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Nature Communications Jul 2021Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan...
Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.
Topics: Amino Acid Motifs; Bacterial Capsules; Catalytic Domain; Cryoelectron Microscopy; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Mass Spectrometry; Membrane Proteins; Models, Molecular; Periplasm; Phosphorylation; Polysaccharides, Bacterial; Protein Conformation, alpha-Helical; Protein-Tyrosine Kinases; Tyrosine
PubMed: 34272394
DOI: 10.1038/s41467-021-24652-1 -
Emerging Infectious Diseases Mar 2018Encapsulated Haemophilus influenzae strains belong to type-specific genetic lineages. Reliable capsule typing requires PCR, but a more efficient method would be useful....
Encapsulated Haemophilus influenzae strains belong to type-specific genetic lineages. Reliable capsule typing requires PCR, but a more efficient method would be useful. We evaluated capsule typing by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Isolates of all capsule types (a-f and nontypeable; n = 258) and isogenic capsule transformants (types a-d) were investigated. Principal component and biomarker analyses of mass spectra showed clustering, and mass peaks correlated with capsule type-specific genetic lineages. We used 31 selected isolates to construct a capsule typing database. Validation with the remaining isolates (n = 227) showed 100% sensitivity and 92.2% specificity for encapsulated strains (a-f; n = 61). Blinded validation of a supplemented database (n = 50) using clinical isolates (n = 126) showed 100% sensitivity and 100% specificity for encapsulated strains (b, e, and f; n = 28). MALDI-TOF mass spectrometry is an accurate method for capsule typing of H. influenzae.
Topics: Bacterial Capsules; Bacterial Typing Techniques; Evolution, Molecular; Genetic Linkage; Haemophilus Infections; Haemophilus influenzae; Multilocus Sequence Typing; Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 29460728
DOI: 10.3201/eid2403.170459 -
PloS One 2021The Campylobacter jejuni capsule type HS1 complex is one of the most common serotypes identified worldwide, and consists of strains typing as HS1, HS1/44, HS44 and...
The Campylobacter jejuni capsule type HS1 complex is one of the most common serotypes identified worldwide, and consists of strains typing as HS1, HS1/44, HS44 and HS1/8. The capsule structure of the HS1 type strain was shown previously to be composed of teichoic-acid like glycerol-galactosyl phosphate repeats [4-)-α-D-Galp-(1-2)-Gro-(1-P-] with non-stoichiometric fructose branches at the C2 and C3 of Gal and non-stoichiometric methyl phosphoramidate (MeOPN) modifications on the C3 of the fructose. Here, we demonstrate that the capsule of an HS1/44 strain is identical to that of the type strain of HS1, and the capsule of HS1/8 is also identical to HS1, except for an additional site of MeOPN modification at C6 of Gal. The DNA sequence of the capsule locus of an HS44 strain included an insertion of 10 genes, and the strain expressed two capsules, one identical to the HS1 type strain, but with no fructose branches, and another composed of heptoses and MeOPN. We also characterize a HS1 capsule biosynthesis gene, HS1.08, as a fructose transferase responsible for the attachment of the β-D-fructofuranoses residues at C2 and C3 of the Gal unit. In summary, the common component of all members of the HS1 complex is the teichoic-acid like backbone that is likely responsible for the observed sero-cross reactivity.
Topics: Bacterial Capsules; Campylobacter jejuni; Carbohydrate Sequence; Multigene Family; Mutation; Polysaccharides, Bacterial; Sequence Analysis, DNA; Serogroup
PubMed: 33621246
DOI: 10.1371/journal.pone.0247305 -
Journal of Bacteriology Jun 2009Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the...
Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 microM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.
Topics: Antimicrobial Cationic Peptides; Bacterial Adhesion; Bacterial Capsules; Bacterial Proteins; Cathelicidins; Drug Resistance, Bacterial; Endotoxins; Epithelial Cells; Gene Expression Regulation, Bacterial; Humans; Meningitis, Meningococcal; Microbial Viability; Neisseria meningitidis
PubMed: 19376861
DOI: 10.1128/JB.01313-08 -
MBio May 2020(pneumococcus) is a major human pathogen producing structurally diverse capsular polysaccharides. Widespread use of highly successful pneumococcal conjugate vaccines...
(pneumococcus) is a major human pathogen producing structurally diverse capsular polysaccharides. Widespread use of highly successful pneumococcal conjugate vaccines (PCVs) targeting pneumococcal capsules has greatly reduced infections by the vaccine types but increased infections by nonvaccine serotypes. Herein, we report a new and the 100th capsule type, named serotype 10D, by determining its unique chemical structure and biosynthetic roles of all capsule synthesis locus () genes. The name 10D reflects its serologic cross-reaction with serotype 10A and appearance of cross-opsonic antibodies in response to immunization with 10A polysaccharide in a 23-valent pneumococcal vaccine. Genetic analysis showed that 10D has three large regions syntenic to and highly homologous with loci from serotype 6C, serotype 39, and an oral streptococcus strain ( SK145). The 10D region syntenic to SK145 is about 6 kb and has a short gene fragment of α at the 5' end. The presence of this nonfunctional α fragment provides compelling evidence for a recent interspecies genetic transfer from oral streptococcus to pneumococcus. Since oral streptococci have a large repertoire of loci, widespread PCV usage could facilitate the appearance of novel serotypes through interspecies recombination. The polysaccharide capsule is essential for the pathogenicity of pneumococcus, which is responsible for millions of deaths worldwide each year. Currently available pneumococcal vaccines are designed to elicit antibodies to the capsule polysaccharides of the pneumococcal isolates commonly causing diseases, and the antibodies provide protection only against the pneumococcus expressing the vaccine-targeted capsules. Since pneumococci can produce different capsule polysaccharides and therefore reduce vaccine effectiveness, it is important to track the appearance of novel pneumococcal capsule types and how these new capsules are created. Herein, we describe a new and the 100th pneumococcal capsule type with unique chemical and serological properties. The capsule type was named 10D for its serologic similarity to 10A. Genetic studies provide strong evidence that pneumococcus created 10D capsule polysaccharide by capturing a large genetic fragment from an oral streptococcus. Such interspecies genetic exchanges could greatly increase diversity of pneumococcal capsules and complicate serotype shifts.
Topics: Bacterial Capsules; Cross-Sectional Studies; Humans; Immune Sera; Immunization; Phagocytosis; Pneumococcal Vaccines; Polysaccharides, Bacterial; Serogroup; Streptococcus pneumoniae
PubMed: 32430472
DOI: 10.1128/mBio.00937-20 -
Infection and Immunity Jun 2016Kingella kingae is a common cause of invasive disease in young children and was recently found to produce a polysaccharide capsule containing N-acetylgalactosamine...
Kingella kingae is a common cause of invasive disease in young children and was recently found to produce a polysaccharide capsule containing N-acetylgalactosamine (GalNAc) and β-3-deoxy-d-manno-octulosonic acid (βKdo). Given the role of capsules as important virulence factors and effective vaccine antigens, we set out to determine the genetic determinants of K. kingae encapsulation. Using a transposon library and a screen for nonencapsulated mutants, we identified the previously identified ctrABCD (ABC transporter) operon, a lipA (kpsC)-like gene, a lipB (kpsS)-like gene, and a putative glycosyltransferase gene designated csaA (capsule synthesis type a gene A). These genes were found to be present at unlinked locations scattered throughout the genome, an atypical genetic arrangement for Gram-negative bacteria that elaborate a capsule dependent on an ABC-type transporter for surface localization. The csaA gene product contains a predicted glycosyltransferase domain with structural homology to GalNAc transferases and a predicted capsule synthesis domain with structural homology to Kdo transferases, raising the possibility that this enzyme is responsible for alternately linking GalNAc to βKdo and βKdo to GalNAc. Consistent with this conclusion, mutation of the DXD motif in the GalNAc transferase domain and of the HP motif in the Kdo transferase domain resulted in a loss of encapsulation. Examination of intracellular and surface-associated capsule in deletion mutants and complemented strains further implicated the lipA (kpsC)-like gene, the lipB (kpsS)-like gene, and the csaA gene in K. kingae capsule production. These data define the genetic requirements for encapsulation in K. kingae and demonstrate an atypical organization of capsule synthesis, assembly, and export genes.
Topics: Acetylgalactosamine; Bacterial Capsules; Bacterial Proteins; DNA Transposable Elements; Escherichia coli; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Genome, Bacterial; Glycosyltransferases; Kingella kingae; Mutation; Operon; Polysaccharides, Bacterial; Protein Domains; Recombinant Proteins; Sugar Acids
PubMed: 27045037
DOI: 10.1128/IAI.00128-16 -
PloS One 2018Bacillus cereus strain G9241 was isolated from a patient with pneumonia who had an anthrax-like illness. Like Bacillus anthracis, the virulence of G9241 is dependent on...
Bacillus cereus strain G9241 was isolated from a patient with pneumonia who had an anthrax-like illness. Like Bacillus anthracis, the virulence of G9241 is dependent on two large plasmids. In G9241 those plasmids are pBCXO1 and pBC210. There is a multi-gene capsule locus on each of these virulence plasmids, and both capsules are produced by G9241 in vitro and in mice. The hasACB operon on pBCXO1 is responsible for production of a hyaluronic acid (HA) capsule. The locus on pBC210 encodes a putative tetrasaccharide (TS) capsule that assembles in a Wzy-dependent manner. We found that the pBC210 capsule locus is transcribed as two operons and identified the promoter regions responsible for transcription. We constructed isogenic mutants to assess the role of genes in the two TS capsule operons in production of the capsule. Spores of strains deficient in production of either the HA or TS capsule were inoculated subcutaneously or intranasally into A/J and C57BL/6 mice to determine the lethal dose 50% of each bacterial mutant by each route of infection. The loss of the HA capsule attenuated G9241 more than the loss of the TS capsule for both infection routes in both mouse strains. Overall, our data further characterize the unique TS capsule on pBC210 and demonstrate that the two capsules do not have the same impact on virulence of G9241.
Topics: Animals; Bacillus cereus; Bacterial Capsules; Gene Expression Regulation, Bacterial; Glycosyltransferases; Humans; Mice; Mice, Inbred C57BL; Mutation; Operon; Plasmids; Pneumonia; Polysaccharides, Bacterial; Virulence Factors
PubMed: 30133532
DOI: 10.1371/journal.pone.0202701 -
Virulence 2011The innate immune system plays a critical role in host defense against mucosal bacteria. Campylobacter jejuni is a major cause of human gastroenteritis that usually...
The innate immune system plays a critical role in host defense against mucosal bacteria. Campylobacter jejuni is a major cause of human gastroenteritis that usually resolves spontaneously within several days, suggesting that innate mechanisms are important to control the infection. However, the specific means by which this occurs is not well understood. While diarrheal isolates of C. jejuni usually are susceptible to human serum, we found that a systemic strain of C. jejuni, isolated from the cerebrospinal fluid of an infant with meningitis, is relatively more resistant to human serum, the Bactericidal/Permeability-Increasing Protein (BPI), an endogenous cationic antimicrobial protein, and the cationic peptide antibiotic polymyxin B. To test the hypothesis that the surface properties of this strain contributed to its ability to withstand these innate host defenses, we constructed isogenic mutants in capsule (kpsM) and lipooligosaccharide (waaF) and complemented these mutants by insertion of the complementation construct in trans into hipO, a chromosomal locus. We found that capsule expression was essential for serum resistance, whereas lipooligosaccharide played no substantial role. In contrast, the lipooligosaccharide mutant showed increased sensitivity to polymyxin B, α-defensins, cathelicidins, and BPI. These findings suggest that the polysaccharides of C. jejuni strains contribute differently to resistance against host innate immunity; whereby capsule is more important for resisting human complement and lipooligosaccharide is more important for protection against killing mediated by cationic antimicrobial peptides and proteins.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Capsules; Blood Bactericidal Activity; Campylobacter Infections; Campylobacter jejuni; Drug Resistance, Bacterial; Humans; Lipopolysaccharides; Serum
PubMed: 21266840
DOI: 10.4161/viru.2.1.14752 -
Virulence 2018Whole genome sequencing analysis of 100 Neisseria meningitidis serogroup A isolates has revealed that the csaABCD-ctrABCD-ctrEF capsule polysaccharide synthesis locus...
Whole genome sequencing analysis of 100 Neisseria meningitidis serogroup A isolates has revealed that the csaABCD-ctrABCD-ctrEF capsule polysaccharide synthesis locus represents a spontaneous point mutation hotspot. Structural and functional properties of the capsule of 11 carriage and two disease isolates with non-synonymous point mutations or stop codons in capsule synthesis genes were analyzed for their capsular polysaccharide expression, recognition by antibodies and sensitivity to bactericidal killing. Eight of eleven carriage isolates presenting capsule locus mutations expressed no or reduced amounts of capsule. One isolate with a stop codon in the O-acetyltransferase gene expressed non-O-acetylated polysaccharide, and was not recognized by anti-capsule antibodies. Capsule and O-acetylation deficient mutants were resistant to complement deposition and killing mediated by anti-capsular antibodies, but not by anti-lipopolysaccharide antibodies. Two capsule polymerase mutants, one carriage and one case isolate, showed capsule over-expression and increased resistance against bactericidal activity of both capsule- and lipopolysaccharide-specific antibodies. Meningococci have developed multiple strategies for changing capsule expression and structure, which is relevant both for colonization and virulence. Here we show that point mutations in the capsule synthesis genes substantially contribute to the repertoire of genetic mechanisms in natural populations leading to variability in capsule expression.
Topics: Animals; Antibodies, Bacterial; Bacterial Capsules; Burkina Faso; Carrier State; Genetic Variation; Genome, Bacterial; Ghana; Humans; Immunity, Herd; Meningitis, Meningococcal; Mice; Neisseria meningitidis, Serogroup A; Point Mutation; Polysaccharides, Bacterial; Virulence
PubMed: 30067453
DOI: 10.1080/21505594.2018.1467710 -
Research in Microbiology 2021An extensive morphological analysis of the Neisseria meningitidis cell envelope, including serogroup B capsule and outer membrane, based on atomic force microscopy (AFM)...
An extensive morphological analysis of the Neisseria meningitidis cell envelope, including serogroup B capsule and outer membrane, based on atomic force microscopy (AFM) together with mechanical characterization by force spectroscopic measurements, has been carried out. Three meningococcal strains were used: the encapsulated serogroup B strain B1940, and the isogenic mutants B1940 siaD(+C) (lacking capsule), and B1940 cps (lacking both capsule and lipooligosaccharide outer core). AFM experiments with the encapsulated strain B1940 provided unprecedented images of the meningococcal capsule, which seems to be characterized by protrusions ("bumps") with the lateral dimensions of about 30 nm. Measurement of the Young's modulus provided quantitative assessment of the property of the capsule to confer resistance to mechanical stress. Moreover, Raman spectroscopy gave a fingerprint by which it was possible to identify the specific molecular species of the three strains analyzed, and to highlight major differences between them.
Topics: Bacterial Capsules; Bacterial Outer Membrane; Elastic Modulus; Microscopy, Atomic Force; Neisseria meningitidis, Serogroup B; Polysaccharides, Bacterial; Spectrum Analysis, Raman; Stress, Mechanical; Surface Properties
PubMed: 34284091
DOI: 10.1016/j.resmic.2021.103865