Diabetes is associated with impaired immune function and increased susceptibility to severe bacterial infections, yet the pathogen-encoded mechanisms that exacerbate disease in this context remain poorly defined. Streptococcus pyogenes (group A Streptococcus [GAS]) causes invasive skin and soft tissue infections that are disproportionately severe in individuals with diabetes, often accompanied by delayed healing, excessive inflammation, and polymicrobial overgrowth. Here, we investigated how the GAS ClpX-dependent regulatory pathway (CDRP), a global virulence regulator, interacts with diabetic immune dysfunction to shape infection outcomes. Using two murine models of type I diabetes, we show that diabetic mice develop more severe and persistent GAS skin infections characterized by increased bacterial burden, exaggerated inflammatory responses, impaired neutrophil recruitment, excessive neutrophil extracellular trap (NET) accumulation, and frequent polymicrobial infections. Deletion of clpX significantly attenuated GAS virulence in both diabetic and non-diabetic hosts; however, the pathogenic consequences of CDRP were markedly amplified in the diabetic environment. In diabetic mice, CDRP promoted sustained inflammation, altered neutrophil behavior, impaired NET clearance, and enhanced tissue damage, leading to delayed resolution of infection. Mechanistically, ClpX-dependent virulence was associated with dysregulated protease-nuclease activity, excessive NET persistence, and defective neutrophil chemotaxis within diabetic lesions. Together, these findings indicate that while ClpX is a core regulator of GAS virulence, its downstream effects are disproportionately deleterious when host immune clearance is compromised. IMPORTANCE:Diabetic patients experience disproportionately severe bacterial infections, yet the microbial mechanisms that exacerbate disease in this immunocompromised context remain incompletely understood. This study demonstrates that the Streptococcus pyogenes ClpX-dependent regulatory pathway, a central regulator of virulence, amplifies tissue damage and inflammatory dysfunction during diabetic skin infection. ClpX-dependent regulation exacerbates disease by intensifying neutrophil dysregulation, excessive NET accumulation, and impaired resolution of infection in an already compromised host environment. These findings underscore the importance of host-pathogen interactions in shaping infection severity and suggest that targeting pathogen regulatory pathways may be particularly effective in settings of immune dysfunction such as diabetes.

Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding-responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.

Introduction The widespread use of antibiotics, in both healthcare and agriculture, has led to the emergence of antibioticresistant bacteria, decreasing our ability to effectively treat common infections. With predictions of antibiotic resistance reaching a tipping point, it is imperative that we develop novel, antibiotic-sparing medicines to avoid a future of increasing mortality due to currently treatable common infections. In the United States, 15% of antibiotics are prescribed for the treatment of urinary tract infections (UTIs) (1) affecting millions of women annually. For those suffering acute UTI, 25% experience recurrent UTIs (rUTIs) (1), involving several infections per year, that require multiple antibiotic courses. Recent history of a UTI is a known risk factor for rUTI (2), yet the mechanisms leading to recurrence are mostly unknown. In addition, antibiotic resistance is making UTIs harder to treat and often necessitates using broad-spectrum antibiotics. Ironically, antibiotic use is also a significant risk factor for a UTI (3), possibly due to associated deleterious effects on the gut microbiota, among which most uropathogens reside. That approximately 50% of rUTIs are caused by the same strain that caused the initial infection (4) argues for a host-associated reservoir that is not adequately cleared by current treatments. Thus, there is an urgent need to better understand uropathogen dynamics within host-associated reservoirs to develop treatment options that limit morbidity and [...] Viewpoint

Recurrent urinary tract infections (rUTIs) are a major health burden worldwide, with history of infection being a significant risk factor. While the gut is a known reservoir for uropathogenic bacteria, the role of the microbiota in rUTI remains unclear. We conducted a year-long study of women with (n = 15) and without (n = 16) history of rUTI, from whom we collected urine, blood and monthly faecal samples for metagenomic and transcriptomic interrogation. During the study 24 UTIs were reported, with additional samples collected during and after infection. The gut microbiome of individuals with a history of rUTI was significantly depleted in microbial richness and butyrate-producing bacteria compared with controls, reminiscent of other inflammatory conditions. However, Escherichia coli gut and bladder populations were comparable between cohorts in both relative abundance and phylogroup. Transcriptional analysis of peripheral blood mononuclear cells revealed expression profiles indicative of differential systemic immunity between cohorts. Altogether, these results suggest that rUTI susceptibility is in part mediated through the gut–bladder axis, comprising gut dysbiosis and differential immune response to bacterial bladder colonization, manifesting in symptoms. Multi-omics analyses of faecal, urine and blood samples from women with and without recurrent urinary tract infections reveal that gut dysbiosis and differential immune responses may play a role in risk of infection via the gut–bladder axis.

Exosomes are an important mechanism of cell-cell interaction in the cardiovascular system, both in maintaining homeostasis and in stress response. Interindividual differences that alter content in exosomes may play a role in cardiovascular disease pathology. To study the effect of interindividual cardiomyocyte (CM) variation, we characterized exosomal content in phenotypically diverse human induced pluripotent stem cell-derived CMs (hiPSC-CMs). Cell lines were generated from six participants in the HyperGEN cohort: three with left ventricular hypertrophy (LVH) and three with normal left ventricular mass (LVM). Sequence analysis of the intracellular and exosomal RNA populations showed distinct expression pattern differences between hiPSC-CM lines derived from individuals with LVH and those with normal LVM. Functional analysis of hiPSC-endothelial cells (hiPSC-ECs) treated with exosomes from both hiPSC-CM groups showed significant variation in response, including differences in tube formation, migration, and proliferation. Overall, treatment of hiPSC-ECs with exosomes resulted in significant expression changes associated with angiogenesis and endothelial cell vasculogenesis. However, the hiPSC-ECs treated with exosomes from the LVH-affected donors exhibited significantly increased proliferation but decreased tube formation and migration, suggesting angiogenic dysregulation.NEW & NOTEWORTHY The intracellular RNA and the miRNA content in exosomes are significantly different in hiPSC-CMs derived from LVH-affected individuals compared with those from unaffected individuals. Treatment of endothelial cells with these exosomes functionally affects cellular phenotypes in a donor-specific manner. These findings provide novel insight into underlying mechanisms of hypertrophic cell signaling between different cell types. With a growing interest in stem cells and exosomes for cardiovascular therapeutic use, this also provides information important for regenerative medicine.

Background: Indices of left ventricular (LV) structure and geometry represent useful intermediate phenotypes related to LV hypertrophy (LVH), a predictor of cardiovascular (CV) disease (CVD) outcomes. Methods and Results: We conducted an exome-wide association study of LV mass (LVM) adjusted to height2.7, LV internal diastolic dimension (LVIDD), and relative wall thickness (RWT) among 1,364 participants of African ancestry (AAs) in the Hypertension Genetic Epidemiology Network (HyperGEN). Both single-variant and gene-based sequence kernel association tests were performed to examine whether common and rare coding variants contribute to variation in echocardiographic traits in AAs. We then used a data-driven procedure to prioritize and select genes for functional validation using a human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) model. Three genes [myosin VIIA and Rab interacting protein (MYRIP), trafficking protein particle complex 11 (TRAPPC11), and solute carrier family 27 member 6 (SLC27A6)] were prioritized based on statistical significance, variant functional annotations, gene expression in the hiPSC-CM model, and prior biological evidence and were subsequently knocked down in the hiPSC-CM model. Expression profiling of hypertrophic gene markers in the knockdowns suggested a decrease in hypertrophic expression profiles. MYRIP knockdowns showed a significant decrease in atrial natriuretic factor (NPPA) and brain natriuretic peptide (NPPB) expression. Knockdowns of the heart long chain fatty acid (FA) transporter SLC27A6 resulted in downregulated caveolin 3 (CAV3) expression, which has been linked to hypertrophic phenotypes in animal models. Finally, TRAPPC11 knockdown was linked to deficient calcium handling. Conclusions: The three genes are biologically plausible candidates that provide new insight to hypertrophic pathways.

Recurrent bacterial infections are a major health burden worldwide, yet the mechanisms dictating host susceptibility to recurrence are poorly understood. Here we demonstrate that an initial bacterial infection of the urinary bladder with uropathogenic E. coli (UPEC) can induce sustained epigenetic changes in the bladder epithelial (urothelial) stem cells that reprogram the differentiated urothelium. We established urothelial stem cell (USC) lines from isogenic mice with different urinary tract infection histories (naïve, chronic or self-resolving). Differentiation of the USC lines in Transwell culture resulted in polarized urothelial cultures that recapitulated distinct remodeling morphologies seen in vivo. In addition, we discovered differences in chromatin accessibility that segregated by disease history, resulting in differences in gene expression upon differentiation of the USC lines in vitro, based on ATAC-seq analysis of the USC lines. Differential basal expression of Caspase-1 led to divergent susceptibilities to inflammatory cell death upon UPEC infection. In mice with a history of chronic infection, enhanced caspase 1-mediated inflammatory cell death was found to be a protective response that enhanced bacterial clearance upon challenge infection. Thus, UPEC infection reshapes the epigenome leading to epithelial-intrinsic remodeling that trains the mucosal immune response to subsequent infection. These findings may have broad implications for the prevention of chronic/recurrent bacterial infections.

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

Recurrent urinary tract infections (rUTIs) are a major health burden worldwide, with history of infection being a significant risk factor. While the gut is a known reservoir for uropathogenic bacteria, the role of the microbiota in rUTI remains unclear. We conducted a year-long study of women with and without history of rUTIs, from whom we collected urine, blood and monthly fecal samples for multi-omic interrogation. The rUTI gut microbiome was significantly depleted in microbial richness and butyrate-producing bacteria compared to controls, reminiscent of other inflammatory conditions, though Escherichia coli gut and bladder dynamics were comparable between cohorts. Blood samples revealed signals of differential systemic immunity, leading us to hypothesize that rUTI susceptibility is in part mediated through a syndrome involving the gut-bladder axis, comprising gut dysbiosis and differential immune response to bacterial bladder colonization, manifesting in symptoms. This work highlights the potential for microbiome therapeutics to prevent and treat rUTIs.

Posttranscriptional regulation of gene expression, typically effected by RNA-binding proteins, microRNAs (miRNAs), and translation initiation factors, is essential for normal germ cell function. Numerous miRNAs have been detected in the germline; however, the functions of specific miRNAs remain largely unknown. Functions of miRNAs have been difficult to determine as miRNAs often modestly repress target mRNAs and are suggested to sculpt or fine tune gene expression to allow for the robust expression of cell fates. In Caenorhabditis elegans hermaphrodites, cell fate decisions are made for germline sex determination during larval development when sperm are generated in a short window before the switch to oocyte production. Here, analysis of newly generated mir-44 family mutants has identified a family of miRNAs that modulate the germline sex determination pathway in C. elegans. Mutants with the loss of mir-44 and mir-45 produce fewer sperm, showing both a delay in the specification and formation of sperm as well as an early termination of sperm specification accompanied by a premature switch to oocyte production. mir-44 and mir-45 are necessary for the normal period of fog-1 expression in larval development. Through genetic analysis, we find that mir-44 and mir-45 may act upstream of fbf-1 and fem-3 to promote sperm specification. Our research indicates that the mir-44 family promotes sperm cell fate specification during larval development and identifies an additional posttranscriptional regulator of the germline sex determination pathway.

As whole exome sequencing (WES) studies identify a large number of potential disease relevant genes and variants, a major challenge will be the subsequent functional analysis and interpretation. We performed WES in 1,382 African American subjects from NHLBI’s HyperGEN cohort to identify novel genes for Left ventricular (LV) hypertrophy. Using criteria that combine significant prior linkage/association signals, expression QTL information, database annotation, and literature search, we prioritized the candidate genes to further scrutinize those with higher functional potentials. As a result, we selected four genes ( FATP6, PDE12, HAX1, TRAPPC11 ) that had not been directly linked to LVH for experimental functional follow up using human iPSC-derived cardiomyocytes (hiPSC-CM). Each gene was knocked-down (KD) using siRNAs in triplicate followed by mRNA sequencing. Differential expression and pathway analyses were performed to evaluate influences of the candidate genes. The KDs of FATP6 and PDE12 , whose variants show significant association with increased LV internal diastolic-dimension, showed regulation changes in cardiac hypertrophy and β-adrenergic signaling pathways (p-value <0.05). In FATP6 KDs we observed 2,696 genes to have significant differential expression (adj. p-value ≤0.05). Furthermore, FATP6 KDs also lead to the upregulation of cholesterol biosynthesis pathways (p-value 1.97E-4) that are important for caveolae formation and signal transduction. Additionally, FATP6 KDs had a moderate increase in FATP1 expression (p-value 0.0024) that has been reported to cause lipotoxicity and increase LV internal diastolic diameter in mice. In PDE12 KDs we observed significant differential expression of 3,276 genes (adj. p-value ≤0.05) and saw significant decreases in Ca2+ channel expression (adj. p-value ≤0.05) that likely lead to decreases in Ca2+ handling. In summary, our study demonstrates the hiPSC-CM based knockdown model as an effective approach to functionally test and characterize candidate genes identified through WES. Further development of the approach using hiPSC derived cells will offer a scalable platform for high-throughput, multi-omics analyses using genome-matched, disease-relevant tissue types.

Small molecule tyrosine kinase inhibitors (TKIs) are a valuable class of therapeutics with widespread clinical utility against multiple cancers. However, there is strong evidence that TKIs are associated with cardiotoxicity and adverse cardiovascular events. Our understanding of the underlying mechanisms related to TKI induced cardiotoxicity is limited. Human iPSC derived cardiomyocytes (hiPSC-CMs) provide a flexible platform and unique model to study the underlying molecular mechanisms associated with TKI associated cardiotoxicity. In this study we describe the gene expression profile between hiPSC-CM cell lines which exhibit susceptibility vs. resistance. RNA-seq analysis was performed in hiPSC-CM cell lines from six participants in the NHLBI HyperGEN study (A to F). Experiments were performed in triplicate using sunitinib (SUN), vandetanib (VAN), gefitinib (GEF) and nilotinib (NIL). We analyzed beat rate, cell index and ATP viability as physiological measurements of CM toxicity and defined a 20% change from the normalized control as TKI susceptibility. Differential gene expression analysis was performed using DESeq2. We observed significant physiological differences between the different hiPSC-CMs after TKI treatment (beat rate, cell index and ATP viability). The most variable cell index and beat rate response was observed for NIL. Based on cell index, lines B, D, E were resistant while A, C, F were significantly more susceptible to NIL. Principal component analysis showed that the variance in gene expression was the highest after NIL treatment when compared to controls (16% for NIL; 11% for VAN; 6% for SUN and 5% for GEF). A total of 567 genes exhibited significant differential expression changes (adj. p-value ≤ 0.1) after NIL treatment in susceptible versus resistant lines. Pathway analysis showed significant enrichment for cardiotoxicity including pathways implicated in cardiac infarction, fibrosis, hypertrophy, and congestive cardiac failure. Taken together, our results identify unique gene expression changes associated with TKI cardiotoxicity. Furthermore, the variability in TKI susceptibility between different hiPSC-CM lines highlights the need to comprehensively assess cardiotoxicity in a diverse set of lines on a physiological and molecular level.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in animal development and physiology, though functions for most miRNAs remain unknown. Worms with reduced miRNA biogenesis due to loss of Drosha or Pasha/DGCR8 activity are sterile and fail to ovulate, indicating that miRNAs are required for the process of oocyte maturation and ovulation. Starting with this penetrant sterile phenotype and using new strains created to perform tissue specific RNAi, we characterized the roles of the C. elegans Pasha, pash-1, and two miRNA-specific Argonautes, alg-1 and alg-2, in somatic gonad cells and in germ cells in the regulation of ovulation. Conditional loss of pash-1 activity resulted in a reduced rate of ovulation and in basal and ovulatory sheath contractions. Similarly, knockdown of miRNA-specific Argonautes in the cells of the somatic gonad by tissue-specific RNAi results in a reduction of the ovulation rate and in basal and ovulatory sheath contractions. Reduced miRNA pathway gene activity resulted in a range of defects, including oocytes that were pinched upon entry of the oocyte into the distal end of the spermatheca in about 42% of the ovulation events observed following alg-1 RNAi. This phenotype was not observed on worms exposed to control RNAi. In contrast, knockdown of alg-1 and alg-2 in germ cells results in few defects in oocyte maturation and ovulation. These data identify specific steps in the process of ovulation that require miRNA pathway gene activity in the somatic gonad cells.