Intracellular Positioning Research Articles

Peripheral Lysosomal Positioning in Inflamed Odontoblasts Facilitates Mineralization.

Odontoblasts, terminally differentiated dentin-producing cells, critically rely on lysosomal functions for intracellular recycling and renewal. Beyond their traditional degradative role, lysosomes actively orchestrate cellular responses to external stimuli through precise and rapid intracellular trafficking and positioning. This study aimed to explore the influence of lysosomal positioning on odontoblast mineralization and the underlying mechanisms implicated in carious inflammation. Human dental pulp stem cells (hDPSCs) were induced to differentiate into human odontoblast-like cells (hOBLCs). hOBLCs were treated with various doses of LPS (0.1, 1, 5 μg/mL) to mimic carious inflammation. Lysosomal positioning was examined by immunofluorescence staining of LAMP1 in healthy and carious human teeth, LPS-treated hOBLCs, mouse lower incisors at postnatal day 2.5, and mineralization medium cultured hDPSCs. Lysosomal positioning was manipulated by knockdown or overexpression of SNAPIN or ARL8B. Mineralization was assessed by ARS staining and expression of DSPP and DMP1. Lysosomal exocytosis was examined by detection of lysosomal-plasma membrane fusion, surface exposure of LAMP1 luminal epitopes (1D4B) and extracellularly released lysosomal enzymes. Peripheral lysosomal positioning was markedly increased in odontoblasts within moderate and extensive carious lesions (P < .001) and in hOBLCs following LPS treatment. Increased peripheral dispersion of lysosomes was similarly observed during odontoblastic differentiation in vivo and in vitro. Moreover, peripheral lysosomal positioning promoted mineralization in inflamed hOBLCs, potentially via mTORC1 signaling pathway and lysosomal exocytosis. Inflammatory stimuli prompted a relocation of lysosomes in odontoblasts, redistributing them from perinuclear location towards the cell periphery, which in turn facilitated mineralization, potentially via mTORC1 signaling and lysosomal exocytosis.

MIRO1 controls energy production and proliferation of smooth muscle cells.

The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown. An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC). MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I. Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.

In vivo assembly of bacterial partition condensates on circular supercoiled and linear DNA.

In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles invivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2 Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Finally, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.

Bacterial Pathogenesis: Assessment of Intracellular Positioning of Pathogen-Containing Vacuoles During Infection.

Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.

Enucleation of the C. elegans embryo revealed dynein-dependent spacing between microtubule asters.

The intracellular positioning of the centrosome, a major microtubule-organizing center, is important for cellular functions. One of the features of centrosome positioning is the spacing between centrosomes; however, the underlying mechanisms are not fully understood. To characterize the spacing activity in Caenorhabditis elegans embryos, a genetic setup was developed to produce enucleated embryos. The centrosome was duplicated multiple times in the enucleated embryo, which enabled us to characterize the chromosome-independent spacing activity between sister and non-sister centrosome pairs. We found that the timely spacing depended on cytoplasmic dynein, and we propose a stoichiometric model of cortical and cytoplasmic pulling forces for the spacing between centrosomes. We also observed dynein-independent but non-muscle myosin II-dependent movement of centrosomes in the later cell cycle phase. The spacing mechanisms revealed in this study are expected to function between centrosomes in general, regardless of the presence of a chromosome/nucleus between them, including centrosome separation and spindle elongation.

Mechanistic Insights into the Interactions of Arl8b with the RUN Domains of PLEKHM1 and SKIP

Arl8b, a specific Arf-like family GTPase present on lysosome, and plays critical roles in many lysosome-related cellular processes such as autophagy. The active Arl8b can be specifically recognized by the RUN domains of two Arl8b-effectors PLEKHM1 and SKIP, thereby regulating the autophagosome/lysosome membrane fusion and the intracellular lysosome positioning, respectively. However, the mechanistic bases underlying the interactions of Arl8b with the RUN domains of PLEKHM1 and SKIP remain elusive. Here, we report the two high-resolution crystal structures of the active Arl8b in complex with the RUN domains of PLEKHM1 and SKIP. In addition to elucidating the detailed molecular mechanism governing the specific interactions of the active Arl8b with the RUN domains of PLEKHM1 and SKIP, the determined complex structures also reveal a general binding mode shared by the PLEKHM1 and SKIP RUN domains for interacting with the active Arl8b. Furthermore, we uncovered a competitive relationship between the RUN domains of PLEKHM1 and SKIP in binding to the active Arl8b as well as a unique small GTPase-binding mode adopted by the PLEKHM1 and SKIP RUN domains, thereby enriching the repertoire of the RUN domain/small GTPase interaction modes. In all, our findings provide new mechanistic insights into the interactions of the active Arl8b with PLEKHM1 and SKIP, and are valuable for further understanding the working modes of these proteins in relevant cellular processes.

NN-RNALoc: Neural network-based model for prediction of mRNA sub-cellular localization using distance-based sub-sequence profiles.

The localization of messenger RNAs (mRNAs) is a frequently observed phenomenon and a crucial aspect of gene expression regulation. It is also a mechanism for targeting proteins to a specific cellular region. Moreover, prior research and studies have shown the significance of intracellular RNA positioning during embryonic and neural dendrite formation. Incorrect RNA localization, which can be caused by a variety of factors, such as mutations in trans-regulatory elements, has been linked to the development of certain neuromuscular diseases and cancer. In this study, we introduced NN-RNALoc, a neural network-based method for predicting the cellular location of mRNA using novel features extracted from mRNA sequence data and protein interaction patterns. In fact, we developed a distance-based subsequence profile for RNA sequence representation that is more memory and time-efficient than well-known k-mer sequence representation. Combining protein-protein interaction data, which is essential for numerous biological processes, with our novel distance-based subsequence profiles of mRNA sequences produces more accurate features. On two benchmark datasets, CeFra-Seq and RNALocate, the performance of NN-RNALoc is compared to powerful predictive models proposed in previous works (mRNALoc, RNATracker, mLoc-mRNA, DM3Loc, iLoc-mRNA, and EL-RMLocNet), and a ground neural (DNN5-mer) network. Compared to the previous methods, NN-RNALoc significantly reduces computation time and also outperforms them in terms of accuracy. This study's source code and datasets are freely accessible at https://github.com/NeginBabaiha/NN-RNALoc.

Intracellular positioning of mesophyll chloroplasts following to aggregative movement in Setaria viridis analysed three-dimensionally with a confocal laser scanning microscope

Chloroplasts can change their intracellular position responding to environmental conditions. In addition to the well-known photorelocation movements, i.e., accumulation and avoidance movements in response to light, mesophyll chloroplasts in C4 plants change their intracellular positioning from the cell periphery to the bundle sheath cell side (aggregative movement) in response to environmental stresses such as drought, salinity, and hyperosmosis, under light. Previous studies on the aggregative movement have been limited in two-dimensional (2D) information on the transverse sections of leaves, because aggregated mesophyll chloroplasts gather inside the leaf tissues, which need to be cut for observation. However, the 2D analysis on the cross sections is difficult to investigate accurately the aggregated chloroplasts overlapped each other in the depth direction. Therefore, there are few studies examining the anatomical features of the aggregated chloroplasts at the cellular level. Here in this study, we established the workflow for three-dimensional (3D) observation using a confocal laser scanning microscope (CLSM), which can investigate a thick section as a stack of optical sections, followed by 3D reconstruction of mesophyll cells and chloroplasts. Using this method, we visualized the 3D representations of mesophyll cells of green foxtail (Setaria viridis), which is a model of C4 plant, and investigated the chloroplasts individually and quantified their structures or intracellular positions before and after the aggregative movement. The 3D data of individual chloroplasts in a whole cell revealed that the aggregated positioning is independent with chloroplast volume or surface area, and that chloroplasts did not change their shape before and after the movement.

Stiffness of Confinement Controls the Localization of an Object under Crowding: Macroscale Real-World and Microscale Numerical Modelings on the Specificity of Intracellular Positioning

Macroscopic systems that mimic microscopic systems can provide fundamental understanding of how macromolecules (e.g., cellular organelles) are organized in a confined space. We studied the behavior of a large particle interacting with several small particles confined in dishes with hard/soft boundary conditions under mechanical vibration using a cm-scale model. We also performed a numerical simulation for the micro-scale system under Brownian fluctuation with fluctuation–dissipation, as a simple model of living cellular cytoplastic crowding. Under a hard boundary condition, the large sphere preferred the boundary at low crowding but tended to localize to the interior under high crowding. Conversely, when the boundary was soft, the large sphere localized to the interior under low crowding and tended to migrate near the boundary when crowding increased. Interestingly, numerical modeling reproduced similar results for both the experimental hard and soft boundary conditions. Our models revealed that membrane stiffness can affect the organization of biopolymers within a confined space and may help explain cellular dynamics.

Opto-katanin, an optogenetic tool for localized, microtubule disassembly

Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.

Biogenesis of a bacterial metabolosome for propanediol utilization

Bacterial metabolosomes are a family of protein organelles in bacteria. Elucidating how thousands of proteins self-assemble to form functional metabolosomes is essential for understanding their significance in cellular metabolism and pathogenesis. Here we investigate the de novo biogenesis of propanediol-utilization (Pdu) metabolosomes and characterize the roles of the key constituents in generation and intracellular positioning of functional metabolosomes. Our results demonstrate that the Pdu metabolosome undertakes both “Shell first” and “Cargo first” assembly pathways, unlike the β-carboxysome structural analog which only involves the “Cargo first” strategy. Shell and cargo assemblies occur independently at the cell poles. The internal cargo core is formed through the ordered assembly of multiple enzyme complexes, and exhibits liquid-like properties within the metabolosome architecture. Our findings provide mechanistic insight into the molecular principles driving bacterial metabolosome assembly and expand our understanding of liquid-like organelle biogenesis.

Normal skeletal pattern formation in chick limb bud with a mesenchymal hole is mediated by adjustment of cellular properties along the anterior–posterior axis in the limb bud

The chick limb bud has plasticity to reconstruct a normal skeletal pattern after a part of mesenchymal mass is excised to make a hole in its early stage of development. To understand the details of hole closure and re-establishment of normal limb axes to reconstruct a normal limb skeleton, we focused on cellular and molecular changes during hole repair and limb restoration. We excised a cube-shaped mass of mesenchymal cells from the medial region of chick hindlimb bud (stage 23) and observed the following morphogenesis. The hole had closed by 15 ​h after excision, followed by restoration of the limb bud morphology, and the cartilage pattern was largely restored by 48 ​h. Lineage analysis of the mesenchymal cells showed that cells at the anterior and posterior margins of the hole were adjoined at the hole closure site, whereas cells at the proximal and distal margins were not. To investigate cell polarity during hole repair, we analyzed intracellular positioning of the Golgi apparatus relative to the nuclei. We found that the Golgi apparatus tended to be directed toward the hole among cells at the anterior and posterior margins but not among cells at identical positions in normal limb buds or cells at the proximal and distal hole margins. In the manipulated limb buds, the frequency of cell proliferation was maintained compared with the control side. Tbx3 expression, which was usually restricted to anterior and posterior margins of the limb bud, was temporarily expanded medially and then reverted to a normal pattern as limb reconstruction proceeded, with Tbx3 negative cells reappearing in the medial regions of the limb buds. Thus, mesenchymal hole closure and limb reconstruction are mainly mediated by cells at the anterior and posterior hole margins. These results suggest that adjustment of cellular properties along the anteroposterior axis is crucial to restore limb damage and reconstruct normal skeletal patterns.

Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein–protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.

Interdependent Polar Localization of FlhF and FlhG and Their Importance for Flagellum Formation of Vibrio parahaemolyticus.

Failure of the cell to properly regulate the number and intracellular positioning of their flagella, has detrimental effects on the cells’ swimming ability. The flagellation pattern of numerous bacteria is regulated by the NTPases FlhF and FlhG. In general, FlhG controls the number of flagella produced, whereas FlhF coordinates the position of the flagella. In the human pathogen Vibrio parahaemolyticus, its single flagellum is positioned and formed at the old cell pole. Here, we describe the spatiotemporal localization of FlhF and FlhG in V. parahaemolyticus and their effect on swimming motility. Absence of either FlhF or FlhG caused a significant defect in swimming ability, resulting in absence of flagella in a ΔflhF mutant and an aberrant flagellated phenotype in ΔflhG. Both proteins localized to the cell pole in a cell cycle-dependent manner, but displayed different patterns of localization throughout the cell cycle. FlhF transitioned from a uni- to bi-polar localization, as observed in other polarly flagellated bacteria. Localization of FlhG was strictly dependent on the cell pole-determinant HubP, while polar localization of FlhF was HubP independent. Furthermore, localization of FlhF and FlhG was interdependent and required for each other’s proper intracellular localization and recruitment to the cell pole. In the absence of HubP or FlhF, FlhG forms non-polar foci in the cytoplasm of the cell, suggesting the possibility of a secondary localization site within the cell besides its recruitment to the cell poles.

A septin GTPase scaffold of dynein-dynactin motors triggers retrograde lysosome transport.

The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase-based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein-dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.

Reshaping cytoskeleton: different acts of modulatory compounds

The eukaryotic cytoskeleton is composed of filamentous structures, namely microfilaments, microtubules and intermediate filaments. The cytoskeleton is an essential component of cells due to its role in various cellular functions, such as intracellular transport, organelle positioning, chromosome segregation and cytokinesis. Abnormalities in cytoskeleton, as well as associated proteins and regulatory pathways, have been shown to contribute to disease pathomechanisms including cancer and neurodegenerative diseases. Therefore, cytoskeleton is an important therapeutic target and many compounds have been identified or developed to modulate the cytoskeleton. In this review, we focused on cytoskeleton modulatory compounds and summarized their mechanisms of action.

Miro1-mediated mitochondrial positioning supports subcellular redox status

Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca2+ and reactive oxygen species (ROS, i.e. hydrogen peroxide, H2O2). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular H2O2 levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded H2O2-responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular H2O2 map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral H2O2 levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of H2O2 in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral H2O2 or PRX2 oxidation but rather lead to increased nuclear H2O2 and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1+/+ MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular H2O2 levels and local cellular responses dependent on mitochondrial ROS.

Physical Modeling of a Sliding Clamp Mechanism for the Spreading of ParB at Short Genomic Distance from Bacterial Centromere Sites.

SummaryBacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation are cytosine triphosphate (CTP)-dependent molecular switches. CTP-binding converts ParB dimers to DNA clamps, allowing unidimensional diffusion along the DNA. This sliding property has been proposed to explain the ParB spreading over large distances from parS centromere sites where ParB is specifically loaded. We modeled such a “clamping and sliding” mechanism as a typical reaction-diffusion system, compared it to the F plasmid ParB DNA binding pattern, and found that it can account neither for the long range of ParB binding to DNA nor for the rapid assembly kinetics observed in vivo after parS duplication. Also, it predicts a strong effect on the F plasmid ParB binding pattern from the presence of a roadblock that is not observed in ChIP-sequencing (ChIP-seq). We conclude that although “clamping and sliding” can occur at short distances from parS, another mechanism must apply for ParB recruitment at larger genomic distances.

Vibrionaceae core, shell and cloud genes are non-randomly distributed on Chr 1: An hypothesis that links the genomic location of genes with their intracellular placement

BackgroundThe genome of Vibrionaceae bacteria, which consists of two circular chromosomes, is replicated in a highly ordered fashion. In fast-growing bacteria, multifork replication results in higher gene copy numbers and increased expression of genes located close to the origin of replication of Chr 1 (ori1). This is believed to be a growth optimization strategy to satisfy the high demand of essential growth factors during fast growth. The relationship between ori1-proximate growth-related genes and gene expression during fast growth has been investigated by many researchers. However, it remains unclear which other gene categories that are present close to ori1 and if expression of all ori1-proximate genes is increased during fast growth, or if expression is selectively elevated for certain gene categories.ResultsWe calculated the pangenome of all complete genomes from the Vibrionaceae family and mapped the four pangene categories, core, softcore, shell and cloud, to their chromosomal positions. This revealed that core and softcore genes were found heavily biased towards ori1, while shell genes were overrepresented at the opposite part of Chr 1 (i.e., close to ter1). RNA-seq of Aliivibrio salmonicida and Vibrio natriegens showed global gene expression patterns that consistently correlated with chromosomal distance to ori1. Despite a biased gene distribution pattern, all pangene categories contributed to a skewed expression pattern at fast-growing conditions, whereas at slow-growing conditions, softcore, shell and cloud genes were responsible for elevated expression.ConclusionThe pangene categories were non-randomly organized on Chr 1, with an overrepresentation of core and softcore genes around ori1, and overrepresentation of shell and cloud genes around ter1. Furthermore, we mapped our gene distribution data on to the intracellular positioning of chromatin described for V. cholerae, and found that core/softcore and shell/cloud genes appear enriched at two spatially separated intracellular regions. Based on these observations, we hypothesize that there is a link between the genomic location of genes and their cellular placement.

Multiple Salmonella-pathogenicity island 2 effectors are required to facilitate bacterial establishment of its intracellular niche and virulence.

The pathogenesis of Salmonella Typhimurium depends on the bacterium’s ability to survive and replicate within host cells. The formation and maintenance of a unique membrane-bound compartment, termed the Salmonella-containing vacuole (SCV), is essential for S. Typhimurium pathogenesis. SCV-bound S. Typhimurium induces formation of filamentous tubules that radiate outwards from the SCV, termed Salmonella-induced filaments (SIFs). SIF formation is concomitant with the onset of replication within host epithelial cells. SIF biogenesis, formation and maintenance of the SCV, and the intracellular positioning of the SCV within the host cell requires translocation of bacterial proteins (effectors) into the host cell. Effectors secreted by the type III secretion system encoded on Salmonella pathogenicity island 2 (T3SS2) function to interfere with host cellular processes and promote both intracellular survival and replication of S. Typhimurium. Seven T3SS2-secreted effectors, SifA, SopD2, PipB2, SteA, SseJ, SseF, and SseG have previously been implicated to play complementary, redundant, and/or antagonistic roles with respect to SIF biogenesis, intracellular positioning of the SCV, and SCV membrane dynamics modulation during infection. We undertook a systematic study to delineate the contribution of each effector to these processes by (i) deleting all seven of these effectors in a single S. Typhimurium strain; and (ii) deleting combinations of multiple effectors based on putative effector function. Using this deletion mutant library, we show that each of SIF biogenesis, intracellular SCV localization, intramacrophage replication, colonization, and virulence depends on the activities of multiple effectors. Together, our data demonstrates the complex interplay between these seven effectors and highlights the necessity to study T3SS2-secreted effectors as groups, rather than studies of individual effectors.