A novel stoploss mutation CYB5R3 c.906A>G(p.*302Trpext*42) involved in the pathogenesis of hereditary methemoglobinemia.

Recessive congenital methemoglobinemia (RCM) is a hereditary autosomal disorder with an extremely low incidence rate. Here, we report a case of methemoglobinemia type I in a patient with congenital persistent cyanosis. The condition was attributed to a novel compound heterozygous mutation in CYB5R3, characterized by elevated methemoglobin levels (13.4 % of total hemoglobin) and undetectable NADH cytochrome b5 reductase (CYB5R3) activity. Whole-exome sequencing (WES) revealed two heterozygous mutations in CYB5R3: a previously reported pathogenic missense mutation c.611G>A(p.Cys204Tyr) inherited from the father, and a novel stop codon mutation c.906A>G(p.*302Trpext*42) from the mother, the latter mutation assessed as likely pathogenic according to ACMG guidelines. In cells overexpressing the CYB5R3 c.906A>G mutant construct, the CYB5R3 mRNA level was significantly lower than in cells overexpressing the wild-type (WT) CYB5R3 construct. However, there was no significant difference in protein expression levels between the mutant and WT constructs. Notably, an additional protein band of approximately 55 kDa was detected in the mutant cells. Immunofluorescence localization showed that, compared to wild-type CYB5R3, the subcellular localization of the CYB5R3 p.*302Trpext*42 mutant protein did not show significant changes and remained distributed in the endoplasmic reticulum and mitochondria. However, the c.906A>G(p.*302Trpext*42) mutation resulted in increased intracellular reactive oxygen species (ROS) levels and decreased NAD+/NADH ratio, suggesting impaired CYB5R3 function and implicating this novel mutation as likely pathogenic.

Analyzing three pedigrees in X-linked Alport syndrome with the presentation of nephrotic syndrome.

Background: Alport syndrome (AS) is a common cause of end-stage renal disease (ESRD) with various clinical symptoms and incomplete manifestation. Patients with AS and other renal disorders are often misdiagnosed. This study reported three X-linked dominant Alport syndrome (XLAS) pedigrees with nephrotic syndrome (NS) as the predominant phenotype and analyzed COL4A5 gene alterations. Methods: Three Han Chinese XLAS pedigrees were recruited, and clinical phenotypes were obtained. The pre-certified individuals' peripheral blood DNA was taken, and whole-genome next-generation sequencing (NGS) was performed for candidate genes and mutation screening, followed by NGS or Sanger sequencing of suspected mutant types in participating family members. Results: Both probands A and B were diagnosed with NS through biochemical tests, and X-linked Alport syndrome-associated renal injury was diagnosed by renal biopsy. The biopsy revealed focal foamy cells in the renal interstitium, tearing and delamination changes in the glomerular basement membrane, and negative α3 and α5 chains of type IV collagen. Proband C, who was earlier diagnosed with NS, has now advanced to ESRD, along with his mother and proband A's mother. Genetic sequencing of all three pedigrees identified three mutations, namely, c.5020C>T, c.4435_4445del, and c.1584_1587+6del in the X-linked dominant gene COL4A5 (NM_000495.5). These mutations lead to the production of shortened proteins, potentially impacting the function of COL4A5 and causing pathogenic effects. Conclusion: The novel c.4435_4445del and c.1584_1587+6del mutations not only enrich the spectrum of mutations in the COL4A5 gene but also indicate that carriers of both mutation sites and those with mutation c.5020C>T may present NS as their primary clinical manifestation.

In vitro study of ATP1A3 p.Ala275Pro mutant causing alternating hemiplegia of childhood and rapid-onset dystonia-parkinsonism.

Introduction: We previously reported that ATP1A3 c.823G>C (p.Ala275Pro) mutant causes varying phenotypes of alternative hemiplegia of childhood and rapid-onset dystonia-parkinsonism in the same family. This study aims to investigate the function of ATP1A3 c.823G>C (p.Ala275Pro) mutant at the cellular and zebrafish models. Methods: ATP1A3 wild-type and mutant Hela cell lines were constructed, and ATP1A3 mRNA expression, ATP1A3 protein expression and localization, and Na+-K+-ATPase activity in each group of cells were detected. Additionally, we also constructed zebrafish models with ATP1A3 wild-type overexpression (WT) and p.Ala275Pro mutant overexpression (MUT). Subsequently, we detected the mRNA expression of dopamine signaling pathway-associated genes, Parkinson's disease-associated genes, and apoptosisassociated genes in each group of zebrafish, and observed the growth, development, and movement behavior of zebrafish. Results: Cells carrying the p.Ala275Pro mutation exhibited lower levels of ATP1A3 mRNA, reduced ATP1A3 protein expression, and decreased Na+-K+-ATPase activity compared to wild-type cells. Immunofluorescence analysis revealed that ATP1A3 was primarily localized in the cytoplasm, but there was no significant difference in ATP1A3 protein localization before and after the mutation. In the zebrafish model, both WT and MUT groups showed lower brain and body length, dopamine neuron fluorescence intensity, escape ability, swimming distance, and average swimming speed compared to the control group. Moreover, overexpression of both wild-type and mutant ATP1A3 led to abnormal mRNA expression of genes associated with the dopamine signaling pathway and Parkinson's disease in zebrafish, and significantly upregulated transcription levels of bad and caspase-3 in the apoptosis signaling pathway, while reducing the transcriptional level of bcl-2 and the bcl-2/bax ratio. Conclusion: This study reveals that the p.Ala275Pro mutant decreases ATP1A3 protein expression and Na+/K+-ATPase activity. Abnormal expression of either wild-type or mutant ATP1A3 genes impairs growth, development, and movement behavior in zebrafish.

A pedigree analysis of Rotor hyperbilirubinemia combined with hepatitis B virus infection in a SLCO1B1 and SLCO1B3 gene mutations patient.

Background: Rotor syndrome (RS, OMIM#237450) is an extremely rare autosomal digenic recessive disorder characterized by mild non-hemolytic hereditary conjugated hyperbilirubinemia, caused by biallelic variation of SLCO1B1 and SLCO1B3 genes that resulted in OATP1B1/B3 dysfunction in the sinusoidal membrane leading to impaired bilirubin reuptake ability of hepatocytes. Methods: One RS pedigree was recruited and clinical features were documented. Whole genome second-generation sequencing was used to screen candidate genes and mutations, Sanger sequencing confirmed predicted mutations. Results: This study detected a homozygous nonsense variant c.1738C > T (p.R580*) in the coding region of the SLCO1B1 (NM006446) gene in a family with RS and hepatitis B virus infection by Variants analysis and Sanger sequencing, and confirmed by Copy Number Variation (CNV) analysis and Long Range PCR that there was a homozygous insertion of intron 5 of the SLCO1B3 gene into intron 5 of long-interspersed element 1 (LINE1). A few cases of such haplotypes have been reported in East Asian populations. A hepatitis B virus infection with fatty liver disease was indicated by pathology, which revealed mild-moderate lobular inflammation, moderate lobular inflammation, moderate hepatocellular steatosis, and fibrosis stage 1-2 (NAS score: 4 points/S1-2) alterations. Heterozygotes carrying p.R580* and LINE1 insertions were also detected in family members (I1, I2, III2, III3), but they did not develop conjugated hyperbilirubinemia. Conclusion: The mutations may be the molecular genetic foundation for the presence of SLCO1B1 c.1738C > T(p.R580*) and SLCO1B3 (LINE1) in this RS pedigree.

A novel splicing mutation DNAH5 c.13,338 + 5G > C is involved in the pathogenesis of primary ciliary dyskinesia in a family with primary familial brain calcification.

BACKGROUND: Primary ciliary dyskinesia (PCD) is an autosomal recessive hereditary disease characterized by recurrent respiratory infections. In clinical manifestations, DNAH5 (NM_001361.3) is one of the recessive pathogenic genes. Primary familial brain calcification (PFBC) is a neurodegenerative disease characterized by bilateral calcification in the basal ganglia and other brain regions. PFBC can be inherited in an autosomal dominant or recessive manner. A family with PCD caused by a DNAH5 compound heterozygous variant and PFBC caused by a MYORG homozygous variant was analyzed. METHODS: In this study, we recruited three generations of Han families with primary ciliary dyskinesia combined with primary familial brain calcification. Their clinical phenotype data were collected, next-generation sequencing was performed to screen suspected pathogenic mutations in the proband and segregation analysis of families was carried out by Sanger sequencing. The mutant and wild-type plasmids were constructed and transfected into HEK293T cells instantaneously, and splicing patterns were detected by Minigene splicing assay. The structure and function of mutations were analyzed by bioinformatics analysis. RESULTS: The clinical phenotypes of the proband (II10) and his sister (II8) were bronchiectasis, recurrent pulmonary infection, multiple symmetric calcifications of bilateral globus pallidus and cerebellar dentate nucleus, paranasal sinusitis in the whole group, and electron microscopy of bronchial mucosa showed that the ciliary axoneme was defective. There was also total visceral inversion in II10 but not in II8. A novel splice variant C.13,338 + 5G > C and a frameshift variant C.4314delT (p. Asn1438lysfs *10) were found in the DNAH5 gene in proband (II10) and II8. c.347_348dupCTGGCCTTCCGC homozygous insertion variation was found in the MYORG of the proband. The two pathogenic genes were co-segregated in the family. Minigene showed that DNAH5 c.13,338 + 5G > C has two abnormal splicing modes: One is that part of the intron bases where the mutation site located is translated, resulting in early translation termination of DNAH5; The other is the mutation resulting in the deletion of exon76. CONCLUSIONS: The newly identified DNAH5 splicing mutation c.13,338 + 5G > C is involved in the pathogenesis of PCD in the family, and forms a compound heterozygote with the pathogenic variant DNAH5 c.4314delT lead to the pathogenesis of PCD.

Pedigree Analysis of Nonclassical Cholesteryl Ester Storage Disease with Dominant Inheritance in a LIPA I378T Heterozygous Carrier.

BACKGROUND: Cholesterol ester storage disorder (CESD; OMIM: 278,000) was formerly assumed to be an autosomal recessive allelic genetic condition connected to diminished lysosomal acid lipase (LAL) activity due to LIPA gene abnormalities. CESD is characterized by abnormal liver function and lipid metabolism, and in severe cases, liver failure can occur leading to death. In this study, one Chinese nonclassical CESD pedigree with dominant inheritance was phenotyped and analyzed for the corresponding gene alterations. METHODS: Seven males and eight females from nonclassical CESD pedigree were recruited. Clinical features and LAL activities were documented. Whole genome Next-generation sequencing (NGS) was used to screen candidate genes and mutations, Sanger sequencing confirmed predicted mutations, and qPCR detected LIPA mRNA expression. RESULTS: Eight individuals of the pedigree were speculatively thought to have CESD. LAL activity was discovered to be lowered in four living members of the pedigree, but undetectable in the other four deceased members who died of probable hepatic failure. Three of the four living relatives had abnormal lipid metabolism and all four had liver dysfunctions. By liver biopsy, the proband exhibited diffuse vesicular fatty changes in noticeably enlarged hepatocytes and Kupffer cell hyperplasia. Surprisingly, only a newly discovered heterozygous mutation, c.1133T>C (p. Ile378Thr) on LIPA, was found by gene sequencing in the proband. All living family members who carried the p.I378T variant displayed reduced LAL activity. CONCLUSIONS: Phenotypic analyses indicate that this may be an autosomal dominant nonclassical CESD pedigree with a LIPA gene mutation.

Adult type I Gaucher disease with splenectomy caused by a compound heterozygous GBA1 mutation in a Chinese patient: a case report.

Gaucher disease (GD) is an autosomal recessive ailment resulting from glucocerebrosidase deficiency caused by a mutation in the GBA1 gene, leading to multi-organ problems in the liver, spleen, and bone marrow. In China, GD is extremely uncommon and has a lower incidence rate than worldwide. In this study, we report the case of an adult male with an enlarged spleen for 13 years who presented with abdominal distension, severe loss of appetite and weight, reduction of the three-line due to hypersplenism, frequent nosebleeds, and bloody stools. Regrettably, the unexpected discovery of splenic pathology suggestive of splenic Gaucher disease was only made after a splenectomy due to a lack of knowledge about rare disorders. Our patient's delayed diagnosis may have been due to the department where he was originally treated, but it highlights the need for multidisciplinary consultation in splenomegaly of unknown etiology. We then investigated the patient's clinical phenotypes and gene mutation features using genetically phenotypical analysis. The analysis of the GBA1 gene sequence indicated that the patient carried a compound heterozygous mutation consisting of two potentially disease-causing mutations: c.907C > A (p. Leu303Ile) and c.1448 T > C (p. Leu483Pro). While previous research has linked the p. Leu483Pro mutation site to neurologic GD phenotypes (GD2 and GD3), the patients in this investigation were identified as having non-neuronopathic GD1. The other mutation, p. Leu303Ile, is a new GD-related mutation not indexed in PubMed that enriches the GBA1 gene mutation spectrum. Biosignature analysis has shown that both mutations alter the protein's three-dimensional structure, which may be a pathogenic mechanism for GD1 in this patient.

[Evaluation of the Short-Term Efficacy and Safety of Orelabrutinib Combined with High-Dose Methotrexate in the First-line Treatment of Elderly Patients with High Risk Primary Central Nervous System Lymphoma].

OBJECTIVE: To explore the short-term efficacy and adverse reactions of orelabrutinib combined with high-dose methotrexate (HD-MTX) in the first-line treatment of elderly high-risk primary central nervous system lymphoma (PCNSL), as well as the survival of patients. METHODS: Twenty-five elderly patients with high-risk primary central nervous system diffuse large B-cell lymphoma admitted to Fujian Provincial Hospital from June 2016 to June 2022 were enrolled in this study, and complete clinical data from all patients were collected retrospectively, and the cut-off for follow-up was December 2022. 15 patients had received temmozolomide combined with HD-MTX regimen for at least four cycles, sequential lenalidomide maintenance therapy, while 10 patients had received orelabrutinib combined with HD-MTX regimen for at least four cycles, sequential orelabrutinib maintenance therapy. The short-term efficacy and adverse reactions of the two groups of patients after treatment were observed. Kaplan-Meier was used to analyze the progression-free survival (PFS) and time to progression (TTP). RESULTS: The objective response rate (ORR) and 2-year median FPS of orelabrutinib combined with HD-MTX regimen group were similar to the temozolomide combined with HD-MTX regimen group (ORR: 100% vs 66.7%; 2-year median PFS: 16 months vs 15 months, P>0.05). The 2-year median TTP of the orelabrutinib+HD-MTX regimen group was better than that of the temozolomide+HD-MTX regimen group (not reached vs 12 months, P<0.05). There were no significant differences in adverse reactions such as gastrointestinal reactions, bone marrow suppression, liver and kidney damage, cardiotoxicity, pneumonia and bleeding between these two groups (P>0.05). CONCLUSION: For elderly patients with high-risk PCNSL, orelabrutinib combined with HD-MTX has reliable short-term efficacy, good safety, and tolerable adverse reactions, which is worthy of clinical promotion.

T2SS-peptidase XcpA associated with LasR evolutional phenotypic variations provides a fitness advantage to Pseudomonas aeruginosa PAO1.

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa possesses hierarchical quorum sensing (QS) systems. The intricate QS network of P. aeruginosa synchronizes a suite of virulence factors, contributing to the mortality and morbidity linked to the pathogenicity of this bacterium. Previous studies have revealed that variations in the lasR gene are frequently observed in chronic isolates of cystic fibrosis (CF). Specifically, LasRQ45stop was identified as a common variant among CF, lasR mutants during statistical analysis of the clinical lasR mutants in the database. In this study, we introduced LasRQ45stop into the chromosome of P. aeruginosa PAO1 through allelic replacement. The social traits of PAO1 LasRQ45stop were found to be equivalent to those of PAO1 LasR-null isolates. By co-evolving with the wild-type in caseinate broth, elastase-phenotypic-variability variants were derived from the LasRQ45stop subpopulation. Upon further examination of four LasRQ45stop sublines, we determined that the variation of T2SS-peptidase xcpA and mexT genes plays a pivotal role in the divergence of various phenotypes, including public goods elastase secretion and other pathogenicity traits. Furthermore, XcpA mutants demonstrated a fitness advantage compared to parent strains during co-evolution. Numerous phenotypic variations were associated with subline-specific genetic alterations. Collectively, these findings suggest that even within the same parental subline, there is ongoing microevolution of individual mutational trajectory diversity during adaptation.

PhcX Is a LqsR-family response regulator that contributes to Ralstonia solanacearum virulence and regulates multiple virulence factors.

IMPORTANCE: The bacterial wilt caused by the soil-borne phytopathogen Ralstonia solanacearum is one of the most destructive crop diseases. To achieve a successful infection, R. solanacearum has evolved an intricate regulatory network to orchestrate the expression of an arsenal of virulence factors and fine-tune the allocation of energy. However, despite the wealth of knowledge gained in the past decades, many players and connections are still missing from the network. The importance of our study lies in the identification of PhcX, a novel conserved global regulator with critical roles in modulating the virulence and metabolism of R. solanacearum. PhcX affects many well-characterized regulators and exhibits contrasting modes of regulation from the central regulator PhcA on a variety of virulence-associated traits and genes. Our findings add a valuable piece to the puzzle of how the pathogen regulates its proliferation and infection, which is critical for understanding its pathogenesis and developing disease control strategies.

Potential regulatory role of the Nrf2/HMGB1/TLR4/NF-κB signaling pathway in lupus nephritis.

OBJECTIVES: Systemic lupus erythematosus is an autoimmune disease that involves multiple organ systems. One of its major complications, lupus nephritis (LN), is associated with a high mortality rate, and children-onset LN have a more severe course and worse prognosis than adults. Oxidative stress and inflammatory responses are involved in LN development and pathogenesis. Thus, this study aimed to explore the role of signaling regulation of the Nrf2/HMGB1/TLR/NF-κB pathway in LN pathogenesis and unravel the expression of TLR4+CXCR4+ plasma cells subset (PCs) in LN. METHODS: C57BL/6 and MRL/lpr mice were divided into four groups: control, model, vector control, and Nrf2 overexpression groups. The vector control and Nrf2 overexpression groups were injected with adenoviral vectors into the kidney in situ. Pathological changes in kidney tissues were observed by hematoxylin-eosin staining. The expression of Nrf2, HMGB1, TLR4, NF-κB, and downstream inflammatory factors in kidney samples was analyzed by quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The ratios of TLR4+CXCR4+ PC subsets in the blood and kidneys of mice were determined by flow cytometry. RESULTS: In MRL/lpr mice, Nrf2 was downregulated while HMGB1/TLR4/NF-κB pathway proteins were upregulated. Nrf2 overexpression decreased the expression of HMGB1, TLR4, NF-κB, and its downstream inflammatory cytokines (IL-1ß and TNFα). These cytokines were negatively correlated with an increase in Nrf2 content. PC and TLR4 + CXCR4 + PCs in the blood and kidney samples were significantly increased in MRL/lpr mice; however, they were decreased upon Nrf2 overexpression. CONCLUSION: This study showed severe kidney injury in an LN mouse model and an increased ratio of TLR4 + CXCR4 + PCs. Furthermore, we observed that Nrf2 regulates LN immune response through the Nrf2/HMGB1/TLR4/NF-κB pathway, which can be considered an important target for LN treatment. The clinical value of the findings of our study requires further investigation.

Derivative of cinnamic acid inhibits T3SS of Xanthomonas oryzae pv. oryzae through the HrpG-HrpX regulatory cascade.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has a significant impact on rice yield and quality worldwide. Traditionally, bactericide application has been commonly used to control this devastating disease. However, the overuse of fungicides has led to a number of problems such as the development of resistance and environmental pollution. Therefore, the development of new methods and approaches for disease control are still urgent. In this paper, a series of cinnamic acid derivatives were designed and synthesized, and three novel T3SS inhibitors A10, A12 and A20 were discovered. Novel T3SS inhibitors A10, A12 and A20 significantly inhibited the hpa1 promoter activity without affecting Xoo growth. Further studies revealed that the title compounds A10, A12 and A20 significantly impaired hypersensitivity in non-host plant tobacco leaves, while applications on rice significantly reduced symptoms of bacterial leaf blight. RT-PCR showed that compound A20 inhibited the expression of T3SS-related genes. In summary, this work exemplifies the potential of the title compound as an inhibitor of T3SS and its efficacy in the control of bacterial leaf blight.

Clinical phenotype and genetic function analysis of a rare family with hereditary leiomyomatosis and renal cell carcinoma complicated with Birt-Hogg-Dubé syndrome.

To date, over 200 families with hereditary leiomyomatosis and renal cell carcinoma (HLRCC) and over 600 families with Birt-Hogg-Dubé (BHD) syndrome have been reported, with low incidence. Here, we describe a patient with suspected rare HLRCC complicated by BHD syndrome. The proband (II1) had characteristic cutaneous leiomyoma-like protrusions on the neck and back, a left renal mass and multiple right renal, liver and bilateral lung cysts. Three family members (I1, II2, II3) had a history of renal cancer and several of the aforementioned clinical features. Two family members (II1, II3) diagnosed with fumarate hydratase (FH)-deficient papillary RCC via pathological biopsy carried two heterozygous variants: FH (NM_000143.3) missense mutation c.1189G>A (p.Gly397Arg) and FLCN (NM_144997.5) frameshift mutation c.1579_1580insA (p.Arg527Glnfs*75). No family member carrying a single variant had renal tumours. In HEK293T cells transfected with mutant vectors, mRNA and protein expression after FLCN p.Arg527Glnfs*75 and FH p.Gly397Arg mutations were significantly lower than those in wild-type (WT) cells. Cell immunofluorescence showed altered protein localisation and reduced protein expression after FLCN p.Arg527Glnfs*75 mutation. The FH WT was uniformly distributed in the cytoplasm, whereas FH protein expression was reduced after the p.Gly397Arg mutation and scattered sporadically with altered cell localisation. Patients with two variants may have a significantly increased penetrance of RCC.

Pseudomonas aeruginosa based concurrent degradation of beta-cypermethrin and metabolite 3-phenoxybenzaldehyde, and its bioremediation efficacy in contaminated soils.

Beta-cypermethrin is one of the widely used pyrethroid insecticides, and problems associated with the accumulation of its residues have aroused public attention. Thus, there is an urgent need to effectively remove the beta-cypermethrin that is present in the environment. Biodegradation is considered a cost-effective and environmentally friendly method for removing pesticide residues. However, the beta-cypermethrin-degrading microbes that are currently available are not optimal. In this study, Pseudomonas aeruginosa PAO1 was capable of efficiently degrading beta-cypermethrin and its major metabolite 3-phenoxybenzaldehyde in water/soil environments. Strain PAO1 could remove 91.4% of beta-cypermethrin (50 mg/L) in mineral salt medium within 120 h. At the same time, it also possesses a significant ability to metabolize 3-phenoxybenzaldehyde-a toxic intermediate of beta-cypermethrin. The Andrews equation showed that the maximum substrate utilization concentrations of beta-cypermethrin and 3-phenoxybenzaldehyde by PAO1 were 65.3558 and 49.6808 mg/L, respectively. Box-Behnken design-based response surface methodology revealed optimum conditions for the PAO1 strain-based degradation of beta-cypermethrin as temperature 30.6 °C, pH 7.7, and 0.2 g/L inoculum size. The results of soil remediation experiments showed that indigenous micro-organisms helped to promote the biodegradation of beta-cypermethrin in soil, and beta-cypermethrin half-life in non-sterilized soil was 6.84 days. The bacterium transformed beta-cypermethrin to produce five possible metabolites, including 3-phenoxybenzyl alcohol, methyl 2-(4-hydroxyphenoxy)benzoate, diisobutyl phthalate, 3,5-dimethoxyphenol, and 2,2-dimethyl-1-(4-phenoxyphenyl)propanone. Among them, methyl 2-(4-hydroxyphenoxy)benzoate and 3,5-dimethoxyphenol were first identified as the intermediate products during the beta-cypermethrin degradation. In addition, we propose a degradation pathway for beta-cypermethrin that is metabolized by strain PAO1. Beta-cypermethrin could be biotransformed firstly by hydrolysis of its carboxylester linkage, followed by cleavage of the diaryl bond and subsequent metabolism. Based on the above results, P. aeruginosa PAO1 could be a potent candidate for the beta-cypermethrin-contaminated environmental bioremediation.

Quorum quenching by a type IVA secretion system effector.

Proteobacteria primarily utilize acyl-homoserine lactones (AHLs) as quorum-sensing signals for intra-/interspecies communication to control pathogen infections. Enzymatic degradation of AHL represents the major quorum-quenching mechanism that has been developed as a promising approach to prevent bacterial infections. Here we identified a novel quorum-quenching mechanism revealed by an effector of the type IVA secretion system (T4ASS) in bacterial interspecies competition. We found that the soil antifungal bacterium Lysobacter enzymogenes OH11 (OH11) could use T4ASS to deliver the effector protein Le1288 into the cytoplasm of another soil microbiome bacterium Pseudomonas fluorescens 2P24 (2P24). Le1288 did not degrade AHL, whereas its delivery to strain 2P24 significantly impaired AHL production through binding to the AHL synthase PcoI. Therefore, we defined Le1288 as LqqE1 (Lysobacter quorum-quenching effector 1). Formation of the LqqE1-PcoI complex enabled LqqE1 to block the ability of PcoI to recognize/bind S-adenosy-L-methionine, a substrate required for AHL synthesis. This LqqE1-triggered interspecies quorum-quenching in bacteria seemed to be of key ecological significance, as it conferred strain OH11 a better competitive advantage in killing strain 2P24 via cell-to-cell contact. This novel quorum-quenching also appeared to be adopted by other T4ASS-production bacteria. Our findings suggest a novel quorum-quenching that occurred naturally in bacterial interspecies interactions within the soil microbiome by effector translocation. Finally, we presented two case studies showing the application potential of LqqE1 to block AHL signaling in the human pathogen Pseudomonas aeruginosa and the plant pathogen Ralstonia solanacearum.

Identification and characterization of two SERPINC1 mutations causing congenital antithrombin deficiency.

BACKGROUND: Antithrombin (AT) is the main physiological anticoagulant involved in hemostasis. Hereditary AT deficiency is a rare autosomal dominant thrombotic disease mainly caused by mutations in SERPINC1, which was usually manifested as venous thrombosis and pulmonary embolism. In this study, we analyzed the clinical characteristics and screened for mutant genes in two pedigrees with hereditary AT deficiency, and the functional effects of the pathogenic mutations were evaluated. METHODS: Candidate gene variants were analyzed by next-generation sequencing to screen pathogenic mutations in probands, followed by segregation analysis in families by Sanger sequencing. Mutant and wild-type plasmids were constructed and transfected into HEK293T cells to observe protein expression and cellular localization of SERPINC1. The structure and function of the mutations were analyzed by bioinformatic analyses. RESULTS: The proband of pedigree A with AT deficiency carried a heterozygous frameshift mutation c.1377delC (p.Asn460Thrfs*20) in SERPINC1 (NM000488.3), a 1377C base deletion in exon 7 resulting in a backward shift of the open reading frame, with termination after translation of 20 residues, and a different residue sequence translated after the frameshift. Bioinformatics analysis suggests that the missing amino acid sequence caused by the frameshift mutation might disrupt the disulfide bond between Cys279 and Cys462 and affect the structural function of the protein. This newly discovered variant is not currently included in the ClinVar and HGMD databases. p.Arg229* resulted in a premature stop codon in exon 4, and bioinformatics analysis suggests that the truncated protein structure lost its domain of interaction with factor IX (Ala414 site) after the deletion of nonsense mutations. However, considering the AT truncation protein resulting from the p.Arg229* variant loss a great proportion of the molecule, we speculate the variant may affect two functional domains HBS and RCL and lack of the corresponding function. The thrombophilia and decreased-AT-activity phenotypes of the two pedigrees were separated from their genetic variants. After lentiviral plasmid transfection into HEK293T cells, the expression level of AT protein decreased in the constructed c.1377delC mutant cells compared to that in the wild-type, which was not only reduced in c.685C > T mutant cells but also showed a significant band at 35 kDa, suggesting a truncated protein. Immunofluorescence localization showed no significant differences in protein localization before and after the mutation. CONCLUSIONS: The p.Asn460Thrfs*20 and p.Arg229* variants of SERPINC1 were responsible for the two hereditary AT deficiency pedigrees, which led to AT deficiency by different mechanisms. The p.Asn460Thrfs*20 variant is reported for the first time.

RdmA Is a Key Regulator in Autoinduction of DSF Quorum Quenching in Pseudomonas nitroreducens HS-18.

Diffusible signal factor (DSF) represents a family of widely conserved quorum-sensing (QS) signals which regulate virulence factor production and pathogenicity in numerous Gram-negative bacterial pathogens. We recently reported the identification of a highly potent DSF-quenching bacterial isolate, Pseudomonas nitroreducens HS-18, which contains an operon with four DSF-inducible genes, digABCD, or digA-D, that are responsible for degradation of DSF signals. However, the regulatory mechanisms that govern the digA-D response to DSF induction have not yet been characterized. In this study, we identified a novel transcriptional regulator we designated RdmA (regulator of DSF metabolism) which negatively regulates the expression of digA-D and represses DSF degradation. In addition, we found that a gene cluster located adjacent to rdmA was also negatively regulated by RdmA and played a key role in DSF degradation; this cluster was hence named dmg (DSF metabolism genes). An electrophoretic mobility shift assay and genetic analysis showed that RdmA represses the transcriptional expression of the dmg genes in a direct manner. Further studies demonstrated that DSF acts as an antagonist and binds to RdmA, which abrogates RdmA binding to the target promoter and its suppression on transcriptional expression of the dmg genes. Taken together, the results from this study have unveiled a central regulator and a gene cluster associated with the autoinduction of DSF degradation in P. nitroreducens HS-18, and this will aid in the understanding of the genetic basis and regulatory mechanisms that govern the quorum-quenching activity of this potent biocontrol agent. IMPORTANCE DSF family quorum-sensing (QS) signals play important roles in regulation of bacterial physiology and virulence in a wide range of plant and human bacterial pathogens. Quorum quenching (QQ), which acts by either degrading QS signals or blocking QS communication, has proven to be a potent disease control strategy, but QQ mechanisms that target DSF family signals and associated regulatory mechanisms remain largely unknown. Recently, we identified four autoinduced DSF degradation genes (digABCD) in P. nitroreducens HS-18. By using a combination of transcriptome and genetic analysis, we identified a central regulator that plays a key role in autoinduction of dig expression, as well as a new gene cluster (dmgABCDEFGH) involved in DSF degradation. The significance of our study is in unveiling the autoinduction mechanism that governs DSF signal quorum quenching for the first time, to our knowledge, and in identification of new genes and enzymes responsible for DSF degradation. The findings from this study shed new light on our understanding of the DSF metabolism pathway and the regulatory mechanisms that modulate DSF quorum quenching and will provide useful clues for design and development of a new generation of highly potent QQ biocontrol agents against DSF-mediated bacterial infections.

A novel bacterial strain Burkholderia sp. F25 capable of degrading diffusible signal factor signal shows strong biocontrol potential.

Vast quantities of synthetic pesticides have been widely applied in various fields to kill plant pathogens, resulting in increased pathogen resistance and decreased effectiveness of such chemicals. In addition, the increased presence of pesticide residues affects living organisms and the environment largely on a global scale. To mitigate the impact of crop diseases more sustainably on plant health and productivity, there is a need for more safe and more eco-friendly strategies as compared to chemical prevention. Quorum sensing (QS) is an intercellular communication mechanism in a bacterial population, through which bacteria adjust their population density and behavior upon sensing the levels of signaling molecules in the environment. As an alternative, quorum quenching (QQ) is a promising new strategy for disease control, which interferes with QS by blocking intercellular communication between pathogenic bacteria to suppress the expression of disease-causing genes. Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is associated with the diffusible signal factor (DSF). As detailed in this study, a new QQ strain F25, identified as Burkholderia sp., displayed a superior ability to completely degrade 2 mM of DSF within 72 h. The main intermediate product in the biodegradation of DSF was identified as n-decanoic acid, based on gas chromatography-mass spectrometry (GC-MS). A metabolic pathway for DSF by strain F25 is proposed, based on the chemical structure of DSF and its intermediates, demonstrating the possible degradation of DSF via oxidation-reduction. The application of strain F25 and its crude enzyme as biocontrol agents significantly attenuated black rot caused by Xcc, and inhibited tissue maceration in the host plant Raphanus sativus L., without affecting the host plant. This suggests that agents produced from strain F25 and its crude enzyme have promising applications in controlling infectious diseases caused by DSF-dependent bacterial pathogens. These findings are expected to provide a new therapeutic strategy for controlling QS-mediated plant diseases.

Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.

Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.