Enteroendocrine cells (EEs) are interspersed between enterocytes and stem cells in the Drosophila intestinal epithelium. Like enterocytes, EEs express components of the immune deficiency (IMD) innate ...immune pathway, which activates transcription of genes encoding antimicrobial peptides. The discovery of large lipid droplets in intestines of IMD pathway mutants prompted us to investigate the role of the IMD pathway in the host metabolic response to its intestinal microbiota. Here we provide evidence that the short-chain fatty acid acetate is a microbial metabolic signal that activates signaling through the enteroendocrine IMD pathway in a PGRP-LC-dependent manner. This, in turn, increases transcription of the gene encoding the endocrine peptide Tachykinin (Tk), which is essential for timely larval development and optimal lipid metabolism and insulin signaling. Our findings suggest innate immune pathways not only provide the first line of defense against infection but also afford the intestinal microbiota control over host development and metabolism.
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•The innate immune deficiency (IMD) pathway regulates tachykinin transcription•Microbial activation of enteroendocrine IMD signaling mediates metabolic homeostasis•Acetate restores IMD pathway activation and metabolic homeostasis in axenic flies•Acetate signaling through IMD depends on the membrane-associated receptor PGRP-LC
Here Kamareddine et al. show that an innate immune pathway in enteroendocrine cells of the Drosophila melanogaster intestine is activated by the intestinal microbiota and the microbial metabolite acetate. Activation of this pathway increases expression of the endocrine peptide tachykinin to promote host metabolic homeostasis.
SARS-CoV-2 is one of three recognized coronaviruses (CoVs) that have caused epidemics or pandemics in the 21st century and that likely emerged from animal reservoirs. Differences in nucleotide and ...protein sequence composition within related β-coronaviruses are often used to better understand CoV evolution, host adaptation, and their emergence as human pathogens. Here we report the comprehensive analysis of amino acid residue changes that have occurred in lineage B β-coronaviruses that show covariance with each other. This analysis revealed patterns of covariance within conserved viral proteins that potentially define conserved interactions within and between core proteins encoded by SARS-CoV-2 related β-coronaviruses. We identified not only individual pairs but also networks of amino acid residues that exhibited statistically high frequencies of covariance with each other using an independent pair model followed by a tandem model approach. Using 149 different CoV genomes that vary in their relatedness, we identified networks of unique combinations of alleles that can be incrementally traced genome by genome within different phylogenic lineages. Remarkably, covariant residues and their respective regions most abundantly represented are implicated in the emergence of SARS-CoV-2 and are also enriched in dominant SARS-CoV-2 variants.
Pathogens adapt to the host environment by altering their patterns of gene expression. Microarray-based and genetic techniques used to characterize bacterial gene expression during infection are ...limited in their ability to comprehensively and simultaneously monitor genome-wide transcription. We used massively parallel cDNA sequencing (RNA-seq) techniques to quantitatively catalog the transcriptome of the cholera pathogen, Vibrio cholerae, derived from two animal models of infection. Transcripts elevated in infected rabbits and mice relative to laboratory media derive from the major known V. cholerae virulence factors and also from genes and small RNAs not previously linked to virulence. The RNA-seq data was coupled with metabolite analysis of cecal fluid from infected rabbits to yield insights into the host environment encountered by the pathogen and the mechanisms controlling pathogen gene expression. RNA-seq-based transcriptome analysis of pathogens during infection produces a robust, sensitive, and accessible data set for evaluation of regulatory responses driving pathogenesis.
Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti.
We ...used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of 23 diverse strains of V. cholerae to assess the likely origin of the cholera outbreak in Haiti.
Both single-nucleotide variations and the presence and structure of hypervariable chromosomal elements indicate that there is a close relationship between the Haitian isolates and variant V. cholerae El Tor O1 strains isolated in Bangladesh in 2002 and 2008. In contrast, analysis of genomic variation of the Haitian isolates reveals a more distant relationship with circulating South American isolates.
The Haitian epidemic is probably the result of the introduction, through human activity, of a V. cholerae strain from a distant geographic source. (Funded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute.).
Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the ...absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.
Defense systems that recognize viruses provide important insights into both prokaryotic and eukaryotic innate immunity mechanisms. Such systems that restrict foreign DNA or trigger cell death have ...recently been recognized, but the molecular signals that activate many of these remain largely unknown. Here, we characterize one such system in pandemic Vibrio cholerae responsible for triggering cell density-dependent death (CDD) of cells in response to the presence of certain genetic elements. We show that the key component is the Lamassu DdmABC anti-phage/plasmid defense system. We demonstrate that signals that trigger CDD were palindromic DNA sequences in phages and plasmids that are predicted to form stem-loop hairpins from single-stranded DNA. Our results suggest that agents that damage DNA also trigger DdmABC activation and inhibit cell growth. Thus, any infectious process that results in damaged DNA, particularly during DNA replication, can in theory trigger DNA restriction and death through the DdmABC abortive infection system.
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•DdmABC targeting and degradation of plasmids are dependent on certain cis-acting sequences•DNA sequences that induce DdmABC-based plasmid instability form hairpins•Palindromic sequences include those from related phages that are also restricted by DdmABC•DdmABC is blocked by a PriA allele
Robins et al. report on the identification of cis-acting palindromic DNA sequences in plasmids and phages targeted by the V. cholerae Lamassu DdmABC defense system. A single mutation in the PriA DNA repair-replication protein blocks DdmABC activity. Moreover, DdmABC expression increases UV sensitivity, suggesting DNA repair is one signal for activation.
Vibrio cholerae is lethal to the model host Drosophila melanogaster through mechanisms not solely attributable to cholera toxin. To examine additional virulence determinants, we performed a genetic ...screen in V. cholerae-infected Drosophila and identified the two-component system CrbRS. CrbRS controls transcriptional activation of acetyl-CoA synthase-1 (ACS-1) and thus regulates the acetate switch, in which bacteria transition from excretion to assimilation of environmental acetate. The resultant loss of intestinal acetate leads to deactivation of host insulin signaling and lipid accumulation in enterocytes, resulting in host lethality. These metabolic effects are not observed upon infection with ΔcrbS or Δacs1 V. cholerae mutants. Additionally, uninfected flies lacking intestinal commensals, which supply short chain fatty acids (SCFAs) such as acetate, also exhibit altered insulin signaling and intestinal steatosis, which is reversed upon acetate supplementation. Thus, acetate consumption by V. cholerae alters host metabolism, and dietary acetate supplementation may ameliorate some sequelae of cholera.
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•V. cholerae CrbRS is a two-component system that activates acetate consumption•Consumption of acetate within the intestine deactivates host insulin signaling•Acetate consumption within the intestine disrupts fat metabolism•V. cholerae-infected and germ-free flies develop intestinal steatosis
Intestinal short chain fatty acids (SCFAs) direct carbohydrate and fat metabolism. Hang et al. show that the Vibrio cholerae two-component system CrbRS activates SCFA uptake, leading to interruption of host insulin signaling and anomalous lipid transport. A diet promoting SCFA synthesis may mitigate metabolic disruption and the sequelae of cholera.
Abstract
The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, ...fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum.
Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short ...read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.
The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. ...The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Thermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.
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