Effects of Maternal Diabetes and Diet on Gene Expression in the Murine Placenta

Adverse exposures during pregnancy have been shown to contribute to susceptibility for chronic diseases in offspring. Maternal diabetes during pregnancy is associated with higher risk of pregnancy complications, structural birth defects, and cardiometabolic health impairments later in life. We showed previously in a mouse model that the placenta is smaller in diabetic pregnancies, with reduced size of the junctional zone and labyrinth. In addition, cell migration is impaired, resulting in ectopic accumulation of spongiotrophoblasts within the labyrinth. The present study had the goal to identify the mechanisms underlying the growth defects and trophoblast migration abnormalities. Based upon gene expression assays of 47 candidate genes, we were able to attribute the reduced growth of diabetic placenta to alterations in the Insulin growth factor and Serotonin signaling pathways, and provide evidence for Prostaglandin signaling deficiencies as the possible cause for abnormal trophoblast migration. Furthermore, our results reinforce the notion that the exposure to maternal diabetes has particularly pronounced effects on gene expression at midgestation time points. An implication of these findings is that mechanisms underlying developmental programming act early in pregnancy, during placenta morphogenesis, and before the conceptus switches from histiotrophic to hemotrophic nutrition.

Plant trans-golgi network/early endosome pH regulation requires cation chloride cotransporter (CCC1)

Plant cells maintain a low luminal pH in the Trans-Golgi-Network/Early Endosome (TGN/EE), the organelle in which the secretory and endocytic pathways intersect. Impaired TGN/EE pH regulation translates into severe plant growth defects. The identity of the proton pump and proton/ion antiporters that regulate TGN/EE pH have been determined, but an essential component required to complete the TGN/EE membrane transport circuit remains unidentified - a pathway for cation and anion efflux. Here, we have used complementation, genetically encoded fluorescent sensors, and pharmacological treatments to demonstrate that Arabidopsis Cation Chloride Cotransporter (CCC1) is this missing component necessary for regulating TGN/EE pH and function. Loss of CCC1 function leads to alterations in TGN/EE-mediated processes including endocytic trafficking, exocytosis and response to abiotic stress, consistent with the multitude of phenotypic defects observed in ccc1 knockout plants. This discovery places CCC1 as a central component of plant cellular function.

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

Genomic analyses have revealed how the gut microbiota impacts human health. However, knowledge about the physiology of most gut commensals is largely lacking. Here, we sorted cells from a pooled library to construct an ordered collection of transposon-insertion mutants in the model commensal Bacteroides thetaiotaomicron. We applied a pooling strategy with barcode sequencing to locate mutants and created a condensed collection with single insertions in 2,565 genes. This effort enabled the development of an accurate model for progenitor-collection assembly, which identified strain-abundance biases and multi-insertion strains as important factors that limit coverage. To demonstrate the potential for phenotypic screening, we analyzed growth dynamics and morphology of the condensed collection and identified growth defects and altered cell shape in the sphingolipid-synthesis gene BT0870 and the thiamine-scavenging gene BT2397. Analyses of this collection and utilization of the platform described herein to construct future ordered libraries will increase understanding of gut commensal physiology and colonization strategies.

Candida glabrata MSH2 deletion increases antifungal tolerance in vitro and during intra-abdominal candidiasis (IAC), it does not impact pathogenesis of peritonitis or intra-abdominal abscesses

Background: IAC is the second most common type of invasive Candidiasis, but its pathogenesis is poorly understood. We have shown that Candida albicans DNA damage response genes are strongly induced within intra-abdominal abscesses. Deletion of C. glabrata MSH2, A DNA mismatch repair (MMR) gene, results in a mutator phenotype that facilitates multidrug resistance in vitro and in mouse gastrointestinal tracts. Our goal was to determine if CGMSH2 Contributed to pathogenesis or resistance to the new antifungal rezafungin during IAC. Methods: We createdΔMSH2 in BG2 using SAT-Flipper, and tested virulence and rezafungin responses in a mouse model of IAC. Results: ΔMSH2 displayed no growth defects at 30°C in liquid (YPD, Ypglycerol) or solid media (YPD+0.02% MMS, 1MM H2O2, 1M NACL, 20 UG/ML CW, 250 UG/ML OR 0.02% SDS). ΔMSH2 longevity in YPD was comparable to BG2. Caspofungin-, Rezafungin- and Fluconazole-resistant mutants arose 24-, 16- and 3-fold more often, respectively, for ΔMSH2 than BG2 (108-106 CFU overnight in YPD, selected on 8XMIC-Containing plates). However, respective minimum inhibitory concentrations (MICS) were not different, nor were rezafungin time-kills.ΔMSH2 was comparable to BG2 in peritonitis and abscess burdens in mouse IAC.ΔMSH2 demonstrated significantly greater caspofungin- and fluconazole-tolerance than BG2 in abscesses. Rezafungin reduced peritonitis and abscess burdens ofΔMSH2,BG2 ANDFKS mutant strains to similar extents. Conclusions: CgMSH2 deletionincreased the frequency of spontaneously-arising echinocandin- and fluconazole-resistant colonies in vitro and tolerance in intra-abdominal abscesses, but it did not attenuate virulence or rezafungin responses during IAC.

LRRFIP2 modulates the response to hypoxia during embryonic cardiogenesis

Oxygen is crucial for appropriate embryonic and fetal development, including cardiogenesis. The heart is the first organ formed in the embryo and is required to provide oxygen and nutrients to all cells in the body. Embryonic cardiogenesis is a complex process finely regulated and prone to congenital malformations. It takes place in a hypoxic environment that activates the HIF-1a; signaling pathway which mediates cellular and systemic adaptations to low oxygen levels. Since inhibition or overactivation of the HIF-1a; signaling pathway in the myocardium lead to severe cardiac malformations and embryonic lethality, it is important that the cellular response to hypoxia be precisely regulated. While many gene regulatory networks involved in embryonic cardiogenesis have been characterized in detail, the modulation of the response of cardiomyocytes (CM) to hypoxia has remained less studied. We identified LRRFIP2 as a new negative cofactor of HIF-1a;. Indeed, we have shown that the absence of Lrrfip2 expression in a mouse KI model led to an enhance of many HIF-1a; target genes including Igfbp3, Bnip3 and Ndufa4l2 in embryonic CM during development. As results, the absence of Lrrfip2 led to the inhibition of the PI3K/Akt survival pathway, growth defects, mitochondrial dysfunction and to a precocious maturation of the embryonic CMs. Altogether, these defects led to the formation of a smaller heart unable to provide sufficient oxygen to the embryo and finally to a severe hypoxia and a precocious lethality.

Forced association of SARS-CoV-2 proteins with the yeast proteome perturb vesicle trafficking

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the highly infectious coronavirus disease COVID-19. Extensive research has been performed in recent months to better understand how SARS-CoV-2 infects and manipulates its host to identify potential drug targets and support patient recovery from COVID-19. However, the function of many SARS-CoV-2 proteins remains uncharacterised. Here we used the Synthetic Physical Interactions (SPI) method to recruit SARS-CoV-2 proteins to most of the budding yeast proteome to identify conserved pathways which are affected by SARS-CoV-2 proteins. The set of yeast proteins that result in growth defects when associated with the viral proteins have homologous functions that overlap those identified in studies performed in mammalian cells. Specifically, we were able to show that recruiting the SARS-CoV-2 NSP1 protein to HOPS, a vesicle-docking complex, is sufficient to perturb membrane trafficking in yeast consistent with the hijacking of the endoplasmic-reticulum–Golgi intermediate compartment trafficking pathway during viral infection of mammalian cells. These data demonstrate that the yeast SPI method is a rapid way to identify potential functions of ectopic viral proteins.

FtsZ-Ring Regulation and Cell Division Are Mediated by Essential EzrA and Accessory Proteins ZapA and ZapJ in Streptococcus pneumoniae

The bacterial FtsZ-ring initiates division by recruiting a large repertoire of proteins (the divisome; Z-ring) needed for septation and separation of cells. Although FtsZ is essential and its role as the main orchestrator of cell division is conserved in most eubacteria, the regulators of Z-ring presence and positioning are not universal. This study characterizes factors that regulate divisome presence and placement in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn), focusing on FtsZ, EzrA, SepF, ZapA, and ZapJ, which is reported here as a partner of ZapA. Epi-fluorescence microscopy (EFm) and high-resolution microscopy experiments showed that FtsZ and EzrA co-localize during the entire Spn cell cycle, whereas ZapA and ZapJ are late-arriving divisome proteins. Depletion and conditional mutants demonstrate that EzrA is essential in Spn and required for normal cell growth, size, shape homeostasis, and chromosome segregation. Moreover, EzrA(Spn) is required for midcell placement of FtsZ-rings and PG synthesis. Notably, overexpression of EzrA leads to the appearance of extra Z-rings in Spn. Together, these observations support a role for EzrA as a positive regulator of FtsZ-ring formation in Spn. Conversely, FtsZ is required for EzrA recruitment to equatorial rings and for the organization of PG synthesis. In contrast to EzrA depletion, which causes a bacteriostatic phenotype in Spn, depletion of FtsZ results in enlarged spherical cells that are subject to LytA-dependent autolysis. Co-immunoprecipitation and bacterial two-hybrid assays show that EzrA(Spn) is in complexes with FtsZ, Z-ring regulators (FtsA, SepF, ZapA, MapZ), division proteins (FtsK, StkP), and proteins that mediate peptidoglycan synthesis (GpsB, aPBP1a), consistent with a role for EzrA at the interface of cell division and PG synthesis. In contrast to the essentiality of FtsZ and EzrA, ZapA and SepF have accessory roles in regulating pneumococcal physiology. We further show that ZapA interacts with a non-ZapB homolog, named here as ZapJ, which is conserved in Streptococcus species. The absence of the accessory proteins, ZapA, ZapJ, and SepF, exacerbates growth defects when EzrA is depleted or MapZ is deleted. Taken together, these results provide new information about the spatially and temporally distinct proteins that regulate FtsZ-ring organization and cell division in Spn.

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1,000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX rescued this phenotype. These studies show that NAD+ regeneration, rather than oxidative phosphorylation, represents the primary role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

Spermidine Is Critical for Growth, Development, Environmental Adaptation, and Virulence in Fusarium graminearum

Putrescine, spermidine, and spermine are the most common natural polyamines. Polyamines are ubiquitous organic cations of low molecular weight and have been well characterized for the cell function and development processes of organisms. However, the physiological functions of polyamines remain largely obscure in plant pathogenic fungi. Fusarium graminearum causes Fusarium head blight (FHB) and leads to devastating yield losses and quality reduction by producing various kinds of mycotoxins. Herein, we genetically analyzed the gene function of the polyamine biosynthesis pathway and evaluated the role of the endogenous polyamines in the growth, development, and virulence of F. graminearum. Our results found that deletion of spermidine biosynthesis gene FgSPE3 caused serious growth defects, reduced asexual and sexual reproduction, and increased sensitivity to various stresses. More importantly, ΔFgspe3 exhibited significantly decreased mycotoxin deoxynivalenol (DON) production and weak virulence in host plants. Additionally, the growth and virulence defects of ΔFgspe3 could be rescued by exogenous application of 5 mM spermidine. Furthermore, RNA-seq displayed that FgSpe3 participated in many essential biological pathways including DNA, RNA, and ribosome synthetic process. To our knowledge, these results indicate that spermidine is essential for growth, development, DON production, and virulence in Fusarium species, which provides a potential target to control FHB.