1 Microbiology Group, School of Pharmacy, 2939 Brunswick Square, London WC1N 1AX, UK; 2 Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK
Received 4 January 2005; returned 25 February 2005; revised 28 February 2005; accepted 26 April 2005
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Abstract |
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Methods: Bacteraemia was established in Wistar rats by induction of gastrointestinal colonization with the virulent K1 strain A192PP; colonization preceded a lethal bacteraemia. Decreasing single doses of endoE were administered intraperitoneally. Macrophage engulfment of K1 strain A192PP was evaluated by staining and microscopy in the presence and absence of endoE.
Results: A192PP colonized the gastrointestinal tract of all 2-day-old animals and produced bacteraemia in over 90%. A single endoE dose of 0.25 µg curtailed bacteraemia and prevented death in at least 80% of infected animals. Older animals (up to 5 days of age) were less susceptible to systemic infection following intestinal colonization. EndoE-mediated removal of K1 capsular polysaccharide led to increased ingestion by macrophages.
Conclusions: A small single dose of capsule-depolymerizing enzyme has therapeutic utility in lethal systemic infection in a non-invasive model that has characteristics of the infectious process in humans. We propose that the enzyme reduces the virulence of E. coli K1 by rapid removal of the protective capsular polysaccharide, sensitizing the pathogen to host defences such as phagocytosis by macrophages. Thus, whilst endoE-mediated therapy may not be a viable approach to the treatment of systemic infection in humans, it does support the concept that alteration of the cell wall phenotype is a valid therapeutic strategy.
Keywords: bacteraemia , endosialidase , phenotype modification , polysaccharide capsule , enzyme therapy
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Introduction |
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A bacteriophage-encoded enzyme, endosialidase E (endoE), which selectively degrades the linear homopolymeric -2,8-linked N-acetylneuraminic acid capsule associated with the capacity of Escherichia coli K1 strains to cause severe infection in the newborn infant, has been identified and produced by recombinant DNA technology.57 This capsular serotype is responsible for about 85% of cases of E. coli neonatal bacterial sepsis and meningitis8 and is associated with unacceptably high rates of mortality and morbidity. Systemic infection can be induced in neonatal rats by feeding cultures of E. coli K1, where intestinal colonization is followed by spontaneous translocation of the bacteria to the blood compartment and further dissemination of the bacteria leads to the development of a fatal infection.9 A single intraperitoneal dose of 20 µg of endoE prevented the development of bacteraemia and ensured the survival of virtually all treated rat pups, presumably by stripping away the protective capsule of the invading bacteria. In support of this contention, we also demonstrated that treatment of E. coli K1 strains in vitro with small amounts of endoE led to a large increase in their susceptibility to killing by complement.9
In the neonatal rat model, the experimental infection follows the natural route of spontaneous bacteraemia, sepsis and meningitis found in the human neonate.10 In addition, both the human spontaneous infection10 and experimental infection in the rat11 are markedly age-dependent. In this study, we determined the effect of dose frequency and dose size on the course of the infection, examined the effect of endoE exposure on susceptibility to murine macrophages and investigated other aspects of the model.
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Materials and methods |
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E. coli LP1674 (serotype O7:K1) was isolated from a patient with urinary tract infection.12 K1 strains A53 (18ac:K1:H7) and A192 (O18:K1) were isolated from patients with neonatal meningitis and septicaemia, respectively.13 RS228 (18ac:K1:H-) was a faecal isolate from the same study. These three strains were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany as DSM Nos. 10723, 10719 and 10809. A192, A53 and RS228 were passaged in neonatal rats and colonies isolated from blood cultures were designated A192PP, A53P and RS228P; as indicated by the suffix, A192 was subjected to two rounds of recovery from the animals whereas the other two strains were passaged once.
Enzymes
His6-tagged endoE was produced in E. coli BL21 (DE3) and purified using Ni affinity chromatography as described previously.9 Recovered protein was pure as judged by SDSPAGE and immunoblotting, with no proteins detectable other than the 76 kDa recombinant endoE fusion product. Kinetic properties and stability of the recombinant enzyme are reported elsewhere.7,9 For intraperitoneal administration, doses of endoE were formulated in 0.1 mL of PBS. His6-tagged glutathione S-transferase (GST) was produced in E. coli BL21 (DE3) and purified in the same fashion as His6-endoE.
Infection of neonatal rats
Procedures involving animals conformed to national and European legislation and were approved in full by the institutional ethics committee. The infection model used was adapted9 from studies by Glode et al.11 and Pluschke et al.14 Wistar rat pups were retained with the natural mothers after birth, in a single cage. Rat pups up to 4 days of age, comprising a single litter of 1014 individuals, were fed 0.02 mL of E. coli K1 (26 x 106 cfu at 37°C) using an Eppendorf micropipette. Intestinal colonization was assessed by culture of swabs taken from the perianal area on MacConkey agar; the presence of the K1 capsule expressed by isolated colonies was determined using K1-specific bacteriophages.15 Samples were taken daily beginning one day after feeding of bacterial cultures. Bacteraemia was detected by culture on MacConkey agar of blood samples taken daily by tail vein puncture using a microlance or by cardiac puncture. Lactose-fermenting colonies were probed for the presence of K1 using K1-specific bacteriophages.
Uptake of K1 by rat peritoneal macrophages
Macrophages were obtained from 5- to 10-day-old Wistar rats; 1 mL of Medium 199 (Gibco, Paisley, UK) was injected into the peritoneum, the area gently massaged and the contents of the peritoneal cavity withdrawn. Cells were collected by low-speed centrifugation and suspended in Medium B (Medium 199 containing 10% fetal calf serum and gentamicin 10 µg/mL) to a concentration of 1 x 104 cells/mL. The cells were allowed to adhere to coverslips in 12-well plates (1 mL per well) at 37°C overnight. The medium was removed, cells washed twice with PBS and 1 mL of fresh Medium B containing 1 x 106 mid-logarithmic (E578 = 0.6) K1 bacteria, with or without 10 µg of endoE, added. At suitable time intervals, coverslips were removed, washed with PBS, stained with 2.5% glutaraldehyde and mounted cell-side-up on glass slides. The slides were stained with 1% Crystal Violet and the proportion of ingested bacteria determined by microscopic examination of 20 macrophages per slide.
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Results |
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In order to select a K1 strain that elicited a consistently high incidence of gastrointestinal colonization and bacteraemia following ingestion of bacterial culture, four E. coli K1 strains from various sources were used to infect litters of 2-day-old rats (Table 1). In general, the pups were efficiently colonized within 24 h of ingestion, with frequencies between 54% and 100%. The incidence of bacteraemia detected over a period of 6 days following colonization was, however, much lower, ranging from 50% for A53 to 15% for RS228. To increase the incidence of bacteraemia, we removed blood from bacteraemic pups by cardiac puncture and examined single colony isolates in the neonatal rat model. Passage of A192 and A53 produced colony forms with enhanced virulence; A53P, obtained from a single passage of A53, caused a higher rate of bacteraemia (92%) than the parent strain (50%) and the enhancement was even more marked with A192PP, which produced bacteraemia in all animals of the litter tested. Strain A192PP was therefore selected for all further studies and in over 50 experiments has continued to give an incidence of bacteraemia in the range 80100%. In these preliminary studies, and in all subsequent experiments, gut colonization always preceded invasion of the bloodstream by at least 24 h; viable counts of E. coli K1 in blood samples were between 102 and 107 per mL.
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We have previously shown that a single intraperitoneal dose of 20 µg of endoE given 24 h after feeding of E. coli A192PP could reduce mortality due to the infection from 80100% to 010% in animals monitored for 7 days.9 A 20 µg dose given on day 3 was less effective. We therefore determined the minimum effective single intraperitoneal dose of endoE, given on day 1, in rats infected 2 days after birth (Figure 2). A progressive reduction of the dose over the range 200.125 µg indicated that a dose as low as 0.25 µg was as effective in preventing death as larger doses; 0.125 µg had little effect on survival. Death was always preceded by bacteraemia. Intraperitoneal administration of endoE had no effect on the degree or duration of colonization with E. coli K1 in comparison to control animals: colonization was evident in all animals 2448 h after feeding of A192PP and persisted through the course of the experiments. His6-tagged GST (20 µg, intraperitoneal), produced in the same E. coli expression system as endoE, had no effect on the outcome of the infection.
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The endoE-mediated removal of the polysialic acid capsule from complement-resistant K1 strains sensitizes them to the bactericidal action of serum and facilitates deposition of both C3 and C9 onto the bacterial surface.9 As K1 strains are known to be more resistant to uptake and killing by macrophages, particularly in the absence of classical complement pathway activation,16 we examined the effect of endoE on the uptake of E. coli K1 strain A192PP by peritoneal macrophages from young (510 days old) rats in the presence of fetal calf serum. At a multiplicity of infection of 100:1, A192PP had a high degree of resistance to macrophage uptake over a 3 h period, but addition of a small quantity of endoE (10 µg) to the incubation mixture resulted in a large increase in the number of bacteria within macrophages (Figure 3). In contrast to uptake of untreated bacteria, endoE treatment resulted in progressive uptake of A192PP over a 3 h period (Figure 3b).
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Discussion |
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In this study and in a previous investigation,9 we have presented evidence that modification of the phenotype can form the basis for an alternative approach to the treatment of infection. EndoE had no effect on either the viability or growth rate of E. coli K1 strains but sensitized these bacteria to the bactericidal action of complement and to ingestion by macrophages. Peritoneal administration of small doses, as low as 0.25 µg, to infant rats early in the infection process ensured that the majority survived a normally lethal systemic infection. Similar doses administered later in the infection process, after 3 days, were less effective and may limit the utility of targeting bacterial capsule expression. The therapeutic effect is endoE-specific, as a 20 µg dose of his6-GST had no effect on the course of infection. We surmise that the infection is resolved due to endoE-mediated stripping of the K1 capsule from the bacterial surface and we are currently using histological and immunohistochemical procedures to establish in vivo the basis for attenuation. The K1 polysaccharide is a key determinant of the capacity of strains to cause extraintestinal infections. The loss of K1, the endoE substrate, by mutation renders strains apathogenic2 and resistance to K1-specific bacteriophages and to endoE is invariably due to defects in K1 biosynthesis.18 As structural modifications, such as non-stoichiometric O-acetylation, do not affect hydrolysis by endoE,5 it is difficult to envisage endoE resistance other than through K1 antigen loss, an event that would prevent development of systemic infection if it occurred in vivo. Such interdependence may delay or completely abrogate the emergence of endoE-resistant, virulent genotypes. The K1 antigen is a critical determinant of virulence in neuroinvasive strains of E. coli and protects the bacterial cell from both complement-induced killing and phagocytosis.2 We have shown in this study that endoE-mediated capsule removal in vitro has a profound effect on the rate of uptake of bacteria by peritoneal macrophages; this complements our earlier observation that endoE sensitizes K1 strains to complement and increases the deposition of key complement proteins involved in bactericidal action.9
As noted by others,11,14,19 the introduction of E. coli K1 isolates from a variety of sources into the gastrointestinal system of neonatal rats leads to systemic infection in a proportion of the cohort. We have determined the presence of often large numbers of bacteria in the blood compartment and others have shown that meningitis also occurs, as indicated by the presence of K1 bacteria in the cerebrospinal compartment.11,14 We felt that, in order to meaningfully evaluate the therapeutic efficacy of endoE, it would be necessary to refine the model in such a way that a large proportion of individuals in a litter would develop systemic infection. This was achieved by passage of human isolates of K1 in neonatal rat pups. Passaged strain A192PP induced bacteraemia in a large majority of colonized animals and this remained a consistent feature throughout this study.
The animal model that we employed has features in common with infection in the human host. Bacteraemia occurred shortly after gastrointestinal colonization and was dependent on the age of the host. Two-day-old pups were uniformly susceptible to bacteraemia following colonization with A192PP and 3- and 5-day-old animals less so, even though they were efficiently colonized. This mirrors the infection in human neonates, who are most susceptible to K1 infection during the early neonatal period.20 Intestinal colonization always preceded systemic infection. EndoE administration prevented bacteraemia and death in colonized animals but had no effect on colonization; assuming that the enzyme gained access to the gut, this indicates no role for the K1 capsule in the maintenance of K1 strains in the gut.
Our observations suggest that severe infections could be successfully treated by agents that have no effect on the viability of the pathogen but modify the phenotype of the bacterial cell to reduce its capacity to survive at the site of infection. Although a limited number of capsular types are responsible for the large majority of cases of neonatal and childhood sepsis and meningitis,10,21 there are severe barriers to the implementation of enzyme therapy to the treatment of infection: therapeutic proteins are immunogenic and usually possess a poor pharmacokinetic profile. However, recent work22,23 has implicated histidine and tyrosine protein kinases in the regulation of capsular polysaccharide expression at the E. coli surface; therefore, inhibitors of the reversible phosphorylation of histidine and tyrosine residues could provide a novel approach to the treatment of systemic infections due to encapsulated bacteria.
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Acknowledgements |
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