Ammonia enhanced dark respiration in Chlorella vulgaris is related to collapse of a transmembrane pH gradient

Abstract We obtained, by different methods, isogenic lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen) mutants from Klebsiella pneumoniae strains able to induce experimental infections (cytitis and pyelonephritis) in rats. We compared the induction of experimental infections in rats by wild-type strains and the lipopolysaccharide and capsular polysaccharide mutants. The high-molecular mass lipopolysaccharide of K. pneumoniae is clearly implicated in the infection process of the rat urinary tract, whilst the capsular polysaccharide seems not to be involved to the same extent.     

Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3.

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage.     

Lipopolysaccharide O1 antigen contributes to the virulence in Klebsiella pneumoniae causing pyogenic liver abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K(1) (-) O(1)) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain.     

Extracellular polysaccharide production by Klebsiella pneumoniae and its relationship to virulence

Klebsiella pneumoniae serotype 1 and serotype 2 and their capsular variants were examined for production of cell-associated capsular polysaccharides and extracellular capsular polysaccharides. The virulence of these organisms in experimental animals was examined via intraperitoneal injection in mice and transtracheal inoculation into the lungs of rats. It was found that the production of either polysaccharide component correlated with the observed virulence. The extracellular polysaccharides were purified by ethanol precipitation, electrodialysis, extraction with quaternary ammonium salts, and gel filtration. These purification steps allowed for the separation and purification of both the extracellular lipopolysaccharide and the extracellular capsular polysaccharide. Purified extracellular capsular polysaccharide and extracellular lipopolysaccharide were co-injected with K. pneumoniae intraperitoneally into mice to determine if either of these substances would produce an effect on the natural course of infection in these animals. These studies showed that only purified extracellular lipopolysaccharide enhanced the virulence of K. pneumoniae when co-injected into mice, and this virulence enhancement correlated with the content of extracellular lipopolysaccharide, but not extracellular capsular polysaccharide in mixtures of these polysaccharides. Saponification of K. pneumoniae serotype 1 extracellular polysaccharides significantly decreased their virulence-enhancing capabilities in mice, further suggesting that extracellular lipopolysaccharide may play a role in these infections.     

Genetic and biochemical evidence of a Campylobacter jejuni capsular polysaccharide that accounts for Penner serotype specificity

Campylobacter jejuni, a Gram-negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post-infection neurological complications of the Miller-Fisher and Guillain-Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one-third of strains also producing high-molecular-weight LPS (referred to as O-antigen). In this report, we demonstrate the presence of the high-molecular-weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O-antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O-antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.     

The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to alpha-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an alpha-D-galactopyranosyluronic acid (alpha-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.     

The structural basis for the serospecificity of Actinobacillus suis serogroup O:2.

Actinobacillus suis is an important bacterial pathogen of healthly pigs. An O-antigen (lipopolysaccharide; LPS) serotyping system is being developed to study the prevalence and distribution of representative isolates from both healthy and diseased pigs. In a previous study, we reported that A. suis serogroup O:1 strains express LPS with a (1-->6)-beta-D-glucan O-antigen chain polysaccharide that is similar in structure to a key cell-wall component in yeasts, such as Saccharomyces cerevisiae and Candida albicans. This study describes the O-antigen polysaccharide chemical structure of an O:2 serogroup strain, A. suis H91-0380, which possesses a tetrasaccharide repeating block with the structure: -->3)-beta-D-Galp-(1-->4)-[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->6)-beta-D-GlcpNAc-(1-->. Studies have shown that A. suis serogroup O:2 strains are associated with severely diseased animals; therefore, work on the synthesis of a glycoconjugate vaccine employing O:2 O-antigen polysaccharide to vaccinate pigs against A. suis serogroup O:2 strains is currently underway.     

Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor

The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed.     

Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens.

The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K(LPS). By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K(LPS) antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K(LPS), as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K(LPS) with longer polysaccharide chains. Collectively, these results suggested that group IA K(LPS) is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K(LPS) results from the absence of Rol activity in these strains.     

Role for Rhizobium rhizogenes K84 cell envelope polysaccharides in surface interactions

Rhizobium rhizogenes strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a wcbD ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of wcbD in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the rkpK gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The rkpK mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms.     

Contribution of capsular and lipopolysaccharide antigens to the pathogenesis of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> respiratory tract infection

The role of Klebsiella pneumoniae K- and O-polysaccharide antigens was determined in a rat model of lobar pneumonia. The induction of experimental infection in rats by wild-type strain, and its lipopolysaccharide- and capsular polysaccharide-deficient mutants was compared. Though the mutant lacking both antigens (K- O-) induced infection it could not successfully establish itself in the rat lung. It caused only mild infection, as compared to the wild type strain (K+ O+) and the strain lacking CPS alone (K- O+). Besides capsular polysaccharide, the lipopolysaccharide antigen was shown to be an important factor in pathogenesis of K. pneumoniae acute respiratory tract infection.     

Structural characterization of the antigenic capsular polysaccharide and lipopolysaccharide O-chain produced by Actinobacillus pleuropneumoniae serotype 15.

The specific capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 15 was determined to be a high-molecular-mass polymer having [alpha]D + 69 degrees (water) and composed of a linear backbone of phosphate diester linked disaccharide units of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc) and 2-acetamido-2-deoxy-D-galactose (D-GalNAc) residues (1:1). Thirty percent of the D-GalNAc residues were substituted at O-4 by beta-D-galactopyranose (beta-D-Galp) residues. Through the application of chemical and NMR methods, the capsule, which defines the serotype specificity of the bacterium, was found to have the structure [structure: see text]. The O-polysaccharide (O-PS) component of the A. pleuro pneumoniae serotype 15 lipopolysaccharide (LPS) was characterized as a linear unbranched polymer of repeating pentasaccharide units composed of D-glucose (2 parts) and D-galactose (3 parts), shown to have the structure [structure: see text]. The O-PS was chemically identical with the O-antigen previously identified in the LPSs produced by A. pleuro pneumoniae serotypes 3 and 8.     

Occurrence of a capsule in Aeromonas salmonicida

Aeromonas salmonicida grown in a medium with excess glucose as carbon source produces both capsular and exocellular polysaccharides. The capsular polysaccharide is composed of glucose, mannose, rhamnose, N-acetylmannosamine and mannuronic acid in the molar ratios of approximately 5:3:0.75:2:1. The extracellular polysaccharide is similarly constituted, but in the molar ratios of approximately 4.75:10.5:1.5:2:1. The capsular and exocellular polysaccharides did not cross-react with monoclonal antibodies against the A-layer or the O-antigen lipopolysaccharide.     

Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30)

The group 1 K30 antigen from Escherichia coli (O9a:K30) is present on the cell surface as both a capsular structure composed of high-molecular-weight K30 polysaccharide and as short K30 oligosaccharides linked to lipid A-core in a lipopolysaccharide molecule (K30LPS). To determine the molecular processes that are responsible for the two forms of K antigen, the 16 kb chromosomal cps region has been characterized. This region encodes 12 gene products required for the synthesis, polymerization and translocation of the K30 antigen. The gene products include four glycosyltransferases responsible for synthesis of the K30 repeat unit; a PST (1) exporter (Wzx), required to transfer lipid-linked K30 units across the plasma membrane to the periplasmic space; and a K30-antigen polymerase (Wzy). These gene products are typical of those seen in O-antigen biosynthesis gene clusters and they interact with the lipopolysaccharide translocation pathway to express K30LPS on the cell surface. The same gene products also provide the biosynthetic intermediates for the capsule assembly pathway, although they are not in themselves sufficient for synthesis of the K30 capsule. Three additional genes, wza, wzb and wzc, encode homologues to proteins that are encoded by gene clusters involved in expression of a variety of bacterial exopolysaccharides. Mutant analysis indicates that Wza and Wzc are required for wild-type surface expression of the capsular structure but are not essential for polymerization and play no role in the translocation of K30LPS. These surface expression components provide the key feature that distinguishes the assembly systems for O antigens and capsules.     

Modification of lipopolysaccharide with colanic acid (M-antigen) repeats in Escherichia coli

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS.MLPS biosynthesis is dependent upon (i) CA induction, (ii) LPS core biosynthesis, and (iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the L-glycero-D-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides.     

Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobcter freundii and Citrobacter sedlakii

A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E. coli O157 O-antigen. A strain of Citrobacter freunndii showing similar cross-reaction with E. coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure [ 2)-alpha-D Rhap-(1-3)-beta-D-Rhap-(1-4)-beta-D-Glcp-(1-]. This O-antigen differs from that of the E. coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E. coli O157 antisera. The C freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen.     

Characterization of an O-antigen bacteriophage from Aeromonas hydrophila.

A unique bacteriophage of Aeromonas hydrophila serotype O:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the O-antigen polysaccharide component of lipopolysaccharide specific to serotype O:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in O antigen) was fully able to inactivate bacteriophage PM1. Phage-resistant mutants of A. hydrophila O:34 were isolated and found to be specifically devoid of lipopolysaccharide O antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PM1 was found to be very narrow, producing plaques only on A. hydrophila strains from serotype O:34.     

Fermentation and evaluation of Klebsiella pneumoniae and K. oxytoca on the production of 2,3-butanediol

Klebsiella is one of the genera that has shown unbeatable production performance of 2,3-butanediol (2,3-BD), when compared to other microorganisms. In this study, two Klebsiella strains, K. pneumoniae (DSM 2026) and K. oxytoca (ATCC 43863), were selected and evaluated for 2,3-BD production by batch and fed-batch fermentations using glucose as a carbon source. Those strains' morphologies, particularly their capsular structures, were analyzed by scanning electron microscopy (SEM). The maximum titers of 2,3-BD by K. pneumoniae and K. oxytoca during 10 h batch fermentation were 17.6 and 10.9 g L(-1), respectively; in fed-batch cultivation, the strains showed the maximum titers of 50.9 and 34.1 g L(-1), respectively. Although K. pneumoniae showed higher productivity, SEM showed that it secreted large amounts of capsular polysaccharide, increasing pathogenicity and hindering the separation of cells from the fermentation broth during downstream processing.     

Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface

Monoclonal antibodies were produced against the capsular antigen of Escherichia coli serotype K(A)30, using a mouse hybridoma system. The antibodies also recognised the chemically identical capsular polysaccharide produced by Klebsiella K20. Chemical modification of the K30 polysaccharide indicated that the glucuronic acid residues found in the E. coli K30 capsular antigen were important in the epitope recognised by these antibodies. Use of the antibodies as molecular probes revealed the presence of two discrete forms of the K30 antigen. One form was comprised of high molecular weight polysaccharide, present as a surface capsular layer. The second form of the antigen was of low molecular weight and was associated with lipopolysaccharide fractions from cell surface polysaccharide extracts. Separation of lipopolysaccharide fractions using gel chromatography in the presence of detergent showed that the low molecular weight K-antigenic fraction comigrated with a lipopolysaccharide lipid A core fraction present in encapsulated E. coli K30 bacteria but absent in acapsular mutants.     

Multiplex-PCR assay for identification of Klebsiella pneumoniae isolates carrying the cps loci for K1 and K2 capsule biosynthesis.

Multiplex-PCR assay for identification of Klebsiella pneumoniae isolates carrying gene clusters for biosynthesis of capsular polysaccharide (CPS) types K1 and K2 was developed. Genes wzc and orf10 of the cps cluster were applied as K1 and K2 specific markers respectively. The assay specificity was confirmed using 147 isolates of Klebsiella spp. including 77 K-antigen reference strains. The multiplex-PCR assay was found simple and cost-effective tool for identification of K. pneumoniae clinical isolates of K1 and K2 geno-serotypes.