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SummaryMaintaining intestinal macrophage (MP) heterogeneity is critical to ensure tissue homeostasis and host defense. The gut microbiota and host factors are thought to synergistically shape colonic MP development, although there remains a fundamental gap in our understanding of the details of such collaboration. Here, we report tertiary lymphoid organs (TLOs), enriched in group 3 innate lymphoid cells (ILC3s), as a microbiota-operated intestinal niche for the development of monocyte-derived MPs. ILC3-derived colony stimulating factor 2 (CSF2) serves as a developmental and functional determinant for MPs and required microbe-derived extracellular adenosine 5’-triphosphate (ATP) as a trigger. Microbial communities rich in extracellular ATP promoted MP turnover via ILC3 activity in an NLRP3-dependent fashion. Single cell RNA-sequencing of MPs revealed unique TLO-associated, CSF2-dependent MP populations critical for anti-microbial defense against enteric infection. Collectively, these findings describe a fundamental framework that constitutes an intestinal MP niche fueled by microbial energy metabolism.

OBJECTIVES: Seasonal influenza A virus (IAV) infection impacts both respiratory and intestinal microbiome homeostasis. However, it is not well understood the extent to which the gut-lung axis plays the role in innate immunity and acute inflammation during IAV. Xanthophylls are fat-soluble, oxygenized carotenoids with potent antioxidant properties. We recently reported that xanthophylls can promote gut microbiome homeostasis and is associated with attenuation of intestinal and systemic inflammation. Here, we sought to investigate the protective effects of xanthophylls, e.g., zeaxanthin (Z) and astaxanthin (A) in IAV pneumonia by regulation of the host gut microbiome. METHODS: Six-week-old male and female 129S6 wild type (WT) and beta-carotene oxygenase 2 (BCO2) knockout mice were fed with AIN93M chow diets supplemented with or without Z (0.02% w/w) and A (0.02 w/w) (e.g., A + Z). After 6 weeks of the dietary intervention, mice were intranasally infected with 100 pfu H1N1 PR8 virus. Animal body weight and phenotypes were monitored daily. Animals were sacrificed 6 days post-infection. Blood and lung tissues were collected for experiments. H & E staining, gut microbiota 16S rRNA sequencing, immunohistochemistry, and immunoblotting were used for clinical, histopathological, and other biochemical assessments. RESULTS: Depletion of BCO2, the xanthophyll cleavage enzyme, made mice more resistant to IAV infection. Administration of A + Z caused A + Z accumulation and enhanced resistance to IAV in BCO2 KO but not WT mice, as demonstrated by histological lung damage and colon and ileum inflammation. Gut microbiome profiling results showed that α–diversity and β–diversity were significantly altered in these experimental groups. In particular, A + Z accumulation is positively associated with Bacteroides abundance. The increases in Bacteroides abundance were even greater in BCO2 KO mice, compared to the WT. Furthermore, Akkermansia abundance was significantly increased in BCO2 KO mice after IAV infection. CONCLUSIONS: Association of xanthophyll accumulation with the gut microbiota shift could protect animals from IAV infection by reducing local inflammation. Bacteroides potentially plays a beneficial role in this process. FUNDING SOURCES: USDA/NIFA 2021-67018-34023 and 2020-67017-30842.

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening infections. Previously, we showed that elevated calcium (Ca(2+)) levels increase the production of virulence factors in P. aeruginosa. In an effort to characterize the Ca(2+) regulatory network, we identified a Ca(2+)–regulated β-propeller protein, CarP, and showed that expression of the encoding gene is controlled by the Ca(2+)-regulated two-component system CarSR. Here, by using a Galleria melonella model, we showed that CarP plays a role in regulating P. aeruginosa virulence. By using transcriptome sequencing (RNA-Seq), reverse transcription (RT)-PCR, quantitative RT-PCR (RT-qPCR), and promoter fusions, we determined that carP is transcribed into at least two transcripts and regulated by several bacterial and host factors. The transcription of carP is elevated in response to Ca(2+) in P. aeruginosa cystic fibrosis isolates and PAO1 laboratory strain. Elevated Fe(2+) also induces carP. The simultaneous addition of Ca(2+) and Fe(2+) increased the carP promoter activity synergistically, which requires the presence of CarR. In silico analysis of the intergenic sequence upstream of carP predicted recognition sites of RhlR/LasR, OxyR, and LexA, suggesting regulation by quorum sensing (QS) and oxidative stress. In agreement, the carP promoter was activated in response to stationary-phase PAO1 supernatant and required the presence of elevated Ca(2+) and CarR but remained silent in the triple mutant lacking rhlI, lasI, and pqsA synthases. We also showed that carP transcription is regulated by oxidative stress and that CarP contributes to P. aeruginosa Ca(2+)-dependent H(2)O(2) tolerance. The multifactorial regulation of carP suggests that CarP plays an important role in P. aeruginosa adaptations to host environments. IMPORTANCE P. aeruginosa is a human pathogen causing life-threatening infections. It is particularly notorious for its ability to adapt to diverse environments within the host. Understanding the signals and the signaling pathways enabling P. aeruginosa adaptation is imperative for developing effective therapies to treat infections caused by this organism. One host signal of particular importance is calcium. Previously, we identified a component of the P. aeruginosa calcium-signaling network, CarP, whose expression is induced by elevated levels of calcium. Here, we show that carP plays an important role in P. aeruginosa virulence and is upregulated in P. aeruginosa strains isolated from sputa of patients with cystic fibrosis. We also identified several bacterial and host factors that regulate the transcription of carP. Such multifactorial regulation highlights the interconnectedness between regulatory circuits and, together with the pleotropic effect of CarP on virulence, suggests the importance of this protein in P. aeruginosa adaptations to the host.

BACKGROUND: Astaxanthin is a red lipophilic carotenoid that is often undetectable in human plasma due to the limited supply in typical Western diets. Despite its presence at lower than detectable concentrations, previous clinical feeding studies have reported that astaxanthin exhibits potent antioxidant properties. OBJECTIVE: We examined astaxanthin accumulation and its effects on gut microbiota, inflammation, and whole-body metabolic homeostasis in wild-type C57BL/6 J (WT) and β-carotene oxygenase 2 (BCO2) knockout (KO) mice. METHODS: Six-wk-old male and female BCO2 KO and WT mice were provided with either nonpurified AIN93M (e.g., control diet) or the control diet supplemented with 0.04% astaxanthin (wt/wt) ad libitum for 8 wk. Whole-body energy expenditure was measured by indirect calorimetry. Feces were collected from individual mice for short-chain fatty acid assessment. Hepatic astaxanthin concentrations and liver metabolic markers, cecal gut microbiota profiling, inflammation markers in colonic lamina propria, and plasma samples were assessed. Data were analyzed by 3-way ANOVA followed by Tukey's post hoc analysis. RESULTS: BCO2 KO but not WT mice fed astaxanthin had ∼10-fold more of this compound in liver than controls (P

Fumarate was previously known to serve as an anaerobic electron acceptor by E. coli when colonizing the mammalian intestine, but the source of that fumarate was elusive. In this issue, Unden and coworkers demonstrate that l-aspartic acid is the source of fumarate that drives anaerobic respiration by colonized E. coli (Schubert et al., 2021). Moreover, Schubert et al., establish that E. coli is able to grow anaerobically by using aspartate as a sole source of nitrogen. These groundbreaking findings indicate that a single amino acid - aspartate - supports anaerobic respiration and acquisition of nitrogen by E. coli in the intestine.

OBJECTIVES: Zeaxanthin, an oxygenized carotenoid, exerts antioxidant properties in human nutrition and metabolism. Likely other carotenoids, zeaxanthin is poorly absorbed in the small intestine. The large portion of zeaxanthin reaches the colon and is not fully recovered in the colon. In this study, we aimed to investigate the association of zeaxanthin intake with the gut microbiome homeostasis and metabolomic responses in mice. METHODS: Six-week-old male and female C57BL/6J wild type (WT), beta-carotene oxygenase 2 (BCO2) knockout mice were fed with AIN93M chow diets supplemented with or without zeaxanthin (0.02% w/w) for 10 weeks. At the termination of the study, mice were fasted for 3 hrs prior to euthanization. Cecal contents, colon, serum, feces, and other tissues were collected for laboratory assessments.16S rRNA sequencing and LC-MS/MS were performed for gut microbiota profiling and serum and fecal metabolomics analysis, respectively. RESULTS: Significant zeaxanthin accumulation occurred in BCO2 KO, but not WT mice. Zeaxanthin accumulation was associated with the alteration of colonic gut microbiota composition, for example, zeaxanthin-increased abundance in Lachnospiraceae, Proteobacteria, and Parabacteroides, indicating enhanced short-chain production, improved intestinal integrity, and anaerobic bacterial colonization. The results of fecal and serum metabolomics revealed that zeaxanthin significantly altered tyrosine metabolism, branched-chain fatty acid oxidation, fatty acid biosynthesis, and phospholipid biosynthesis, and suppressed levels of kynurenine and trimethylamine N-oxide (TMAO). CONCLUSIONS: The results suggested that zeaxanthin accumulation promotes gut microbiome homeostasis and alters the gut microbial metabolites as signals in stimulating the host-gut microbe interplay. FUNDING SOURCES: USDA/NIFA 2021-67018-34023 and 2020-67017-30842.

A novel in vitro gut model was developed to better understand the interactions between Escherichia coli and the mouse cecal mucus commensal microbiota. The gut model is simple and inexpensive while providing an environment that largely replicates the nonadherent mucus layer of the mouse cecum. 16S rRNA gene profiling of the cecal microbial communities of streptomycin-treated mice colonized with E. coli MG1655 or E. coli Nissle 1917 and the gut model confirmed that the gut model properly reflected the community structure of the mouse intestine. Furthermore, the results from the in vitro gut model mimic the results of published in vivo competitive colonization experiments. The gut model is initiated by the colonization of streptomycin-treated mice, and then the community is serially transferred in microcentrifuge tubes in an anaerobic environment generated in anaerobe jars. The nutritional makeup of the cecum is simulated in the gut model by using a medium consisting of porcine mucin, mouse cecal mucus, HEPES-Hanks buffer (pH 7.2), Cleland’s reagent, and agarose. Agarose was found to be essential for maintaining the stability of the microbial community in the gut model. The outcome of competitions between E. coli strains in the in vitro gut model is readily explained by the “restaurant hypothesis” of intestinal colonization. This simple model system potentially can be used to more fully understand how different members of the microbiota interact physically and metabolically during the colonization of the intestinal mucus layer. IMPORTANCE Both commensal and pathogenic strains of Escherichia coli appear to colonize the mammalian intestine by interacting physically and metabolically with other members of the microbiota in the mucus layer that overlays the cecal and colonic epithelium. However, the use of animal models and the complexity of the mammalian gut make it difficult to isolate experimental variables that might dictate the interactions between E. coli and other members of the microbiota, such as those that are critical for successful colonization. Here, we describe a simple and relatively inexpensive in vitro gut model that largely mimics in vivo conditions and therefore can facilitate the manipulation of experimental variables for studying the interactions of E. coli with the intestinal microbiota.

We present the first high-resolution determination of transcriptome architecture in the priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3′ end of coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of TSS enabled prediction of −10 and −35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation of craA expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as ‘AcinetoCom’ (http://bioinf.gen.tcd.ie/acinetocom/).

As in other ecosystems, competition for resources drives the microbial community structure within the intestine. For so many species to coexist within the intestine, each uses at least one limiting nutrient better than all others, according to the late Rolf Freter of the University of Michigan and his collaborators. Accordingly, if residents within that community consume the nutrients that an invader needs, this potential pathogen will not find essential nutrients and will fail to become established.

Escherichia coli MG1655, a K-12 strain, uses glycolytic nutrients exclusively to colonize the intestines of streptomycin-treated mice when it is the only E. coli strain present or when it is confronted with E. coli EDL933, an O157:H7 strain. In contrast, E. coli EDL933 uses glycolytic nutrients exclusively when it is the only E. coli strain in the intestine but switches in part to gluconeogenic nutrients when it colonizes mice precolonized with E. coli MG1655 (R. L. Miranda et al., Infect Immun 72:1666–1676, 2004, http://dx.doi.org/10.1128/IAI.72.3.1666-1676.2004 ). Recently, J. W. Njoroge et al. (mBio 3:e00280-12, 2012, http://dx.doi.org/10.1128/mBio.00280-12 ) reported that E. coli 86-24, an O157:H7 strain, activates the expression of virulence genes under gluconeogenic conditions, suggesting that colonization of the intestine with a probiotic E. coli strain that outcompetes O157:H7 strains for gluconeogenic nutrients could render them nonpathogenic. Here we report that E. coli Nissle 1917, a probiotic strain, uses both glycolytic and gluconeogenic nutrients to colonize the mouse intestine between 1 and 5 days postfeeding, appears to stop using gluconeogenic nutrients thereafter in a large, long-term colonization niche, but continues to use them in a smaller niche to compete with invading E. coli EDL933. Evidence is also presented suggesting that invading E. coli EDL933 uses both glycolytic and gluconeogenic nutrients and needs the ability to perform gluconeogenesis in order to colonize mice precolonized with E. coli Nissle 1917. The data presented here therefore rule out the possibility that E. coli Nissle 1917 can starve the O157:H7 E. coli strain EDL933 of gluconeogenic nutrients, even though E. coli Nissle 1917 uses such nutrients to compete with E. coli EDL933 in the mouse intestine.

RNA sequencing has emerged as the premier approach to study bacterial transcriptomes. While the earliest published studies analyzed the data qualitatively, the data are readily digitized and lend themselves to quantitative analysis. High-resolution RNA sequence (RNA-seq) data allows transcriptional features (promoters, terminators, operons, among others) to be pinpointed on any bacterial transcriptome. Once the transcriptome is mapped, the activity of transcriptional features can be quantified. Here we highlight how quantitative transcriptome analysis can reveal biological insights and briefly discuss some of the challenges to be faced by the field of bacterial transcriptomics in the near future.

We present the first high-resolution determination of transcriptome architecture in the priority pathogenAcinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification inA. baumanniiof sRNAs encoded at the 3’ end of coding genes. Most sRNAs were conserved among sequencedA. baumanniigenomes, but were only weakly conserved or absent in otherAcinetobacterspecies. Single nucleotide mapping of TSS enabled prediction of −10 and −35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance,craA. The twocraApromoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation ofcraAexpression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as “AcinetoCom” (http://bioinf.gen.tcd.ie/acinetocom/).

OBJECTIVES: Watermelon is a delicious and healthy fruit that contains low calories and is rich in carotenoids, vitamin A, vitamin C, citrulline, and other bioactive compounds. The health benefits of watermelon in diabetes are poorly understood. In the current study, we sought to determine the effects of watermelon juice on gut microbiome profile and blood glucose management in type 1 and type 2 diabetic mice. METHODS: Six-week-old male C57BL/6 J wild type (WT), db/db type 2 diabetic (db/db), and streptozocin (STZ)-induced type 1 (STZ) diabetic mice were fed a chow diet and given 50% or 100% watermelon juice or tape water during night cycle for 8 weeks. At the termination of the study, mice were fasted for 3 hrs prior to euthanization. Blood, cecal contents, and other tissues were collected for laboratory assessments. Plasma metabolic parameters and pro-inflammatory cytokines were monitored by a clinical analyzer and ELISA, respectively. Cecal microbiome was profiled by 16S rRNA sequencing and followed by bioinformatic analysis. RESULTS: Consumption of watermelon juice significantly lowered fasting blood glucose levels in both diabetic mouse models. The fasting insulin level was significantly decreased in db/db consuming watermelon juice, though it was undetectable in STZ mice, with or without watermelon juice. Drinking watermelon juice tremendously changed the gut microbiome composition. At the phylum level, the Firmicutes/Bacteroidetes ratio was significantly associated with genotype (e.g., WT vs STZ vs db/db) and diet (e.g., watermelon juice vs tap water). At the genus level, abundances of Ruminiclostridium_9, Parasutterella, and Clostridium_sensu_stricto_1 were increased in STZ mice with watermelon, and abundances of Oscillibacter and Ruminiclostridium were decreased in db/db mice with watermelon. Watermelon juice induced gut microbiome compositional changes also occurred at the species level. CONCLUSIONS: Watermelon juice intervention causes a decrease in blood glucose level and shifts of the gut microbiome in both type 1 and type 2 mice. FUNDING SOURCES: National watermelon board grant.