Late Gene Research Articles

Fever-like temperature bursts promote competence development via an HtrA-dependent pathway in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is well known for its ability to develop competence for natural DNA transformation. Competence development is regulated by an autocatalytic loop driven by variations in the basal level of transcription of the comCDE and comAB operons. These genes are part of the early gene regulon that controls expression of the late competence genes known to encode the apparatus of transformation. Several stressful conditions are known to promote competence development, although the induction pathways are remain poorly understood. Here we demonstrate that transient temperature elevation induces an immediate increase in the basal expression level of the comCDE operon and early genes that, in turn, stimulates its full induction, including that of the late competence regulon. This thermal regulation depends on the HtrA chaperone/protease and its proteolytic activity. We find that other competence induction stimulus, like norfloxacin, is not conveyed by the HtrA-dependent pathway. This finding strongly suggests that competence can be induced by at least two independent pathways and thus reinforces the view that competence is a general stress response system in the pneumococcus.

IHerd: an integrative hierarchical graph representation learning framework to quantify network changes and prioritize risk genes in disease.

Different genes form complex networks within cells to carry out critical cellular functions, while network alterations in this process can potentially introduce downstream transcriptome perturbations and phenotypic variations. Therefore, developing efficient and interpretable methods to quantify network changes and pinpoint driver genes across conditions is crucial. We propose a hierarchical graph representation learning method, called iHerd. Given a set of networks, iHerd first hierarchically generates a series of coarsened sub-graphs in a data-driven manner, representing network modules at different resolutions (e.g., the level of signaling pathways). Then, it sequentially learns low-dimensional node representations at all hierarchical levels via efficient graph embedding. Lastly, iHerd projects separate gene embeddings onto the same latent space in its graph alignment module to calculate a rewiring index for driver gene prioritization. To demonstrate its effectiveness, we applied iHerd on a tumor-to-normal GRN rewiring analysis and cell-type-specific GCN analysis using single-cell multiome data of the brain. We showed that iHerd can effectively pinpoint novel and well-known risk genes in different diseases. Distinct from existing models, iHerd's graph coarsening for hierarchical learning allows us to successfully classify network driver genes into early and late divergent genes (EDGs and LDGs), emphasizing genes with extensive network changes across and within signaling pathway levels. This unique approach for driver gene classification can provide us with deeper molecular insights. The code is freely available at https://github.com/aicb-ZhangLabs/iHerd. All other relevant data are within the manuscript and supporting information files.

The Human Cytomegalovirus Latency-Associated Gene Product Latency Unique Natural Antigen Regulates Latent Gene Expression.

Human cytomegalovirus (HCMV) infection can lead to either lytic or latent infection, which is dependent on the regulation of the viral major immediate early promoter (MIEP). Suppression of the MIEP is a pre-requisite for latency and is driven by repressive epigenetic modifications at the MIEP during latent infection. However, other viral genes are expressed during latency and this is correlated with activatory epigenetic modifications at latent gene promoters. Yet the molecular basis of the differential regulation of latent and lytic gene expression by epigenetics is unclear. LUNA, a latent viral transcript, has been suggested to be important for HCMV latency and has also been shown to be important for efficient reactivation likely through its known deSUMOylase activity. Intriguingly, we and others have also observed that LUNA enhances latency-associated expression of the viral UL138 gene. Here, we show that in the absence of LUNA, the expression of multiple latency-associated transcripts is reduced during latent infection, which is correlated with a lack of activatory marks at their promoters. Interestingly, we also show that LUNA interacts with the hematopoietic transcription factor GATA-2, which has previously been shown to bind to a number of latency-associated gene promoters, and that this interaction is dependent on the deSUMOylase domain of LUNA. Finally, we show that the deSUMOylase activity of LUNA is required for the establishment and/or maintenance of an open chromatin configuration around latency-associated gene promoters. As such, LUNA plays a key role in efficient latency-associated viral gene expression and carriage of viral genome during latent carriage.

Inferring single-cell transcriptomic dynamics with structured latent gene expression dynamics

Gene expression dynamics provide directional information for trajectory inference from single-cell RNA sequencing data. Traditional approaches compute RNA velocity using strict modeling assumptions about transcription and splicing of RNA. This can fail in scenarios where multiple lineages have distinct gene dynamics or where rates of transcription and splicing are time dependent. We present "LatentVelo," an approach to compute a low-dimensional representation of gene dynamics with deep learning. LatentVelo embeds cells into a latent space with a variational autoencoder and models differentiation dynamics on this "dynamics-based" latent space with neural ordinary differential equations. LatentVelo infers a latent regulatory state that controls the dynamics of an individual cell to model multiple lineages. LatentVelo can predict latent trajectories, describing the inferred developmental path for individual cells rather than just local RNA velocity vectors. The dynamics-based embedding batch corrects cell states and velocities, outperforming comparable autoencoder batch correction methods that do not consider gene expression dynamics.

Establishment of the Effectiveness of Early Versus Late Stem Cell Gene Therapy in Mucopolysaccharidosis II for Treating Central Versus Peripheral Disease.

Mucopolysaccharidosis type II (MPSII) is a rare pediatric X-linked lysosomal storage disease, caused by heterogeneous mutations in the iduronate-2-sulfatase (IDS) gene, which result in accumulation of heparan sulfate (HS) and dermatan sulfate within cells. This leads to severe skeletal abnormalities, hepatosplenomegaly, and cognitive deterioration. The progressive nature of the disease is a huge obstacle to achieve full neurological correction. Although current therapies can only treat somatic symptoms, a lentivirus-based hematopoietic stem cell gene therapy (HSCGT) approach has recently achieved improved central nervous system (CNS) neuropathology in the MPSII mouse model following transplant at 2 months of age. In this study, we evaluate neuropathology progression in 2-, 4- and 9-month-old MPSII mice, and using the same HSCGT strategy, we investigated somatic and neurological disease attenuation following treatment at 4 months of age. Our results showed gradual accumulation of HS between 2 and 4 months of age, but full manifestation of microgliosis/astrogliosis as early as 2 months. Late HSCGT fully reversed the somatic symptoms, thus achieving the same degree of peripheral correction as early therapy. However, late treatment resulted in slightly decreased efficacy in the CNS, with poorer brain enzymatic activity, together with reduced normalization of HS oversulfation. Overall, our findings confirm significant lysosomal burden and neuropathology in 2-month-old MPSII mice. Peripheral disease is readily reversible by LV.IDS-HSCGT regardless of age of transplant, suggesting a viable treatment for somatic disease. However, in the brain, higher IDS enzyme levels are achievable with early HSCGT treatment, and later transplant seems to be less effective, supporting the view that the earlier patients are diagnosed and treated, the better the therapy outcome.

Hemin treatment drives viral reactivation and plasma cell differentiation of EBV latently infected B cells.

Epstein-Barr virus (EBV) and Plasmodium falciparum have a well described role in the development of endemic Burkitt lymphoma (BL), yet the mechanisms involved remain unknown. A major hallmark of malarial disease is hemolysis and bystander eryptosis of red blood cells, which causes release of free heme in large quantities into peripheral blood. We hypothesized that heme released during malaria infection drives differentiation of latently infected EBV-positive B cells, resulting in viral reactivation and release of infectious virus. To test this hypothesis, we used the EBV-positive Mutu I B-cell line and treated with hemin (the oxidized form of heme) and evaluated evidence of EBV reactivation. Hemin treatment resulted in the expression of EBV immediate early, early and late lytic gene transcripts. In addition, expression of CD138, a marker of plasma cells was co-expressed with the late lytic protein gp350 on hemin treated Mutu I cells. Finally, DNase-resistant EBV DNA indicative of virion production was detected in supernatant. To assess the transcriptional changes induced by hemin treatment, RNA sequencing was performed on mock- and hemin-treated Mutu I cells, and a shift from mature B cell transcripts to plasma cell transcripts was identified. To identify the mechanism of hemin-induced B cell differentiation, we measured levels of the plasma cell transcriptional repressor, BACH2, that contains specific heme binding sites. Hemin treatment caused significant degradation of BACH2 by 24 hours post-treatment in four BL cell lines (two EBV positive, two EBV negative). Knockdown of BACH2 in Mutu I cells using siRNAs significantly increased CD138+gp350+ cells to levels similar to treatment with hemin. This suggested that hemin induced BACH2 degradation was responsible for plasma cell differentiation and viral reactivation. Together, these data support a model where EBV reactivation can occur during malaria infection via heme modulation, providing a mechanistic link between malaria and EBV.

Kar4 is required for the normal pattern of meiotic gene expression.

Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also required for a second seemingly non-catalytic function during meiosis. Overexpression of the early meiotic transcription factor, IME1, can bypass the requirement for Kar4p in meiotic entry but the additional overexpression of the translational regulator, RIM4, is required to permit sporulation in kar4Δ/Δ. Using microarray analysis and RNA sequencing, we sought to determine the impact of removing Kar4p and consequently mRNA methylation on the early meiotic transcriptome in a strain background (S288c) that is sensitive to the loss of early meiotic regulators. We found that kar4Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1, and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is likely the result of the loss of mRNA methylation and is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. The requirement for RIM4 led us to predict that the non-catalytic function of Kar4p, like methyltransferase complex orthologs in other systems, may function at the level of translation. Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1, but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation.

Euo is a developmental regulator that represses late genes and activates midcycle genes in Chlamydia trachomatis.

In this study, we developed a correlative approach that combined DNA immunoprecipitation-seq and RNA-seq analyses to define the regulon of the Chlamydia trachomatis transcription factor Euo. We confirmed the proposed role of Euo as a transcriptional repressor of late chlamydial genes but also showed that Euo activates transcription of a subset of midcycle genes and autoregulates its own expression via negative feedback. This study validates and expands the role of Euo as an important developmental regulator in C. trachomatis. In addition, this genome-wide correlative approach can be applied to study transcription factors in other pathogenic bacteria.

Epigenetics of Epstein Barr virus — A review

Epstein Barr is the first-in-human oncogenic virus, closely related to numerous lymphoproliferative and malignant diseases, including HL, BL, NPC, and GC. EBV establishes life-long persistence infection portraying a biphasic viral life cycle: latent period and lytic replication. B-cells serve as critical regions for EBV latent genes, wherein viral gene expression is suppressed, promoting viral genome maintenance and immune recognition evasion. Upon its lytic reactivation, viral gene expression induces its replication, progeny production, and transmission. Dysregulations of epigenetic regulation in expressions of TSGs lead to carcinogenesis. Several studies reveal that EBV is associated with aberrant viral DNA and host genome methylation patterns, promoting immune monitoring, recognition evasiveness and host cell persistence. Among other epigenetic modifications, DNA methylation suppresses the majority of viral latent gene promoters, sparing a few, and acts as a prerequisite for activating EBV's lytic cycle, giving rise to viral progeny. It affects the host's epigenome via reprogramming cells to oncogenic, long-lasting phenotypes, as evident in several malignancies. At each phase of its life cycle, EBV exploits cellular mechanisms of epigenetic regulation, implying its unique host-pathogen relationship. This review summarized the DNA methylation's regulatory roles on several EBV-related promoter regions, along with the host genome in pathological conditions, highlights viral genes involved in a latent, lytic and latent-lytic phase of EBV infection. Moreover, it provides diagrammatic insights into methylation-based pathways in EBV.

Identification of two novel papillomaviruses in belugas.

Papillomaviruses (PVs) can cause hyperplasia in the skin and mucous membranes of humans, mammals, and non-mammalian animals, and are a significant risk factor for cervical and genital cancers. Using next-generation sequencing (NGS), we identified two novel strains of papillomavirus, PV-HMU-1 and PV-HMU-2, in swabs taken from belugas (Delphinapterus leucas) at Polar Ocean Parks in Qingdao and Dalian. We amplified the complete genomes of both strains and screened ten belugas and one false killer whale (Pseudorca crassidens) for the late gene (L1) to determine the infection rate. In Qingdao, 50% of the two sampled belugas were infected with PV-HMU-1, while the false killer whale was negative. In Dalian, 71% of the eight sampled belugas were infected with PV-HMU-2. In their L1 genes, PV-HMU-1 and PV-HMU-2 showed 64.99 and 68.12% amino acid identity, respectively, with other members of Papillomaviridae. Phylogenetic analysis of combinatorial amino acid sequences revealed that PV-HMU-1 and PV-HMU-2 clustered with other known dolphin PVs but formed distinct branches. PVs carried by belugas were proposed as novel species under Firstpapillomavirinae. The discovery of these two novel PVs enhances our understanding of the genetic diversity of papillomaviruses and their impact on the beluga population.

The pattern of stability of African swine fever virus in leeches

African swine fever virus (ASFV) can accumulate and survive in leeches for a long time. The reasons for the survival of ASFV in leeches are not entirely clear. Here, we elucidate the virus survival pathway in infected leeches. One of the questions reported previously is addressed in this article. How the virus concentration in the body of the leech is equal to or higher than in the water infected with ASFV? Examination of blood swallowed by leeches reveals that the blood cells retain their morphological characteristics for several weeks. It can explain the long-term persistence of the high levels of ASFV in the leeches that ingested ASFV-infected pig blood. qRT-PCR assay showed the transcription of ASFV genes in infected leeches. However, the infectious particles of the virus measured by HADU haven’t increased. Quantitative studies of the ASFV revealed a high content of both viral genes and infectious particles in the skin of leeches compared with other body parts. Electron microscopy analysis revealed the ability of the ASFV to effectively bind to the skin surface of the leeches, which explained the high concentrations of ASFV in the leeches’ skin. A significant difference in the transcriptional activity between early and late viral genes indicates that the virus entered the initial stage of replication, but for some reason failed to complete it, which is typical of abortive infections.

Proliferating cell nuclear antigen inhibitors block distinct stages of herpes simplex virus infection.

Proliferating cell nuclear antigen (PCNA) forms a homotrimer that encircles replicating DNA and is bound by DNA polymerases to add processivity to cellular DNA synthesis. In addition, PCNA acts as a scaffold to recruit DNA repair and chromatin remodeling proteins to replicating DNA via its interdomain connecting loop (IDCL). Despite encoding a DNA polymerase processivity factor UL42, it was previously found that PCNA associates with herpes simplex virus type 1 (HSV-1) replication forks and is necessary for productive HSV-1 infection. To define the role that PCNA plays during viral DNA replication or a replication-coupled process, we investigated the effects that two mechanistically distinct PCNA inhibitors, PCNA-I1 and T2AA, have on the HSV-1 infectious cycle. PCNA-I1 binds at the interface between PCNA monomers, stabilizes the homotrimer, and may interfere with protein-protein interactions. T2AA inhibits select protein-protein interactions within the PCNA IDCL. Here we demonstrate that PCNA-I1 treatment results in reduced HSV-1 DNA replication, late gene expression, and virus production, while T2AA treatment results in reduced late viral gene expression and infectious virus production. To pinpoint the mechanisms by which PCNA inhibitors affect viral processes and protein recruitment to replicated viral DNA, we performed accelerated native isolation of proteins on nascent DNA (aniPOND). Results indicate that T2AA inhibits recruitment of the viral uracil glycosylase UL2 and transcription regulatory factors to viral DNA, likely leading to a defect in viral base excision repair and the observed defect in late viral gene expression and infectious virus production. In addition, PCNA-I1 treatment results in decreased association of the viral DNA polymerase UL30 and known PCNA-interacting proteins with viral DNA, consistent with the observed block in viral DNA replication and subsequent processes. Together, we conclude that inhibitors of cellular PCNA block recruitment of key viral and cellular factors to viral DNA to inhibit viral DNA synthesis and coupled processes.

Expression of EBV-encoded genes in children with asymptomatic infection detected by sensitive methods

Epstein–Barr virus (EBV) is an usually harmless virus whose oncogenic properties in vitro are related to its ability to transform lymphoid cells, and, in consequence, it can be associated with lymphomas. Since a few studies detected EBV presence in supposedly EBV-negative lymphomas, our aim was to evaluate EBV presence by sensitive gene expression assays in the tonsils from healthy pediatric donors from a region with high incidence of EBV-associated lymphomas. EBERs transcripts were detected by View RNA ISH in all cases, even in cases assessed negative by widely used in situ hybridization. The presence of LMP1 transcripts was proved in 93% of cases, co-expressed with EBNA2 in 30%. In this study, evidence for the expression of different latent and lytic viral genes in a population of young age of primary infection, detected with more sensitive methods, in particular at the germinal center, where most EBV-associated lymphomas originate, was provided.

Interactive effects of light quality and nitrate supply on growth and metabolic processes in two lettuce cultivars (Lactuca sativa L.)

Light quality and nutrient parameters alter plant growth and metabolic pathways in leafy greens, therefore being primarily optimized in indoor precision agriculture. While their individual effects were studied extensively with large-scale phenomics and transcriptomics, the combinatorial effects remain poorly understood. Here, we explored the hidden interactions among light- and nitrate-responsive transcriptomes and their associations with six growth and biochemical traits, using the full factorial experimental design with three lights (red (R), blue (B) and a 1:1 mixture of red and blue (RB)), two nitrate concentrations and two cultivars (Green oak-leaf and Red oak-leaf). Multifactor analysis of 36 RNA-sequencing data revealed their largely independent but additive effects that are associated with MYB, bHLH and GATA family transcription factors. Red light and sufficient nitrate supply additively increased the primary metabolite pathway and bHLH and GATA genes, leading to a high growth rate. Blue and RB lights, together with nitrate deficiency, induced flavonoid pathway and MYB genes. Variety-specific deviations were detected in late anthocyanin biosynthetic genes and specific MYB transcription factors that would explain anthocyanin accumulations in the Red oak-leaf cultivar. Taken together, our large-scale transcriptomic and physiologic results suggest that the two environmental parameters (i.e., light quality and nitrate level) (1) utilize independent transcriptional regulatory mechanisms to achieve similar phenotypic and metabolic changes, and (2) induce the variety-specific anthocyanin accumulations via a small number of genes that show different gene expression profiles between the two cultivars.

Cytomegalovirus infection induces Alzheimer's disease-associated alterations in tau.

Alzheimer's disease (AD) manifests with loss of neurons correlated with intercellular deposition of amyloid (amyloid plaques) and intracellular neurofibrillary tangles of hyperphosphorylated tau. However, targeting AD hallmarks has not as yet led to development of an effective treatment despite numerous clinical trials. A better understanding of the early stages of neurodegeneration may lead to development of more effective treatments. One underexplored area is the clinical correlation between infection with herpesviruses and increased risk of AD. We hypothesized that similar to work performed with herpes simplex virus 1 (HSV1), infection with the cytomegalovirus (CMV) herpesvirus increases levels and phosphorylation of tau, similar to AD tauopathy. We used murine CMV (MCMV) to infect mouse fibroblasts and rat neuronal cells to test our hypothesis. MCMV infection increased steady-state levels of primarily high molecular weight forms of tau and altered the patterns of tau phosphorylation. Both changes required viral late gene products. Glycogen synthase kinase 3 beta (GSK3β) was upregulated in the HSVI model, but inhibition with lithium chloride suggested that this enzyme is unlikely to be involved in MCMV infection mediated tau phosphorylation. Thus, we confirm that MCMV, a beta herpes virus, like alpha herpes viruses (e.g., HSV1), can promote tau pathology. This suggests that CMV infection can be useful as another model system to study mechanisms leading to neurodegeneration. Since MCMV infects both mice and rats as permissive hosts, our findings from tissue culture can likely be applied to a variety of AD models to study development of abnormal tau pathology.

Variation in the Calyx Color in Two Styrax japonicus Varieties Is Attributed to Varied Anthocyanin Levels as Revealed by Integrated Metabolomic and Transcriptomic Analyses

Styrax japonicus is a small ornamental tree with medicinal value. An S. japonicus variety with purplish red calyxes and white petals has higher ornamental value. The mechanism underlying calyx pigmentation in S. japonicus is still unclear. In this study, metabolome data combined with transcriptome profiling were used to explore the molecular mechanisms underlying the difference in the color of calyx in two varieties of S. japonicus, namely, Red Linglong (RA; purplish red calyx) and Green Linglong (GA; green calyx). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the levels of delphinidin 3,5-diglucoside, delphinidin 3-O-glucoside, and malvidin 3-O-glucoside when enriched in delphinidin biosynthesis were significantly higher in the RA calyx than in the GA calyx. These key differentially accumulated metabolites were highly correlated with five late biosynthetic genes that were enriched in the anthocyanin biosynthesis pathway. MYB1, MYB82, and MYB113 were the three probable transcription factors responsible for anthocyanin accumulation. This study provides novel insights into secondary metabolism pathways, their regulators, and the changes in the transcription and metabolite levels in the calyx of S. japonicus regulating sepal color. The results provide a theoretical basis for exploring the mechanism of calyx color formation in S. japonicus and provide genetic material and a reference for molecular breeding to obtain desired flower colors in the future.

Identification of Late Flavonoid Biosynthesis Genes of Moso Bamboo Reveals the Potential Function of PeANR4 Involved in Osmotic and Salt Stress

Flavonoids are important secondary metabolites in plants, and their biosynthesis includes various enzymes. Although bamboo is a potential resource with abundant flavonoids, its flavonoids biosynthesis is still unclear. Based on the genome and transcriptome data of moso bamboo (Phyllostachys edulis), 24 late flavonoid biosynthesis genes (LFBGs) were identified. Further molecular characteristics analyses suggested they may have different biological functions in flavonoids biosynthesis. Sixteen differentially expressed genes were identified according to transcriptome data from different-height shoots, including five PeANSs, four PeANRs, three PeLARs, and PeDFR1. PeANR4 expressed continuously under drought stress was selected for further analysis. A co-expression network of PeANR4 and 27 differentially expressed transcription factors (DETFs) was constructed, and the regulatory relationship of four DETFs and PeANR4 was validated by Y1H assays. Furthermore, PeANR4 was ectopically expressed in Arabidopsis, and the transgenic lines had darker seed coat color and higher fresh, dry weight and proanthocyanidin (PA) content than the wild type and mutant. Moreover, the transgenic lines had higher germination rate and longer primary root than the wild type and mutant under osmotic and salt stress. These results provide a full understanding and lay a foundation for further functional studies on the LFBGs of bamboo.

An Endogenous Staphylococcus aureus CRISPR-Cas System Limits Phage Proliferation and Is Efficiently Excised from the Genome as Part of the SCCmec Cassette.

CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-Cas; however, in the human pathogen Staphylococcus aureus, CRISPR-Cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-Cas in genomes of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Denmark. Only 2.9% of the strains carried CRISPR-Cas systems, but for strains of sequence type ST630, over half were positive. All CRISPR-Cas loci were type III-A and located within the staphylococcal cassette chromosome mec (SCCmec) type V(5C2&5), conferring β-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-Cas positive strains, and almost identical SCCmec cassettes, CRISPR arrays, and cas genes are present in staphylococcal species other than S. aureus, suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCCmec cassette containing CRISPR-Cas is excised from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage phiIPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or circumvented by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in S. aureus is active against targeted phages, albeit with low efficacy. This suggests that native S. aureus CRISPR-Cas offers only partial immunity and in nature may work in tandem with other defense systems. IMPORTANCE CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of Staphylococcus aureus, CRISPR-Cas is rare, but when present, it is located within the SCCmec element, which encodes resistance to methicillin and other β-lactam antibiotics. We show that the element is excisable, suggesting that the CRISPR-Cas locus is transferable. In support of this, we found almost identical CRISPR-Cas-carrying SCCmec elements in different species of non-S. aureus staphylococci, indicating that the system is mobile but only rarely acquires new spacers in S. aureus. Additionally, we show that in its endogenous form, the S. aureus CRISPR-Cas is active but inefficient against lytic phages that can overload the system or form escape mutants. Thus, we propose that CRISPR-Cas in S. aureus offers only partial immunity in native systems and so may work with other defense systems to prevent phage-mediated killing.

#6456 DSA-NEGATIVE MICROVASCULAR INFLAMMATION IN KIDNEY TRANSPLANT BIOPSIES: GENE EXPRESSION COMPARISON WITH NATIVE AND TRANSPLANT KIDNEY CONTROLS

Abstract Background and Aims Microvascular inflammation (MVI) in kidney transplant (KT) biopsies from patients without detectable anti-HLA donor-specific antibodies (DSA) presents a diagnostic and therapeutic dilemma. This study aimed to further understand the significance of these changes by characterizing their molecular phenotype compared to other native and transplant kidney biopsies. Method The NanoString B-HOT panel (770 genes) was used to measure the expression of six literature-derived gene sets in 195 archival FFPE kidney biopsies from four centers, including transplant biopsies with MVI (g+ptc>1) but no detectable DSA (MVI, n = 47), antibody-mediated rejection with DSA (ABMR, n = 42), pure T-cell mediated rejection without DSA (TCMR, n = 25), mixed MVI and TCMR without DSA (MVI+TCMR, n = 47), normal implant biopsies (Normal, n = 11), and native kidney biopsies with either endocapillary proliferative glomerulonephritis (GN, n = 12) or minimal change disease (MCD, n = 11). The evaluated gene sets included transcripts previously associated with ABMR, DSA (DSAST), endothelial injury (ENDAT), TCMR, early injury, and late injury. Gene expression was compared between groups using principal component and class comparison analyses. Results Principal component analysis demonstrated significant molecular overlap between sample groups (Fig. 1A). However, gene set analysis showed lower expression of ABMR-related, DSAST (Fig. 1B), and ENDAT gene sets in DSA-negative MVI compared to ABMR. DSAST and ENDAT gene set expression was similar between MVI, MVI+TCMR, and TCMR groups; but higher than native biopsies (p ≤ 0.002). TCMR and early injury gene set expression was higher in TCMR than all other groups (p ≤ 0.002), except MVI+TCMR. Late injury gene set expression was lower in MVI compared to ABMR, MVI+TCMR, and TCMR groups (p ≤ 0.031); but higher than the Normal and MCD groups (p ≥ 0.016). Conclusion These results suggest that DSA-negative MVI displays a lower expression of ABMR-related genes than ABMR, but similar to MVI+TCMR and higher than native kidney biopsies with or without glomerulonephritis. Further work is underway to evaluate the potential role of non-HLA DSA and recognition of missing self in these cases.

#6352 CLINICAL AND MOLECULAR SPECTRUM OF V-LESION

Abstract Background and Aims Isolated v-lesion is an increasingly recognized but clinically challenging entity. Some studies suggest that isolated v+ early post-transplant may be caused by non-alloimmune etiologies such as ischemic injury. We aimed to characterize the allograft outcomes of isolated v-lesions according to post-transplant time (early: ≤1 month vs. late: >1 month) and further understand the significance of this lesion by characterizing its molecular phenotype in comparison with other forms of rejection v+. Method The NanoString B-HOT panel (770 genes) was used to measure the expression of six literature-derived gene sets in 92 archival FFPE kidney biopsies from two centers, including transplant biopsies with isolated v-lesion (n = 23), antibody-mediated rejection (ABMR) with v+ (n = 26), pure T-cell mediated rejection (TCMR) with v+ (n = 10), mixed rejection v+ (n = 23), and normal implant biopsies (Normal, n = 10). The evaluated gene sets included transcripts previously associated with ABMR, DSA (DSAST), endothelial injury (ENDAT), TCMR, early injury, and late injury. Gene expression was compared between groups using principal component and class comparison analyses. Death-censored graft survival according to diagnosis and time after KT was assessed. Results Isolated v+ early conferred the worst death-censored graft survival one year after the biopsy (40%) when compared to isolated v+ late (100%) or other forms of rejection (≥82%, p = 0.034). The principal component analysis demonstrated significant molecular overlap between sample groups (PC1: 29.4%, PC2: 8.1%). However, gene set analysis showed lower expression of TCMR-related genes in isolated v+ groups compared to TCMR and mixed rejection (p<0.001). Both Isolated v+ early and late had lower ABMR-related genes than ABMR, mixed rejection, and TCMR groups (p ≤ 0.022). Moreover, isolated v+ late showed lower DSAST and ENDAT gene set expression than ABMR (p ≤ 0.046); and lower early injury gene set expression than isolated v+ early, ABMR, TCMR, and mixed rejection (p ≤ 0.026). Late injury gene set expression was highest in TCMR and mixed rejection compared to the other groups (p ≥ 0.034). Conclusion These results suggest that early and late isolated v+ lesions display lower expression of TCMR-related genes than TCMR and mixed rejection and lower expression of ABMR-related genes than ABMR. Isolated v+ early confers a bad prognosis and is associated with higher expression of early injury genes compared to isolated v+ late, suggesting a different ethology.