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Microbiology Spectrum Sep 2021The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and...
The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.
Topics: Bacterial Capsules; Bacteriophages; Cryoelectron Microscopy; Klebsiella pneumoniae; Polysaccharide-Lyases; Viral Proteins
PubMed: 34431721
DOI: 10.1128/Spectrum.01023-21 -
Frontiers in Cellular and Infection... 2023The polysaccharide capsule of plays a major role in virulence, adherence to epithelial cells, and overall survival of the bacterium in the human host. Galactose,...
BACKGROUND
The polysaccharide capsule of plays a major role in virulence, adherence to epithelial cells, and overall survival of the bacterium in the human host. Galactose, mannose, and N-acetylglucosamine (GlcNAc) are likely to be relevant for metabolization in the nasopharynx, while glucose is the primary carbon source in the blood. In this study, we aim to further the understanding of the influence of carbon sources on pneumococcal growth, capsule biosynthesis, and subsequent adherence potential.
METHODS
We tested the growth behavior of clinical wild-type and capsule knockout strains, using galactose, GlcNAc, mannose, and glucose as carbon source for growth. We measured capsule thickness and quantified capsule precursors by fluorescein isothiocyanate (FITC)-dextran exclusion assays and P-nuclear magnetic resonance measurements, respectively. We also performed epithelial adherence assays using Detroit 562 cells and performed a transcriptome analysis (RNA sequencing).
RESULTS
We observed a reduced growth in galactose, mannose, and GlcNAc compared to growth in glucose and found capsular size reductions in mannose and GlcNAc compared to galactose and glucose. Additionally, capsular precursor measurements of uridine diphosphate-(UDP)-glucose and UDP-galactose showed less accumulation of precursors in GlcNAc or mannose than in glucose and galactose, indicating a possible link with the received capsular thickness measurements. Epithelial adherence assays showed an increase in adherence potential for a pneumococcal strain, when grown in mannose compared to glucose. Finally, transcriptome analysis of four clinical isolates revealed not only strain specific but also common carbon source-specific gene expression.
CONCLUSION
Our findings may indicate a careful adaption of the lifestyle of according to the monosaccharides encountered in the respective human niche.
Topics: Humans; Streptococcus pneumoniae; Galactose; Carbon; Mannose; Glucose; Uridine Diphosphate; Bacterial Capsules
PubMed: 38094742
DOI: 10.3389/fcimb.2023.1279119 -
Nature Chemical Biology Jul 2023Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and...
Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.
Topics: Infant; Child; Humans; Haemophilus influenzae type b; Haemophilus Infections; Haemophilus Vaccines; Bacterial Capsules; Gram-Negative Bacteria
PubMed: 37277468
DOI: 10.1038/s41589-023-01324-3 -
Scientific Reports Nov 2020Streptococcus pneumoniae (pneumococcus) is a principal cause of bacterial middle ear infections, pneumonia, and meningitis. Capsule-targeted pneumococcal vaccines have...
Streptococcus pneumoniae (pneumococcus) is a principal cause of bacterial middle ear infections, pneumonia, and meningitis. Capsule-targeted pneumococcal vaccines have likely contributed to increased carriage of nonencapsulated S. pneumoniae (NESp). Some NESp lineages are associated with highly efficient DNA uptake and transformation frequencies. However, NESp strains lack capsule that may increase disease severity. We tested the hypothesis that NESp could acquire capsule during systemic infection and transform into more virulent pneumococci. We reveal that NESp strains MNZ67 and MNZ41 are highly transformable and resistant to multiple antibiotics. Natural transformation of NESp when co-administered with heat-killed encapsulated strain WU2 in a murine model of systemic infection resulted in encapsulation of NESp and increased virulence during bacteremia. Functional capsule production increased the pathogenic potential of MNZ67 by significantly decreasing complement deposition on the bacterial surface. However, capsule acquisition did not further decrease complement deposition on the relatively highly pathogenic strain MNZ41. Whole genome sequencing of select transformants demonstrated that recombination of up to 56.7 kbp length occurred at the capsule locus, along with additional recombination occurring at distal sites harboring virulence-associated genes. These findings indicate NESp can compensate for lack of capsule production and rapidly evolve into more virulent strains.
Topics: Animals; Anti-Bacterial Agents; Bacteremia; Bacterial Capsules; Disease Models, Animal; Drug Resistance, Bacterial; Mice; Pneumococcal Infections; Streptococcus pneumoniae; Transformation, Bacterial; Virulence; Whole Genome Sequencing
PubMed: 33144660
DOI: 10.1038/s41598-020-75988-5 -
The Journal of Biological Chemistry Sep 2023The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit...
The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-β-D-Galf2Ac-(1→3)-β-D-Galp-(1→3)-α-D-Galp-(1→3)-β-D-Galf-(1→3)-β-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F.
Topics: Antibodies, Bacterial; Pilot Projects; Pneumococcal Infections; Pneumococcal Vaccines; Polysaccharides; Serogroup; Streptococcus pneumoniae; Vaccines, Conjugate; Bacterial Capsules; Genes, Bacterial; Gene Silencing; Transferases
PubMed: 37495106
DOI: 10.1016/j.jbc.2023.105085 -
Applied and Environmental Microbiology Mar 2011We studied the interaction between capsule production and hydrodynamic growth conditions on the internal and macroscopic structure of biofilms and spontaneously formed...
We studied the interaction between capsule production and hydrodynamic growth conditions on the internal and macroscopic structure of biofilms and spontaneously formed aggregates of Klebsiella pneumoniae. Wild-type and capsule-deficient strains were studied as biofilms and under strong and mild hydrodynamic conditions. Internal organization of multicellular structures was determined with a novel image-processing algorithm for feature extraction from high-resolution confocal microscopy. Measures included interbacterial spacing and local angular alignment of individual bacteria. Macroscopic organization was measured via the size distribution of aggregate populations forming under various conditions. Compared with wild-type organisms, unencapsulated mutant organisms formed more organized aggregates with less variability in interbacterial spacing and greater interbacterial angular alignment. Internal aggregate structure was not detectably affected by the severity of hydrodynamic growth conditions. However, hydrodynamic conditions affected both wild-type and mutant aggregate size distributions. Bacteria grown under high-speed shaking conditions (i.e., at Reynolds' numbers beyond the laminar-turbulent transition) formed few multicellular aggregates while clumpy growth was common in bacteria grown under milder conditions. Our results indicate that both capsule and environment contribute to the structure of communities of K. pneumoniae, with capsule exerting influence at an interbacterial length scale and fluid dynamic forces affecting overall particle size.
Topics: Bacterial Adhesion; Bacterial Capsules; Biofilms; Klebsiella pneumoniae; Microscopy, Confocal
PubMed: 21239544
DOI: 10.1128/AEM.01752-10 -
Critical Reviews in Biochemistry and... 2014Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative... (Review)
Review
Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of the most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last 10 years in the discovery and development of novel inhibitors targeting their biogenesis.
Topics: Bacterial Capsules; Cell Membrane; Glycoconjugates; Glycoproteins; Humans; Models, Biological; Mycobacterium tuberculosis
PubMed: 24915502
DOI: 10.3109/10409238.2014.925420 -
MSphere Mar 2021Capsular polysaccharide (CPS) heterogeneity within carbapenem-resistant (CR-) strain sequence type 258 (ST258) must be considered when developing CPS-based vaccines....
Capsular polysaccharide (CPS) heterogeneity within carbapenem-resistant (CR-) strain sequence type 258 (ST258) must be considered when developing CPS-based vaccines. Here, we sought to characterize CPS-specific antibody responses elicited by CR--infected patients. Plasma and bacterial isolates were collected from 33 hospital patients with positive CR- cultures. Isolate capsules were typed by sequencing. Reactivity and measures of efficacy of patient antibodies were studied against 3 prevalent CR- CPS types (, , and ). High IgG titers against and CPS were documented in 79% of infected patients. Patient-derived (PD) IgGs agglutinated CR- and limited growth better than naive IgG and promoted phagocytosis of strains across the serotype isolated from their donors. Additionally, poly-IgG from and patients promoted phagocytosis of nonconcordant CR- serotypes. Such effects were lost when poly-IgG was depleted of CPS-specific IgG. Additionally, mice infected with , , and CR- strains preopsonized with patient-derived IgG exhibited lower lung CFU than controls. Depletion of antibodies (Abs) reversed this effect in and infections, whereas Ab depletion reduced poly-IgG efficacy against CR- We are the first to report cross-reactive properties of CPS-specific Abs from CR- patients through both and models. Carbapenem-resistant is a rapidly emerging public health threat that can cause fatal infections in up to 50% of affected patients. Due to its resistance to nearly all antimicrobials, development of alternate therapies like antibodies and vaccines is urgently needed. Capsular polysaccharides constitute important targets, as they are crucial for pathogenesis. Capsular polysaccharides are very diverse and, therefore, studying the host's capsule-type specific antibodies is crucial to develop effective anti-CPS immunotherapies. In this study, we are the first to characterize humoral responses in infected patients against carbapenem-resistant expressing different capsule types. This study is the first to report the efficacy of cross-reactive properties of CPS-specific Abs in both and models.
Topics: Adult; Aged; Aged, 80 and over; Anti-Bacterial Agents; Antibodies, Bacterial; Bacterial Capsules; Carbapenem-Resistant Enterobacteriaceae; Carbapenems; Female; Genotype; Humans; Klebsiella pneumoniae; Male; Middle Aged; Polysaccharides, Bacterial; Serogroup; Virulence; Young Adult
PubMed: 33658281
DOI: 10.1128/mSphere.01335-20 -
Genes Aug 2020is the causative agent of a multitude of diseases, and further study into its pathogenies is vital. The pneumococcus is genetically malleable, and several tools are...
is the causative agent of a multitude of diseases, and further study into its pathogenies is vital. The pneumococcus is genetically malleable, and several tools are available to manipulate this pathogen. In this study, we attempted to utilize one such tool, the Sweet Janus cassette, to replace the capsule locus with other capsule loci in our strain background and found that the efficiency of allelic replacement was low and the number of revertant false-positive colonies was high. We determined that the capacity to recombine capsule varied by the initial isolated colony, suggesting that frequency of reversion is dependent on the bacterial clone. Alternative selection markers may further expand the application of Sweet Janus. We created novel cassettes that utilized chlorinated phenylalanine as an alternative counter-selection agent in conjunction with the Janus or Sweet Janus cassette, providing a new dual or triple selection marker. Moreover, we created cassettes that do not require engineered resistance in the background strain, including both single and dual selection markers. We were able to utilize all constructs in allelic replacement of the capsule loci. These novel constructs provide a new means for generating gene deletions in that expand experimental applications.
Topics: Alleles; Bacterial Capsules; Drug Resistance, Bacterial; Gene Conversion; Gene Deletion; Genetics, Microbial; Plasmids; Recombination, Genetic; Selection, Genetic; Streptococcus pneumoniae
PubMed: 32825523
DOI: 10.3390/genes11090965 -
Molecular Microbiology Sep 2013Protein glycosylation and capsular polysaccharide formation are increasingly recognized as playing central roles in the survival and virulence of bacterial pathogens. In...
Protein glycosylation and capsular polysaccharide formation are increasingly recognized as playing central roles in the survival and virulence of bacterial pathogens. In this issue of Molecular Microbiology, structural analysis in Acinetobacter baumannii 17978 revealed that a pentasaccharide that decorates glycoproteins is formed of the same building blocks used for capsule biosynthesis demonstrating split roles for this glycan. Disruption of PglC, the initiating glycosyltransferase responsible for attachment of the first sugar to undecaprenylphosphate abolished glycoprotein production and capsule biosynthesis. Both pathways are demonstrated to be important in biofilm formation and pathogenesis, and disabling their synthesis should provide a useful route for antimicrobial design. Shared polysaccharide usage reduces the genetic and metabolic burden in a bacterial cell and is an emerging theme among bacterial pathogens that need to be energy efficient for their streamlined lifestyle.
Topics: Acinetobacter baumannii; Animals; Bacterial Capsules; Metabolic Networks and Pathways; Protein Processing, Post-Translational
PubMed: 23844892
DOI: 10.1111/mmi.12328