Method for inducing bacterial growth using autoinducers

Details Method for inducing bacterial growth using autoinducers Method for inducing bacterial growth using autoinducers

2001-07-12

2003-11-18

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C424S093400, C424S780000, C435S243000, C435S252800, C435S822000, C435S849000, C435S879000

Reexamination Certificate

active

06649398

ABSTRACT:

BACKGROUND
1. Field of the Invention
The present invention concerns bacterial autoinducers of growth, methods for their purification, autoinducers purified by such methods, and their use to induce the growth of bacteria, both the source organism and other species.
2. Description of the Related Art
Signalling events between bacteria and host cells are an integral component of the dynamic and complex process of infection and disease. It has recently become clear that signalling between bacteria is also of importance to this process.
Low molecular weight, diffusible signal molecules produced by bacteria, termed autoinducers (AI), play a crucial role in the development of bacterial infections, of both plants and animals. These autoinducers may determine whether or not an initial infection, often involving only a very few bacteria, will succumb to the many defence mechanisms of a host or whether these host defences are overcome, and bacterial growth and disease occur.
One class of autoinducers has already been well-characterised, the N-acyl homoserine lactones, which are composed of derivatives of amino acid and fatty acid molecules. This family of molecules play a key role in the mechanisms by which Gram negative bacteria monitor population densities, factors which are important in virulence of a number species. However, despite the fact that N-acyl homoserine lactone-type sensing systems have been shown to exist in
E. coli
, there is so far no evidence that N-acyl homoserine lactones themselves are made by, or play a role in the pathogenesis of this organism. In addition, no evidence has been so far been presented to suggest a role for these autoinducers in the pathogenensis of Salmonella.
The existence of an additional class of autoinducer molecule has been shown, the AI being different from the homoserine lactones. These also appear to play an important role in pathogenesis.
A purported bacterial AI was isolated by Lyte, M. et al. (1996, FEMS Microbiology Letters, 139: 155-159) having a molecular weight of approximately 10,000 Da (see also, Lyte, M., 1993, Journal of Endocrinology, 137: 343-345; U.S. Pat. No. 5,629,349).
BRIEF SUMMARY OF THE INVENTION
The present inventors have succeeded in isolating, purifying and characterising a novel autoinducer from
E. coli
and
Hafnia alvei.
According to the present invention there is provided a bacterial autoinducer, characterised in that it has substantially the following properties:
i) it is produced in response to noradrenaline in serum SAPI medium;
ii) it is heat stable;
iii) it is stable to lyophilisation;
iv) it has a negative charge;
v) it is polar;
vi) it is hydrophilic;
vii) it will not partition into organic solvents;
viii) it is capable of binding positively charged metal ions; and
ix) it has a molecular weight of about 300-1500 daltons
The bacterium may be
E. coli
or
Hafnia alveii.
The bacterium may be Salmonella, for example
S. enteriditis
or
S. typhimurium.
The autoinducer is distinct from N-acyl homoserine lactones and the molecule of Lyte et al. (1996, supra) (for example, the molecular weight of an autoinducer according to the present invention is less than 1000 Da, compared to the 10,000 Da of Lyte et al.). Similarly it is not a peptide pheremone nor is it a known siderophore such as enterochelin which, amongst other things, is stable to acidification, soluble in organic solvents such as ethanol and upon crystallisation forms white needle-like crystals. Experiments (below) show that the autoinducers of the present invention appear to form a novel family of highly-related molecules.
The autoinducer has a wide range of possible uses, essentially including anything in which the growth of a bacterium or the production of a desired molecule is to be stimulated or assayed. For example, it may be used in fermentation processes, in culture media for diagnostic and environmental monitoring or in the drug discovery process in order to find agents which will inhibit autoinducer-mediated bacterial stimulation. In fermentation processes, the autoinducer may be used to stimulate starting cultures or to shorten and synchronise lag phases; in fermentation processes to stimulate the production of secondary metablolites such as antibiotics, chemicals for biological screening, and recombinant proteins; in culture media to shorten turn-around times or to assay viable but non-culturable organisms. Other uses of the autoinducer will be readily apparent to one skilled in the art.
The
E. coli
autoinducer is a low molecular weight diffusible signal molecule, initially found as a bacterial response to physiologically relevant concentrations of noradrenaline, such as those found in the gastrointestinal tract of mammalian hosts. This effect is not nutritionally mediated. The half-life of activity of intestinal nor-adrenaline is quite short lived—the hormone is active for only a few hours, before becoming irreversibly sulphonated. However, this transient exposure to nor-adrenaline is sufficient to induce the bacteria to synthesize their own growth stimulus, the autoinducer, which has much greater stability. The autoinducer acts by effecting both accelerated growth rate, increased bacterial cell numbers and the production of virulence factors, such as toxins and adhesins, the activity being cross-species specific.
The apparent molecular weight of the
E. coli
autoinducer is dependent upon the elution conditions used (see ‘Experimental’ below), due to the substantial charge the molecule has. Experiments (below) have shown the charge on the molecule to be greater than that on ATP. The molecule has also been found to be polar. It is heat stable and is capable of being autoclaved at 121° C. Similarly it is capable of withstanding lyophilisation. The molecule is also capable of inducing cross-species stimulation.
The above list of characteristics may be considered the “core” characteristics of the family of autoinducers. Other characteristics have been identified as detailed in the experimental section below and the autoinducer may have at least one of the following characteristics:
i) it has absorbtion maxima at 255,325 and 500-550 nm; and
ii) it is stable in prolonged storage in a dried state and/or in solution.
Additional characteristics (which may be specific to the
E. coli
, Salmonella or Hafnia autoinducers) of which the autoinducer may have at least one are:
i) it is produced in substantially smaller quantities by bacteria grown in LURIA broth, Tryptone soya broth, M9 minimal medium and Davis-Mingioli minimal medium than by the same bacteria grown in serum SAPI medium;
ii) it has a reddish-pink colour, reversibly decolorisable by reducing the pH to <4;
iii) it contains serine;
iv) its synthesis involves the entA and entB gene products;
v) its synthesis is not stimulated by conditions of Fe starvation;
vi) it is synthesised in conditions of excess Fe;
vii) its entry into bacteria occurs via a tonB dependent receptor;
viii) it is inactivated by oxidation;
ix) it is inactivated by extreme pH; and
x) it is resistant to degradation by ribonuclease, deoxyribonuclease, trypsin, pepsin, V8 protease, proteinase K, acid phosphatases, alkaline phosphates and phosphodiesterase.
Also provided according to the present invention is a method for isolating and purifying a bacterial autoinducer from a sample comprising the steps of:
i) collecting a sample containing the autoinducer;
ii) fractionating the sample to isolate fractions corresponding to molecular weights of approximately 300-1500 Daltons; and
iii) eluting the isolate of (ii) on an anion-exchange chromatographic column and selecting the fraction containing the autoinducer.
It may comprise the additional step of performing gel filtration chromatography upon the fraction containing the autoinducer selected in (iii) and selecting the fraction containing the autoinducer.
It may comprise the additional step of concentrating the sample prior to fractionation.
Concentration may be achieved by means of ultrafiltration. Such ultra-filtration may be performed with a membrane molecular weight cu

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