Wild-type Research Articles

Exploration of the Interactions Between Xanthomonas citri subsp. citri-Agrobacterium-Citrus to Improve Agrobacterium-Mediated Transient Expression in Plants.

Agrobacterium-mediated transient expression (AMTE) is an important tool in plant genetics study and biotechnology. AMTE remains problematic in citrus and many plant species. Previous study has shown that pretreatment of citrus leaves with Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, significantly improves the AMTE efficacy. Here we have shown that Xcc promotes AMTE mainly through triggering cell division and upregulating plant cell wall degrading enzymes. We demonstrate that Xcc improves AMTE via PthA, a transcription activator-like effector (TALE) known to trigger cell division in citrus, and mutation of pthA4 abolished the promoting effect of Xcc. Mutation of the effector (PthA4) binding element (EBE) in the promoter region and coding region of CsLOB1, which is known to be directly activated by PthA4, significantly reduced Xcc promotion of AMTE. Mutation of PthA4 significantly reduced expression of cell division related genes (CDKA, CDKB1-2 and CDKB2-2) compared to wild type Xcc and the complemented strain. Cell division inhibitor mimosine but not colchicine also significantly decreased Xcc promoting of AMTE. In addition, PthA4 is known to upregulate plant growth hormones auxin (IAA), gibberellin and cytokinin, as well as cell wall degrading enzymes (e.g., cellulase). Exogenous application of IAA, cytokinin, and cellulase but not gibberellin significantly improved AMTE in leaves of sweet orange, pummelo, Meiwa kumquat, lucky bamboo and rose mallow. Our study provides a mechanistic understanding regarding how Xcc promotes AMTE and develops practical measures to improve AMTE via pretreatment with plant hormones (i.e., auxin and cytokinin) and cellulase.

A modulatory role of CG methylation on gene expression in soybean implicates its potential utility in breeding.

Cytosine methylation (mCG) is an important heritable epigenetic modification, yet its functions remain to be fully defined in important crops. This study investigates mCG in soybean following the loss-of-function mutation of two GmMET1 genes. We generated knockout mutants of GmMET1s by CRISPR-Cas9 and conducted comprehensive methylome and transcriptome analyses. Our findings unravel the functional redundancy of the two GmMET1s, with GmMET1b being more critically involved in maintaining mCG levels, and complete knockout of both copies is lethal. We establish that genome-wide mCG levels scale with aggregated expression of GmMET1s. We identify a set of mCG-regulated genes whose expression levels were quantitatively modulated by upstream, body, or downstream mCG. Moreover, we find genes that were negatively regulated by upstream or body mCG are enriched in specific biological processes such as that of jasmonic acid metabolism. Notably, >80% of the differentially methylated genes (DMGs) in the mutants also exist as DMGs in natural soybean populations. Phenotypically, mutants that are heterozygous for GmMET1a and homozygous for GmMET1b knockouts (GmMET1a+/-GmMET1b-/-) exhibited early flowering, which was inherited by their selfed progeny (GmMET1a+/+GmMET1b-/-) with otherwise normal growth and development. Moreover, mutation of either GmMET1s, with slight reduction of mCG levels and similar phenotypes compared to the wild type under normal conditions, showed enhanced tolerance to cold and drought stresses. Together, our results underscore highly orchestrated regulatory effects of mCG on gene expression in soybean, which dictates growth, development and stress responses, implicating its utility in the improvement of soybean for better adaptability and higher yield.

Photosynthesis in Synechocystis sp. PCC 6803 is not optimally regulated under very high CO2

One strategy for CO2 mitigation is using photosynthetic microorganisms to sequester CO2 under high concentrations, such as in flue gases. While elevated CO2 levels generally promote growth, excessively high levels inhibit growth through uncertain mechanisms. This study investigated the physiology of the cyanobacterium Synechocystis sp. PCC 6803 under very high CO2 concentrations and yet stable pH around 7.5. The growth rate of the wild type (WT) at 200 µmol photons m−2 s−1 and a gas phase containing 30% CO2 was 2.7-fold lower compared to 4% CO2. Using a CRISPR interference mutant library, we identified genes that, when repressed, either enhanced or impaired growth under 30% or 4% CO2. Repression of genes involved in light harvesting (cpc and apc), photochemical electron transfer (cytM, psbJ, and petE), and several genes with little or unknown functions promoted growth under 30% CO2, while repression of key regulators of photosynthesis (pmgA) and CO2 capture and fixation (ccmR, cp12, and yfr1) increased growth inhibition under 30% CO2. Experiments confirmed that WT cells were more susceptible to light inhibition under 30% than under 4% CO2 and that a light-harvesting-impaired ΔcpcG mutant showed improved growth under 30% CO2 compared to the WT. These findings suggest that enhanced fitness under very high CO2 involves modifications in light harvesting, electron transfer, and carbon metabolism, and that the native regulatory machinery is insufficient, and in some cases obstructive, for optimal growth under 30% CO2. This genetic profiling provides potential targets for engineering cyanobacteria with improved photosynthetic efficiency and stress resilience for biotechnological applications.Key points• Synechocystis growth was inhibited under very high CO2.• Inhibition of growth under very high CO2 was light dependent.• Repression of photosynthesis genes improved growth under very high CO2.Graphical

Dectin-1 stimulating β-glucans inhibit Chlamydia infections both in vitro and in vivo.

Chlamydia trachomatis and Candida albicans are common inhabitants of the female genital tract. C. albicans can impact viability and pathogenesis of some bacteria. Previously, we investigated physical interactions between C. trachomatis elementary bodies (EB) and C. albicans. This work indicated that EB bind to C. albicans and become noninfectious by 24 hours post binding. Here, we continue our investigation of these interkingdom, polymicrobial interactions. C. albicans adheres to bacteria or host surfaces via agglutinin-like sequence (Als) or heat shock 70 (Ssa) proteins. C. trachomatis EB did not bind C. albicans Ssa2 deficient strains as efficiently as wild type or complemented strains, indicating a role for this protein in chlamydial adherence to Candida. Additionally, C. albicans β-glucans inhibit chlamydial infection when exposure occurs during EB adsorption onto cervical cells. Laminarin (Lam), a β-glucan agonist of the C-type lectin receptor Dectin-1, inhibited chlamydial infection both cervical epithelial cells and mice when exposure occurred prior to, during, or immediately following EB inoculation. Conversely, a Dectin-1 antagonist laminarin (Lam-Ant) did not inhibit infection in vitro, suggesting β-glucan inhibition of C. trachomatis requires CTLR signaling. Overall, our data demonstrate that β-glucans from multiple species, including Candida albicans, inhibits Chlamydia via stimulation of host signaling pathways.

A rapid genome-proteome approach to identify rate-limiting steps in the butyrate production pathway in probiotic Clostridium butyricum, CBM588.

Clostridium butyricum ferments non-digested dietary fibre in the colon to produce butyric acid. Butyrate is a four-carbon, short-chain fatty acid (SCFA) which has multiple health benefits. Many microbial products of pharmaceutical or industrial interest, such as butyrate, are produced in low quantities due to rate-limiting steps in their metabolic pathway, including low abundance or low activity of one or more enzymes in the pathway. By identifying the former, appropriate enzymes can be over-expressed to increase product yields, however, methods to determine these enzymes are laborious. To improve butyrate production in C. butyricum probiotic strain, CBM588, a novel rapid genome-proteome approach was deployed. First, whole genome sequencing was performed and the 8 genes involved in butyrate production identified on the chromosome. Second, the relative abundance of these enzymes was investigated by liquid chromatography-mass spectrometry (LC-MS) analysis of total cytosolic proteins from early stationary phase cultures. Phosphotransbutyrylase (Ptb), butyrate kinase (Buk) and crotonase (Crt) were found to be the least abundant. Over-expression episomally of the corresponding genes individually or of the ptb-buk bicistron led to significant increases in butyrate titre per density of culture from 10 to 24 hours, compared to the wild type. Our findings pave the way for over-expressing these genes chromosomally to generate a recombinant probiotic with improved butyrate production and potentially enhanced gut health properties.

Dose escalation pre-clinical trial of novel DOK7-AAV in mouse model of DOK7 congenital myasthenia

Abstract Congenital myasthenic syndromes are a group of inherited disorders characterised by defective neuromuscular transmission and fatigable muscle weakness. Causative mutations have been identified in over 30 genes, including DOK7, a gene encoding a post-synaptic protein crucial in the formation and stabilisation of the neuromuscular junction. Mutations in this gene is one of the leading three most prevalent causes of congenital myasthenia in diverse populations across the globe. The majority of DOK7 congenital myasthenic patients experience varying degrees of disability despite receiving optimised treatment (usually salbutamol), necessitating the development of improved therapeutic approaches. Here we executed a dose escalation pre-clinical trial using a DOK7 congenital myasthenic syndrome mouse model to assess the efficacy of AMP-101, an innovative recombinant adeno-associated viral gene replacement therapy. This mouse model harbours a duplication in the Dok7 gene which corresponds to the mutation most commonly found in DOK7 congenital myasthenia patients, c.1124-1127dupTGCC. The model has a much more severe phenotype than patients, and lives for only a few days. AMP-101 is based on AAVrh74 and contains human DOK7 cDNA under the control of a muscle-restricted promoter. Three doses of AMP-101 (2x1013vg/kg, 6x1013vg/kg or 1x1014 vg/kg) were administered intra-peritoneally at four days of age. We show that the two higher doses of 6x1013vg/kg and 1x1014 vg/kg generated enlarged neuromuscular junctions and rescued the very severe phenotype of the model. Treated mice became at least as strong as wild type littermates, as demonstrated by using an inverted screen hang test, a rotarod test, and a grip strength test. EMG showed that the treated model mice had decrement of compound muscle action potential on repetitive nerve stimulation, which indicates defective signalling at the neuromuscular junction. However, male models treated with 1x1014 vg/kg showed the least decrement which was not statistically different from wild type littermates. Western blot analysis demonstrated robust expression of DOK7 in the diaphragm and tibialis anterior muscles. This data shows that AMP-101 is an effective treatment in a mouse model for DOK7 congenital myasthenia, and suggests that AMP-101 is a promising candidate to move forward to clinic trials as a gene therapy for patients.

A Model of Zymogen Factor XII: Insights into Protease Activation.

In plasma, the zymogens factor XII (FXII) and prekallikrein reciprocally convert each other to the proteases FXIIa and plasma kallikrein (PKa). PKa cleaves high-molecular-weight kininogen (HK) to release bradykinin, which contributes to regulation of blood vessel tone and permeability. Plasma FXII is normally in a "closed" conformation that limits activation by PKa. When FXII binds to a surface during contact activation it assumes an "open" conformation that increases the rate of activation by PKa. Mutations in FXII that disrupt the closed conformation have been identified in patients with conditions associated with excessive bradykinin formation. Using FXII structures predicted by AlphaFold, we generated models for the closed form of human FXII that we tested with site-directed mutagenesis. The best model predicts multiple interactions between the fibronectin type 2, kringle and catalytic domains involving highly conserved amino acids that restrict access to the FXII activation cleavage sites. Based on the model, we expressed FXII with single amino acid substitutions and studied their effects on FXII activation by PKa. Replacements for Arg36 in the fibronectin type 2 domain; Glu225, Asp253 or Trp268 in the kringle domain, or Lys346 near the activation cleavage site were activated >10-fold faster by PKa than wild type FXII. Adding these proteins to plasma resulted in rapid HK cleavage due to markedly enhanced reciprocal activation with PK. The results support a model that explains the behavior of FXII in solution. Conformational changes involving the identified amino acids likely occur when FXII binds to a surface to facilitate activation.

Chrebp Deletion and Mild Protein Restriction Additively Decrease Muscle and Bone Mass and Function

Background/Objectives: Carbohydrate and protein restriction are associated with sarcopenia and osteopenia, but the underlying mechanisms remain unclear. We aimed to determine whether mild protein restriction affects muscle and bone function in wild-type (WT) and homozygous carbohydrate response element binding protein (Chrebp) knockout (KO) mice. Methods: Eighteen-week-old male wild-type and homozygous carbohydrate response element binding protein (Chrebp) knockout (KO) mice were fed a control diet (20% protein) or a low-protein diet (15% protein) for 12 weeks. We estimated the muscle weight and limb grip strength as well as the bone mineral density, bone structure, and bone morphometry. Results: Chrebp deletion and a low-protein diet additively decreased body weight (WT control–KO low-protein: mean difference with 95% CI, 8.7 [6.3, 11.0], p < 0.0001) and epidydimal fat weight (1.0 [0.7, 1.2], p < 0.0001). Chrebp deletion and a low-protein diet additively decreased tibialis anterior muscle weight (0.03 [0.01, 0.05], p = 0.002) and limb grip strength (63.9 [37.4, 90.5], p < 0.0001) due to a decrease in insulin/insulin-like growth factor 1 mRNA and an increase in myostatin mRNA. In contrast, Chrebp deletion increased bone mineral density (BMD) (WT control–KO control: –6.1 [–1.0, –2.3], p = 0.0009), stiffness (–21.4 [–38.8, –4.1], p = 0.011), cancellous bone BV/TV (–6.517 [–10.99, –2.040], p = 0.003), and the number of trabeculae (–1.1 [–1.8, –0.5], p = 0.0008). However, in KO mice, protein restriction additively decreased BMD (KO control–KO low-protein: 8.1 [4.3, 11.9], p < 0.0001), bone stiffness (38.0 [21.3, 54.7], p < 0.0001), cancellous bone BV/TV (7.7 [3.3, 12.2], p = 0.006), and the number of trabeculae (1.2 [0.6, 1.9], p = 0.0004). The effects of mild protein restriction on bone formation parameters (osteoid volume (WT control–WT low-protein: –1.7 [–2.7, –0.7], p = 0.001) and the osteoid surface (–11.2 [–20.8, –1.5], p = 0.02) were observed only in wild-type (WT) mice. The levels of bone resorption markers, such as the number of osteoclasts on the surface, the number of osteoclasts, and surface erosion, did not differ between the groups. Conclusions: Both Chrebp deletion and protein restriction led to a decrease in muscle and bone function; therefore, an adequate intake of carbohydrates and proteins is important for maintaining muscle and bone mass and function. Further studies will be needed to elucidate the mechanisms by which ChREBP deletion and a low-protein diet cause osteosarcopenia.

Overexpression of triadin in adipose tissue causes cardiac dysfunction

Abstract Introduction As a member of the protein complex that regulates the ryanodine receptor (RyR) , triadin plays a critical role in intracellular calcium homeostasis. Our previous results demonstrate that the NAD-dependent deacetylase sirtuin-1 (SIRT1) downregulates the expression of triadin isoform Trisk 51 in adipose tissue. A transgenic mouse model with adipose tissue selective overexpression of Trisk 51 (Adipo-TRDN) was established. Aim The present study is designed to investigate the role of SIRT1-triadin signalling in the development of dilated cardiomyopathy/heart failure with reduced ejection fraction. Methods Wild type (WT) littermates, Adipo-TRDN without or with human SIRT1 overexpressed in adipose tissue (Adipo-TRDN/SIRT1) were used for this study. All mice were provided with standard chow diet, normal drinking water and kept in a 12/12 light-dark cycle. Echocardiography was performed in 30 weeks old male mice using Visual Sonics Vevo 2100 Imaging System to measure systolic and diastolic function using B-mode, M-mode, Color Doppler and Tissue Doppler parameters. Results Systolic cardiac impairment was present in the Adipo-TRDN mice compared with their littermate controls. Left ventricular internal diameter during systole (LVID, s) was increased in Adipo-TRDN compared with WT littermates (3.0031 vs. 2.1878 mm, p-value: 0.0059). Changes in the Left ventricular anterior wall thickness (LVAW) during systole and diastole were found to be non-significant (1.6180 vs. 1.3530 mm, 1.1443 vs. 1.0455mm). Although fractional shortening (FS %) differences were non-significant (27.64% vs. 34.83%), there was a significant reduction in ejection fraction (EF %) in Adipo-TRDN mice compared with their WT controls (49.41% vs. 64.73%, p-value: 0.0472). No differences were found in the Left ventricular mass (LV) (106.80 vs.84.10 mg). Left ventricular volume during systole (LV Vol; s) was also significantly enhanced in Adipo-TRDN compared with their WT controls (35.25 vs.16.30 μL, p-value: 0.0047). Overexpression of human SIRT1 lead to partial improvement in the pathogenesis of dilated cardiomyopathy in Adipo-TRDN mice. Ejection fraction and fractional shortening were both increased in the Adipo-TRDN/SIRT1 compared with Adipo-TRDN mice (70.70% vs. 49.41%, p-value: 0.0017, 39.60% vs. 27.64% p-value: 0.0151). LV mass and LV Vol;s were reduced in the Adipo-TRDN/SIRT1 compared with Adipo-TRDN mice (75.83 vs. 106.80 mg, p-value: 0.0543) (14.02 vs. 35.25 μL, p-value: 0.0005). LVID (s), LVAW (d) and LVAW (s) were also reduced in Adipo-TRDN/SIRT1 compared with Adipo- TRDN mice (2.1641 vs. 3.0031mm, p-value: 0.0041, 0.8728 vs. 1.1443, p-value: 0.0319, 1.2369 vs. 1.6180mm, p-value: 0.0018). Conclusion Development of dilated cardiomyopathy induced by adipocyte specific overexpression of Triadin is partially prevented by adipose SIRT1.

P-1342. LiaX Decreases Killing By β-Lactam Antibiotics: Enterococcal Tolerance Revisited

Abstract Background Compared to other Firmicutes, enterococci are often tolerant to many antibiotics; combination therapy is used for severe, deep-seated infections. We have previously reported the LiaFSR system, a three-component cell-envelop stress regulatory system, mediates resistance and tolerance to daptomycin (DAP). The main effector, LiaX, harbors distinct N- and C-terminal domains. The N-terminal portion of LiaX is released into the extracellular milieu where it recognizes DAP and other antimicrobial peptides functioning as a sentinel and signal transduction protein to decrease DAP-mediated killing. LiaX likely acts through LiaF, a transmembrane protein necessary for the LiaX effect. We postulated that the LiaX-LiaF relay system may be involved in the β-lactam tolerance shown by E. faecalis (Efs).Figure 1.N-terminal domain of LiaX protects E. faecalis from ampicillin and ceftriaxone. Methods We used wild type (WT) Efs OG117 and derivatives: OG117liaF*11-14 (liaF deletion) and OG117 liaF*11-14::liaF (liaF complemented). Ampicillin (AMP) and ceftriaxone (CRO) broth-microdilution MICs were performed. N-terminal LiaX1-275 was expressed in E. coli and purified. Killing assays (5 replicates) with 0.5 X MICs of AMP and CRO were done, with and without LiaX1-275 (80 nM). Bacteria were diluted (∼1x105 CFU/mL) and used to inoculate test media containing antibiotic alone, antibiotic+LiaX1-275, and no antibiotic. After 2-hours of incubation at 37°C, samples were plated on BHI agar for enumeration. Results The CRO MICs for WT and complemented strains were 64 μg/mL, whereas it was 2-fold higher (128 μg/mL) for OG117liaF*11-14. All strains showed AMP MICs of 1 μg/mL. In the AMP killing assay, OG117 survival increased with the addition of LiaX1-275 (p < .005). The addition of LiaX1-275 failed to increase survival of OG117liaF*11-14 in the presence of AMP compared to WT (p = 0.4). The survival advantage was restored in the complemented strain (p < .0001). CRO showed a similar trend, i.e., increased survival in OG117 (p < .01) and complemented strain (p < .001), but not in OG117liaF*11-14 (p = 0.29). Conclusion We have discovered a novel pathway in Efs for antibiotic tolerance. The LiaF-LiaX signal relay, part of the enterococcal response to antibiotics, boosts organism survival, contributing to tolerance to multiple anti-enterococcal drugs. Disclosures William R. Miller, M.D., Merck: Grant/Research Support|UptoDate: Royalties

P-100. Durable immune response induced by self-amplifying mRNA (samRNA) SARS-CoV-2 vaccine candidates against variants of concern (VOC) in HIV-negative and people living with HIV (PLWH) populations in South Africa, irrespective of prior SARS-CoV-2 vaccination

Abstract Background Protection against COVID-19 from authorized SARS-CoV-2 vaccines diminishes after six months, so it is evident we need a second-generation vaccine that provides durable cross-variant protection through durable antibody responses and/or T cell responses to conserved epitopes. CORAL-CEPI (NCT05435027) is a Phase I study conducted in South Africa evaluating three self-amplifying mRNA (samRNA)-based SARS-CoV-2 vaccine candidates.Figure 1Vaccine Cassette Design GRT-R914 encodes full length SpikeBeta and T cell epitopes (TCE) from conserved viral proteins (TCE9). GRT-R912 encodes full length SpikeBeta, full length Nucleocapsid, and a TCE cassette (TCE11). GRT-R918 encodes full length SpikeOmicron BA.1, full length Nucleocapsid, and a TCE cassette (TCE11). SGP: Sub-genomic promoter; T2A: Ribosomal skip-site Methods Vaccine candidates GRT-R914, GRT-R912, and GRT-R918 encode full-length Spike (Beta or Omicron BA.1), Nucleocapsid (full-length or selected T cell epitopes [TCEs]), and non-Spike TCE’s from conserved viral proteins beyond Spike (Fig.1). GRT-R914 and GRT-R912 were evaluated in HIV-negative and PLWH populations who were SARS-CoV-2 anti-Spike and anti-Nucleocapsid seronegative or seropositive at baseline (Parts A/B/C). In Part D, GRT-R918 was evaluated in adults who were either previously vaccinated against SARS-CoV-2 or vaccine naïve (Fig.2). Primary objectives included safety assessments (reactogenicity and adverse events [AEs]). Secondary objectives assessed ancestral Spike-specific binding IgG (bAb) and neutralizing antibodies (nAbs) to SARS-CoV-2 variants as well as T cell responses against Spike and TCEs.Figure 2Study DesignGO-012 Study Schema (Parts A, B, C, and D): 341 participants received either GRT-R914, GRT-R912, or GRT-R918 vaccine candidates. 140 participants received GRT-R914 (3mcg, 10mcg, or 30mcg), 102 participants received GRT-R912 (3mcg or 10mcg), and 99 participants received GRT-R918 (10mcg) Results Among 341 vaccinated participants, most reported reactogenicity events were grade 1 or 2 and transient in nature. No vaccine-related serious AEs or severe COVID-19 cases were reported. Twenty-five out of 341 participants reported grade 3 solicited AE’s which resolved within 1-4 days. IgG titers against WT and nAb titers against Beta, Delta, and Omicron BA.1 were increased after administration of any of the three vaccine candidates and were durable up to at least 12 months in both HIV negative individuals and PLWH (Fig. 3 and 4). Spike and/or TCE-specific T cell responses were increased or maintained in the majority of participants after vaccination.Figure 3ResultsSpikeWT IgG bAbs and nAb titers against Alpha, Beta, Delta, Gamma, and Omicron BA.1 variants are induced and maintained through at least 12-months in HIV-negative participants irrespective of SARS-CoV-2 serostatus or vaccination status at baseline Conclusion All dose levels of all samRNA vaccine candidates were generally well-tolerated. Administration of any of the three vaccine candidates induced robust and durable IgG binding antibodies to Wild Type (WT) and nAbs against variants of concern and T cell responses to both Spike and TCE epitopes. Complete study results will be presented.Figure 4ResultsIgG levels to SpikeWT and nAb titers to Alpha, Beta, Delta, Gamma, and Omicron BA.1 variants are increased and remain durable through 12 months in PLWH following 10µg dose(s) of GRT-R914, GRT-R912, or GRT-R918 Disclosures Atul Nagare, MBBS, Gritstone bio: employee Elizabeth Martin, DO, MPH, MBA, Gritstone bio: employee M. Marrali, PhD, Gritstone bio: employee Meghan Hart, MS, Gritstone bio: employee Harshni Venkatraman, MS, Gritstone bio: employee Jason Jaroslavsky, MS, Gritstone bio: employee Jenchun Kuan, PhD, Gritstone bio: employee Sonia Kounlavouth, BS, Gritstone bio: employee Enrique Podaza, PhD, Gritstone bio: employee Mathieu Le Gars, PhD, Gritstone bio: employee A. Allen, MBBS, PhD, Gritstone bio: Board Member|Gritstone bio: Stocks/Bonds (Public Company) Karin Jooss, PhD, Gritstone bio: employee|Gritstone bio: Stocks/Bonds (Public Company)

Phosphatidylserine-binding receptor, CD300f, on macrophages mediates host invasion of pathogenic and non-pathogenic rickettsiae.

Some arthropod-borne obligate intracellular rickettsiae are among the most virulent human pathogens. Rickettsia species modulate immune (e.g., macrophages; MΦ) and non-immune cell (e.g., endothelial cells) responses to create a habitable environment for host colonization. In particular, MΦ play a crucial role in either terminating an infection at an early stage or succumbing to bacterial replication and colonization. However, our understanding on how Rickettsia species invade host cells, including MΦ, remain poorly defined. In this study, we describe a mechanism of host invasion by Rickettsia species, involving rickettsial phosphatidylserine (PS), as a ligand, and the CD300f receptor on MΦ. Using bone marrow-derived macrophages (BMDMΦ) from wild-type (WT) and CD300f-/- mice, we demonstrated that engulfment of both pathogenic R. typhi (the etiologic agent of murine typhus) and R. rickettsii (the etiologic agent of Rocky Mountain spotted fever) species as well as the non-pathogenic R. montanensis was significantly reduced in CD300f-/- BMDMΦ as compared to that of WT BMDMΦ. Furthermore, our mechanistic analysis suggests bacterial PS as the potential source for the CD300f-mediated rickettsiae engulfment by MΦ. In vivo infection studies using WT and CD300f-/- C57BL/6J mice showed that CD300f-/- animals were protected against R. typhi- or R. rickettsii-induced fatal rickettsiosis, which correlated with levels of bacterial burden detected in the spleens of mice. Adoptive transfer studies further revealed that CD300f-expressing MΦ are important mediators to control rickettsiosis in vivo. Collectively, our findings describe a previously unappreciated role for the efferocytic receptor, CD300f, to facilitate engulfment of rickettsiae within the host.

P-487. [18F]-labeled J3 Nanobody is a Specific PET Tracer for Molecular Imaging of HIV-1 gp120

Abstract Background Targeted molecular imaging of HIV-1 can potentially identify viral reservoirs and provide a better understanding of disease pathogenesis and management. Nanobody-based radiolabeled probes offer several advantages over full antibodies, including smaller size, deep tissue penetration, and high stability. In this study, three fluorine-18 labeled nanobodies targeting the well-conserved CD4-binding site on HIV-1 envelope protein (gp120) were evaluated in gp120-expressing flank tumor models. Methods Binding affinities to gp120 protein were determined by bio-layer interferometry. For in vivo PET imaging, an animal model with wild type (WT) and/or gp120-expressing HEK293T cells (gp120+) flank tumors was developed in nude mice. Radiolabeled nanobodies were prepared by an indirect labeling method using 6-fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[18F]FPy-TFP) prosthetic group. Dynamic PET/CT images up to 60 minutes and static images at 120 minutes were obtained. Tumors were evaluated ex vivo using immunofluorescent staining. Unpaired t test was used for statistical analysis. Results J3 nanobody showed higher binding affinity to gp120 compared to 3E3 and B9, with KD values of 1.1nM, 3.4nM, and 23.7nM, respectively. PET/CT imaging revealed selective binding (measured as SUVpeak) to gp120+ compared to WT tumors with J3 (0.64 ± 0.14 vs. 0.29 ± 0.08) and 3E3 (0.61 ± 0.27 vs. 0.22 ± 0.12) but not with B9 (0.21 ± 0.14 vs 0.25 ± 0.12). After 2 hours, J3 and 3E3 had minimal background signal, whereas B9 showed poor ligand clearance and off target binding (Fig. 1). [18F]J3 time activity curves (TACs) from dynamic and static scans showed selective binding to gp120+ tumors compared to WT tumors (Fig. 2). To validate our PET findings, ex vivo gp120 tumor expression was confirmed by immunofluorescence staining. J3 nanobody showed specific immunoreactivity that colocalized with VRC01, an anti-gp120 full antibody (Fig. 3). Conclusion We evaluated the binding characteristics of three 18F-labeled nanobodies to gp120+ cells. Overall, J3 nanobody had higher specificity and binding to its target compared to 3E3 and B9, demonstrating a higher potential for non-invasive identification of HIV-infected cells in infected models. Disclosures All Authors: No reported disclosures

Decreased SynMuv B gene activity in response to viral infection leads to activation of the antiviral RNAi pathway in C. elegans.

RNA interference (RNAi) mediates antiviral defense in many eukaryotes. Caenorhabditis elegans mutants that disable RNAi are more sensitive to viral infection. Many mutants that enhance RNAi have also been identified; these mutations may reveal genes that are normally down-regulated in antiviral defense. About one-third of the score of mutants that enhance RNAi are in synMuv B genes, identified 30 years ago in unrelated screens for increased growth factor signaling. Many synMuv B genes encode dREAM complex chromatin-regulatory proteins found in nearly all animals and plants. We show that mRNAs which are highly induced in synMuv B mutants are congruent with those induced by Orsay RNA virus infection, suggesting that the enhanced RNAi of synMuv B mutants may also be triggered by down-regulation of synMuvB gene activity in an Orsay virus infection of wild type. The multivulval (Muv) phenotype of synMuv B mutants requires the presence of a second nematode-specific synMuv A gene mutation, but the enhanced RNAi of synMuv B mutants does not require a second synMuv A mutation. To test if Orsay viral infection down-regulates synMuv B gene activity, we infected a single synMuv A mutant with Orsay virus and found that a Muv phenotype could be induced. Thus, decreased synMuv B gene activity is part of the normal C. elegans viral defense response. In support of the model that decreased syn- Muv B gene activity enhances antiviral response, we found that synMuv B mutants have 50 to 100× lower viral RNA levels during an Orsay virus infection than wild type. Thus down-regulation of synMuv B activity to enhance RNAi is a key component in the defense response to viral infection. Small RNA deep sequencing analysis of dREAM complex mutants revealed siRNA profiles indicative of such a response. Thus, the pan-eukaryotic synMuv B genes constitute an element in C. elegans antiviral defense which is conserved across many eukaryotes where it also may act in viral defense. The enhanced RNAi and conservation of the dREAM complex mutants suggests new therapeutic avenues to boost antiviral defenses.

P-1823. Development of a Nocardia Reporter Strain to Study Cell-type Specific Phagocytosis and Killing in vivo

Abstract Background Nocardia infections are associated with substantial morbidity and mortality. Little is known about how the immune system defends against Nocardia. Methods Nocardia was transformed to contain a genomic copy of mScarlet. The cell wall was then labelled with AlexaFluor633 (AF633) to generate dually labelled (mScarlet+ AF633+) Nocardia. Cybb knockout (KO) mice (chronic granulomatous disease or CGD) and wildtype (WT) controls were inoculated with 5-10 million colony forming units (CFU) via oropharyngeal aspiration. High-dimensional flow cytometry (HDFC) and bacterial burden were determined in lung and bronchoalveolar lavage (BAL) fluid obtained 24 hours post-infection. Phagocytic index (PI) was defined as % of cells that are AF633+ cells. Killing capacity (KI) was defined as % of AF633+ cells that are mScarlet–. Results Live cultures of dually labelled Nocardia were positive for both mScarlet and AF633. mScarlet but not AF633 fluorescence decreased to undetectable levels upon killing of Nocardia. HDFC demonstrated that alveolar macrophages (AM), interstitial macrophages (IM), neutrophils, monocytes, and type 2 dendritic cells (cDC2) are the primary cell types that interact with Nocardia in vivo. PI was highest in AM and IM. PI was greater in cells from BAL versus lung (P< 0.01) while the opposite was true for other cell types. KI was lower in neutrophils versus other cell types (P< 0.05). Mortality and bacterial burden in lung were higher in CGD mice compared to WT controls (P< 0.01 for both comparisons). CGD mice demonstrated reduced PI in neutrophils, AM, IM (P< 0.05 for both). KI was impaired in AM and neutrophils. Conclusion Use of a dually labelled Nocardia reporter strain demonstrated cell-type specific defects in both phagocytosis and killing of Nocardia in CGD mice. This might explain the underlying predisposition to nocardiosis seen in humans with CGD. Ongoing studies aim to elucidate the molecular basis for the susceptibility to nocardiosis in humans and mice with CGD. Disclosures All Authors: No reported disclosures

Trace amine-associated receptors (TAARs)2-9 knockout mice exhibit reduced wakefulness and disrupted REM sleep

IntroductionThis study aimed to investigate the role of TAAR2-9 in sleep/wake regulation, given TAAR1's known involvement in modulating neurotransmitter release and sleep patterns.MethodsMale TAAR2-9 knockout (KO) and wild-type (WT) mice were compared using baseline sleep/wake patterns, responses to sleep deprivation, effects of TAAR1 agonists, and dopaminergic markers. EEG recordings and tyrosine hydroxylase immunohistochemistry were used for analysis.ResultsKO mice exhibited lower delta and theta power and higher gamma power, with fragmented sleep characterized by 16% more NREM sleep during the dark phase and 23% more REM sleep during the light phase compared to WT mice. High doses of the TAAR1 agonist RO5256390 increased wakefulness and reduced NREM sleep, while both RO5256390 and the partial agonist RO5263397 suppressed REM sleep in KO mice. Elevated tyrosine hydroxylase levels in the ventral tegmental area suggested dopaminergic involvement in these altered sleep patterns.DiscussionTAAR2-9 modulates sleep/wake states and interacts with TAAR1. These findings highlight the therapeutic potential of targeting TAARs 2-9 in sleep-related neuropsychiatric disorders. Further research is needed to elucidate their roles.

P-1061. In vitro Activity of Gepotidacin against Klebsiella pneumoniae, Including Molecularly Characterized Fluoroquinolone not Susceptible Subsets Causing Urinary Tract Infections in the United States (2019-2022)

Abstract Background Gepotidacin (GEP) is a novel, bactericidal, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a unique mechanism of action, distinct binding site and provides well-balanced inhibition (for most uUTI uropathogens) of two different type II topoisomerase enzymes. This study reports the activity of GEP and other oral antibiotics against K. pneumoniae, including molecularly characterized fluoroquinolone (FQ) not susceptible (NS) isolates collected from urinary tract infection (UTI) patients in the US. Methods A total of 2,001 K. pneumoniae were collected from 73 sites. CLSI methods were used for both susceptibility (S) testing and MIC interpretation. Isolates with MIC≥0.5 mg/L for ciprofloxacin (NS) and/or ≥1 mg/L for levofloxacin (NS) were screened for FQ-resistance (R) mechanisms (plasmid-mediated quinolone resistance [PMQR]), QRDR mutations, and expression of acrAB and oqxAB by qRT-PCR. Results GEP inhibited 94.9% of all these isolates at ≤16 mg/L (MIC50/90 = 4/16mg/L). GEP MIC50/90 against FQ-S isolates was 4/8 mg/L, and other agents showed activity against ≥90% of isolates, except nitrofurantoin (37.4%S). 14.5% (291/2,001) of isolates were screened for FQ-R mechanisms. GEP MIC50/90 against FQ-NS strains was 16/32 mg/L, whereas other agents had S of 5.8–48.5%. GEP had generally similar activity (MIC50/90, 16-32/32-64 mg/L) against isolates with wildtype QRDR and with or without PMQR genes. Most of the isolates (95.2%) without PMQR genes overexpressed oqxAB and/or acrAB, against which amoxicillin-clavulanate and cefazolin (90.3–95.2%S) were active. GEP had consistent MIC90 of 16 mg/L against isolates with distinct QRDR mutations and absence of PMQR genes, whereas other agents had S of ≤68%. Conclusion GEP showed activity against FQ-S and FQ-NS K. pneumoniae UTI isolates from the US, in particular against FQ-NS isolates carrying QRDR mutations, where standard oral antibiotics showed limited activity. GEP MICs were not substantially affected by QRDR mutations. Highest MICs were seen against FQ-NS isolates with wildtype QRDR and PMQR genes and/or overexpression of efflux-pump genes. These data support the development of GEP for the treatment of uUTI caused by K. pneumoniae. Disclosures Rodrigo E. Mendes, PhD, GSK: Grant/Research Support Renuka Kapoor, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Didem Torumkuney, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company)

P-290. Clinical and microbiological characteristics of patients infected with ceftazidime/avibactam-resistant Klebsiella pneumoniae carbapenemase-producing K. pneumoniae strains

Abstract Background Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae with ceftazidime/avibactam (CZA) resistance emerged after the widely use of CZA. The most common resistance mechanism was mutations in blaKPC. We aimed to study the clinical and microbiological characteristics in patients infected with CZA-resistant KPC-producing K. pneumoniae, and focus on the comparison between the strains with or without KPC variants (wild-type KPC). Methods A retrospective study was conducted in Taipei Veterans General Hospital from February 2019 to July 2023. Hospitalized unique adult patients infected with CZA-resistant KPC-producing K. pneumoniae were included. Clinical characteristics and outcomes were collected, and genes encoding carbapenemases were detected by polymerase chain reaction followed by Sanger sequencing. Results A total of 44 cases with CZA-resistant KPC-producing K. pneumoniae infections were identified, and most of them were critically ill with the median APACHE II score 23.0 (IQR, 19.3-29.8). Pneumonia was the most common infection (n=18). Only 15 patients (34.1%) received appropriate treatment regimens, and tigecycline (n=8) and meropenem-based therapy (n=5) were the common regimens. Twenty-seven strains (61.4%) harbored KPC variants, and a total of 16 different types were identified. KPC-33 (n=10) was the most common, followed by KPC-35 (n=3). The overall 28-day mortality was similar between patients infected with and without KPC variants (22.2% vs 23.5%, p=1.000). A previous isolation of carbapenem-resistant K. pneumoniae and exposure to CZA were more common in KPC variants group compared with wild-type KPC group (92.6% vs 58.8%, p=0.017, 92.6% vs 41.2%, p< 0.001, respectively). KPC variants-producing strains also had a higher proportion of CZA MIC > 16 mg/L (85.2% vs 41.2%, p=0.002) and restored meropenem susceptibility (MIC≤ 4 mg/L) (70.4% vs 5.9%, p< 0.001) than that in wild-type KPC strain. Conclusion CZA-resistant KPC variants-producing K. pneumoniae has the tendency of restored meropenem susceptibility and higher level of CZA resistance compared with the wild type KPC-producing strain. The optimal therapy in these patients needs further study. Disclosures All Authors: No reported disclosures

Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to lethality in mice.

Bone morphogenetic protein4 (BMP4) plays numerous roles during embryogenesis and can signal either alone as a homodimer, or together with BMP7 as a more active heterodimer. BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments. In humans, heterozygous mutations within the prodomain of BMP4 are associated with birth defects. We studied the effect of two of these mutations (p.S91C and p.E93G), which disrupt a conserved FAM20C phosphorylation motif, on ligand activity. We compared the activity of ligands generated from BMP4, BMP4S91C or BMP4E93G in Xenopus embryos and found that these mutations reduce the activity of BMP4 homodimers but not BMP4/7 heterodimers. We generated Bmp4S91C and Bmp4E93G knock-in mice and found that Bmp4S91C/S91C mice die by E11.5 and display reduced BMP activity in multiple tissues including the heart. Most Bmp4E93G/E93G mice die before weaning and Bmp4-/E93G mutants die prenatally with reduced or absent eyes, heart and ventral body wall closure defects. Mouse embryonic fibroblasts (MEFs) isolated from Bmp4S91C and Bmp4E93G embryos show accumulation of BMP4 precursor protein, reduced levels of cleaved BMP ligand and reduced BMP activity relative to MEFs from wild type littermates. Because Bmp7 is not expressed in MEFs, the accumulation of unprocessed BMP4 precursor protein in mice carrying these mutations most likely reflects an inability to cleave BMP4 homodimers, leading to reduced levels of ligand and BMP activity in vivo. Our results suggest that phosphorylation of the BMP4 prodomain is required for proteolytic activation of BMP4 homodimers, but not heterodimers.

P-2340. Single-Center Inpatient Measles Analysis During a Congregate Setting Outbreak; Comparing Vaccine History between Measles Positive and Negative Cohorts

Abstract Background Chicago’s 2024 measles outbreak affected many new arrivals living in congregate settings with unknown or poorly documented vaccination history. Despite post-exposure prophylaxis provided at shelters, many families still developed measles. Frequency of measles exposure in congregate settings certainly plays a role in infection, but vaccination history and source should also be evaluated.Table.Characteristics of Measles Patients Under Investigation (PUI) stratified by PCR result * One case received IGIM and five received 1 MMR post-exposure prophylaxis dose given at the shelter between 10—30 days prior to rash onset (median: 11 days), ∞ including vaccine strain case, # n=7, ^n=10, ^^Countries of origin: Venezuela, Peru, Ecuador Methods Data was abstracted from 12 measles PCR positive and 13 measles PCR negative patient charts from 3/10/24—4/23/24 including measles vaccination history, IgG antibody titers, concurrent infections and measles complications. Relative risks and p-values were calculated using MedCalc Software Ltd (Version 22.023). Results From 3/10/24—4/23/24, 12 measles PCR positive patients were admitted with median age 16 years (range 13 months—43 years) including 3 parent-child dyads; serotyping through the Illinois Department of Public Health reported one vaccine strain and 11 wild type cases. Of the 11 wild type measles cases, 3 (27%) had concurrent infections; (2 of 3 group A streptococcus culture positive (oropharynx); 1 of 3 was rhinovirus/enterovirus positive (nasopharyngeal PCR); 8 of 11 (73%) had at least 1 measles complication including dehydration (4), acute otitis media (3), pneumonia (1), and hepatitis (2). Comparing vaccine history between measles negative and positive cohorts, there was a non-significant trend towards history of international vaccination in the measles positive cohort. History of current congregate setting residence and exposure to a known measles case were significantly associated with measles positivity with a relative risk of 2.2. Conclusion Inpatient data is lacking during the 2024 international measles surge and new arrivals' vaccination history documentation is inconsistent. A trend towards international measles vaccine receipt in a measles positive cohort highlights the importance of US public health efforts to offer MMR vaccination to new arrivals. Limitations of this report include small cohort sizes and recall bias. Additional data is needed to describe the impact of improper vaccine storage and handling on vaccine effectiveness in the current international measles epidemic. Disclosures All Authors: No reported disclosures