ABSTRACT
Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.
Subject(s)
Bacterial Capsules , Bacterial Proteins , Gene Expression Regulation, Bacterial , Klebsiella Infections , Klebsiella pneumoniae , RNA, Bacterial , RNA, Small Untranslated , Klebsiella pneumoniae/pathogenicity , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Animals , Virulence/genetics , Mice , Klebsiella Infections/microbiology , RNA, Small Untranslated/genetics , RNA, Small Untranslated/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Capsules/metabolism , Bacterial Capsules/genetics , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Humans , Female , Host Factor 1 Protein/metabolism , Host Factor 1 Protein/geneticsABSTRACT
Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.
Subject(s)
Galectins , Immunity, Innate , Klebsiella Infections , Klebsiella pneumoniae , Sialic Acid Binding Immunoglobulin-like Lectins , Klebsiella pneumoniae/immunology , Klebsiella pneumoniae/metabolism , Humans , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/immunology , Galectins/metabolism , Galectins/immunology , Klebsiella Infections/immunology , Klebsiella Infections/microbiology , Bacterial Capsules/immunology , Bacterial Capsules/metabolism , Lectins/metabolism , Lectins/immunology , Protein BindingABSTRACT
Escherichia coli (E. coli) is a major cause of urinary tract infections, bacteraemia, and sepsis. CFT073 is a prototypic, urosepsis isolate of sequence type (ST) 73. This laboratory, among others, has shown that strain CFT073 is resistant to serum, with capsule and other extracellular polysaccharides imparting resistance. The interplay of such polysaccharides remains under-explored. This study has shown that CFT073 mutants deficient in lipopolysaccharide (LPS) O-antigen and capsule display exquisite serum sensitivity. Additionally, O-antigen and LPS outer core mutants displayed significantly decreased surface K2 capsule, coupled with increased unbound K2 capsule being detected in the supernatant. The R1 core and O6 antigen are involved in the tethering of K2 capsule to the CFT073 cell surface, highlighting the importance of the R1 core in serum resistance. The dependence of capsule on LPS was shown to be post-transcriptional and related to changes in cell surface hydrophobicity. Furthermore, immunofluorescence microscopy suggested that the surface pattern of capsule is altered in such LPS core mutants, which display a punctate capsule pattern. Finally, targeting LPS biosynthesis using sub-inhibitory concentrations of a WaaG inhibitor resulted in increased serum sensitivity and decreased capsule in CFT073. Interestingly, the dependency of capsule on LPS has been observed previously in other Enterobacteria, indicating that the synergy between these polysaccharides is not just strain, serotype or species-specific but may be conserved across several pathogenic Gram-negative species. Therefore, using WaaG inhibitor derivatives to target LPS is a promising therapeutic strategy to reduce morbidity and mortality by reducing or eliminating surface capsule.
Subject(s)
Bacterial Capsules , Lipopolysaccharides , Lipopolysaccharides/metabolism , Bacterial Capsules/metabolism , Bacterial Capsules/genetics , Humans , Extraintestinal Pathogenic Escherichia coli/genetics , Extraintestinal Pathogenic Escherichia coli/drug effects , Extraintestinal Pathogenic Escherichia coli/metabolism , O Antigens/genetics , O Antigens/metabolism , Escherichia coli Infections/microbiology , Escherichia coli/genetics , Escherichia coli/drug effects , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , MutationABSTRACT
AIMS: Hypervirulent Klebsiella pneumoniae (hvKp) causes invasive community-acquired infections in healthy individuals, and hypermucoviscosity (HMV) is the main phenotype associated with hvKp. This study investigates the impact of microaerobic environment availability on the mucoviscosity of K. pneumoniae. METHODS AND RESULTS: By culturing 25 clinical strains under microaerobic and aerobic environments, we observed a notable reduction in mucoviscosity in microaerobic environments. RNA sequencing and qRT-PCR revealed downregulated expressions of capsule synthesis genes (galf, orf2, wzi, wza, wzb, wzc, wcaj, manC, manB, and ugd) and regulatory genes (rmpA, rmpD, and rmpC) under microaerobic conditions. Transmission electron microscopy and Indian ink staining analysis were performed, revealing that the capsular thickness of K. pneumoniae decreased by half in microaerobic conditions compared to aerobic conditions. Deletion of rmpD and rmpC caused the loss of the HMV phenotype in both aerobic and microaerobic conditions. However, compared to wild-type strain in microaerobic condition, only rmpD overexpression strain, and not rmpC overexpression strain, displayed a significant increase in capsule thickness in microaerobic conditions. CONCLUSIONS: Microaerobic conditions can suppress the mucoviscosity of K. pneumoniae, but this suppression can be overcome by altering the expression of rmpD, indicating a specific function for rmpD in the oxygen environmental adaptation of K. pneumoniae.
Subject(s)
Bacterial Proteins , Klebsiella pneumoniae , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Aerobiosis , Humans , Gene Expression Regulation, Bacterial , Phenotype , Klebsiella Infections/microbiology , Bacterial Capsules/metabolism , Bacterial Capsules/genetics , Virulence/geneticsABSTRACT
Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.
Subject(s)
Bacterial Proteins , Glycoside Hydrolases , Lipopolysaccharides , Macrophages , Mannans , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/growth & development , Lipopolysaccharides/metabolism , Mannans/metabolism , Macrophages/metabolism , Macrophages/microbiology , Glycoside Hydrolases/metabolism , Bacterial Proteins/metabolism , Animals , Mice , Humans , Phosphatidylinositols/metabolism , Bacterial Capsules/metabolismABSTRACT
Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages. IMPORTANCE: Phage resistance is a direct threat to phage therapy, and understanding phage-host interactions, especially how bacteria block phage infection, is essential for developing successful phage therapy. In the present study, we demonstrate for the first time that Vibrio alginolyticus uses quorum sensing (QS) to promote capsular polysaccharide (CPS)-specific phage infection by upregulating ugd expression, which is necessary for the synthesis of Autographiviridae phage receptor CPS. Although increased CPS-specific phage susceptibility is a novel trade-off mediated by QS, it results in the upregulation of virulence factors, promoting biofilm development and enhanced capsular polysaccharide production in V. alginolyticus. This suggests that inhibiting QS may improve the effectiveness of antibiotic treatment, but it may also reduce the efficacy of phage therapy.
Subject(s)
Quorum Sensing , Vibrio alginolyticus , Vibrio alginolyticus/virology , Vibrio alginolyticus/physiology , Bacteriophages/physiology , Bacteriophages/genetics , Homoserine/analogs & derivatives , Homoserine/metabolism , Bacterial Capsules/metabolism , Podoviridae/genetics , Podoviridae/physiology , Biofilms/growth & development , Polysaccharides, Bacterial/metabolismABSTRACT
Objective: To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods: The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δwza were detected by measuring the optical density OD600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δwza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δwza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δwza mutant strain. Results: The PCR results revealed that the Δwza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δwza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δwza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δwza mutant strain (45.963±2.795) µg/ml compared with wild-type strain W14 (138.800±5.201) µg/ml. There was a statistical difference between the two groups (t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion: Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.
Subject(s)
Bacterial Capsules , Bacteriophages , Gene Deletion , Klebsiella pneumoniae , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/virology , Bacteriophages/genetics , Bacterial Capsules/genetics , Bacterial Proteins/geneticsABSTRACT
Infections caused by antimicrobial-resistant Escherichia coli are the leading cause of death attributed to antimicrobial resistance (AMR) worldwide, and the known AMR mechanisms involve a range of functional proteins. Here, we employed a pan-genome wide association study (GWAS) approach on over 1,000 E. coli isolates from sick dogs collected across the US and Canada and identified a strong statistical association (empirical P < 0.01) of AMR, involving a range of antibiotics to a group 1 capsular (CPS) gene cluster. This cluster included genes under relaxed selection pressure, had several loci missing, and had pseudogenes for other key loci. Furthermore, this cluster is widespread in E. coli and Klebsiella clinical isolates across multiple host species. Earlier studies demonstrated that the octameric CPS polysaccharide export protein Wza can transmit macrolide antibiotics into the E. coli periplasm. We suggest that the CPS in question, and its highly divergent Wza, functions as an antibiotic trap, preventing antimicrobial penetration. We also highlight the high diversity of lineages circulating in dogs across all regions studied, the overlap with human lineages, and regional prevalence of resistance to multiple antimicrobial classes. IMPORTANCE: Much of the human genomic epidemiology data available for E. coli mechanism discovery studies has been heavily biased toward shiga-toxin producing strains from humans and livestock. E. coli occupies many niches and produces a wide variety of other significant pathotypes, including some implicated in chronic disease. We hypothesized that since dogs tend to share similar strains with their owners and are treated with similar antibiotics, their pathogenic isolates will harbor unexplored AMR mechanisms of importance to humans as well as animals. By comparing over 1,000 genomes with in vitro antimicrobial susceptibility data from sick dogs across the US and Canada, we identified a strong multidrug resistance association with an operon that appears to have once conferred a type 1 capsule production system.
Subject(s)
Anti-Bacterial Agents , Dog Diseases , Drug Resistance, Multiple, Bacterial , Escherichia coli Infections , Escherichia coli , Dogs , Animals , Escherichia coli/genetics , Escherichia coli/drug effects , Dog Diseases/microbiology , Escherichia coli Infections/veterinary , Escherichia coli Infections/microbiology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Canada , Genome-Wide Association Study , Genome, Bacterial , United States , Bacterial Capsules/genetics , Multigene Family , Evolution, Molecular , Genomics , Escherichia coli Proteins/geneticsABSTRACT
Analysis of pneumococcal polysaccharides (PnPs) has been an arduous task, especially in similar serotypes. Pneumococci invades the host immune response by modulating capsule structure with small genetic changes making them indistinguishable from similar serotypes by conventional modes of analysis. The new serotype 24F causing invasive pneumococcal-resistant infection is an analytical challenge for its analysis as related serotypes 24A and 24B Ps share a common backbone. The difference in the branched chain which contains arabinitol and ribitol in 24F and 24B respectively are stereoisomers making their identification even more challenging. The composition analysis by GC-MS revealed distinct peaks for arabinitol in 24F and 24A Ps and ribitol in Pn 24B serotype polysaccharide. The mass spectral analysis confirmed their identification along with a heterologous cross-reactivity which confirmed anti-Pn-24F mAb reactive to Pn 24B than Pn 24A. The quantitative analysis of pneumococcal 24A, 24B and 24F using GC-MS showed sensitive analysis over the concentration range 3.125-200 µg/mL with regression coefficient >0.99 making ideal modality for the characterization, identification, and quantitation of pneumococcal 24A, 24B and 24F similar serotypes.
Subject(s)
Gas Chromatography-Mass Spectrometry , Polysaccharides, Bacterial , Serogroup , Streptococcus pneumoniae , Gas Chromatography-Mass Spectrometry/methods , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/immunology , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Bacterial Capsules/immunology , Bacterial Capsules/chemistryABSTRACT
Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.
Subject(s)
Bacterial Capsules , Ducks , Flavobacteriaceae Infections , Riemerella , Riemerella/genetics , Riemerella/pathogenicity , Riemerella/metabolism , Animals , Ducks/microbiology , Bacterial Capsules/genetics , Bacterial Capsules/metabolism , Flavobacteriaceae Infections/microbiology , Flavobacteriaceae Infections/veterinary , Poultry Diseases/microbiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Polysaccharides, Bacterial/biosynthesis , Virulence Factors/genetics , Gene DeletionABSTRACT
BACKGROUND: Streptococcus pneumoniae serotype 3 remains a problem globally. Malawi introduced 13-valent pneumococcal conjugate vaccine (PCV13) in 2011, but there has been no direct protection against serotype 3 carriage. We explored whether vaccine escape by serotype 3 is due to clonal expansion of a lineage with a competitive advantage. METHODS: The distribution of serotype 3 Global Pneumococcal Sequence Clusters (GPSCs) and sequence types (STs) globally was assessed using sequences from the Global Pneumococcal Sequencing Project. Whole-genome sequences of 135 serotype 3 carriage isolates from Blantyre, Malawi (2015-2019) were analyzed. Comparative analysis of the capsule locus, entire genomes, antimicrobial resistance, and phylogenetic reconstructions were undertaken. Opsonophagocytosis was evaluated using serum samples from vaccinated adults and children. RESULTS: Serotype 3 GPSC10-ST700 isolates were most prominent in Malawi. Compared with the prototypical serotype 3 capsular polysaccharide locus sequence, 6 genes are absent, with retention of capsule polysaccharide biosynthesis. This lineage is characterized by increased antimicrobial resistance and lower susceptibility to opsonophagocytic killing. CONCLUSIONS: A serotype 3 variant in Malawi has genotypic and phenotypic characteristics that could enhance vaccine escape and clonal expansion after post-PCV13 introduction. Genomic surveillance among high-burden populations is essential to improve the effectiveness of next-generation pneumococcal vaccines.
Subject(s)
Bacterial Capsules , Phylogeny , Pneumococcal Infections , Pneumococcal Vaccines , Serogroup , Streptococcus pneumoniae , Humans , Pneumococcal Vaccines/immunology , Pneumococcal Vaccines/administration & dosage , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/immunology , Streptococcus pneumoniae/classification , Pneumococcal Infections/prevention & control , Pneumococcal Infections/microbiology , Pneumococcal Infections/immunology , Bacterial Capsules/immunology , Bacterial Capsules/genetics , Malawi , Adult , Whole Genome Sequencing , Child, Preschool , Child , Vaccines, Conjugate/immunology , Male , Genome, Bacterial , Female , Young Adult , Infant , Genotype , Carrier State/microbiologyABSTRACT
BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.
Subject(s)
Bacterial Capsules , Complement System Proteins , Klebsiella Infections , Klebsiella pneumoniae , Phagocytosis , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/immunology , Humans , Klebsiella Infections/immunology , Klebsiella Infections/microbiology , Animals , Bacterial Capsules/immunology , Bacterial Capsules/genetics , Bacterial Capsules/metabolism , Mice , Complement System Proteins/immunology , Mutation , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Whole Genome Sequencing , Reinfection/microbiology , Reinfection/immunology , Bacteremia/microbiology , Bacteremia/immunology , FemaleABSTRACT
Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control Vi polymerization or acetylation is enough to result in different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper Vi capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo Vi capsule variants have primarily been identified in Africa, whereas the hyper Vi capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria.
Subject(s)
Bacterial Capsules , Mutation, Missense , Polysaccharides, Bacterial , Salmonella typhi , Typhoid Fever , Salmonella typhi/genetics , Salmonella typhi/pathogenicity , Animals , Mice , Virulence/genetics , Polysaccharides, Bacterial/genetics , Polysaccharides, Bacterial/biosynthesis , Polysaccharides, Bacterial/metabolism , Bacterial Capsules/genetics , Bacterial Capsules/metabolism , Typhoid Fever/microbiology , Humans , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Virulence Factors/genetics , Virulence Factors/metabolism , Female , Whole Genome SequencingABSTRACT
The disaccharide (ß-D-glucopyranosyluronic acid)-(1â4)-ß-D-glucopyranoside represents a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 3. A conjugate of the disaccharide with BSA (di-BSA conjugate) adjuvanted with aluminum hydroxide induced - in contrast to the non-adjuvanted conjugate - IgG1 antibody production and protected mice against S. pneumoniae serotype 3 infection after intraperitoneal prime-boost immunization. Adjuvanted and non-adjuvanted conjugates induced production of Th1 (IFNγ, TNFα); Th2 (IL-5, IL-13); Th17 (IL-17A), Th1/Th17 (IL-22), and Th2/Th17 cytokines (IL-21) after immunization. The concentration of cytokines in mice sera was higher in response to the adjuvanted conjugate, with the highest level of IL-17A production after the prime and boost immunizations. In contrast, the non-adjuvanted conjugate elicited only weak production of IL-17A, which gradually decreased after the second immunization. After boost immunization of mice with the adjuvanted di-BSA conjugate, there was a significant increase in the number of CD45+/CD19+ B cells, TCR+ γδ T cell, CD5+ Ð1 cells, and activated cells with MHC II+ expression in the spleens of the mice. IL-17A, TCR+ γδ T cells, and CD5+ Ð1 cells play a crucial role in preventing pneumococcal infection, but can also contribute to autoimmune diseases. Immunization with the adjuvanted and non-adjuvanted di-BSA conjugate did not elicit autoantibodies against double-stranded DNA targeting cell nuclei in mice. Thus, the molecular and cellular markers associated with antibody production and protective activity in response to immunization with the di-BSA conjugate adjuvanted with aluminum hydroxide are IL-17A, TCR+ γδ T cells, and CD5+ Ð1 cells against the background of increasing MHC II+ expression.
Subject(s)
Interleukin-17 , Pneumococcal Vaccines , Serum Albumin, Bovine , Streptococcus pneumoniae , Animals , Interleukin-17/immunology , Interleukin-17/metabolism , Streptococcus pneumoniae/immunology , Mice , Serum Albumin, Bovine/immunology , Pneumococcal Vaccines/immunology , Pneumococcal Infections/immunology , Pneumococcal Infections/prevention & control , Disaccharides/immunology , Bacterial Capsules/immunology , Polysaccharides, Bacterial/immunology , Adjuvants, Immunologic/administration & dosage , Female , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Intraepithelial Lymphocytes/immunology , Serogroup , Receptors, Antigen, T-Cell, gamma-delta/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolismABSTRACT
Meningococcal glycoconjugate vaccines sourced from capsular polysaccharides (CPSs) of pathogenic Neisseria meningitidis strains are well-established measures to prevent meningococcal disease. However, the exact structural factors responsible for antibody recognition are not known. CPSs of Neisseria meningitidis serogroups Y and W differ by a single stereochemical center, yet they evoke specific immune responses. Herein, we developed specific monoclonal antibodies (mAbs) targeting serogroups C, Y, and W and evaluated their ability to kill bacteria. We then used these mAbs to dissect structural elements responsible for carbohydrate-protein interactions. First, Men oligosaccharides were screened against the mAbs using ELISA to select putative lengths representing the minimal antigenic determinant. Next, molecular interaction features between the mAbs and serogroup-specific sugar fragments were elucidated using STD-NMR. Moreover, X-ray diffraction data with the anti-MenW CPS mAb enabled the elucidation of the sugar-antibody binding mode. Our findings revealed common traits in the epitopes of all three sialylated serogroups. The minimal binding epitopes typically comprise five to six repeating units. Moreover, the O-acetylation of the neuraminic acid moieties was fundamental for mAb binding. These insights hold promise for the rational design of optimized meningococcal oligosaccharides, opening new avenues for novel production methods, including chemical or enzymatic approaches.
Subject(s)
Antibodies, Monoclonal , Meningococcal Vaccines , Neisseria meningitidis , Polysaccharides, Bacterial , Serogroup , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/chemistry , Neisseria meningitidis/immunology , Neisseria meningitidis/chemistry , Meningococcal Vaccines/immunology , Meningococcal Vaccines/chemistry , Polysaccharides, Bacterial/immunology , Polysaccharides, Bacterial/chemistry , Antibodies, Bacterial/immunology , Epitopes/immunology , Epitopes/chemistry , Animals , Mice , Humans , Bacterial Capsules/immunology , Bacterial Capsules/chemistry , Antibody Formation/immunologyABSTRACT
Since the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in Malawi in 2011, there has been persistent carriage of vaccine serotype (VT) Streptococcus pneumoniae, despite high vaccine coverage. To determine if there has been a genetic change within the VT capsule polysaccharide (cps) loci since the vaccine's introduction, we compared 1022 whole-genome-sequenced VT isolates from 1998 to 2019. We identified the clonal expansion of a multidrug-resistant, penicillin non-susceptible serotype 23F GPSC14-ST2059 lineage, a serotype 14 GPSC9-ST782 lineage and a novel serotype 14 sequence type GPSC9-ST18728 lineage. Serotype 23F GPSC14-ST2059 had an I253T mutation within the capsule oligosaccharide repeat unit polymerase Wzy protein, which is predicted in silico to alter the protein pocket cavity. Moreover, serotype 23F GPSC14-ST2059 had SNPs in the DNA binding sites for the cps transcriptional repressors CspR and SpxR. Serotype 14 GPSC9-ST782 harbours a non-truncated version of the large repetitive protein (Lrp), containing a Cna protein B-type domain which is also present in proteins associated with infection and colonisation. These emergent lineages also harboured genes associated with antibiotic resistance, and the promotion of colonisation and infection which were absent in other lineages of the same serotype. Together these data suggest that in addition to serotype replacement, modifications of the capsule locus associated with changes in virulence factor expression and antibiotic resistance may promote vaccine escape. In summary, the study highlights that the persistence of vaccine serotype carriage despite high vaccine coverage in Malawi may be partly caused by expansion of VT lineages post-PCV13 rollout.
Subject(s)
Bacterial Capsules , Pneumococcal Infections , Pneumococcal Vaccines , Serogroup , Streptococcus pneumoniae , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/pathogenicity , Pneumococcal Vaccines/immunology , Humans , Malawi , Bacterial Capsules/genetics , Pneumococcal Infections/microbiology , Pneumococcal Infections/prevention & control , Vaccines, Conjugate , Polysaccharides, Bacterial/genetics , Polysaccharides, Bacterial/immunology , Virulence/genetics , Genotype , Whole Genome Sequencing , Bacterial Proteins/genetics , Virulence Factors/genetics , Child, Preschool , Polymorphism, Single Nucleotide , Infant , MaleABSTRACT
BACKGROUND: Tuberculosis (TB), a global infectious threat, has seen a concerning rise in aminoglycoside-resistant Mycobacterium tuberculosis (M.tb) strains. The potential role of capsule proteins remains largely unexplored. This layer acts as the primary barrier for tubercle bacilli, attempting to infiltrate host cells and subsequent disease development. METHODS: The study aims to bridge this gap by investigating the differentially expressed capsule proteins in aminoglycoside-resistant M.tb clinical isolates compared with drug-sensitive isolates employing two-dimensional gel electrophoresis, mass spectrometry, and bioinformatic approaches. RESULTS: We identified eight proteins that exhibited significant upregulation in aminoglycoside-resistant isolates. Protein Rv3029c and Rv2110c were associated with intermediary metabolism and respiration; Rv2462c with cell wall and cell processes; Rv3804c with lipid metabolism; Rv2416c and Rv2623 with virulence and detoxification/adaptation; Rv0020c with regulatory functions; and Rv0639 with information pathways. Notably, the Group-based Prediction System for Prokaryotic Ubiquitin-like Protein (GPS-PUP) algorithm identified potential pupylation sites within all proteins except Rv3804c. Interactome analysis using the STRING 12.0 database revealed potential interactive partners for these proteins, suggesting their involvement in aminoglycoside resistance. Molecular docking studies revealed suitable binding between amikacin and kanamycin drugs with Rv2462c, Rv3804c, and Rv2623 proteins. CONCLUSION: As a result, our findings illustrate the multifaceted nature of aminoglycoside resistance in M.tb and the importance of understanding how capsule proteins play a role in counteracting drug efficacy. Identifying the role of these proteins in drug resistance is crucial for developing more effective treatments and diagnostics for TB.
Subject(s)
Aminoglycosides , Bacterial Proteins , Drug Resistance, Bacterial , Mycobacterium tuberculosis , Proteomics , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Humans , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Aminoglycosides/pharmacology , Bacterial Capsules/metabolism , Antitubercular Agents/pharmacology , Microbial Sensitivity Tests , Computational Biology , Electrophoresis, Gel, Two-Dimensional , Tuberculosis/microbiologyABSTRACT
Bacteria utilize intercellular communication to orchestrate essential cellular processes, adapt to environmental changes, develop antibiotic tolerance, and enhance virulence. This communication, known as quorum sensing (QS), is mediated by the exchange of small signalling molecules called autoinducers. AI-2 QS, regulated by the metabolic enzyme LuxS (S-ribosylhomocysteine lyase), acts as a universal intercellular communication mechanism across gram-positive and gram-negative bacteria and is crucial for diverse bacterial processes. In this study, we demonstrated that in Streptococcus suis (S. suis), a notable zoonotic pathogen, AI-2 QS enhances galactose utilization, upregulates the Leloir pathway for capsular polysaccharide (CPS) precursor production, and boosts CPS synthesis, leading to increased resistance to macrophage phagocytosis. Additionally, our molecular docking and dynamics simulations suggest that, similar to S. pneumoniae, FruA, a fructose-specific phosphoenolpyruvate phosphotransferase system prevalent in gram-positive pathogens, may also function as an AI-2 membrane surface receptor in S. suis. In conclusion, our study demonstrated the significance of AI-2 in the synthesis of galactose metabolism-dependent CPS in S. suis. Additionally, we conducted a preliminary analysis of the potential role of FruA as a membrane surface receptor for S. suis AI-2.
Subject(s)
Galactose , Quorum Sensing , Streptococcus suis , Streptococcus suis/physiology , Galactose/metabolism , Quorum Sensing/physiology , Virulence , Animals , Bacterial Capsules/metabolism , Lactones/metabolism , Streptococcal Infections/veterinary , Streptococcal Infections/microbiology , Streptococcal Infections/immunology , Homoserine/analogs & derivatives , Homoserine/metabolism , Polysaccharides, Bacterial/metabolismABSTRACT
Among the Acinetobacter genus, Acinetobacter pittii stands out as an important opportunistic infection causative agent commonly found in hospital settings, which poses a serious threat to human health. Recently, the high prevalence of carbapenem-resistant A. pittii isolates has created significant therapeutic challenges for clinicians. Bacteriophages and their derived enzymes are promising therapeutic alternatives or adjuncts to antibiotics effective against multidrug-resistant bacterial infections. However, studies investigating the depolymerases specific to A. pittii strains are scarce. In this study, we identified and characterized a capsule depolymerase, Dpo27, encoded by the bacteriophage IME-Ap7, which targets A. pittii. A total of 23 clinical isolates of Acinetobacter spp. were identified as A. pittii (21.91%, 23/105), and seven A. pittii strains with various K locus (KL) types (KL14, KL32, KL38, KL111, KL163, KL207, and KL220) were used as host bacteria for phage screening. The lytic phage IME-Ap7 was isolated using A. pittii 7 (KL220) as an indicator bacterium and was observed for depolymerase activity. A putative tail fiber gene encoding a polysaccharide-degrading enzyme (Dpo27) was identified and expressed. The results of the modified single-spot assay showed that both A. pittii 7 and 1492 were sensitive to Dpo27, which was assigned the KL220 type. After incubation with Dpo27, A. pittii strain was susceptible to killing by human serum; moreover, the protein displayed no hemolytic activity against erythrocytes. Furthermore, the protein exhibited sustained activity across a wide pH range (5.0-10.0) and at temperatures between 20 and 50°C. In summary, the identified capsule depolymerase Dpo27 holds promise as an alternative treatment for combating KL220-type A. pittii infections.