Soluble Programmed Cell Death Ligand-1 (sPD-L1) Levels in Various Cancer Types and Normal Populations.

BACKGROUND: To date, PD-L1 expression in tumor tissue, assessed by immunohistochemical stain, is clinically applicable as a predictive companion biomarker for PD-1/PD-L1 inhibitor which has been highlighted over the past several years. Before blood-based sPD-L1 enters clinical use, it is critical to establish the reference range. This study was designed to investigate soluble sPD-L1 levels in various cancer patients and normal population. METHODS: For the detection of sPD-L1, 4 cancer groups (hepatocellular carcinoma, lung cancer, bladder cancer, gastric cancer) and healthy volunteers' samples were analyzed using an ELISA kit. Using a receiver operating characteristic curve, optimal sPD-L1 cutoff levels were determined. RESULTS: The mean serum sPD-L1 level of the normal population was 59.97 pg/mL (range; 23.780 - 115.2 pg/mL). In various cancer types, serum sPD-L1 levels ranged from 38.696 pg/mL to 228.77 pg/mL, and cutoff values under AUC ranged from 60.307 pg/mL to 64.371 pg/mL. CONCLUSIONS: sPD-L1 can be used as a screening biomarker in various cancer patients referring to optimal cutoff levels suggested.

Trend Analysis of the Profiles of 12 Sexually Transmitted Disease Pathogens in the Republic of Korea in 2019.

BACKGROUND: Sexually transmitted diseases (STDs) are common infectious diseases in humans transmitted through unprotected sexual activities. In South Korea, despite the high annual incidence of STDs, detailed examinations of pathogen-specific factors and causes for delays in diagnosis and treatment are still lacking. Furthermore, STD prevalence patterns and important pathogen-specific factors remain unclear. Herein, we retrospectively analyzed the epidemiology of STDs in South Korea in 2019 by analyzing the association of pathogen-specific infection patterns with factors such as sex, age, region, and month. METHODS: We obtained the STD test results of 172,973 individuals from the Seoul Clinic Laboratory in 2019, most of whom had multiple infections; hence, 275,296 STD-positive cases were included in this analysis. Through deoxyribonucleic acid (DNA) amplification, they were categorized by pathogen type. Subsequently, they were further classified by month, region, and age while concurrently being stratified according to sex. RESULTS: Among the 12 pathogens detected in this study, Gardnerella vaginalis had the highest prevalence, with 92,490 cases in both sex groups; moreover, many of them were concurrently infected by two or more pathogens. The prevalence of STDs did not differ according to month or region. Conversely, the pathogen-specific prevalence rates significantly differed according to age. Older adults had higher prevalence rates of Chlamydia trachomatis, Trichomonas vaginalis, Candida albicans, and herpes simplex virus type 1 infections than younger adults. CONCLUSION: These pathogen-specific prevalence patterns provide information that helps to understand population vulnerability according to region and age and helps develop STD prevention and treatment strategies in South Korea.

Two DNA Methyltransferases for Site-Specific 6mA and 5mC DNA Modification in Xanthomonas euvesicatoria.

Xanthomonas euvesicatoria (Xe) is a gram-negative phytopathogenic bacterium that causes bacterial spot disease in tomato/pepper leading to economic losses in plantations. DNA methyltransferases (MTases) are critical for the survival of prokaryotes; however, their functions in phytopathogenic bacteria remain unclear. In this study, we characterized the functions of two putative DNA MTases, XvDMT1 and XvDMT2, in Xe by generating XvDMT1- and XvDMT2-overexpressing strains, Xe(XvDMT1) and Xe(XvDMT2), respectively. Virulence of Xe(XvDMT2), but not Xe(XvDMT1), on tomato was dramatically reduced. To postulate the biological processes involving XvDMTs, we performed a label-free shotgun comparative proteomic analysis, and results suggest that XvDMT1 and XvDMT2 have distinct roles in Xe. We further characterized the functions of XvDMTs using diverse phenotypic assays. Notably, both Xe(XvDMT1) and Xe(XvDMT2) showed growth retardation in the presence of sucrose and fructose as the sole carbon source, with Xe(XvDMT2) being the most severely affected. In addition, biofilm formation and production of exopolysaccharides were declined in Xe(XvDMT2), but not Xe(XvDMT1). Xe(XvDMT2) was more tolerant to EtOH than Xe(XvDMT1), which had enhanced tolerance to sorbitol but decreased tolerance to polymyxin B. Using single-molecule real-time sequencing and methylation-sensitive restriction enzymes, we successfully predicted putative motifs methylated by XvDMT1 and XvDMT2, which are previously uncharacterized 6mA and 5mC DNA MTases, respectively. This study provided new insights into the biological functions of DNA MTases in prokaryotic organisms.

Profiling Differentially Abundant Proteins by Overexpression of Three Putative Methyltransferases in Xanthomonas axonopodis pv. glycines.

Methyltransferases (MTases) are enzymes that modify specific substrates by adding a methyl group using S-adenosyl-l-methionine. Functions of MTases have been extensively studied in eukaryotic organisms and animal pathogenic bacteria. Despite their importance, mechanisms underlying MTase function in plant pathogenic bacteria have not been studied in depth, as is the case of Xanthomonas axonopodis pv. glycines (Xag) that causes bacterial pustule disease in soybean crops worldwide. Here, the association between Xag proteome alterations and three MTase-overexpressing strains, Xag(XgMT1), Xag(XgMT2), and Xag(XgMT3), compared to Xag carrying an empty vector, Xag(EV) is reported. Using label-free shotgun comparative proteomic analysis, proteins are identified in all three biological replicates of the four strains and ranged from 1004 to 1082. In comparative analyses, 124, 135, and 134 proteins are differentially changed (over twofold) by overexpression of XgMT1, XgMT2, and XgMT3, respectively. These proteins are also categorized using cluster of orthologous group (COG) analyses, allowing postulation of biological mechanisms associated with three MTases in Xag. COGs reveal that the three MTases may play distinct roles, although some functions may overlap. These results are expected to allow new insight into understanding and predicting the biological functions of MTases in plant pathogenic bacteria. Data are available via ProteomeXchange (Identifier PXD012590).

Functional characterization of a putative DNA methyltransferase, EadM, in Xanthomonas axonopodis pv. glycines by proteomic and phenotypic analyses.

Xanthomonas axonopodis pv. glycines (Xag) is a phytopathogenic bacterium causing bacterial pustule disease in soybean. Functions of DNA methyltransferases have been characterized in animal pathogenic bacteria, but are poorly understood in plant pathogens. Here, we report that functions of a putative DNA methyltransferase, EadM, in Xag. An EadM-overexpressing strain, Xag(EadM), was less virulent than the wild-type carrying an empty vector, Xag(EV). Interestingly, the viable cell numbers of Xag(EadM) were much lower (10-fold) than those of Xag(EV) at the same optical density. Comparative proteomic analysis revealed that proteins involved in cell wall/membrane/envelope and iron-transport were more abundant. Based on proteomic analysis we carried out diverse phenotypic assays. Scanning electron microscopy revealed abnormal bacterial envelopes in Xag(EadM). Additionally, Xag(EadM) showed decreased stress tolerance against ciprofloxacin and sorbitol, but enhanced resistance to desiccation. Exopolysaccharide production in Xag(EadM) was also decreased. Production of siderophores, which are iron-chelators, was much higher in Xag(EadM). As in Xag, Escherichia coli expressing EadM showed significantly reduced (1000-fold) viable cell numbers at the same optical density. Thus, EadM is associated with virulence, envelope biogenesis, stress tolerance, exopolysaccharide production, and siderophore production. Our results provide valuable and fundamental information regarding DNA methyltransferase functions and their related cellular mechanisms in plant pathogenic bacteria.

A LysR-Type Transcriptional Regulator LcrX Is Involved in Virulence, Biofilm Formation, Swimming Motility, Siderophore Secretion, and Growth in Sugar Sources in Xanthomonas axonopodis Pv. glycines.

Xanthomonas axonopodis pv. glycines (Xag) is a Gram-negative bacterium that causes bacterial pustule disease in soybean. To acclimate to new environments, the expression of genes in bacteria is controlled directly or indirectly by diverse transcriptional factors. Among them, LysR type transcriptional regulators are well-characterized and abundant in bacteria. In a previous study, comparative proteomic analysis revealed that LysR type carbohydrate-related transcriptional regulator in Xag (LcrX) was more abundant in XVM2, which is a minimal medium, compared with a rich medium. However, the functions of LcrX in Xag have not been characterized. In this study, we generated an LcrX-overexpressing strain, Xag(LcrX), and the knockout mutant strain, XagΔlcrX(EV), to elucidate the functions of LcrX. Bacterial multiplication of Xag(LcrX) in soybean was significantly impaired, indicating that LcrX is related to virulence. Comparative proteomic analysis revealed that LcrX is mainly involved in carbohydrate metabolism/transport and inorganic ion transport/metabolism. Based on the results of proteomics analysis, diverse phenotypic assays were carried out. A gel electrophoresis mobility shift assay demonstrated that LcrX specifically bound to the putative promoter regions of genes encoding putative fructose 1,6-bisphosphatase and protease. Through a 96-well plate assay under various conditions, we confirmed that the growth of Xag(LcrX) was dramatically affected in the presence of various carbon sources, while the growth of XagΔlcrX(EV) was only slightly changed. Biofilm formation activity was reduced in Xag(LcrX) but enhanced in XagΔlcrX(EV). The production of siderophores was also decreased in Xag(LcrX) but not altered in XagΔlcrX(EV). In contrast, LcrX was not associated with exopolysaccharide production, protease activity, or bacterial motility. These findings provide new insights into the functions of a carbohydrate-related transcriptional regulator in Xag.

Elucidating Functions of FleQ in Xanthomonas oryzae pv. oryzae by Comparative Proteomic and Phenotypic Analyses.

To acclimate to different environments, gene expression has to be controlled using diverse transcriptional activators. FleQ activates σ54-dependent transcription initiation and regulates flagellar biosynthesis and other mechanisms in several bacteria. Xanthomonas oryzae pv. oryzae (Xoo), which is a causal agent of bacterial leaf blight on rice, lacking FleQ loses swimming motility and virulence is not altered. However, other biological mechanisms related with FleQ in Xoo are unknown. In this study, we generated the FleQ-overexpressing strain, Xoo(FleQ), and knockout mutant, XooΔfleQ. To predict the mechanisms affected by FleQ, label-free shotgun comparative proteomics was carried out. Based on proteomic results, we performed diverse phenotypic assays. Xoo(FleQ) had reduced ability to elicit disease symptoms and exopolysaccharide production. Additionally, the ability of XooΔfleQ(EV) (empty vector) and Xoo(FleQ) to form biofilm was decreased. Swarming motility of XooΔfleQ(EV) was abolished, but was only reduced for Xoo(FleQ). Additionally, abnormal twitching motility was observed in both strains. Siderophore production of Xoo(FleQ) was enhanced in iron-rich conditions. The proteomic and phenotypic analyses revealed that FleQ is involved in flagellar-dependent motility and other mechanisms, including symptom development, twitching motility, exopolysaccharide production, biofilm formation, and siderophore production. Thus, this study provides fundamental information about a σ54-dependent transcription activator in Xoo.

Deciphering the functions of the outer membrane porin OprBXo involved in virulence, motility, exopolysaccharide production, biofilm formation and stress tolerance in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a Gram-negative bacterium causing bacterial leaf blight disease in rice. Previously, proteomic analysis has shown that the outer membrane protein B in Xoo (OprBXo) is more abundant in the wildtype strain than is the outer membrane protein 1 in the Xoo (Omp1X) knockout mutant. OprBXo shows high homology with OprB, which has been well characterized as a carbohydrate-selective porin in X. citri ssp. citri and Pseudomonas species. However, the functions of OprBXo in Xoo have not yet been documented. To elucidate the functions of OprBXo, we generated the OprBXo-overexpressing mutant, Xoo(OprBXo), and the knockout mutant, XooΔoprBXo(EV). We found that the virulence and migration of Xoo(OprBXo), but not XooΔoprBXo(EV), were markedly reduced in rice. To postulate the mechanisms affected by OprBXo, comparative proteomic analysis was performed. Based on the results of proteomics, we employed diverse phenotypic assays to characterize the functions of OprBXo. Abnormal twitching motility and reduction in swarming motility were observed in Xoo(OprBXo). Moreover, Xoo(OprBXo) decreased, but XooΔoprBXo(EV) enhanced, exopolysaccharide production and biofilm formation. The chemotactic ability of XooΔoprBXo(EV) was dramatically lower than that of Xoo(EV) in the presence of glucose and xylose. Xoo(OprBXo) was resistant to sodium dodecylsulphate and hydrogen peroxide, but XooΔoprBXo(EV) was highly sensitive compared with Xoo(EV). Thus, OprBXo is not only essential for chemotaxis and stress tolerance, but also for motility, biofilm formation and exopolysaccharide production, which may contribute to the virulence of Xoo. These results will lead to new insights into the functions of a sugar-selective porin in Xoo.

Functional and Proteomic Analyses Reveal That ScpBXv Is Involved in Bacterial Growth, Virulence, and Biofilm Formation in Xanthomonas campestris pv. vesicatoria.

Segregation and condensation protein B (ScpB) is essential for replication and segregation in living organisms. Here, we reported the functions of ScpBXv (ScpB-like protein in Xanthomonas campestris pv. vesicatoria) using phenotypic and proteomic analyses. Growth of XcvΔscpBXv (ScpBXv knockout mutant) was reduced under both slow and fast growth conditions in rich medium, but comparable to this of the wild-type in plant-mimic conditions. Interestingly, the mutant was significantly less virulent than the wild-type in tomato, indicating that ScpBXv is involved in virulence. To investigate ScpBXv-associated mechanisms, comparative proteomic analyses were carried out and the abundance of 187 proteins was altered. Among them, diverse transcriptional regulators involved in biofilm formation and virulence were abundant in the wild-type. We further showed that biofilm formation of XcvΔscpBXv was reduced. This study provides new insights into the functions of ScpBXv in bacterial replication and biofilm formation, which may contribute to the virulence of Xcv.

Comparative Proteomic Analysis of Three Xanthomonas spp. Cultured in Minimal and Rich Media.

Bacteria change their gene expression when exposed to different nutrient conditions. The levels of proteins do not always correlate with those of RNAs, hence proteomic analysis is required for understanding how bacteria adapt to different conditions. Herein, differentially abundant proteins from Xanthomonas oryzae pv. oryzae (Xoo), X. campestris pv. vesicatoria (Xcv), and X. axonopodis pv. glycines (Xag), which were cultured in rich media and in minimal media, were determined using label-free shotgun proteomic analysis and clusters of orthologous groups classification. The detected proteins from all three species ranged from 1190 to 1187. Among them, 702, 584, and 529 proteins from Xoo, Xcv, and Xag, respectively, were more (> twofold) abundant depending on the media, indicating that about 11.4-13.8% of proteins from the three species were differentially expressed. The levels of abundant proteins in minimal media were significantly higher than those in rich media for all three species, demonstrating how Xanthomonas species actively change their protein expression in different nutrient conditions. These results will lead to new insights in elucidation of cellular mechanisms involved in virulence and adaption of bacteria to harsh environments for further studies. The MS proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD006310.

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana.

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium-mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa, Psa-NZ V13 and Psa-NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana. Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium-mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1, NDR1, or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana. In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta.

Methylome Analysis of Two Xanthomonas spp. Using Single-Molecule Real-Time Sequencing.

Single-molecule real-time (SMRT) sequencing allows identification of methylated DNA bases and methylation patterns/motifs at the genome level. Using SMRT sequencing, diverse bacterial methylomes including those of Helicobacter pylori, Lactobacillus spp., and Escherichia coli have been determined, and previously unreported DNA methylation motifs have been identified. However, the methylomes of Xanthomonas species, which belong to the most important plant pathogenic bacterial genus, have not been documented. Here, we report the methylomes of Xanthomonas axonopodis pv. glycines (Xag) strain 8ra and X. campestris pv. vesicatoria (Xcv) strain 85-10. We identified N6-methyladenine (6mA) and N4-methylcytosine (4mC) modification in both genomes. In addition, we assigned putative DNA methylation motifs including previously unreported methylation motifs via REBASE and MotifMaker, and compared methylation patterns in both species. Although Xag and Xcv belong to the same genus, their methylation patterns were dramatically different. The number of 4mC DNA bases in Xag (66,682) was significantly higher (29 fold) than in Xcv (2,321). In contrast, the number of 6mA DNA bases (4,147) in Xag was comparable to the number in Xcv (5,491). Strikingly, there were no common or shared motifs in the 10 most frequently methylated motifs of both strains, indicating they possess unique species- or strain-specific methylation motifs. Among the 20 most frequent motifs from both strains, for 9 motifs at least 1% of the methylated bases were located in putative promoter regions. Methylome analysis by SMRT sequencing technology is the first step toward understanding the biology and functions of DNA methylation in this genus.

Deciphering the Role of Tyrosine Sulfation in Xanthomonas oryzae pv. oryzae Using Shotgun Proteomic Analysis.

A bacterial tyrosine sulfotransferase, RaxST, is required for activation of rice XA21-mediated immunity, and it catalyzes sulfation of tyrosine residues of Omp1X and RaxX in Xanthomonas oryzae pv. oryzae, a causal agent of bacterial blight in rice. Although RaxST is biochemically well-characterized, biological functions of tyrosine sulfation have not been fully elucidated. We compared protein expression patterns between the wildtype and a raxST knockout mutant using shotgun proteomic analysis. Forty nine proteins displayed a more than 1.5-fold difference in their expression between the wildtype and the mutant strains. Clusters of orthologous groups analysis revealed that proteins involved in cell motility were most abundant, and phenotypic observation also showed that the twitching motility of the mutant was dramatically changed. These results indicate that tyrosine sulfation by RaxST is essential for Xoo movement, and they provide new insights into the biological roles of RaxST in cellular processes.

Proteomic and functional analyses of a novel porin-like protein in Xanthomonas oryzae pv. oryzae.

Proteomic analysis is a useful technique for postulating and elucidating protein functions. In the present work, a shotgun proteomic analysis was used to identify functions of the PXO_03968 gene (previously known as the ax21) from Xanthomonas oryzae pv. oryzae (Xoo), a causal agent for bacterial blight disease in rice. Structural prediction performed on the protein sequence encoded by PXO_03968 reveals that it encodes a putative porin-like protein, possessing a ß-barrel domain with 10 ß-strands and a signal peptide at the N-terminus. We renamed the gene as an omp1X (outer membrane protein 1 in Xoo), generated its knock out mutant (XooΔomp1X), and compared the protein expression level in the mutant to that in the wild type. A total of 106 proteins displayed more than 1.5-fold difference in expression between the mutant and the wild type strains. COG analysis revealed that these proteins are involved in cell motility as well as signal transduction. In addition, phenotypic analysis demonstrated that motility and biofilm formation in XooΔomp1X are lower than the wild type. These results provide new insights into the functions of outer membrane proteins in Gram-negative bacteria.

Functional and proteomic analyses reveal that wxcB is involved in virulence, motility, detergent tolerance, and biofilm formation in Xanthomonas campestris pv. vesicatoria.

The bacterial envelope possesses diverse functions, including protection against environmental stress and virulence factors for host infection. Here, we report the function of wxcB in Xanthomonas campestris pv. vesicatoria (Xcv), a causal agent of bacterial leaf spot disease in tomato and pepper. To characterize roles of wxcB, we generated a knockout mutant (XcvΔwxcB) and found that the virulence of the mutant was weaker than that of the wild type in tomato plants. To predict the mechanism affected by wxcB, we compared protein expressions between the wild type and the mutant. Expression of 152 proteins showed a greater than 2-fold difference. Proteins involved in motility and cell wall/membrane were the most abundant. Through phenotypic assays, we further demonstrated that the mutant displayed reduced motility and tolerance to treatment, but it showed increased biofilm formation. Interestingly, the LPS profile was unchanged. These results lead to new insights into the functions of wxcB that is associated with cell wall/membrane functions, which contributes to pathogen virulence.

Draft genome sequence of Xanthomonas axonopodis pv. glycines 8ra possessing transcription activator-like effectors used for genetic engineering.

Xanthomonas axonopodis pv. glycines 8ra is a causal agent of bacterial pustule disease in soybean. This bacterium possesses transcription activator-like (TAL) effectors which are useful for genetic/protein engineering applications in higher organisms including plants and humans. Here, we report that the draft genome sequence consists of 5,337,885-bp double-stranded DNA encoding 4674 open reading frames (ORFs) in 13 different contigs. This genome sequence would be useful in applications of TAL effectors in genetic engineering and in elucidating virulence factors against plants.