Neisseria gonorrhoeae is a Gram-negative bacterium that causes the sexually transmitted infection gonorrhea. N. gonorrhoeae has progressively developed resistance to all currently prescribed ...antibiotics, and no vaccine is available. Here, we report the closed, completed, annotated genome sequences for seven N. gonorrhoeae strains obtained by single-molecule real-time (SMRT) long-read genome sequencing.
► Phosphorylcholine biosynthesis does not require exogenous choline in Neisseria meningitidis. ► Pilin is the only substrate for phosphorylcholine modification in N. meningitidis. ► The sequence ...requirement for phosphorylcholine is D–A–S. ► Structural features and charge residues in the sequence are important for substrate recognition.
Neisseria meningitidis is a human pathogen that can cause life threatening meningitis and sepsis. Pili of Neisseria are one of the major virulence factors in host–pathogen interaction. Pilin of N.meningitidis is post-translationally modified by a glycan and two phosphorylcholines (ChoP). ChoP modifications have been found to have an important role in bacterial colonisation and invasion. Unlike N. gonorrhoeae, ChoP modifications on pili seem to be restricted to the C-terminus of pilin protein in N. meningitidis. In this study, we investigate the substrate recognition of phosphorylcholine transferase. We found that a single sequence of D–A–S after the disulphide bond of pilin protein is able to form a motif for ChoP modifications and the charge residue in this motif and the local structure are essential for the substrate recognition.
Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N
6
-adenine DNA methyltransferases (Mod) alter global ...methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions.
Neisseria meningitidis
phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N
6
-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key
N. meningitidis
methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGY
m6
A
G-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-AC
m6
A
CC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CC
m6
A
GC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGY
m6
A
G-3′. The observed efficiency of methylation in the
modA11
strain (MC58) genome ranged from 4.6% at 5′-GCGC
m6
A
GG-3′ sites, to 100% at 5′-ACGT
m6
A
GG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.
Invasive streptococcal disease (ISD) and toxic shock syndrome (STSS) result in over 160,000 deaths each year. We modelled these in HLA-transgenic mice infected with a clinically lethal isolate ...expressing Streptococcal pyrogenic exotoxin (Spe) C and demonstrate that both SpeC and streptococcal M protein, acting cooperatively, are required for disease. Vaccination with a conserved M protein peptide, J8, protects against STSS by causing a dramatic reduction in bacterial burden associated with the absence of SpeC and inflammatory cytokines in the blood. Furthermore, passive immunotherapy with antibodies to J8 quickly resolves established disease by clearing the infection and ablating the inflammatory activity of the M protein, which is further enhanced by addition of SpeC antibodies. Analysis of 77 recent isolates of
causing ISD, demonstrated that anti-J8 antibodies theoretically recognize at least 73, providing strong support for using antibodies to J8, with or without antibodies to SpeC, as a therapeutic approach.
causes the sexually transmitted infection gonorrhea. High-coverage (∼3,300-fold) transcriptome sequencing data have been collected from multidrug-resistant
strain WHO Z grown in the presence and ...absence of PBT2.
Liposomes and enzymes: Liposome formulations improved solubility of a lipid‐modified lactose enkephalin and, when used in enzymatic transformation, led to a twofold increase in glycosylation in ...comparison to substrate solubilised in 5 % dimethyl sulfoxide (DMSO; see figure).
Phosphorylcholine (ChoP) can be found in all life forms. Although this molecule was first thought to be uncommon in bacteria, it is now appreciated that many bacteria express ChoP on their surface. ...ChoP is usually attached to a glycan structure, but in some cases, it is added as a post-translation modification to proteins. Recent findings have demonstrated the role ChoP modification and phase variation (ON/OFF switching) in bacterial pathogenesis. However, the mechanisms of ChoP synthesis are still unclear in some bacteria. Here, we review the literature and examine the recent developments in ChoP-modified proteins and glycolipids and of ChoP biosynthetic pathways. We discuss how the well-studied Lic1-pathway exclusively mediates ChoP attachment to glycans, but not to proteins. Finally, we provide a review of the role of ChoP in bacterial pathobiology and the role of ChoP in modulating the immune response.
•Sugar and lipid modified enkephalin peptides were chemically synthesized.•LgtC galactosyltransferase catalyzed conversion of enkephalin derivatives.•Galactosylated products were purified in 13–71% ...yields.•5% DMSO increased solubility and enzymatic conversion of lipidated enkephalins.•LgtC displayed unexpectedly high substrate specificity toward galactosyl moieties.
Glycosylation of therapeutic peptides has been reported to improve delivery and targeting of various vaccines and drugs to specific cells/tissues. However, chemical synthesis of complex oligosaccharide derivatives via conventional methods can be challenging due to the need for several orthogonal hydroxyl group protections. Liposaccharyl galactosyltransferase C, a naturally occurring glycosyltransferase enzyme from Neisseria meningitidis, was found to have the ability to transfer a galactosyl moiety to glyco(lipo)peptides. An enzymatic glycosylation of Leu-enkephalin glyco(lipo)peptides was developed and optimized in this study in order to prepare pain regulating peptides with potentially improved central nervous system delivery.
Abstract
Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many ...host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.
Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N(6)-adenine DNA methyltransferases (Mod) alter global ...methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N(6)-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY M6A: G-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC M6A: CC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC M6A: GC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY M6A: G-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC M6A: GG-3' sites, to 100% at 5'-ACGT M6A: GG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.
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