Integration of spatial and single-cell transcriptomics localizes epithelial cell–immune cross-talk in kidney injury

Single-cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single-nuclear sequencing data sets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell-type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of 2 murine AKI models: ischemia/reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single-cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature, and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by indEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complemented single-cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations

Size, shape, and direction matters: Matching secondary genital structures in male and female mites using multiple microscopy techniques and 3D modeling

Studies of female genital structures have generally lagged behind comparable studies of male genitalia, in part because of an assumption of a lower level of variability, but also because internal genitalia are much more difficult to study. Using multiple microscopy techniques, including video stereomicroscopy, fluorescence microscopy, low-temperature scanning electron microscopy (LT-SEM), and confocal laser scanning microscopy (CLSM) we examined whether the complex sperm transfer structures in males of Megalolaelaps colossus (Acari: Mesostigmata) are matched by similarly complex internal structures in the female. While both LT-SEM and CLSM are well suited for obtaining high-quality surface images, CLSM also proved to be a valuable technique for observing internal anatomical structures. The long and coiled sperm transfer organ on the chelicera of the males (spermatodactyl) largely matches an equally complex, but internal, spiral structure in the females in shape, size, and direction. This result strongly suggests some form of genital coevolution. A hypothesis of sexual conflict appears to provide the best fit for all available data (morphology and life history)

Histological Atlas of the Internal Anatomy of Female \u3ci\u3eVarroa destructor\u3c/i\u3e (Mesostigmata: Varroidae) Mites in Relation to Feeding and Reproduction

Histochemical staining of histological sections of Varroa destructor (Anderson and Trueman, 2000) mites reveal the internal body plan and are used to contrast the internal organs associated with feeding and reproduction of starved versus recently fed female mites. The gnathosoma is comprised of a powerful sucking pharynx, which employs 11 alternating dilator and constrictor muscles, the chelicerae, the salivary ducts, and the salivarium. Coronally, the esophagus is visible through the synganglion between its supraesophageal and subesophageal regions and connects posteriorly to the midgut. The midgut is devoid of food particles in starved mites, but in fed mites, the midgut epithelial cells are filled with innumerable globular spheroids replete with lipoproteins, including polyunsaturated lipids, whereas the lumen is filled with saturated lipids or other unidentified nutrients. In the opisthosomal body region of the fed female, the bilobed lyrate organ lies adjacent to the midgut on one side and the ovary on the opposite side, with very close cell to cell linkages that appear to form a syncytium. The fed female ovary contains an enormously enlarged ovum, and numerous elongated nurse cells extending from the lyrate organ. Dyes staining selectively for lipoproteins suggests rapid incorporation of neutral and polyunsaturated lipids and lipoproteins. Also evident near the ovary in fed females is the spermatheca filled with elongated, fully capacitated spermatozoa. The histological and histochemical findings reported in this study provide a fresh insight into the body structure, nutrition, and reproductive activity of the female of this harmful honey bee parasite and disease vector

Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes

The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni. Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species

<i>C</i>. <i>jejuni</i> CsrA regulates FlaA expression by binding to the 5’ end of <i>flaA</i> mRNA.

<p><b>(A)</b> WT and <i>csrA</i> mutant <i>C</i>. <i>jejuni</i> were diluted to an initial OD₆₀₀ = 0.1 and grown at 42°C. Cells were removed at 5, 6, 8 and 10 hours, and were used in western blots with antibodies against FlaA. <b>(B)</b> Electrophoretic Mobility Shift Assay (EMSA) analysis was used to assess the interaction between purified <i>C</i>. <i>jejuni</i> CsrA-His₆ and the 5’ end of <i>flaA</i> mRNA. Purified CsrA-His₆ at concentrations varying from 0–300 nM were incubated with 100 ng of purified ³²P-end-labeled RNA corresponding to the 5’ ends of either <i>C</i>. <i>jejuni flaA</i> (top panel) or <i>E</i>. <i>coli phoB</i> (bottom panel) as a control that does not bind CsrA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156932#pone.0156932.ref015" target="_blank">15</a>]. The positions of bound (B) and free (F) RNA are shown.</p

<i>Campylobacter jejuni</i> CsrA Regulates Metabolic and Virulence Associated Proteins and Is Necessary for Mouse Colonization

<div><p><i>Campylobacter jejuni</i> infection is a leading bacterial cause of gastroenteritis and a common antecedent leading to Gullian-Barré syndrome. Our previous data suggested that the RNA-binding protein CsrA plays an important role in regulating several important phenotypes including motility, biofilm formation, and oxidative stress resistance. In this study, we compared the proteomes of wild type, <i>csrA</i> mutant, and complemented <i>csrA</i> mutant <i>C</i>. <i>jejuni</i> strains in an effort to elucidate the mechanisms by which CsrA affects virulence phenotypes. The putative CsrA regulon was more pronounced at stationary phase (111 regulated proteins) than at mid-log phase (25 regulated proteins). Proteins displaying altered expression in the <i>csrA</i> mutant included diverse metabolic functions, with roles in amino acid metabolism, TCA cycle, acetate metabolism, and various other cell processes, as well as pathogenesis-associated characteristics such as motility, chemotaxis, oxidative stress resistance, and fibronectin binding. The <i>csrA</i> mutant strain also showed altered autoagglutination kinetics when compared to the wild type. CsrA specifically bound the 5’ end of <i>flaA</i> mRNA, and we demonstrated that CsrA is a growth-phase dependent repressor of FlaA expression. Finally, the <i>csrA</i> mutant exhibited reduced ability to colonize in a mouse model when in competition with the wild type, further underscoring the role of CsrA in <i>C</i>. <i>jejuni</i> colonization and pathogenesis.</p></div

Bacterial strains, plasmids, and primers used in this study.

<p>Bacterial strains, plasmids, and primers used in this study.</p

CsrA is involved in mouse colonization.

<p>BALB/c-ByJ mice were orally inoculated with a 1:1 mixture of wild type (closed circles) and <i>csrA</i> mutant (open circles) <i>C</i>. <i>jejuni</i> and fecal pellets were collected at 7, 14, and 21 days post-infection for enumeration of wild type and mutant bacteria. The geometric means of colonization of each group are represented by horizontal lines. The limit of detection (represented by a dashed line) was 10² CFU/g.</p

Proteins with altered expression <i>ΔcsrA</i> at mid-log phase.

<p>Proteins with altered expression <i>ΔcsrA</i> at mid-log phase.</p

The kinetics of autoagglutination are altered in the <i>csrA</i> mutant strain.

<p>Static suspensions of <i>C</i>. <i>jejuni</i> wild type, <i>csrA</i> mutant, and complemented <i>csrA</i> mutant strains were incubated and OD₆₀₀ measurements were taken at 2, 4, 6, and 24 hours. The assay was performed in triplicate on three separate occasions. Statistical significance (p<0.05) is represented by an asterisk. Error bars are present; however, they are too small to be seen.</p