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PloS One 2023In bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine-6-phosphate deaminase (NagB) catalyzes the last step in converting sialic acid into...
In bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine-6-phosphate deaminase (NagB) catalyzes the last step in converting sialic acid into Fructose-6-phosphate. These bacteria then use the Fructose-6-phosphate as an energy source. The enzyme NagB exists as a hexamer in Gram-negative bacteria and is allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of two closely related oligomeric forms. We report here the crystal structures of NagB from two Gram-negative pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm). The Hi-NagB is active as a hexamer, while Pm-NagB is active as a dimer. Both Hi-NagB and Pm-NagB contain the C-terminal helix implicated as essential for hexamer formation. The hexamer is described as a dimer of trimers. In the Pm-NagB dimer, the dimeric interface is conserved. The conservation of the dimer interface suggests that the three possible oligomeric forms of NagB are a monomer, a dimer, and a trimer of dimers. Computational modeling and MD simulations indicate that the residues at the trimeric interface have less stabilizing energy of oligomer formation than those in the dimer interface. We propose that Pm-NagB is the evolutionary link between the monomer and the hexamer forms.
Topics: Aldose-Ketose Isomerases; N-Acetylneuraminic Acid; Polymers; Haemophilus influenzae; Pasteurella multocida; Bacterial Proteins
PubMed: 36598911
DOI: 10.1371/journal.pone.0271654 -
PloS One 2015Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of...
Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.
Topics: Animal Husbandry; Animals; DNA, Bacterial; Housing, Animal; Mice; Pasteurellaceae; RNA, Ribosomal, 16S; Real-Time Polymerase Chain Reaction; Rodent Diseases
PubMed: 26556281
DOI: 10.1371/journal.pone.0142560 -
DNA Research : An International Journal... Apr 2018Aggregatibacter actinomycetemcomitans is a major periodontal pathogen that has several virulence factors such as leukotoxin and cytolethal distending toxin. Although the...
Aggregatibacter actinomycetemcomitans is a major periodontal pathogen that has several virulence factors such as leukotoxin and cytolethal distending toxin. Although the genes responsible for virulence have been identified, little is known about their regulatory mechanisms. Small RNA (sRNA) has been recognized as an important factor for gene regulation. To identify new regulatory mechanisms via sRNA in A. actinomycetemcomitans HK1651, we performed a systematic search for sRNAs by RNA-seq and identified 90 intergenic region sRNAs and 30 antisense sRNAs. Of the 85 analysable sRNAs, we successfully detected and quantified 70 sRNAs by developing an RT-PCR system, and we identified 17 sRNAs that were differentially expressed during different growth phases. In addition, we found notable intraspecies variation in the sRNA repertoire of A. actinomycetemcomitans, thus suggesting that frequent acquisition or deletion of sRNAs occurred during the evolution of this species. The predicted target genes of the intergenic region sRNAs indicated the possibility of sRNA interaction with several virulence genes including leukotoxin and cytolethal distending toxin. Our results should serve as an important genomic and genetic basis for future studies to fully understand the regulatory network in A. actinomycetemcomitans and provide new insights into the intraspecies variation of the bacterial sRNA repertoire in bacteria.
Topics: Aggregatibacter actinomycetemcomitans; Gene Expression Regulation, Bacterial; RNA, Bacterial; RNA, Small Untranslated; Sequence Analysis, RNA; Virulence
PubMed: 29211829
DOI: 10.1093/dnares/dsx050 -
BMC Veterinary Research Nov 2022Gram-negative bacterial infections are a serious problem in beef and dairy cattle. Bacterial outer membrane proteins (OMPs) play a pivotal role in cellular survival and...
BACKGROUND
Gram-negative bacterial infections are a serious problem in beef and dairy cattle. Bacterial outer membrane proteins (OMPs) play a pivotal role in cellular survival and the host-bacterium interaction. Histophilus somni OMP40 was identified as a porin with homology between its N-terminal amino acid sequence and the sequences of porins of other gram-negative bacteria The aim of this study was to produce recombinant H. somni OMP40 (rOMP40), optimize its production and evaluate its immunogenic properties in calves. The cross-reactivity of anti-rOMP40 antibodies were also checked.
RESULTS
The highest overexpression of rOMP40 was demonstrated by Escherichia coli C41 using the autoinduction process. Double immunization of calves (20 μg rOMP40 per animal) induced a significant increase of anti-rOMP40 antibodies in the IgG (P ≤ 0.01) and IgG (P ≤ 0.01, after first immunization only) subclasses, but not IgM. ELISA revealed increased reactivity of the IgG against surface antigens of E. coli and Pasteurella multocida after the second immunization (P < 0.01). Cross reactivity of anti-rOMP40 antibodies with ~ 40 kDa antigens of most common gram-negative pathogens was shown by Western blotting.
CONCLUSION
Immunization with H. somni rOMP40 induced a humoral response in cattle with broad cross-reactivity with similar antigens of other species of Pasteurellaceae and Enterobacteriaceae families and the delayed-type hypersensitivity reaction. The obtained results encourage further study to evaluate the protective effect of the produced protein as a subunit vaccine in cattle.
Topics: Cattle; Animals; Escherichia coli; Antibody Formation; Pasteurellaceae; Recombinant Proteins; Bacterial Outer Membrane Proteins; Immunoglobulin G
PubMed: 36401280
DOI: 10.1186/s12917-022-03515-x -
The Chinese Journal of Dental Research 2019To investigate the immunoinflammatory response in the crosstalk of human oral keratinocytes (HOKs) with selected periodontal commensals and pathogens.
OBJECTIVE
To investigate the immunoinflammatory response in the crosstalk of human oral keratinocytes (HOKs) with selected periodontal commensals and pathogens.
METHODS
Four representative viable oral bacteria, including periodontal commensals (Streptococcus mutans, Sm; and Actinomyces israelii, Ai) and pathogens (Aggregatibacter actinomycetemcomitans, Aa; and Porphyromonas gingivalis, Pg), were selected. A viable bacteria-HOKs interactive model was tested under various conditions of oxygen, antibiotics, duration and multiplicity of infection (MOI). The expression of IL-6 and IL-8 in HOKs was assessed by real-time qPCR and ELISA.
RESULTS
An MOI of 1 was determined to be the appropriate ratio of bacteria and HOKs with substantial amounts of viable bacterial cells and HOKs in an antibiotic-free medium under aerobic conditions for 2 h. Sm and Pg significantly upregulated the expression of IL-6 and IL-8 (P < 0.05), while Ai and Aa could not induce significant levels of these cytokines with reference to the control.
CONCLUSION
Within the limitations of this study, the current findings suggest that periodontal commensals and pathogens may differentially modulate immunoinflammatory response in human oral keratinocytes.
Topics: Aggregatibacter actinomycetemcomitans; Cytokines; Humans; Keratinocytes; Porphyromonas gingivalis; Streptococcus mutans
PubMed: 31172138
DOI: 10.3290/j.cjdr.a42514 -
Scientific Reports Aug 2017Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current... (Comparative Study)
Comparative Study
Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current study, transcriptional profiling was applied to analyse the variation in gene expression of JS0135 and tildipirosin-resistant JS32. The growth curves showed that JS32 had a higher growth rate but fewer bacteria than JS0135. The cell membranes of JS32 and a resistant clinical isolate (HB32) were observed to be smoother than those of JS0135. From the comparative gene expression profile 349 up- and 113 downregulated genes were observed, covering 37 GO and 63 KEGG pathways which are involved in biological processes (11), cellular components (17), molecular function (9), cellular processes (1), environmental information processing (4), genetic information processing (9) and metabolism (49) affected in JS32. In addition, the relative overexpression of genes of the metabolism pathway (HAPS_RS09315, HAPS_RS09320), ribosomes (HAPS_RS07815) and ABC transporters (HAPS_RS10945) was detected, particularly the metabolism pathway, and verified with RT-qPCR. Collectively, the gene expression profile in connection with tildipirosin resistance factors revealed unique and highly resistant determinants of H. parasuis to macrolides that warrant further attention due to the significant threat of bacterial resistance.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Bacterial; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gene Ontology; Gene Regulatory Networks; Haemophilus parasuis; Metabolic Networks and Pathways; Microarray Analysis; Molecular Sequence Annotation; Transcriptome; Tylosin
PubMed: 28790420
DOI: 10.1038/s41598-017-07972-5 -
BMC Microbiology Sep 2020This study evaluated the effect of oral lactobacilli on the cytotoxicity and cytokine release from peripheral blood mononuclear cells (PBMCs) when exposed to...
Oral Lactobacillus strains reduce cytotoxicity and cytokine release from peripheral blood mononuclear cells exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro.
BACKGROUND
This study evaluated the effect of oral lactobacilli on the cytotoxicity and cytokine release from peripheral blood mononuclear cells (PBMCs) when exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro. The supernatants and cell wall extracts (CWEs) of eight A. actinomycetemcomitans strains, representing different subtypes, and three Lactobacillus strains were used. The PBMCs from six blood donors were exposed to supernatants and CWEs of A. actinomycetemcomitans or Lactobacillus strains alone or combinations and untreated cells as control. The cytotoxicity was determined by trypan blue exclusion method and IL-1β secretion by ELISA. TNF-α, IL-6, and IL-8 secretions were measured using Bioplex Multiplex Immunoassay.
RESULTS
Supernatants or CWEs from all bacterial strains showed cytotoxicity and IL-1β secretion and the subtypes of A. actinomycetemcomitans showed generally a significantly higher effect on PBMCs than that of the Lactobacillus strains. Two highly toxic A. actinomycetemcomitans strains (JP2 and JP2-like) induced a higher response than all other strains. When combined, Lactobacillus significantly reduced the toxicity and the IL-1β secretion induced by A. acinomycetemcomitans. The effect varied between the subtypes and the reduction was highest for the JP2 and JP2-like strains. The Lactobacillus paracasei strain SD1 had a higher reducing effect than the other Lactobacillus strains. This strain had a consistent reducing effect on all subtypes of A. actinomycetemcomitans cytotoxicity, and release of IL-1β, IL-6, IL-8, and TNF-α from PBMCs of the blood donors. A strong and significant variation in cytokine release between the six blood donors was noticed.
CONCLUSIONS
Lactobacillus spp. and L. paracasei SD1 in particular, showed a limited but statistically significant reducing interaction with A. actinomycetemcomitans toxicity and release of cytokines in vitro.
Topics: Aggregatibacter actinomycetemcomitans; Cell Wall; Cytokines; Humans; Lactobacillus; Lacticaseibacillus paracasei; Leukocytes, Mononuclear; Mouth; Pasteurellaceae Infections; Probiotics
PubMed: 32917132
DOI: 10.1186/s12866-020-01959-5 -
Bioresource Technology Jan 2022Succinic acid (SA) is a top platform chemical obtainable from biomass. The current study evaluated the potential of Actinobacillus succinogenes for SA production using...
Succinic acid (SA) is a top platform chemical obtainable from biomass. The current study evaluated the potential of Actinobacillus succinogenes for SA production using xylose-rich hemicellulosic fractions of two important lignocellulosic feedstocks, olive pits (OP) and sugarcane bagasse (SCB) and the results were compared with pure xylose. Initial experiments were conducted in shake flask followed by batch and fed-batch cultivation in bioreactor. Further separation of SA from the fermented broth was carried out by adapting direct crystallisation method. During fed-batch culture, maximum SA titers of 36.7, 33.6, and 28.7 g/L was achieved on pure xylose, OP and SCB hydrolysates, respectively, with same conversion yield of 0.27 g/g. The recovery yield of SA accumulated on pure xylose, OP and SCB hydrolysates was 79.1, 76.5, and 75.2%, respectively. The results obtained are of substantial value and pave the way for development of sustainable SA biomanufacturing in an integrated biorefinery.
Topics: Actinobacillus; Fermentation; Succinic Acid; Xylose
PubMed: 34751156
DOI: 10.1016/j.biortech.2021.126224 -
MBio Mar 2020The posttranslational Ca-dependent "clip-and-link" activity of large epeat-in-oin (RTX) proteins starts by Ca-dependent structural rearrangement of a highly conserved...
The posttranslational Ca-dependent "clip-and-link" activity of large epeat-in-oin (RTX) proteins starts by Ca-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface. The Ca-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of , resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.
Topics: Actinobacillus Infections; Actinobacillus pleuropneumoniae; Animals; Bacterial Proteins; Bacterial Toxins; Calcium; Membrane Proteins; Neisseria meningitidis; Protein Processing, Post-Translational; Swine; Virulence
PubMed: 32184239
DOI: 10.1128/mBio.00226-20 -
Nature Communications Apr 2020Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate...
Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 μM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L of SA with the maximum productivity of 21.3 g L h, demonstrating the importance of enzyme optimization in strain development.
Topics: Bacterial Proteins; Bioreactors; Corynebacterium glutamicum; Fermentation; Kinetics; Malate Dehydrogenase; Metabolic Engineering; Oxaloacetic Acid; Pasteurellaceae; Protein Conformation; Recombinant Proteins; Substrate Specificity; Succinic Acid
PubMed: 32327663
DOI: 10.1038/s41467-020-15839-z