Regulation of luminal diameter is critical to the function of small single-celled tubes, of which the seamless tubular excretory canals of Caenorhabditis elegans provide a tractable genetic model. Mutations in several sets of genes exhibit the Exc phenotype, in which canal luminal growth is visibly altered. Here, a focused reverse genomic screen of genes highly expressed in the canals found 18 genes that significantly affect luminal outgrowth or diameter. These genes encode novel proteins as well as highly conserved proteins involved in processes including gene expression, cytoskeletal regulation, and vesicular and transmembrane transport. In addition, two genes act as suppressors on a pathway of conserved genes whose products mediate vesicle movement from early to recycling endosomes. The results provide new tools for understanding the integration of cytoplasmic structure and physiology in forming and maintaining the narrow diameter of single-cell tubules.

Pleomorphism is an evolutionary adaptation by which diverse microorganisms maximize their fitness by transitioning between morphologically distinct forms that perform disparate functions in response to the local microenvironment. Cell division is critical for morphotype transition in many pleomorphic bacterial systems. Anaplasma phagocytophilum , which causes the emerging disease granulocytic anaplasmosis, is a pleomorphic obligate intracellular bacterium that lives in a pathogen-modified vacuole except for when it is exocytically released for dissemination to naïve cells. This bacterium cycles between non-infectious, replicative reticulate cell (RC) and infectious, non-replicative dense-cored (DC) forms. Here, we establish that differential modes of A. phagocytophilum cell division drive morphotype development where RC bacteria divide symmetrically to expand the intravacuolar population after which they switch to sacrificial asymmetric division to produce DCs. A. phagocytophilum MreB is crucial for cell division, specifically septation, and thereby formation of both morphotypes. Inhibition of cell division prevents not only DC formation but also A. phagocytophilum vacuole maturation and infectious progeny release, which suggests that these pathogenic processes are coordinated. This study advances understanding of A. phagocytophilum growth and morphotype development and, thus, pathobiology. It also provides the first evidence linking cell division to morphotype development in the Anaplasmataceae . IMPORTANCE: Anaplasma phagocytophilum , an obligate intracellular bacterial pathogen that lives in a host cell-derived vacuole, causes human and veterinary diseases of global importance. In the pathogen-occupied vacuole, A. phagocytophilum transitions from a replicative, non-infectious morphotype to a non-replicative, infectious morphotype that is released to spread infection. We established that distinct modes of bacterial cell division drive not only A. phagocytophilum replication but also its differentiation to the infectious form and dissemination to naïve cells. How pleomorphism is regulated in most vacuole-adapted bacterial pathogens is poorly understood. Therefore, this study advances fundamental knowledge of vacuole-adapted pleomorphic bacteria pathobiology and could ultimately identify common novel antibiotic targets for treating the diseases they cause.

Nuclear factor-κB (NF-κB) nuclear localization and gene accessibility is mediated by Ser22-phosphorylated lamin A/C (pSer22-lamin A/C). Here, we report that the intracellular bacterium Orientia tsutsugamushi impairs NF-κB nuclear accumulation by targeting lamin A using multiple ankyrin repeat (AR)-containing effectors (Anks). The Anks' immunomodulatory capability requires a conserved hydrophilic α-helical peptide that binds lamin A/pSer22-lamin A and lies between the AR and PRANC (pox proteins repeats of ankyrin C-terminal) domains. Orientia promotes pSer22-lamin A redistribution from the lamina to the nucleoplasm. This phenotype can be recapitulated in uninfected cells by ectopically expressing Anks that carry the lamin A-binding sequence. O. tsutsugamushi also alters chromatin accessibility at sites regulated by lamin A, NF-κB, and the NF-κB coactivator, adapter protein 1. These findings identify a mechanism by which a pathogen synergistically modulates lamin A and chromatin accessibility to counteract NF-κB, reinforce the regulatory link between lamin A and NF-κB, and indicate that pSer22-lamin A enhances chromatin accessibility to NF-κB and its coactivators.

Orientia tsutsugamushi is an obligate intracellular alphaproteobacterium and the causative agent of the potentially fatal rickettsiosis, scrub typhus. During infection, O. tsutsugamushi replicates exclusively in the eukaryotic cytosol near mitochondria and alters host metabolic pathways governed by mitochondria. We report that O. tsutsugamushi induces mitochondrial enzymatic impairment and structural abnormalities without altering mitochondrial abundance or the levels of proteins that maintain mitochondrial homeostasis. Confocal and structured illumination microscopy revealed a selective spatial association between O. tsutsugamushi and the mitochondrial membrane protein, voltage-dependent anion channel (VDAC) but not other mitochondrial proteins. Immunosignal for VDAC paralogs 1 and 3 colocalized with cytosolic O. tsutsugamushi organisms whereas VDAC2 did not. Additionally, the antibody specific for VDAC1 and VDAC3 detected proteins of the expected sizes in Orientia membrane fractions. These findings indicate that O. tsutsugamushi negatively impacts mitochondrial function without overt organelle loss and selectively associates with VDAC1/VDAC3.

Scrub typhus is a potentially fatal infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi . While antibiotic treatment is generally effective, it requires extended treatment, and drug resistance and treatment failures have emerged. O. tsutsugamushi encodes a deubiquitylating enzyme, OtDUB, which interferes with host ubiquitin-dependent pathways. OtDUB cleaves ubiquitin from various substrates, but whether this activity can be selectively targeted by small molecules is unknown. Here we have screened a chemically diverse small-molecule library using a fluorescence-based deubiquitylation assay to identify potential inhibitors of OtDUB. Two compounds, gentisic acid and amiloride hydrochloride, inhibited OtDUB activity at low dosage, with little effect on the related Wolbachia CidB or yeast Ulp1 enzymes. Computational docking predicted the compounds engage regions near the OtDUB catalytic pocket, suggesting a competitive mode of inhibition; this was supported by enzyme kinetic analyses. Neither compound caused detectable cytotoxicity in mammalian cells. Amiloride hydrochloride treatment reduced both total cellular deubiquitylating activity and the O. tsutsugamushi bacterial load in infected cells. While the identified compounds are not optimized inhibitors, they establish that bacterial pathogen-encoded deubiquitylating enzymes can be targeted by small molecules. Overall, our results provide a framework for using selective inhibitors as tools to study DUB function in genetically intractable intracellular bacteria and as potential treatments for scrub typhus.

Anaplasma phagocytophilum is an obligate intracellular gram-negative bacterium and etiologic agent of human granulocytic anaplasmosis. A. phagocytophilum genomic sequencing has historically been performed via short-read platforms. Our optimized bacterial isolation protocol combined with Nanopore sequencing produced a single, closed 1,481,805 bp circular A. phagocytophilum strain NCH-1 chromosome.