MCP Genes Research Articles

Sequence analysis of isolated strains of herpes zoster virus among patients with shingles.

Herpes zoster, or shingles, is caused by the varicella-zoster virus (VZV), which initially presents as chickenpox in children. VZV is a global health concern, especially in winter and spring, affecting 10-20% of adults over 50 and posing a 30% risk for the general population. This study used PCR to detect VZV, confirming results with duplicated DNA samples and identifying 234 bp fragments by targeting the gpB gene. This study examined 50 herpes zoster cases from October 2020 to April 2021, involving 30 males and 20 females aged 10 to 90, diagnosed by dermatologists. Data were collected via a questionnaire. PCR detected VZV by amplifying the gpB and MCP genes from skin lesion samples. Six positive 234-bp PCR products were sequenced at Macrogen Inc. in Seoul, South Korea. Six DNA samples with 234 bp amplicons were sequenced, showing 99-100% similarity to human alpha herpesvirus sequences in the gpB gene. NCBI BLAST matched these sequences to a reference (GenBank acc. MT370830.1), assigning accession numbers LC642111, LC642112, and LC642113. Eight nucleic acid substitutions caused amino acid changes in the gpB protein: isoleucine to threonine, serine to isoleucine, and threonine to Proline. These variants were deposited in NCBI GenBank as gpB3 samples. The study found high sequence similarity to known VZV sequences, identifying six nucleic acid variations and eight SNPs. Notable amino acid changes in the gpB protein were deposited in NCBI GenBank as the gpB3 sample.

Characterization and phylogenetic analysis of bovine gammaherpesvirus 4 isolated in China, 2022.

Bovine gammaherpesvirus 4 (BoHV-4) is a common virus detected in bovine with respiratory disease worldwide. In this study, we identified and characterized a novel BoHV-4 strain, referred as HB-ZJK, in vaginal swabs collected from cattle in China, 2022. The long unique region (LUR) of HB-ZJK is 10,9811bp in length. It shares 99.17% to 99.38% nucleotide identity to five BoHV-4 strains available in GenBank and the highest similarity was seen with BoHV-4V. test (JN133502.1) strain (99.38%). Mutations, insertions or deletions were observed mainly in HB-ZJK gB (ORF8), TK (ORF21), gH (ORF22), MCP (ORF25), PK (ORF36), gM (ORF39), and gL (ORF47) genes compared to its genomic coordinates. Phylogenetic analyses of gB and TK genes showed that HB-ZJK clustered with China 512 (2019), B6010 (2009), and J4034 (2009) strains, demonstrating that the isolated HB-ZJK belongs to genotype 1. This is the first report that has revealed a comprehensive genome profile of BoHV-4 strain in China. This study will provide foundation for epidemiological investigations of BoHV-4 and contribute to the molecular and pathogenic studies of BoHV-4.

Mutations in the Alternative Complement Pathway in Multiple Myeloma Patients with Carfilzomib-Induced Thrombotic Microangiopathy

IntroductionVascular endothelial injury related to treatment with proteasome inhibitors (PI) has been previously described. Carfilzomib is an irreversible PI and has been associated with cardiovascular toxicity, suggesting increased risk of endothelial injury. Thrombotic microangiopathy (TMA) has been described in multiple myeloma (MM) patients receiving PIs; more often with carfilzomib. In the pediatric transplant population, increased risk of TMA was related to heterozygous mutation in the alternative complement pathway. The homozygous deletions enable uncontrolled complement activation and was found three times more frequently in TMA population. We hypothesized that MM patients with complement mutation are at increased risk of PI-related TMA.Materials and MethodsWe identified ten cases of renal TMA in MM patients receiving carfilzomib from two medical institutions in Greece and in the United States. TMA was diagnosed based on either renal biopsy or acute kidney injury, new-onset anemia, thrombocytopenia and increased LDH in the absence of disease progression. We performed targeted sequencing of the twelve genes implicated in TMA: CFH, CFI, MCP, CFB, CFHR5, C3, THBD, DGKE, PLG, ADAMTS13, MMACHC and G6PD genes. Multiplex Ligation-Dependent Probe Amplification (MLPA) was performed for the analysis of the CFH-CFHR5 region.ResultsPatient characteristics and laboratory values at TMA diagnosis are presented in Table 1. The median age of patients was 68 years (range, 47-73), with slight male predominance (60%). Median laboratory values at diagnosis included hemoglobin 9.35 g/dL, platelet count 26,500 x 10 6/L, LDH 356,5 U/L and creatinine 2.25 mg/dL. Patients were treated with carfilzomib doses ranging 20-70 mg/m 2. Regimens included carfilzomib-dexamethasone (Kd, 7 patients), carfilzomib-lenalidomide-dexamethasone (KRd, 1 patient), carfilzomib-pomalidomide-dexamethasone (KPd, 1 patient), and carfilzomib-daratumumab-dexamethasone (DaraKd, 1 patient). All patients had previously received at least one line of therapy and seven patients had previously undergone autologous stem cell transplant (ASCT), 1-12 years prior to TMA diagnosis. The median time between carfilzomib initiation and TMA diagnosis was 4.5 months (range, 1-60 months). Diagnosis was confirmed with renal biopsy in four cases. Carfilzomib was discontinued in all patients and five patients were treated with plasma exchange (PLEX) while one patient received eculizumab. Seven patients demonstrated clinical improvement and resolution of TMA at 1 year after discontinuation of carfilzomib. Two patients progressed to end stage renal disease (ESRD) requiring intermittent hemodialysis, and one patient developed multiorgan failure. Results of genetic panel are shown in Table 2. Deletions of the CFHR3-CFHR5 region were present in seven cases (70%): two patients carrying a homozygous deletion of CFHR3-CFHR1, four patients with a heterozygous deletion of CFHR3-CFHR1, and one patient with heterozygous deletion of CFHR1-CFHR4. Direct gene sequencing revealed identifiable mutations in CD46 (MCP) and CFHR5 in two distinct patients. The functional correlation and clinical significance are yet to be investigated.ConclusionsIn our cohort of ten patients of carfilzomib-induced TMA, deletions of CFHR3-CFHR5 occurred frequently (70%). In the setting of carfilzomib use, heterozygous CFHR3-CFHR1 deletion may represent a risk factor for the development of TMA. Our data set the bases for larger studies assessing complement mutation as a predisposing factor for PI-induced TMA. [Display omitted] DisclosuresKastritis: Amgen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding. Dimopoulos: Janssen: Honoraria; BMS: Honoraria; Takeda: Honoraria; Beigene: Honoraria; Amgen: Honoraria. Richardson: AbbVie: Consultancy; Secura Bio: Consultancy; Sanofi: Consultancy; GlaxoSmithKline: Consultancy; Karyopharm: Consultancy, Research Funding; Regeneron: Consultancy; AstraZeneca: Consultancy; Oncopeptides: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Celgene/BMS: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Red sea bream iridovirus infection in cage farmed Asian sea bass (Lates calcarifer): Insights into the pathology, epizootiology, and genetic diversity

Red sea bream iridovirus (RSIV) is an emerging viral pathogen having significant socio-economic and environmental impacts on marine and brackish aquaculture systems. The study documents certain interesting findings of an RSIV disease outbreak that caused 50% cumulative mortality among cage farmed Asian sea bass in India. The confirmatory diagnosis was made based on internationally approved protocols. The affected fish showed splenomegaly, anaemia, and abnormally enlarged cells within the spleen, liver, kidney, and brain. The consistent presence of two fish pathogens, viz., Vibrio alginolyticus and Vibrio fluvialis, in the liver, along with characteristic vibrio-induced pathologies were noted, indicating the possible role of these bacterial pathogens in RSIV pathogenesis during the outbreak. More strikingly, the surviving fish were found to carry the virus even after two months post-outbreak, warranting future investigations on the carrier status of RSIV survivors. Transmission electron microscopy in the survivors demonstrated atypical virus particles within the degenerated cells of the vital organs, suggesting the role of host immune response in preventing the maturation of RSIV particles in the survivors. Phylogram based on MCP and ATPase genes demonstrated the spread of the same RSIV clone across different countries, regardless of the fish species. Results also revealed the presence of two RSIV subtypes. Both subtypes were mainly distributed in marine demersal fishes of Asian countries. Presently, subtype 2 is the most frequently reported one. Subtype 1 revealed higher genetic diversity with a wider geographical spread and host range. Molecular analyses revealed that MCP and ATPase genes could be targeted in RSIV subtyping, with MCP as a better candidate. A purifying selection was observed on the MCP gene that can be applied for diagnostic protocols. In short, the study generates certain thought-provoking insights on RSIV through the analyses of a recent outbreak along with the genetic and epizootiological data of global strains. Further, the paper also forms the first report on the presence of RSIV in surviving fish post-disease outbreak in the absence of clinical signs. Altogether, the results highlight the necessity of strengthening quarantine and RSIV diagnostic measures to have sustainable aquaculture practices.

Molecular evidence for homologous strains of infectious spleen and kidney necrosis virus (ISKNV) genotype I infecting inland freshwater cultured Asian sea bass (Lates calcarifer) in Thailand.

Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch's postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.

Vfr targets promoter of genes encoding methyl-accepting chemotaxis protein in Pseudomonas syringae pv. tabaci 6605

Virulence factor regulator (Vfr) is an indispensable transcription factor in the expression of virulence in the phytopathogenic bacteria Pseudomonassyringae. However, the function of Vfr is not known so far. The deletion of vfr resulted in the loss of surface swarming motility and reduced the virulence in P. syringae pv. tabaci (Pta) 6605. In order to identify the target genes of Vfr, we screened the sequences that bind to Vfr by chromatin immune precipitation (ChIP) and sequencing methods using the closely related bacterium P. syringae pv. syringae (Pss) B728a. For this purpose we first generated a strain that possesses the recombinant gene vfr::FLAG in Pss B728a, and performed ChIP using an anti-FLAG antibody. Immunoprecipitated DNA was purified and sequenced with Illumina HiSeq. The Vfr::FLAG-specific peaks were further subjected to an electrophoresis mobility-shift assay, and the promoter regions of locus tag for Psyr_0578 , Psyr_1776, and Psyr_2237 were identified as putative target genes of Vfr. These genes encode plant pathogen–specific methyl-accepting chemotaxis proteins (Mcp). These mcp genes seem to be involved in the Vfr-regulated expression of virulence.

Association between thrombotic microangiopathy and activated alternative complement pathway in malignant nephrosclerosis.

Malignant nephrosclerosis, defined as renal microangiopathy in the setting of severe hypertension, remains a critical renal emergency leading to end-stage renal disease despite aggressive anti-hypertensive treatment. Recently, activation of the complement alternative pathway (AP) has been reported to play a prominent role in the pathogenesis of malignant nephrosclerosis. However, subsequent study failed to recapitulate the findings of genetic complement abnormalities in the disease. This study aimed to determine the presence of AP activation and genetic complement defects and establish their correlations to renal microangiopathy lesions, clinical features and prognosis in patients with malignant nephrosclerosis. Fifty patients with malignant hypertension and concomitant thrombotic microangiopathy (TMA) proven by renal biopsy were investigated; 25 cases of kidney donors who received zero-hour allograft biopsies were used as normal controls. Various renal TMA lesions in patients with malignant nephrosclerosis were reviewed and evaluated using a semi-quantitative scoring system. Deposition of C5b-9, C3a, C5a, C4d and mannose-binding lectin was assessed by immunohistochemistry. Co-localization of C5b-9 and CD34 was detected by confocal microscopy. Complement factor B (FB), factor P (FP; properdin), factor D (FD), factor H (FH), C3a and C5a levels were quantified by enzyme-linked immonosorbent assay in plasma and urine samples of patients with malignant nephrosclerosis and controls. Genetic abnormalities of complement components were analysed by whole-exome sequencing. Renal biopsies of malignant nephrosclerosis showed identical histopathological and ultrastructural features to atypical haemolytic uraemic syndrome. C5b-9, C3a and C5a deposits were found along the walls of arteries/arterioles and glomerular capillaries and localized in the endothelial cells. Elevated plasma and urinary levels of FB, FP, FD, C3a and C5a as well as decreased FH levels were observed in patients with malignant nephrosclerosis compared with normal controls. The urinary levels of complement AP components, but not the plasma levels, were correlated with renal functions, prognosis and active TMA lesions except for arteriolar thrombi. Finally, mutations of the MCP, CFB, CFH and CFHR5 genes were identified in 8 of 20 patients with malignant nephrosclerosis. Aberrant complement AP dysregulation was demonstrated and associated with the activity, severity and renal outcomes of malignant nephrosclerosis. This observation warrants screening for complement defects in patients with malignant nephrosclerosis for the potential use of complement regulators and also highlights the need for further investigation of the precise role of AP in the pathogenesis of the disease.

Characterization and Genome Analysis of a Novel Marine Alteromonas Phage P24.

Although Alteromonas is ubiquitous in the marine environment, very little is known about Alteromonas phages, with only ten, thus far, being isolated and reported on. In this study, a novel double-stranded DNA phage, Alteromonas phage P24, which infects Alteromonas macleodii, was isolated from the coastal waters off Qingdao. Alteromonas phage P24 has a siphoviral morphology, with an icosahedral head, 61 ± 1nm in diameter, and a tail length of 105 ± 1nm. Alteromonas phage P24 contains lipids. It has an optimal temperature and pH for growth of 20℃ and 5-7, respectively. A one-step growth curve shows a latent period of 55min, a rise period of 65min, and an average burst size of approximately 147 virions per cell. Alteromonas phage P24 has the genome of 46,945bp with 43.80% GC content and 74 open reading frames (ORFs) without tRNA. The results of the phylogenetic tree, based on the mcp and terL genes, show that Alteromonas phage P24 is closely related to Aeromonas phage phiARM81ld. Meanwhile, phylogenetic analysis based on the whole genome of P24 indicates that it forms a unique viral sub-cluster within Siphoviridae. This study contributes to the understanding of the genomic characteristics and the virus-host interactions of Alteromonas phages.

Rapid and sensitive detection of infectious spleen and kidney necrosis virus by recombinase polymerase amplification combined with lateral flow dipsticks

Mandarin fish (Siniperca chuatsi) is a vital freshwater cultured species in China. Infectious spleen and kidney necrosis virus disease (ISKNVD), caused by infectious spleen and kidney necrosis virus (ISKNV), results in significant financial losses to mandarin fish farming industry. Rapid and convenient detection of ISKNV is urgent demands for diagnosis. In this study, we developed rapid, convenient and sensitive detection methods RPA (recombinase polymerase amplification) and RPA-LFD (combining RPA with lateral flow dipsticks (LFD)), targeting the conserved sequences of MCP and ORF007 genes in ISKNV. The RPA-LFD method could achieve detection within 30 min at 38 °C. Both methods had high sensitivity (102 copies/μL) and were 10 times more sensitive than that in general PCR. And they were highly specific for diagnosis of ISKNV. 24 samples of mandarin fish from farms were tested using RPA and RPA-LFD methods, both of them showed consistent results with the general PCR detection. In short, RPA and RPA-LFD provide a novel alternative for the simple, rapid and sensitive detection of ISKNV and show great potential for diagnosis of ISKNVD in the farm.

Fifteen Marseilleviruses Newly Isolated From Three Water Samples in Japan Reveal Local Diversity of Marseilleviridae

The family Marseilleviridae, defined as a group of icosahedral double-stranded DNA viruses with particle size of approximately 250 nm and genome size of 350–380 kbp, belongs to the nucleo-cytoplasmic family of large DNA viruses. The family Marseilleviridae is currently classified into lineages A–E. In this study, we isolated 12 or 15 new members of the family Marseilleviridae from three sampling locations in Japan. Molecular phylogenetic analysis of the MCP genes showed that the new viruses could be further classified into three groups, hokutoviruses, kashiwazakiviruses, and kyotoviruses. Hokutoviruses were closely related to lineage B, kyotoviruses were related to lineage A, and kashiwazakiviruses were also classified into lineage B but a new putative subgroup of lineage B, revealing the diversity of this lineage. Interestingly, more than two viruses with slightly different MCP genes were isolated from a single water sample from a single location, i.e., two hokutoviruses and one kashiwazakivirus were isolated from a small reservoir, five kashiwazakiviruses from the mouth of a river, and five kyotoviruses from fresh water of a river, suggesting that several milliliters of water samples contain several types of giant viruses. Amoeba cells infected with hokutoviruses or kashiwazakiviruses exhibited a “bunch” formation consisting of normal and infected cells similarly to a tupanvirus, whereas cells infected with kyotoviruses or tokyovirus did not. These results suggest the previously unrecognized local diversity of the family Marseilleviridae in aquatic environments.

Seasonality Drives Microbial Community Structure, Shaping both Eukaryotic and Prokaryotic Host⁻Viral Relationships in an Arctic Marine Ecosystem.

The Arctic marine environment experiences dramatic seasonal changes in light and nutrient availability. To investigate the influence of seasonality on Arctic marine virus communities, five research cruises to the west and north of Svalbard were conducted across one calendar year, collecting water from the surface to 1000 m in depth. We employed metabarcoding analysis of major capsid protein g23 and mcp genes in order to investigate T4-like myoviruses and large dsDNA viruses infecting prokaryotic and eukaryotic picophytoplankton, respectively. Microbial abundances were assessed using flow cytometry. Metabarcoding results demonstrated that seasonality was the key mediator shaping virus communities, whereas depth exerted a diversifying effect within seasonal virus assemblages. Viral diversity and virus-to-prokaryote ratios (VPRs) dropped sharply at the commencement of the spring bloom but increased across the season, ultimately achieving the highest levels during the winter season. These findings suggest that viral lysis may be an important process during the polar winter, when productivity is low. Furthermore, winter viral communities consisted of Operational Taxonomic Units (OTUs) distinct from those present during the spring-summer season. Our data provided a first insight into the diversity of viruses in a hitherto undescribed marine habitat characterized by extremes in light and productivity.

Microbial arsenic oxidation and chemotaxis (Abstract)

We isolated a highly As(III) resistant [minimal inhibitory concentration (MIC) = 25 mM] and As(III)-oxidizing bacterium A. tumefaciens GW4 from As-enriched groundwater sediments. Unlike most of the heterotrophic As(III)-oxidizing bacteria using As(III) oxidation as a detoxification process, the As(III) oxidation of strain GW4 enhanced the bacterial growth, and the strain showed positive chemotaxis toward As(III) towards 0.5-2 mM As(III). Genomic analyses revealed a putative chemoreceptor-encoding gene, mcp, located in the arsenic gene island and having a predicted promoter binding site for the As(III) oxidation regulator AioR. Expression of mcp and other chemotaxis related genes (cheA, cheY2 and fliG) was inducible by As(III), but not in the aioR mutant. Using capillary assays and intrinsic tryptophan fluorescence spectra analysis, Mcp was confirmed to be responsible for chemotaxis towards As(III) and to bind As(III) (but not As(V) nor phosphate) as part of the sensing mechanism. A bacterial one-hybrid system technique and electrophoretic mobility shift assays showed that AioR interacts with the mcp regulatory region in vivo and in vitro, and the precise AioR binding site was confirmed using DNase I foot-printing. Taken together, these results indicate that this Mcp is responsible for the chemotactic response towards As(III) and is regulated by AioR. Additionally, disrupting the mcp gene affected bacterial As(III) oxidation and growth, inferring that Mcp exerts functional connection between As(III) oxidation and As(III) chemotaxis.

Involvement of many chemotaxis sensors in negative chemotaxis to ethanol in Ralstonia pseudosolanacearum Ps29.

Ralstonia pseudosolanacearum Ps29 showed repellent responses to alcohols including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1,3-propanediol and prenol. R. pseudosolanacearum Ps29 possesses 22 putative chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). To identify a MCP involved in negative chemotaxis to ethanol, we measured ethanol chemotaxis of a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29. However, all the mutants showed repellent responses to ethanol comparable to that of the wild-type strain. We constructed a stepwise- and multiple-mcp gene deletion mutant collection of R. pseudosolanacearum Ps29. Analysis of the collection found that an 18-mcp-knockout mutant (strain POC18) failed to respond to ethanol. Complementation analysis using POC18 as the host strain found that introduction of mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 restored the ability of POC18 to respond to ethanol. However, unexpectedly, strain POC10II, harbouring unmarked deletions in 10 mcp genes including mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 showed repellent responses to ethanol comparable to that of wild-type Ps29. We hypothesised that multiple mcp mutations in POC18 led to a shortage of MCPs required for formation of functional chemoreceptor arrays. When pPS16 (encoding McpP involved in phosphate chemotaxis) was introduced into POC18, POC18(pPS16) did not respond to phosphate. This result supports the hypothesis. But, genetic analysis revealed that MCPs (Mcp07, Mcp13, Mcp20 and Mcp21) are not essential for ethanol chemotaxis. Thus, we conclude that many and unspecified MCPs are involved in negative chemotaxis to ethanol in R. pseudosolanacearum Ps29.

Identification of boric acid as a novel chemoattractant and elucidation of its chemoreceptor in Ralstonia pseudosolanacearum Ps29

Chemotaxis enables bacteria to move toward more favorable environmental conditions. We observed chemotaxis toward boric acid by Ralstonia pseudosolanacearum Ps29. At higher concentrations, the chemotactic response of R. pseudosolanacearum toward boric acid was comparable to or higher than that toward L-malate, indicating that boric acid is a strong attractant for R. pseudosolanacearum. Chemotaxis assays under different pH conditions suggested that R. pseudosolanacearum recognizes B(OH)3 (or B(OH3) + B(OH)4−) but not B(OH)4− alone. Our previous study revealed that R. pseudosolanacearum Ps29 harbors homologs of all 22R. pseudosolanacearum GMI1000 mcp genes. Screening of 22 mcp single-deletion mutants identified the RS_RS17100 homolog as the boric acid chemoreceptor, which was designated McpB. The McpB ligand-binding domain (LBD) was purified in order to characterize its binding to boric acid. Using isothermal titration calorimetry, we demonstrated that boric acid binds directly to the McpB LBD with a KD (dissociation constant) of 5.4 µM. Analytical ultracentrifugation studies revealed that the McpB LBD is present as a dimer that recognizes one boric acid molecule.

Negative chemotaxis of Ralstonia pseudosolanacearum to maleate and identification of the maleate chemosensory protein

Ralstonia pseudosolanacearum Ps29 was repelled by maleate. Screening of a complete collection of Ps29 single-methyl-accepting chemotaxis protein (mcp) gene mutants identified the RSp0303 homolog (McpP) as a chemotaxis sensor mediating negative chemotaxis to maleate. Interestingly, the mcpP-deletion mutant was attracted to maleate, indicating that this bacterium expresses a MCP(s) for both positive and negative chemotaxis to maleate. We constructed a Ps29 derivative (designated POC14) harboring deletions in 14 individual mcp genes, including mcpP, to characterize McpP. Introduction of a plasmid harboring the mcpP gene (pPS16) restored the ability to negatively respond to maleate, confirming that McpP is a MCP for negative chemotaxis to maleate. We thought that maleate might be applied to controlling plant infection by R.pseudosolanacearum. To evaluate this possibility, we measured chemotactic responses of seven other virulent R.pseudosolanacearum strains to maleate. We confirmed that they harbored functional mcpP orthologues, but they showed no chemotactic responses to maleate. Quantitative RT-PCR analysis revealed that these seven R.pseudosolanacearum strains did not show negative chemotaxis to maleate because of negligible transcription of the mcpP genes. We compared the chemotactic responses of POC14 and POC14[pPS16] toward various chemicals and found that McpP senses inorganic phosphate as a chemoattractant.

Arsenite oxidation regulator AioR regulates bacterial chemotaxis towards arsenite in Agrobacterium tumefaciens GW4

Some arsenite [As(III)]-oxidizing bacteria exhibit positive chemotaxis towards As(III), however, the related As(III) chemoreceptor and regulatory mechanism remain unknown. The As(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 displays positive chemotaxis towards 0.5–2 mM As(III). Genomic analyses revealed a putative chemoreceptor-encoding gene, mcp, located in the arsenic gene island and having a predicted promoter binding site for the As(III) oxidation regulator AioR. Expression of mcp and other chemotaxis related genes (cheA, cheY2 and fliG) was inducible by As(III), but not in the aioR mutant. Using capillary assays and intrinsic tryptophan fluorescence spectra analysis, Mcp was confirmed to be responsible for chemotaxis towards As(III) and to bind As(III) (but not As(V) nor phosphate) as part of the sensing mechanism. A bacterial one-hybrid system technique and electrophoretic mobility shift assays showed that AioR interacts with the mcp regulatory region in vivo and in vitro, and the precise AioR binding site was confirmed using DNase I foot-printing. Taken together, these results indicate that this Mcp is responsible for the chemotactic response towards As(III) and is regulated by AioR. Additionally, disrupting the mcp gene affected bacterial As(III) oxidation and growth, inferring that Mcp may exert some sort of functional connection between As(III) oxidation and As(III) chemotaxis.

Identification and characterization of chemosensors for d-malate, unnatural enantiomer of malate, in Ralstonia pseudosolanacearum.

Ralstonia pseudosolanacearum Ps29 is attracted by nonmetabolizable d-malate, an unnatural enantiomer. Screening of a complete collection of single-mcp-gene deletion mutants of Ps29 revealed that the RSc1156 homologue is a chemosensor for d-malate. An RSc1156 homologue deletion mutant of Ps29 showed decreased but significant responses to d-malate, suggesting the existence of another d-malate chemosensor. McpM previously had been identified as a chemosensor for l-malate. We constructed an RSc1156 homologue mcpM double deletion mutant and noted that this mutant failed to respond to d-malate; thus, the RSc1156 homologue and McpM are the major chemosensors for d-malate in this organism. To further characterize the ligand specificities of the RSc1156 homologue and McpM, we constructed a Ps29 derivative (designated K18) harbouring deletions in 18 individual mcp genes, including mcpM and RSc1156. K18 harbouring the RSc1156 homologue responded strongly to l-tartrate and d-malate and moderately to d-tartrate, but not to l-malate or succinate. K18 harbouring mcpM responded strongly to l-malate and d-tartrate and moderately to succinate, fumarate and d-malate. Ps29 utilizes l-malate and l-tartrate, but not d-malate. We therefore concluded that l-tartrate and l-malate are natural ligands of the RSc1156 homologue and McpM, respectively, and that chemotaxis toward d-malate is a fortuitous response by the RSc1156 homologue and McpM in Ps29. We propose re-designation of the RSc1156 homologue as McpT. In tomato plant infection assays, the mcpT deletion mutant of highly virulent R. pseudosolanacearum MAFF106611 was as infectious as wild-type MAFF106611, suggesting that McpT-mediated chemotaxis does not play an important role in tomato plant infection.

Turkish pediatric atypical hemolytic uremic syndrome registry: initial analysis of 146 patients

BackgroundAtypical hemolytic uremic syndrome (aHUS) is a devastating disease with significant morbidity and mortality. Its genetic heterogeneity impacts its clinical presentation, progress, and outcome, and there is no consensus on its clinical management.MethodsTo identify the characteristics of aHUS in Turkish children, an industry-independent registry was established for data collection that includes both retrospective and prospective patients.ResultsIn total, 146 patients (62 boys, 84 girls) were enrolled; 53 patients (36.3%) were less than 2 years old at initial presentation. Among the 42 patients (37.1%) whose mutation screening was complete for CFH, CFI, MCP, CFB, C3, DGKE, and CHFR5 genes, underlying genetic abnormalities were uncovered in 34 patients (80.9%). Sixty-one patients (41.7%) had extrarenal involvement. During the acute stage, 33 patients (22.6%) received plasma therapy alone, among them 17 patients (51.5%) required dialysis, and 4 patients (12.1%) were still on dialysis at the time of discharge. In total, 103 patients (70.5%) received eculizumab therapy, 16 of whom (15.5%) received eculizumab as a first-line therapy. Plasma therapy was administered to 84.5% of the patients prior to eculizumab. In this group, renal replacement therapy was administered to 80 patients (77.7%) during the acute period. A total of 3 patients died during the acute stage. A total of 101 patients (77.7%) had a glomerular filtration rate >90 mL/min/1.73 m2 at the 2-year follow-up.ConclusionsThe Turkish aHUS registry will increase our knowledge of patients with aHUS who have different genetic backgrounds and will enable evaluation of the different treatment options and outcomes.

Identification of the mcpA and mcpM genes, encoding methyl-accepting proteins involved in amino acid and l-malate chemotaxis, and involvement of McpM-mediated chemotaxis in plant infection by Ralstonia pseudosolanacearum (formerly Ralstonia solanacearum phylotypes I and III).

Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand.

Pregnancy-Associated Atypical Hemolytic Uremic Syndrome (aHUS), Treated with Eculizumab

Introduction:aHUS is a life-threatening, chronic, progressive disease of complement mediated thrombotic microangiopathy (TMA), commonly associatedwith pregnancy, 10-20% of all cases (P-aHUS), that usually occurs in late pregnancy and postpartum, primiparous women. Plasma exchange (PEX) may transiently maintain hematologic parameters, but does not treat the underlying systemic disease. Clinical studies have shown excellent effectiveness of eculizumab (Soliris®), the only complement inhibitor, used in PNH treatment. In 2011, eculizumab was approved for the treatment of aHUS. We report the 1st case of aHUS treated with eculizumab, in Greece, which was pregnancy related.A 29 years old female, on the 16th week of gestation was referred, due to anemia (Hgb 6.9 g/dL) and thrombocytopenia (plt 78.000/μL). Family history: father with renal transplantation. Clinical examination: no abnormal findings. Laboratory exams: coagulation tests normal, low fibrinogen, 5-6 schistocytes/hpf, retics 12%, normal liver function, LDH 975 IU/L (normal range, nr 0-248), inDBil 2,5 mg/dl, ESR 41, CRP negative. Renal function was mildly deteriorated, urea 50 mg/dL, creatinine 1.3 mg/dL, proteinurea (urine protein 1 g/24h). The patient was diagnosed with pregnancy associated TMA and underwent daily PEX plus steroids. Partial response for the first 3-4 days. IVIgG was added. Rapid relapse with fall in Hgb, Hct, plt, schistocytes 6-8/hpf, decrease in fibrinogen, LDH and inDBil elevation. Pregnancy was terminated on the 8th day of hospitalization, by uterine section. She was transferred to the ICU: diffuse bleeding from operative wound/drains. She had daily PEX and RBCs, PLTs, FFPs, CPs transfusions, schistocytes 12-18/hpf. Rituximab 375 mg/m2/5 days × 5 cycles, begun 2 weeks after PEX initiation.Diagnostic testing: antiphospholipid antibodies (PTTLa, anti-B2GPI, ACAs) negative, APC-R positive, FVL heterozygous, virology tests (anti-HIV, HBsAg, anti-HCV) negative, immunology tests (ANAs, anti-ds-DNA, anti-ENAs) negative, stool cultures negative, Verotoxin/E. Coli 0157 antibodies negative, PNH test by high sensitivity flow cytometrynegative, complement levels at the lower normal, normal ADAMTS13 activity. Post-operatively, daily PEX and RBCs, FFPs transfusions, replenish fibrinogen. Refractory arterial hypertension (furosemide, clonidine, amlodipine). Severe kidney involvement (urine protein 14-44g/24h). Laboratoryresults: Hgb 6.6 mg/dL, plt 52.000/μL, creatinine 1.04 mg/dL, inDBil 2 mg/dL, LDH1100 IU/L. So, the patient showing no response to treatments, was in poor clinical condition, multitransfused, with whole body edema.The diagnosisof aHUS was confirmed by clinical and laboratory findings. The patient was vaccinated against Neisseria meningitidis with tetravalent conjugated, serogroups A, C, W-135, and Y (Menveo®) and serogroup Β strains (Bexsero®) and eculizumab was started,on day 47, in the suggested protocol, 900 mg/week × 4 weeks, 1200 mg, week 5, following maintenance with 1200 mg/2 weeks, indefinitely. She experienced immediate “weaning” from PEX withrapid and stable improvement of all pathological parameters andgradual resolution of proteinuria. Recent laboratory tests: normal blood counts, LDH 161 IU/L, creatinine 0.7 mg/dL, urine protein 1 g/24h. Renal biopsy performed after remission identified morphological (segmental double contour of glomerular basic membrane, focal segmental glomerular sclerosis and mesangiolysis) and immunohistochemical (subendothelial, subepithelial and mesangial IgM, C3d, C1q deposition), all consistent with TMA, HUS type. Molecular genetics by NG Sequencing for CFH, MCP, CFB, C3, CFI, THBD, CFHR5, ADAMTS13, C6A, C9B, C9, CFD and DGKE genes, identified no mutations known as a cause for aHUS, by now. However, aHUS is a genetically complex and heterogeneous disorder.Conclusions: Therapy with eculizumab was life saving in our case, inducing rapid response of all active TMA markers and renal involvement recovery. Only 16% of P-aHUS occurs during the 2nd semester of pregnancy, as in our case. Multiple risk factors during pregnancy may trigger an acute episode of TMA in predisposed women and a contributing effect of hereditary thrombophilia plays a role in the pathophysiology of aHUS. The co-existence of hereditary thrombophilia, FVL heterozygous, might have played a role in the aHUS presentation during pregnancy, in our patient. DisclosuresNo relevant conflicts of interest to declare.