Treatment of experimental Escherichia coli infection with recombinant bacteriophage-derived capsule depolymerase

Naseem Mushtaq1, Maria B. Redpath1, J. Paul Luzio2 and Peter W. Taylor1,*

1 Microbiology Group, School of Pharmacy, 29–39 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


* Corresponding author. Tel/Fax: +44-20-7753-5867; E-mail: peter.taylor{at}ulsop.ac.uk

Received 4 January 2005; returned 25 February 2005; revised 28 February 2005; accepted 26 April 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The aim of this study was to investigate the effect of single doses of the capsule depolymerizing enzyme endosialidase E (endoE) on the course of systemic infection due to Escherichia coli K1 strains in neonatal rats. We also determined the capacity of the enzyme to increase the sensitivity of K1 strains to rat peritoneal macrophages.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many bacterial pathogens produce hydrated, negatively-charged polysaccharide capsules that lie external to the cell wall and confer resistance to immune mechanisms such as engulfment by phagocytes and killing by complement.1,2 Encapsulated bacteria cause severe infections such as septicaemia, meningitis, pneumonia, osteomyelitis, septic arthritis and pyelonephritis, and also less-severe non-invasive infections.3 In many of these cases, the capsule is the major determinant of survival and confers on the pathogen the capacity to cause infection. Removal of the capsule, or inhibition of its biosynthesis may, therefore, represent an alternative to conventional chemotherapy by sensitizing the bacteria to components of the host's defences.4

A bacteriophage-encoded enzyme, endosialidase E (endoE), which selectively degrades the linear homopolymeric {alpha}-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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

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 SDS–PAGE 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 10–14 individuals, were fed 0.02 mL of E. coli K1 (2–6 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
K1 infection

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 80–100%. 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.


View this table:
[in this window]
[in a new window]
 
Table 1. Incidence of gastrointestinal colonization and bacteraemia in 2-day-old Wistar rat pups following ingestion of E. coli K1 strains

 
We studied the effect of the age of the animal on the infection process using E. coli A192PP (Figure 1). This organism was administered to 2-, 3-and 5-day-old rats. Gastrointestinal colonization by K1 was readily achieved in all animals, usually within 24 h of feeding the culture. In 2-day-old pups, colonization led to bacteraemia, which usually resulted in the death of all animals within 7 days of infection. With 3-day-old pups, bacteraemia occurred in around 50–70% of animals and resulted in a 50–70% mortality rate after 7 days. Five-day-old rats were more refractory to systemic infection and fewer deaths were recorded within the 7 day period of the experiments. All further studies were undertaken, therefore, with 2-day-old rat pups. There was no clear difference in the viable counts from the blood of 2- compared with 3- and 5-day-old bacteraemic animals: for positive blood cultures (from living animals only), viable counts ranged from 3 x 102 to 7.8 x 106 (mean 5.5 x 104) for 2-day olds, from 3 x 102 to 1.5 x 106 (mean 9.2 x 103) for 3-day olds and 6 x 103 to 6 x 105 (mean 7.4 x 103) for 5-day olds.



View larger version (12K):
[in this window]
[in a new window]
 
Figure 1. Bacteraemia and death in 2- (a), 3- (b) and 5-day-old (c) Wistar rats following oral administration of 0.02 mL of E. coli A192PP culture. All pups in a litter were fed A192PP on day 0. E. coli colonies expressing the K1 antigen were detected using K1-specific bacteriophage from cultures of perianal swabs (filled diamonds) and blood samples (filled squares). Accumulated deaths are also shown (filled triangles). Data from typical experiments involving single litters, usually 10–14 pups.

 
Effect of intraperitoneal administration of endoE on K1 infection in neonatal rats

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 80–100% to 0–10% 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 20–0.125 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 24–48 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.



View larger version (12K):
[in this window]
[in a new window]
 
Figure 2. Effect of intraperitoneal administration of endoE on mortality after 7 days of E. coli K1 infected 2-day-old rats. A group comprised a single litter of 10–14 animals. The controls received PBS; animals in the treated group were given a single dose of endoE in PBS 1 day after infection by the oral route.

 
Effect of endoE on uptake of K1 strains by macrophages

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 (5–10 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).



View larger version (77K):
[in this window]
[in a new window]
 
Figure 3. Uptake of E. coli A192PP by peritoneal macrophages from young rats. (a) The upper panel shows presence of Crystal Violet-stained bacteria within macrophages following incubation at 37°C for the times indicated. The lower panel shows the effect of addition of 10 µg of endoE to each incubation mixture. (b) Kinetics of uptake over 3 h incubation period. Squares show uptake of untreated bacteria; triangles show uptake after addition of 10 µg of endoE at 0 min. Bacteria within 20 macrophages were enumerated at each time point and data from two separate experiments were combined. Data are shown as means ± 1 SD.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The excessive and often unnecessary use of antibiotics in humans and animals has probably advanced the emergence and spread of drug-resistant bacterial pathogens. Such selective pressure alters the genetic complement of microbial populations and severely reduces the efficacy of conventional antibiotic chemotherapy.17 We are investigating alternative approaches to the treatment of infectious disease in which the phenotype of the infectious agent is modified by a primary agent and thereby sensitized to either a second agent, such as a conventional antibiotic, or a component of the host's immune system.4 Pathogens that are attenuated in this way are less likely to survive at the locus of infection than unmodified bacteria.

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.


    Acknowledgements
 
This work was supported by project grants GA230 and GA382 from the British Society for Antimicrobial Chemotherapy. N. M. is the recipient of a Fellowship from the Maplethorpe Trust.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. Jann B, Jann K. Capsules of Escherichia coli. In: Sussman M, ed. Mechanisms of Virulence. Cambridge University Press, 1997; 113–43.

2. Cross AS. The biologic significance of bacterial encapsulation. Curr Top Microbiol Immunol 1990; 150: 87–95.[ISI][Medline]

3. Moxon ER, Kroll JS. The role of bacterial polysaccharide capsules as virulence factors. Curr Top Microbiol Immunol 1990; 150: 65–85.[ISI][Medline]

4. Taylor PW, Stapleton PD, Luzio JP. New ways to treat bacterial infections. Drug Discov Today 2002; 7: 1086–91.[CrossRef][ISI][Medline]

5. Tomlinson S, Taylor PW. Neuraminidase associated with coliphage E that specifically depolymerises the Escherichia coli K1 capsular polysaccharide. J Virol 1985; 55: 374–8.[ISI][Medline]

6. Long GS, Bryant JM, Taylor PW et al. Complete nucleotide sequence of the gene encoding bacteriophage E endosialidase: implications for K1E endosialidase structure and function. Biochem J 1995; 309: 543–50.[ISI][Medline]

7. Leggate DR, Bryant JM, Redpath MB et al. Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis. Mol Microbiol 2002; 44: 749–60.[CrossRef][ISI][Medline]

8. Korhonen TK, Valtonen MV, Parkkinen J et al. Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis. Infect Immun 1985; 48: 486–91.[ISI][Medline]

9. Mushtaq N, Redpath MB, Luzio JP et al. Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype. Antimicrob Agents Chemother 2004; 48: 1503–8.[Abstract/Free Full Text]

10. Polin RA, Harris MC. Neonatal bacterial meningitis. Semin Neonatol 2001; 6: 157–72.[CrossRef][Medline]

11. Glode MP, Sutton A, Moxon ER et al. Pathogenesis of neonatal Escherichia coli meningitis: induction of bacteremia and meningitis in infant rats fed E. coli K1. Infect Immun 1977; 16: 75–80.[ISI][Medline]

12. Taylor PW. Immunochemical investigations on lipopolysaccharides and acidic polysaccharides from serum-sensitive and serum-resistant strains of Escherichia coli isolated from urinary-tract infections. J Med Microbiol 1976; 9: 405–21.[Abstract]

13. Achtman M, Mercer A, Kusecek B et al. Six widespread bacterial clones among Escherichia coli K1 isolates. Infect Immun 1983; 39: 315–35.[ISI][Medline]

14. Pluschke G, Mercer A, Kusecek B et al. Induction of bacteremia in newborn rats by Escherichia coli K1 is correlated with only certain O (lipopolysaccharide) antigen types. Infect Immun 1983; 39: 599–608.[ISI][Medline]

15. Gross RJ, Cheasty T, Rowe B. Isolation of bacteriophages specific for the K1 polysaccharide antigen of Escherichia coli. J Clin Microbiol 1977; 6: 548–50.[ISI][Medline]

16. Bortolussi R, Ferrieri P, Björksten B et al. Capsular K1 polysaccharide of Escherichia coli: relationship to virulence in newborn rats and resistance to phagocytosis. Infect Immun 1979; 25: 293–8.[ISI][Medline]

17. Monroe S, Polk R. Antimicrobial use and bacterial resistance. Curr Opin Microbiol 2000; 3: 496–501.[CrossRef][ISI][Medline]

18. Pelkonen S. Capsular sialyl chains of Escherichia coli K1 mutants resistant to K1 phage. Curr Microbiol 1990; 21: 23–8.[ISI]

19. Pluschke G, Pelkonen S. Host factors in the resistance of newborn mice to K1 Escherichia coli infection. Microb Pathog 1988; 4: 93–102.[CrossRef][ISI][Medline]

20. Holt DE, Halket S, de Louvois J et al. Neonatal meningitis in England and Wales: 10 years on. Arch Dis Child Fetal Neonatal Ed 2001; 84: F85–9.[Abstract/Free Full Text]

21. Kornelisse RF, de Groot R, Neijens HJ. Bacterial meningitis: mechanisms of disease and therapy. Eur J Pediatr 1995; 154: 85–96.[CrossRef][ISI][Medline]

22. Wugeditsch T, Paiment A, Hocking J et al. Phosphorylation of Wzc, a tyrosine autokinase, is essential for assembly of group 1 capsular polysaccharides in Escherichia coli. J Biol Chem 2001; 276: 2361–71.[Abstract/Free Full Text]

23. Chen MH, Takeda S, Yamada H et al. Characterization of the RcsC->YojN->RcsB phosphorelay signalling pathway involved in capsular synthesis in Escherichia coli. Biosci Biotechnol Biochem 2001; 65: 2364–7.[CrossRef][ISI][Medline]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
56/1/160    most recent
dki177v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Mushtaq, N.
Articles by Taylor, P. W.
PubMed
PubMed Citation
Articles by Mushtaq, N.
Articles by Taylor, P. W.