is a potential therapeutic target for non-alcoholic fatty liver disease management.

Ces3/TGH Deficiency Attenuates Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in developed countries. NAFLD describes a wide range of liver pathologies from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NASH is distinguished from simple steatosis by inflammation, cell death and fibrosis. In this study we found that mice lacking triacylglycerol hydrolase (TGH, also known as carboxylesterase 3 or carboxylesterase 1d) are protected from high-fat diet (HFD) - induced hepatic steatosis via decreased lipogenesis, increased fatty acid oxidation and improved hepatic insulin sensitivity. To examine the effect of the loss of TGH function on the more severe NAFLD form NASH, we ablated Tgh expression in two independent NASH mouse models, Pemt(-/-) mice fed HFD and Ldlr(-/-) mice fed high-fat, high-cholesterol Western-type diet (WTD). TGH deficiency reduced liver inflammation, oxidative stress and fibrosis in Pemt(-/-) mice. TGH deficiency also decreased NASH in Ldlr(-/-) mice. Collectively, these findings indicate that TGH deficiency attenuated both simple hepatic steatosis and irreversible NASH.

Tumor-Induced Hyperlipidemia Contributes to Tumor Growth.

The known link between obesity and cancer suggests an important interaction between the host lipid metabolism and tumorigenesis. Here, we used a syngeneic tumor graft model to demonstrate that tumor development influences the host lipid metabolism. BCR-Abl-transformed precursor B cell tumors induced hyperlipidemia by stimulating very low-density lipoprotein (VLDL) production and blunting VLDL and low-density lipoprotein (LDL) turnover. To assess whether tumor progression was dependent on tumor-induced hyperlipidemia, we utilized the VLDL production-deficient mouse model, carboxylesterase3/triacylglycerol hydrolase (Ces3/TGH) knockout mice. In Ces3/Tgh(-/-) tumor-bearing mice, plasma triglyceride and cholesterol levels were attenuated. Importantly tumor weight was reduced in Ces3/Tgh(-/-) mice. Mechanistically, reduced tumor growth in Ces3/Tgh(-/-) mice was attributed to reversal of tumor-induced PCSK9-mediated degradation of hepatic LDLR and decrease of LDL turnover. Our data demonstrate that tumor-induced hyperlipidemia encompasses a feed-forward loop that reprograms hepatic lipoprotein homeostasis in part by providing LDL cholesterol to support tumor growth.

Roles of Acyl-CoA:Diacylglycerol Acyltransferases 1 and 2 in Triacylglycerol Synthesis and Secretion in Primary Hepatocytes.

OBJECTIVE: Very low-density lipoprotein assembly and secretion are regulated by the availability of triacylglycerol. Although compelling evidence indicates that the majority of triacylglycerol in very low-density lipoprotein is derived from re-esterification of lipolytic products released by endoplasmic reticulum-associated lipases, little is known about roles of acyl-CoA:diacylglycerol acyltransferases (DGATs) in this process. We aimed to investigate the contribution of DGAT1 and DGAT2 in lipid metabolism and lipoprotein secretion in primary mouse and human hepatocytes. APPROACH AND RESULTS: We used highly selective small-molecule inhibitors of DGAT1 and DGAT2, and we tracked storage and secretion of lipids synthesized de novo from [(3)H]acetic acid and from exogenously supplied [(3)H]oleic acid. Inactivation of individual DGAT activity did not affect incorporation of either radiolabeled precursor into intracellular triacylglycerol, whereas combined inactivation of both DGATs severely attenuated triacylglycerol synthesis. However, inhibition of DGAT2 augmented fatty acid oxidation, whereas inhibition of DGAT1 increased triacylglycerol secretion, suggesting preferential channeling of separate DGAT-derived triacylglycerol pools to distinct metabolic pathways. Inactivation of DGAT2 impaired cytosolic lipid droplet expansion, whereas DGAT1 inactivation promoted large lipid droplet formation. Moreover, inactivation of DGAT2 attenuated expression of lipogenic genes. Finally, triacylglycerol secretion was significantly reduced on DGAT2 inhibition without altering extracellular apolipoprotein B levels. CONCLUSIONS: Our data suggest that DGAT1 and DGAT2 can compensate for each other to synthesize triacylglycerol, but triacylglycerol synthesized by DGAT1 is preferentially channeled to oxidation, whereas DGAT2 synthesizes triacylglycerol destined for very low-density lipoprotein assembly.

Endoplasmic reticulum-localized hepatic lipase decreases triacylglycerol storage and VLDL secretion.

Hepatic triacylglycerol levels are governed through synthesis, degradation and export of this lipid. Here we demonstrate that enforced expression of hepatic lipase in the endoplasmic reticulum in McArdle RH7777 hepatocytes resulted in a significant decrease in the incorporation of fatty acids into cellular triacylglycerol and cholesteryl ester accompanied by attenuation of secretion of apolipoprotein B-containing lipoproteins. Hepatic lipase-mediated depletion of intracellular lipid storage increased the expression of peroxisome proliferator-activated receptor α and its target genes and augmented oxidation of fatty acids. These data show that 1) hepatic lipase is active in the endoplasmic reticulum and 2) intracellular hepatic lipase modulates cellular lipid metabolism and lipoprotein secretion.

Ces3/TGH deficiency improves dyslipidemia and reduces atherosclerosis in Ldlr(-/-) mice.

RATIONALE: Carboxylesterase 3/triacylglycerol hydrolase (TGH) has been shown to participate in hepatic very low-density lipoprotein (VLDL) assembly. Deficiency of TGH in mice lowers plasma lipids and atherogenic lipoproteins without inducing hepatic steatosis. OBJECTIVE: To investigate the contribution of TGH to atherosclerotic lesion development in mice that lack low-density lipoprotein receptor (LDLR). METHODS AND RESULTS: Mice deficient in LDL receptor (Ldlr(-/-)) and mice lacking both TGH and LDLR (Tgh(-/-)/Ldlr(-/-)) were fed with a Western-type diet for 12 weeks. Analysis of Tgh(-/-)/Ldlr(-/-) plasma showed an atheroprotective lipoprotein profile with decreased cholesterol in the VLDL and the LDL fractions, concomitant with elevated high-density lipoprotein cholesterol. Significantly reduced plasma apolipoprotein B levels were also observed in Tgh(-/-)/Ldlr(-/-) mice. Consequently, Tgh(-/-)/Ldlr(-/-) mice presented with a significant reduction (54%, P<0.01) of the high-fat, high-cholesterol dieteninduced atherosclerotic plaques when compared with Tgh(+/+)/Ldlr(-/-) mice in the cross-sectional aortic root analysis. TGH deficiency did not further increase liver steatosis despite lowering plasma lipids, mainly due to reduced hepatic lipogenesis. The ameliorated dyslipidemia in Tgh(-/-)/Ldlr(-/-) mice was accompanied with significantly improved insulin sensitivity. CONCLUSIONS: Inhibition of TGH activity ameliorates atherosclerosis development and improves insulin sensitivity in Ldlr(-/-) mice.

Deficiency of carboxylesterase 1/esterase-x results in obesity, hepatic steatosis, and hyperlipidemia.

UNLABELLED: Increased lipogenesis, together with hyperlipidemia and increased fat deposition, contribute to obesity and associated metabolic disorders including nonalcoholic fatty liver disease. Here we show that carboxylesterase 1/esterase-x (Ces1/Es-x) plays a regulatory role in hepatic fat metabolism in the mouse. We demonstrate that Ces1/Es-x knockout mice present with increased hepatic lipogenesis and with oversecretion of apolipoprotein B (apoB)-containing lipoproteins (hepatic very-low density lipoproteins), which leads to hyperlipidemia and increased fat deposition in peripheral tissues. Consequently, Ces1/Es-x knockout mice develop obesity, fatty liver, hyperinsulinemia, and insulin insensitivity on chow diet without change in food intake and present with decreased energy expenditure. Ces1/Es-x deficiency prevents the release of polyunsaturated fatty acids from triacylglycerol stores, leading to an up-regulation of sterol regulatory element binding protein 1c-mediated lipogenesis, which can be reversed with dietary ω-3 fatty acids. CONCLUSION: These studies support a role for Ces1/Es-x in the partitioning of regulatory fatty acids and concomitant control of hepatic lipid biosynthesis, secretion, and deposition.

Liver specific inactivation of carboxylesterase 3/triacylglycerol hydrolase decreases blood lipids without causing severe steatosis in mice.

UNLABELLED: Carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) participates in hepatic very low-density lipoprotein (VLDL) assembly and in adipose tissue basal lipolysis. Global ablation of Ces3/Tgh expression decreases serum triacylglycerol (TG) and nonesterified fatty acid levels and improves insulin sensitivity. To understand the tissue-specific role of Ces3/TGH in lipid and glucose homeostasis, we generated mice with a liver-specific deletion of Ces3/Tgh expression (L-TGH knockout [KO]). Elimination of hepatic Ces3/Tgh expression dramatically decreased plasma VLDL TG and VLDL cholesterol concentrations but only moderately increased liver TG levels in mice fed a standard chow diet. Significantly reduced plasma TG and cholesterol without hepatic steatosis were also observed in L-TGH KO mice challenged with a high-fat, high-cholesterol diet. L-TGH KO mice presented with increased plasma ketone bodies and hepatic fatty acid oxidation. Intrahepatic TG in L-TGH KO mice was stored in significantly smaller lipid droplets. Augmented hepatic TG levels in chow-fed L-TGH KO mice did not affect glucose tolerance or glucose production from hepatocytes, but impaired insulin tolerance was observed in female mice. CONCLUSION: Our data suggest that ablation of hepatic Ces3/Tgh expression decreases plasma lipid levels without causing severe hepatic steatosis.

Immunogenicity and protective efficacy of a DNA vaccine encoding a chimeric protein of avian influenza hemagglutinin subtype H5 fused to CD154 (CD40L) in Pekin ducks.

The potential of CD154 (CD40L) as a powerful immunological adjuvant has been shown in various strategies. In this study we examine the immunogenicity and protective efficacy of a CD40-targeting avian influenza hemagglutinin (HA) subunit DNA vaccine in ducks. DNA constructs encoded the ectodomain of the HA protein of LPAI A/mallard/BC/373/2005 (H5N2) with or without fusion to the ectodomain of duck CD154. CD40-targeting significantly accelerated and enhanced humoral responses to the vector-encoded HA protein. In viral challenge experiments with A/chicken/Vietnam/14/2005 (H5N1), DNA immunization conferred partial protection against the genetically distant HPAI. The observed improved kinetics and magnitude of immune induction suggest that CD40-targeting holds promise for influenza A vaccine development.

Identification of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) isoforms in the Pekin duck.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152) is an inhibitory T cell receptor predominately expressed on activated T cells. The duck CTLA-4 (DuCTLA-4) cDNA and a transcript lacking the predicted transmembrane encoding region (DuCTLA-4DeltaTM) were isolated from splenocytes using RT-PCR. The predicted DuCTLA-4 protein showed an identity of 92%, 49% and 47% with chicken, human and mouse homologues, respectively. Sequence comparison revealed conservation of residues implicated in the B7 ligand binding, disulfide linkages, glycosylation and intracellular signaling. DuCTLA-4 mRNA was predominately expressed in primary and secondary immune organs. DuCTLA-4 and DuCTLA-4DeltaTM transcripts were differentially regulated in PBMCs. Flow cytometric analysis showed constitutive expression of DuCTLA-4 protein on freshly isolated PBMCs and a modest increase upon mitogen stimulation. Our observations suggest that DuCTLA-4 and its isoform DuCTLA-4DeltaTM evolved before the divergence of birds and mammals. Both DuCTLA-4 isoforms have significant structural homology to mammalian CTLA-4 proteins but their individual roles in the regulation of duck immune responses remains to be elucidated.

Loss of intestinal fatty acid binding protein increases the susceptibility of male mice to high fat diet-induced fatty liver.

Mice lacking I-FABP (encoded by the Fabp2 gene) exhibit a gender dimorphic response to a high fat/cholesterol diet challenge characterized by hepatomegaly in male I-FABP-deficient mice. In this study, we determined if this gender-specific modification of liver mass in mice lacking I-FABP is attributable to the high fat content of the diet alone and whether hepatic Fabp1 gene (encodes L-FABP) expression contributes to this difference. Wild-type and Fabp2-/- mice of both genders were fed a diet enriched with either polyunsaturated or saturated fatty acids (PUFA or SFA, respectively) in the absence of cholesterol. Male Fabp2-/- mice, but not female Fabp2-/- mice, exhibited increased liver mass and hepatic triacylglycerol (TG) deposition as compared to corresponding wild-type mice. In wild-type mice that were fed the standard chow diet, there was no difference in the concentration of hepatic L-FABP protein between males and females although the loss of I-FABP did cause a slight reduction of hepatic L-FABP abundance in both genders. The hepatic L-FABP mRNA abundance in both male and female wild-type and Fabp2-/- mice was higher in the PUFA-fed group than in the SFA-fed group, and was correlated with L-FABP protein abundance. No correlation between hepatic L-FABP protein abundance and hepatic TG concentration was found. The results obtained demonstrate that loss of I-FABP renders male mice sensitive to high fat diet-induced fatty liver, and this effect is independent of hepatic L-FABP.

Adaptations to the loss of intestinal fatty acid binding protein in mice.

It was shown previously that the intestinal fatty acid binding protein (I-FABP) is not essential for the absorption of dietary fat. One notable feature of I-FABP deficiency was the enhancement of body weight gain in male mice but not in female mice. To explore a possible cause for this gender dimorphic effect, we examined the changes in expression of genes that encode liver fatty acid binding protein (L-FABP) and ileal lipid binding protein in the small intestine resulting from I-FABP deficiency. The results indicate that both L-FABP and ilbp levels are modestly increased in the small intestine of chow-fed mice lacking I-FABP. There was no discernible alteration of overall morphology or histology in the small intestine but changes in liver histology were evident in I-FABP deficient male mice. Glucose tolerance was also investigated in aged mice. I-FABP deficiency had no effect on glucose tolerance in male mice but it appeared to be improved in female mice. Thus, male and female mice clearly respond differently to the loss of I-FABP from the small intestine but the observed changes in the abundance of L-FABP and ilbp protein do not readily account for this phenomenon.