Surface Antigen Exposure by Bismuth Dimercaprol Suppression of Klebsiella pneumoniae Capsular Polysaccharide

ABSTRACT The bacterial capsule is an important virulence determinant in animal and plant disease. Bacterial capsule and slime can be inhibited by bismuth compounds, especially when complexed with lipophilic thiol chelators. Bismuth dimercaprol (BisBAL) at 1 ppm of Bi3+repressed Klebsiella pneumoniae capsule expression in defined medium by nearly 90%, which exposed subsurface structures. The phagocytic index for BisBAL-treated bacteria increased from <10 to 360 bacteria per 100 neutrophils in the presence of complement and anticapsular or anti-O antigen antiserum. BisBAL treatment also enhanced the reactivity of monoclonal antibodies (MAbs) specific for the O1-antigen lipopolysaccharide (LPS) or the LPS core in a dose-dependent manner as indicated by the results of enzyme-linked immunosorbent assays. When anti-O1 MAb was used, the reactivity increased significantly for fully encapsulated O1:K1 or O1:K2 cells but not for O1:K− cells. Deposition of C3b also increased significantly for BisBAL-treated O1:K1 or O1:K2 cells but not for O1:K− cells. Survival of a serum-sensitive strain was <0.1% when nonimmune human serum absorbed with O1:K1 cells was used and 107% when BisBAL-treated cells were used for absorption. Outer membrane proteins were also more accessible on the surface of K. pneumoniae after BisBAL treatment. Thus, at subinhibitory levels, BisBAL inhibited capsule expression, which promoted phagocytosis, enhanced the reactivity of specific antibodies for LPS O antigen, LPS core epitopes, or outer-membrane proteins, and enhanced complement interaction with encapsulated K. pneumoniae. By unmasking bacterial surface structures and enhancing the immune system reactivity to bacteria, bismuth thiols may prove useful as adjuncts for vaccination.

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Development of a new trend conjugate vaccine for the prevention of Klebsiella pneumoniae

Infectious Disease Reports ◽

10.4081/idr.2012.e33 ◽

2012 ◽

Vol 4 (1) ◽

pp. 33 ◽

Cited By ~ 8

Author(s):

Tarek A. Ahmad ◽

Medhat Haroun ◽

Ahmed A. Hussein ◽

El Sayed H. El Ashry ◽

Laila H. El-Sayed

Keyword(s):

Klebsiella Pneumoniae ◽

Respiratory Diseases ◽

Capsular Polysaccharide ◽

Outer Membrane Proteins ◽

Immunological Methods ◽

Easy Method ◽

O Antigen ◽

Bacterial Endotoxins ◽

Tract Infections ◽

Limited Spectrum

Klebsiella pneumoniae is a major cause of nosocomial pneumonia, septicemia and urinary tract infections, especially in newborns, blood cancer patients, and other immunocompromised candidates. The control of K. pneumoniae is a complicated issue due to its tight pathogenesis. Immuno-prophylactic preparations, especially those directed toward the bacterium O-antigen, showed to be the most successful way to prevent the infection incidence. However, all previously proposed preparations were either of limited spectrum or non-maternal, and hence not targeting the main Klebsiella patients. Moreover, all preparations were directed only to prevent the respiratory diseases due to that pathogen. This article addresses the development of a method originally used to purify the non-capsular bacterial- endotoxins, as a new and easy method for vaccine production against K. pneumoniae. The application of this method was preceded by a biotechnological control of capsular polysaccharide production in K. pneumoniae. The new produced natural conjugate between the bacterial O-antigen and its outer membrane proteins was evaluated by physicochemical and immunological methods to investigate its purity, integrity, safety and immunogenicity. It showed to be pure, stable, safe for use, and able to elicit a protective immunoglobulin titer against different Klebsiella infections. This immune-response proved to be transferable to the offspring of the vaccinated experimental rabbits via placenta.

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Regulated Delayed Expression of rfaH in an Attenuated Salmonellaenterica Serovar Typhimurium Vaccine Enhances Immunogenicity of Outer Membrane Proteins and a Heterologous Antigen

Infection and Immunity ◽

10.1128/iai.00831-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5572-5582 ◽

Cited By ~ 29

Author(s):

Qingke Kong ◽

Qing Liu ◽

Kenneth L. Roland ◽

Roy Curtiss

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Lethal Dose ◽

Enteric Bacteria ◽

Lymphoid Tissues ◽

Heterologous Antigen ◽

O Antigen ◽

Mutant Strains ◽

Serovar Typhimurium

ABSTRACT RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A ΔrfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >108 CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, ΔPrfaH178, was introduced into the attenuated vaccine strain χ9241 (ΔpabA ΔpabB ΔasdA) expressing the pneumococcal surface protein PspA from an Asd+ balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic ΔrfaH derivative or the isogenic RfaH+ parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent S treptococcus pneumoniae WU2. Immunization with ΔPrfaH178 mutant strains led to increased levels of protection compared to that of the parent χ9241 and of a ΔrfaH derivative of χ9241.

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Influence of growth rate and iron limitation on the expression of outer membrane proteins and enterobactin by Klebsiella pneumoniae grown in continuous culture.

Journal of Bacteriology ◽

10.1128/jb.165.2.353-356.1986 ◽

1986 ◽

Vol 165 (2) ◽

pp. 353-356 ◽

Cited By ~ 7

Author(s):

J M Lodge ◽

P Williams ◽

M R Brown

Keyword(s):

Growth Rate ◽

Membrane Proteins ◽

Klebsiella Pneumoniae ◽

Continuous Culture ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Iron Limitation

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Genetics of the (gram-negative) bacterial surface

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1978.0055 ◽

1978 ◽

Vol 202 (1146) ◽

pp. 5-30 ◽

Cited By ~ 25

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Capsular Polysaccharide ◽

Repeat Unit ◽

Outer Membrane Proteins ◽

Missense Mutations ◽

Protein Antigens ◽

Gram Negative ◽

E Coli ◽

S Genes

The surface of a gram-negative bacterium is made up of the lipopolysaccharide (l. p. s.) and protein components of the outer leaflet of its outer membrane, and of capsular polysaccharide, flagella and fimbriae if present. In Salmonella all the special genes needed for synthesis of the O-specific oligosaccharide repeat unit (different in each O group) of the l. p. s. sidechains are found in the rfb cluster, near his . Nearly all so-far identified rfa genes, for synthesis of l. p. s. core, are clustered between cysE and pyrE . Genes for polymerization and modification of O units are scattered: some are part of prophage genomes and some show ‘form variation’ – spontaneous alternation between expression and non-expression, mechanism unknown. Escherichia coli differs by frequent presence of capsular polysaccharides (K antigens), some determined by kps genes, unlinked to l. p. s. genes, others by his -linked genes perhaps homologous with rfb . Expression of some non-l. p. s. polysaccharide genes, but not of l. p. s. genes, is greatly influenced by the environment. Major outer membrane proteins (more than 10 5 molec. /bacterium) include: a lipoprotein, in part covalently joined to the cell wall, perhaps anchoring the outer membrane; and several proteins of molec. mass 30000–40000 (one of them phage-determined), some of which serve to make the outer membrane permeable to small hydrophilic molecules. Genes affecting sensitivity (adsorbing capacity) to various phages and colicins (e. g. tonA, bfe ) specify various ‘minor’ outer membrane proteins concerned with uptake of nutrients (e. g. iron ferrichrome, vitamin B 12 ) when present at very low concentrations. Neither the ‘major’ nor the ‘minor’ protein genes are clustered: their expression is subject to conspicuous regulation by environmental conditions. In E. coli the flagellin and hook protein structural genes are located in different clusters of motility-related genes. Missense mutations in the flagellin gene may cause alteration in flagellar shape or in serological character, which in Salmonella is also affected by gene nml , for methylation of the free amino groups of some lysines of flagellin. Electron microscopy of re-annealed DNA from the relevant region indicates that change of flagellar antigenic phase in Salmonella results from a reversible inversion of a 750 base-pair segment, probably constituting the phase-determinant gene. Production of fimbriae (pili) requires function of several linked pil genes, and is subject to a kind of ‘form variation’ of unknown mechanism. Genes in conjugative plasmids when derepressed cause production of sex pili. E. coli protein antigens K88 and K99, apparently fimbrial, concerned with adhesion to intestinal mucosa and so with enteropathogenicity, are plasmid-determined.

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Factor H Is Bound by Outer Membrane-Displayed Carbohydrate Metabolism Enzymes of Extraintestinal Pathogenic Escherichia coli and Contributes to Opsonophagocytosis Resistance in Bacteria

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.592906 ◽

2021 ◽

Vol 10 ◽

Author(s):

Yu Sun ◽

Bin Xu ◽

Xiangkai Zhuge ◽

Fang Tang ◽

Xuhang Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Outer Membrane ◽

Binding Assay ◽

Outer Membrane Proteins ◽

Factor H ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Metabolism Enzymes ◽

Elisa Plate

Extraintestinal pathogenic Escherichia coli (ExPEC) causes bloodstream infections in humans and animals. Complement escape is a prerequisite for bacteria to survive in the bloodstream. Factor H (FH) is an important regulatory protein of the complement system. In this study, ExPEC was found to bind FH from serum. However, the mechanisms of ExPEC binding to FH and then resistance to complement-mediated attacks remain unclear. Here, a method that combined desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry was used to identify the FH-binding membrane proteins of ExPEC. Seven identified proteins, which all were carbohydrate metabolic enzymes (CMEs), including acetate kinase, fructose-bisphosphate aldolase, fumarate reductase flavoprotein subunit, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase, phosphoenolpyruvate synthase, and pyruvate dehydrogenase, were verified to recruit FH from serum using GST pull-down and ELISA plate binding assay. The ELISA plate binding assay determined that these seven proteins bind to FH in a dose-dependent manner. Magnetic beads coupled with any one of seven proteins significantly reduced the FH recruitment of ExPEC (p < 0.05) Subsequently, immunofluorescence, colony blotting, and Western blotting targeting outer membrane proteins determined that these seven CMEs were located on the outer membrane of ExPEC. Furthermore, the FH recruitment levels and C3b deposition levels on bacteria were significantly increased and decreased in an FH-concentration-dependent manner, respectively (p < 0.05). The FH recruitment significantly enhanced the ability of ExPEC to resist the opsonophagocytosis of human macrophage THP-1 in an FH-concentration-dependent manner (p < 0.05), which revealed a new mechanism for ExPEC to escape complement-mediated killing. The identification of novel outer membrane-displayed CMEs which played a role in the FH recruitment contributes to the elucidation of the molecular mechanism of ExPEC pathogenicity.

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Virulence Factors in Hypervirulent Klebsiella pneumoniae

Frontiers in Microbiology ◽

10.3389/fmicb.2021.642484 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jie Zhu ◽

Tao Wang ◽

Liang Chen ◽

Hong Du

Keyword(s):

Membrane Proteins ◽

Klebsiella Pneumoniae ◽

Outer Membrane ◽

Virulence Factors ◽

Outer Membrane Proteins ◽

Pyogenic Liver Abscess ◽

Secretion System ◽

Asian Pacific ◽

Shed Light ◽

New Strategies

Hypervirulent Klebsiella pneumoniae (hvKP) has spread globally since first described in the Asian Pacific Rim. It is an invasive variant that differs from the classical K. pneumoniae (cKP), with hypermucoviscosity and hypervirulence, causing community-acquired infections, including pyogenic liver abscess, pneumonia, meningitis, and endophthalmitis. It utilizes a battery of virulence factors for survival and pathogenesis, such as capsule, siderophores, lipopolysaccharide, fimbriae, outer membrane proteins, and type 6 secretion system, of which the former two are dominant. This review summarizes these hvKP-associated virulence factors in order to understand its molecular pathogenesis and shed light on new strategies to improve the prevention, diagnosis, and treatment of hvKP-causing infection.

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Antiproliferative effects of porins extracted from Klebsiella pneumoniae on HL-60, NIH/3T3 and Human foreskin fibroblast cell (HFFC) lines measured by ELISA method

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2010.4.1.88 ◽

2010 ◽

Vol 4 (1) ◽

pp. 28-35

Author(s):

Amir H. Al–Shammary ◽

Essam F. Al-Jumaily ◽

Nidhal Abdulmohymen

Keyword(s):

Klebsiella Pneumoniae ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Primary Source ◽

Gram Negative Bacteria ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Gram Negative ◽

Isolation And Purification ◽

Antiproliferative Effects

Approximately, 50% of the dry mass of the outer membrane of gram-negative bacteria consists of proteins, and more than 20 immunochemically distinct proteins (termed outer membrane proteins [OMPs]) have been identified. An identified local strain of Klebsiella pneumoniae was used as a primary source for the isolation and purification of porins. Multiple concentrations of purified porins (5, 10, 15, 20, 25) g/ml were incubated with three different cell lines for (24, 72 , 120) hrs, after the end of the incubation periods, the cells were treated with Cell proliferation ELISA, BrdU (colorimetric) kit to evaluate the antiproliferative effects of porins. The results revealed that porins are potent antiproliferative agent in a time and concentration dependent manner and thus could greatly affect prokaryote-eukaryote interaction as well as the whole inflammatory process resulted after infection with gram negative bacteria.

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Energy-Dependent Accumulation of Fluoroquinolones in Quinolone-Resistant Klebsiella pneumoniaeStrains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1850 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1850-1852 ◽

Cited By ~ 33

Author(s):

Luis Martínez-Martínez ◽

Isabel García ◽

Sofía Ballesta ◽

Vicente Javier Benedí ◽

Santiago Hernández-Allés ◽

...

Keyword(s):

Membrane Proteins ◽

Klebsiella Pneumoniae ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Clinical Isolates ◽

Intracellular Accumulation ◽

Active Efflux ◽

Energy Dependent ◽

Isogenic Strains

The intracellular accumulation of norfloxacin and pefloxacin inKlebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents.

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