Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi.

Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi.

Borrelia burgdorferi , a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/SMC. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that SMC and the SMC-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC . Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi.

The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete.

Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete's length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.

A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi.

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen.IMPORTANCE Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

Stem cell-derived polarized hepatocytes.

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

Fluorescent Proteins, Promoters, and Selectable Markers for Applications in the Lyme Disease Spirochete Borrelia burgdorferi.

Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing, and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirochete Borrelia burgdorferi, is thus evermore pressing. Despite important advances in B. burgdorferi genetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multicolor imaging in B. burgdorferi We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Last, we describe a set of promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities in B. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, a B. burgdorferi lipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites of B. burgdorferi cells, highlighting the complex subcellular organization of this spirochete.IMPORTANCE Genetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium's transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular Systems.

BACKGROUND & AIMS: The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. METHODS: Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. RESULTS: HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. CONCLUSIONS: Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin.

Differential Regulation of Lipoprotein and Hepatitis C Virus Secretion by Rab1b.

Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion.

Green fluorescent protein-tagged apolipoprotein E: A useful marker for the study of hepatic lipoprotein egress.

Apolipoprotein E (ApoE), a component of very-low-density and high-density lipoproteins, participates in many aspects of lipid transport in the bloodstream. Underscoring its important functions, ApoE isoforms have been associated with metabolic and circulatory disease. ApoE is also incorporated into hepatitis C virus (HCV) particles, and promotes their production and infectivity. Live cell imaging analysis of ApoE behavior during secretion from producing cells thus has the potential to reveal important details regarding lipoprotein and HCV particle biogenesis and secretion from cells. However, this approach requires expression of fluorescently tagged ApoE constructs that need to faithfully reproduce known ApoE behaviors. Herein, we evaluate the usefulness of using an ApoE-GFP fusion protein in studying hepatocyte-derived, ApoE-containing lipoproteins and HCV particles. We show that while ApoE-GFP alone is not sufficient to support infectious HCV production, it nonetheless colocalizes intracellularly and associates with secreted untagged lipoprotein components. Furthermore, its rate of secretion from hepatic cells is indistinguishable from that of untagged ApoE. ApoE-GFP thus represents a useful marker for ApoE-containing hepatic lipoproteins.

Hepatitis C virus RNA functionally sequesters miR-122.

Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection showed robust AGO binding on the HCV 5'UTR at known and predicted miR-122 sites. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 "sponge" effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV-induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.

Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and young adults with no history of primary liver disease or cirrhosis. We identified a chimeric transcript that is expressed in FL-HCC but not in adjacent normal liver and that arises as the result of a ~400-kilobase deletion on chromosome 19. The chimeric RNA is predicted to code for a protein containing the amino-terminal domain of DNAJB1, a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed in tumor tissue, and a cell culture assay indicated that it retains kinase activity. Evidence supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100% of the FL-HCCs examined (15/15) suggests that this genetic alteration contributes to tumor pathogenesis.

Growth medium-dependent glycine incorporation into the peptidoglycan of Caulobacter crescentus.

The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

Osmolality-dependent relocation of penicillin-binding protein PBP2 to the division site in Caulobacter crescentus.

The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.

A protein critical for cell constriction in the Gram-negative bacterium Caulobacter crescentus localizes at the division site through its peptidoglycan-binding LysM domains.

During division of Gram-negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast-growth conditions. Under slow-growth conditions, the DeltadipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non-canonical. DipM also carries two tandems of the PG-binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non-septal, monolayered PG.

MreB drives de novo rod morphogenesis in Caulobacter crescentus via remodeling of the cell wall.

MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped Caulobacter crescentus cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of C. crescentus peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.

D-amino acids govern stationary phase cell wall remodeling in bacteria.

In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.