K1 Polysaccharide Research Articles

An investigation of the molecular and cellular mechanisms of in vitro phage therapy in human cells

As the consequences of increasing bacterial resistance to traditional antibiotics become evident, the importance of research and development of therapeutic alternatives is apparent. Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host. However, whether and how bacteriophages can kill intracellular bacteria in a human cell environment remains elusive. E. coli strains displaying the K1 polysaccharide capsule virulence factor, are nosocomial pathogens responsible for urinary tract infections (UTIs), neonatal meningitis and potentialprecursors for septicaemia. These different types of infections were modelled in vitro by infecting human bladder epithelial cells (T24 HTB-4) and humancerebral endothelial cells (hCMEC/D3) with E. coli EV36, a strain displaying the K1 capsule. The infected human cells then received in vitro phage therapy using bacteriophage K1F that specifically targets E. coli strains displaying the K1 capsule. The rate of bacterial infection and the molecular and cellular mechanisms of in vitro phage therapy was analysed by means of flow cytometry, confocal and live microscopy. We show that rfp-tagged E. coli EV36-RFP and gfp-tagged bacteriophage K1F-GFP, enter the human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP can efficiently kill intracellular E. coli EV36-RFP in human urinary bladder epithelial cells and humancerebral endothelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy. Collectively this data contribute evidence-based knowledge for the ongoing development of phage therapy.

Phenotypic Heterogeneity in Expression of the K1 Polysaccharide Capsule of Uropathogenic Escherichia coli and Downregulation of the Capsule Genes during Growth in Urine.

Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTI). The K1 capsule on the surface of UPEC strains is a key virulence factor, and its expression may be important in the onset and progression of UTI. In order to understand capsule expression in more detail, we analyzed its expression in the UPEC strain UTI89 during growth in rich medium (LB medium) and urine and during infection of a bladder epithelial cell line. Comparison of capsule gene transcription using a chromosomal gfp reporter fusion showed a significant reduction in transcription during growth in urine compared to that during growth in LB medium. When examined at the single-cell level, following growth in both media, capsule gene expression appears to be heterogeneous, with two distinct green fluorescent protein (GFP)-expressing populations. Using anti-K1 antibody, we showed that this heterogeneity in gene expression results in two populations of encapsulated and unencapsulated cells. We demonstrated that the capsule hinders attachment to and invasion of epithelial cells and that the unencapsulated cells within the population preferentially adhere to and invade bladder epithelial cells. We found that once internalized, UTI89 starts to produce capsule to aid in its intracellular survival and spread. We propose that this observed phenotypic diversity in capsule expression is a fitness strategy used by the bacterium to deal with the constantly changing environment of the urinary tract.

Analysis of the SDS-PAGE patterns of outer membrane proteins from Escherichia coli strains that have lost the ability to form K1 antigen and varied in the susceptibility to normal human serum

We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1−) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1− strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.

Effects of Lipopolysaccharide Biosynthesis Mutations on K1 Polysaccharide Association with the Escherichia coli Cell Surface

The presence of cell-bound K1 capsule and K1 polysaccharide in culture supernatants was determined in a series of in-frame nonpolar core biosynthetic mutants from Escherichia coli KT1094 (K1, R1 core lipopolysaccharide [LPS] type) for which the major core oligosaccharide structures were determined. Cell-bound K1 capsule was absent from mutants devoid of phosphoryl modifications on L-glycero-D-manno-heptose residues (HepI and HepII) of the inner-core LPS and reduced in mutants devoid of phosphoryl modification on HepII or devoid of HepIII. In contrast, in all of the mutants, K1 polysaccharide was found in culture supernatants. These results were confirmed by using a mutant with a deletion spanning from the hldD to waaQ genes of the waa gene cluster to which individual genes were reintroduced. A nuclear magnetic resonance (NMR) analysis of core LPS from HepIII-deficient mutants showed an alteration in the pattern of phosphoryl modifications. A cell extract containing both K1 capsule polysaccharide and LPS obtained from an O-antigen-deficient mutant could be resolved into K1 polysaccharide and core LPS by column chromatography only when EDTA and deoxycholate (DOC) buffer were used. These results suggest that the K1 polysaccharide remains cell associated by ionically interacting with the phosphate-negative charges of the core LPS.

Differential expression of two pathways of chemokine induction protect CD14- and TLR4-deficient mice from localized E. coli infections (94.16)

Abstract Sepsis, characterized by a systemic bacterial infection, is a life-threatening condition that can lead to multi-organ failure and death from septic shock. We have observed that CD14- and TLR4-deficient mice are resistant to death by sepsis from several different clinical isolates of E. coli injected intra-peritoneally. This resistance has been attributed to the 10-fold lower levels of the proinflammatory cytokines TNF and IL-6 induced in these mice. Surprisingly, however, neither TLR4- nor CD14-deficient mice were resistant to two closely related organisms that differed only in that they expressed a K1 polysaccharide capsule, even though these mice continued to express low levels of the proinflammatory cytokines. These and other observations suggest that the ability to limit bacterial dissemination may be equally or even more important than reducing the levels of these proinflammatory cytokines in preventing death from sepsis. We present evidence here, that the pathogenic role for the CD14/TLR4 pathway in severe infection, whereby it mediates the induction of proinflammatory cytokines, may be secondary to a more important insidious role whereby differential activation of local and systemic pathways of chemokine induction via CD14/TLR4-dependent and independent mechanisms generate chemokine gradients that prevent PMN transmigration from the blood to infected tissue and facilitates the dissemination of bacteria from a localized site of infection.

Polysaccharide Capsule and Sialic Acid-Mediated Regulation Promote Biofilm-Like Intracellular Bacterial Communities during Cystitis

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4(+) and TLR4(-) mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.

Role of CD14 in Responses to Clinical Isolates ofEscherichia coli: Effects of K1 Capsule Expression

Severe bacterial infections leading to sepsis or septic shock can be induced by bacteria that utilize different factors to drive pathogenicity and/or virulence, leading to disease in the host. One major factor expressed by all clinical isolates of gram-negative bacteria is lipopolysaccharide (LPS); a second factor expressed by some Escherichia coli strains is a K1 polysaccharide capsule. To determine the role of the CD14 LPS receptor in the pathogenic effects of naturally occurring E. coli, the responses of CD14-/- and CD14+/+ mice to three different isolates of E. coli obtained from sepsis patients were compared; two isolates express both smooth LPS and the K1 antigen, while the third isolate expresses only LPS and is negative for K1. An additional K1-positive isolate obtained from a newborn with meningitis and a K1-negative isogenic mutant of this strain were also used for these studies. CD14-/- mice were resistant to the lethal effects of the K1-negative isolates. This resistance was accompanied by significantly lower levels of systemic tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) in these mice than in CD14+/+ mice, enhanced clearance of the bacteria, and significantly fewer additional gross symptoms. In contrast, CD14-/- mice were as sensitive as CD14+/+ mice to the lethal effects of the K1-positive isolates, even though they had significantly lower levels of TNF-alpha and IL-6 than CD14+/+ mice. These studies show that different bacterial isolates can use distinctly different mechanisms to cause disease and suggest that new, nonantibiotic therapeutics need to be directed against multiple targets.

Escherichia coli BL21(DE3) chromosome contains a group II capsular gene cluster

During our study of de novo synthesis of Escherichia coli K1 capsular polysaccharides, we found that E. coli BL21(DE3) has a capsular gene cluster, similar to those of group II capsular E. coli strains. Analysis of the nucleotide sequence of the E. coli BL21(DE3) gene cluster showed homologues to all group II regions 1 and 3 genes and the presence of an IS1 element in one of the region 2 ORFs, which likely prevents capsule expression. Complementation analysis showed that region 1 and 3 genes encode functional proteins that are sufficient for the export of newly synthesized polysaccharide. The gene products of Bl21(DE3) kpsC and kpsS supported in vitro de novo synthesis of K1 polysaccharide when co-expressed with K1 NeuE and NeuS. Sequence homology between BL21(DE3) region 2 open reading frames and capsule-related genes in other bacteria such as Haemophilus influenzae serotype b, suggests that the encapsulated ancestor of BL21(DE3) may have produced a ribose/ribitol-phosphate containing polysaccharide.

The Genome of Bacteriophage K1F, a T7-Like Phage That Has Acquired the Ability To Replicate on K1 Strains of Escherichia coli

Bacteriophage K1F specifically infects Escherichia coli strains that produce the K1 polysaccharide capsule. Like several other K1 capsule-specific phages, K1F encodes an endo-neuraminidase (endosialidase) that is part of the tail structure which allows the phage to recognize and degrade the polysaccharide capsule. The complete nucleotide sequence of the K1F genome reveals that it is closely related to bacteriophage T7 in both genome organization and sequence similarity. The most striking difference between the two phages is that K1F encodes the endosialidase in the analogous position to the T7 tail fiber gene. This is in contrast with bacteriophage K1-5, another K1-specific phage, which encodes a very similar endosialidase which is part of a tail gene "module" at the end of the phage genome. It appears that diverse phages have acquired endosialidase genes by horizontal gene transfer and that these genes or gene products have adapted to different genome and virion architectures.

Escherichia coli K1's Capsule Is a Barrier to Bacteriophage T7

Escherichia coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule in E. coli physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structures beneath it and that the K1-specific phages evolved to counter this physical barrier.

Genetic analysis of Escherichia coli K1 gastrointestinal colonization.

Strains of Escherichia coli expressing the K1 polysaccharide capsule colonize the large intestine of newborn infants, and are the leading cause of Gram-negative septicaemia and meningitis in the neonatal period. We used signature-tagged mutagenesis (STM) to identify genes that E. coli K1 requires to colonize the gastrointestinal (GI) tract. A total of 2140 mTn5 mutants was screened for their capacity to colonize the GI tract of infant rats, and 16 colonization defective mutants were identified. The mutants have transposon insertions in genes affecting the synthesis of cell surface structures, membrane transporters, transcriptional regulators, enzymes in metabolic pathways, and in genes of unknown function, designated dgc (defective in GI colonization). Three dgcs are absent from the whole genome sequence of E. coli K-12, although related sequences are found in other pathogenic strains of E. coli and in Shigella flexneri. Additionally, immunohistochemistry was used to define the nature of the colonization defect in five mutants including all dgc mutants. STM was successfully applied to examine the factors involved in E. coli K1 colonization, and the findings are relevant to the pathogenesis of other enteric infections.

Binding diversity of monoclonal antibodies toα(2 → 8) polysialic acid conjugated to outer membrane vesicle via adipic acid dihydrazide

Murine monoclonal antibodies (mAbs) were generated using group B Neisseria meningitidis and Escherichia coli K1 polysaccharides (PSs) conjugated to outer membrane vesicle (OMV) via adipic acid dihydrazide, and were used to identify the immunodeterminants expressed on these capsular PSs. Ten mAbs representative of IgM and all subclasses of IgG were obtained which recognized diverse immunodeterminants on alpha(2-->8) polysialic acid (PSA). The specificity of mAbs to different antigenic determinants was assessed by their differential binding to PSA attached to a solid phase by different methods and confirmed by absorption studies. Two mAbs from the E. coli K1 fusion were directed to the O-acetyl epitope and the rest reacted with both the PSs only when attached to a solid phase by certain means. The methods by which PSA was coated on the solid phase had an impact on the epitope expression and binding pattern. At the concentrations used, the O-acetyl-specific mAbs, IgG1 and IgG3 mAbs were not bactericidal against group B N. meningitidis, whereas other mAbs were. The conjugates B and K1 PSs present to the murine immune system different antigenic determinants, some of which elicit bactericidal antibodies.

Binding diversity of monoclonal antibodies to α(2 → 8) polysialic acid conjugated to outer membrane vesicle via adipic acid dihydrazide

Murine monoclonal antibodies (mAbs) were generated using group B Neisseria meningitidis and Escherichia coli K1 polysaccharides (PSs) conjugated to outer membrane vesicle (OMV) via adipic acid dihydrazide, and were used to identify the immunodeterminants expressed on these capsular PSs. Ten mAbs representative of IgM and all subclasses of IgG were obtained which recognized diverse immunodeterminants on α(2 → 8) polysialic acid (PSA). The specificity of mAbs to different antigenic determinants was assessed by their differential binding to PSA attached to a solid phase by different methods and confirmed by absorption studies. Two mAbs from the E. coli K1 fusion were directed to the O-acetyl epitope and the rest reacted with both the PSs only when attached to a solid phase by certain means. The methods by which PSA was coated on the solid phase had an impact on the epitope expression and binding pattern. At the concentrations used, the O-acetyl-specific mAbs, IgG1 and IgG3 mAbs were not bactericidal against group B N. meningitidis, whereas other mAbs were. The conjugates B and K1 PSs present to the murine immune system different antigenic determinants, some of which elicit bactericidal antibodies.

Capsular sialyl chains ofEscherichia coli K1 mutants resistant to K1 phage

Capsular polysialyl chains of wild-typeEscherichia coli K1 consist of about 200 sialyl units. Factors associated with the degree of polymerization of the K1 polysaccharide were studied by isolation of mutant bacteria resistant to K1 phage. The mutants (n=55) were characterized with respect to the length of their capsular sialyl chains by gel electrophoresis and reactivity with anti-K1 antibodies. No mutants with short sialyl chains were found, although the mutants displayed different phenotypic properties and produced different amounts of sialic acid. Ultrastructural examination revealed phenotypes with accumulations of apparent polysialic acid in the periplasm or cytosol. This observation provides direct support for the previously proposed translocation of the K1 polysaccharide via the periplasmic space during its biosynthesis.

Comparative Infectivity for Axenic and Specific-Pathogen-Free Chickens of O2 Escherichia coli Strains with or without Virulence Factors

Adhesion to epithelial respiratory cells, iron acquisition, and production of K1 polysaccharide capsules have been proposed as potential virulence factors of avian Escherichia coli. These factors were studied by inoculating groups of axenic or specific-pathogen-free (SPF) chickens intratracheally with O2 E. coli strains after previous challenge with a wild strain of infectious bronchitis virus (IBV). In all experiments, the association between IBV and an E. coli strain endowed with the three virulence factors previously mentioned resulted in the most severe pathological effects, as measured by mortality, weight gains, lesions, and reisolation of E. coli from internal organs. An E. coli strain devoid of virulence factors was able only to induce mild pathological effects restricted to the respiratory tract when combined with IBV. Both E. coli strains were more invasive in axenic chickens than in SPF chickens. These results confirm the probable involvement of the three factors studied in the pathogenic properties of avian E. coli. This model can be used to assess the role of virulence factors, by comparing pairs of positive and negative isogenic strains.

Quantitative relationship between capsular content and killing of K1-encapsulated Escherichia coli.

Since there are conflicting reports in the literature on a possible relationship between the K1 capsular polysaccharide (CP) content of Escherichia coli and its susceptibility to killing, we reexamined this issue in a strain that had a smooth lipopolysaccharide (LPS) phenotype (E. coli O18:K1:H7 Bort) and in a strain with a deep rough LPS phenotype (E412, spontaneously agglutinable: K1:H-). When cell-associated K1 capsular content was greater than 90 micrograms of K1 polysaccharide per 10(10) CFU, neither strain was lysed by 20% normal human serum. In contrast, at equivalent but lower levels of K1 CP content, E412 but not strain Bort was lysed by normal human serum. Thus, LPS phenotype is an additional surface determinant that affects bacterial susceptibility to killing. Organisms obtained from very early log phase, when cell-associated K1 CP is greatest, were significantly more virulent for mice than were bacteria harvested in stationary phase, when cell-associated K1 polysaccharide is lowest. We conclude that (i) there is a threshold level of K1 CP needed to confer protection from lysis by serum, and this is usually exceeded under standard growth conditions; (ii) at a given level of K1 CP the LPS phenotype is an important determinant of bacterial killing; and (iii) the loss of capsule at low pH may be an additional mechanism by which hosts defend against invasive infection by K1-encapsulated E. coli.

Surface exposure of the O-antigen in Klebsiella pneumoniae O1:K1 serotype strains

Isogenic non-encapsulated (K −) mutants (O1:K −) were obtained from several different Klebsiella pneumoniae O1:K1 serotype strains. By employing K. pneumoniae bacteriophages FC3-1, FC3-2, and bacteriophage Φ1, the bacterial surface receptors of which, are the O1-antigen of lipopolysaccharide and the K1 capsular polysaccharide (K1) respectively, the K1 polysaccharide was found to completely cover the O1 lipopolysaccharide molecules in each of the O1:K1 strains examined. Exposure of the O-antigen at the cell surface was only observed after growth in the presence of sub-minimum inhibitory concentrations of antibiotics. The implications of these findings for the design of vaccines for the prevention of Klebsiella infections is discussed.

Characterisation and in vivo behaviour of aSalmonella typhimurium aroA strain expressingEscherichia coliK1 polysaccharide

Plasmid pKT274 encoding a determinant for the Escherichia coli K1 polysaccharide was introduced into the Salmonella typhimurium aroA vaccine strain SL3261 and cells harbouring the plasmid were shown to express K1 polysaccharide at their cell surface. SL3261 (pKT274) could be detected in the livers and spleens of BALB/c mice infected by the intravenous route and viable organisms persisted for several weeks. SL3261 (pKT274) was cleared from the livers more rapidly and from the spleens more slowly than SL3261. Unlike mice infected with SL3261 those infected with SL3261 (pKT274) did not exhibit gross splenomegaly during the first three weeks after infection. Mice vaccinated with viable SL3261 (pKT274) were protected against challenge with virulent S. typhimurium but failed to produce detectable levels of humoral anti-K1 polysaccharide antibodies.

The K1 capsular polysaccharide of Escherichia coli.

Epidemiologic, immunologic, and genetic evidence indicate that the K1 capsular polysaccharide confers invasiveness to Escherichia coli. The capsule, an alpha-2----8-linked homopolymer of sialic acid (NeuNAc), provides the bacterium with a physical antiphagocytic barrier. Structural similarities between K1 and human tissue components suggest that immune tolerance may also be a factor in pathogenesis of K1 disease. The molecular and genetic events involved in the synthesis and export of the K1 polysaccharide were examined. The cloned K1 genes encode at least 12 proteins involved in capsule biosynthesis. These genes appear to be coordinately regulated and functionally clustered. One cluster is associated with the synthesis and activation of NeuNAc and includes the gene encoding CMP-NeuNAc synthetase. This enzyme catalyzes the activation of NeuNAc to CMP-NeuNAc. A second region, encoding five proteins, is associated with translocation of polysaccharide to the bacterial surface. The K1 polysaccharide is a poor immunogen in humans, and an understanding of the key reactions involved in K1 synthesis may help in providing an alternative to anticapsular immunity.

The epitope associated with the binding of the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1 to a human monoclonal macroglobulin, IgMNOV.

The fine structure of the combining site of human mAb IgMNOV to poly-alpha(2----8)linked NeuNAc, the epitope of the group B meningococcal and E. coli K1 polysaccharides, has been probed using RIA and ELISA. Inhibition by oligomers ranging from 2 to 12 residues was used to assay binding to IgMNOV by group B meningococcal polysaccharide preparations (GBMP) or by poly(A). The inhibitory properties of the oligomers were almost identical in both assays of the binding of GBMP to horse IgM (H46). This evidence and the finding that both GBMP and poly(A) precipitated IgMNOV equally per unit weight indicated that the epitope of poly(A) must mimic an equivalent epitope on GBMP despite the absence of any apparent common structural features in the two molecules. Unlike most carbohydrate-anticarbohydrate systems in which the site is saturated by oligomers of up to six or seven sugars, all the anti-alpha(2----8)NeuNAc systems above required much larger oligomers. Because these oligomers are larger than the maximum size of an antibody site the epitope must be conformationally controlled, and this has been confirmed by nuclear magnetic resonance spectroscopy. However, despite the above similarities, GBMP and poly(A) were differentiated in that only GBMP bound to H46. Smaller linear molecules obtained by delipidating the GBMP, as well as periodate-oxidized GBMP with its nonreducing end oxidized or linked covalently to BSA, bound to and precipitated IgMNOV and H46. This showed that, despite their differences, terminal nonreducing ends were not involved and that both epitopes were located in the conformationally controlled inner residues of the GBMP. The difference thus must reside in the ability of IgMNOV and H46 to recognize different structural aspects of the same conformationally controlled inner residues. The ELISA data indicate that both IgMNOV and H46 have groove-type sites that bind exclusively to an epitope located on the acidic side of the inner residues. The differences determining the ability of IgMNOV and the failure of H46 to cross-react with poly(A), poly(I), and denatured DNA, may depend on differences in the degree of protonation required by each antibody, and this may be clarified by a study of the effects of pH on the precipitin behavior of IgMNOV and H46.