Identification of genes

Details Identification of genes Identification of genes

1998-12-01

2002-01-29

Schwartzman, Robert A. (Department: 1636)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C435S252100, C435S252400, C530S350000, C536S023700

Reexamination Certificate

active

06342215

ABSTRACT:

The present invention relates to methods for the identification of genes involved in the adaptation of a microorganism to its environment, particularly the identification of genes responsible for the virulence of a pathogenic microorganism.
BACKGROUND OF THE INVENTION
Antibiotic resistance in bacterial and other pathogens is becoming increasingly important. It is therefore important to find new therapeutic approaches to attack pathogenic microorganisms.
Pathogenic microorganisms have to evade the host's defence mechanisms and be able to grow in a poor nutritional environment to establish an infection. To do so a number of “virulence” genes of the microorganism are required.
Virulence genes have been detected using classical genetics and a variety of approaches have been used to exploit transposon mutagenesis for the identification of bacterial virulence genes. For example, mutants have been screened for defined physiological defects, such as the loss of iron regulated proteins (Holland et al, 1992), or in assays to study the penetration of epithelial cells (Finlay et al, 1988) and survival within macrophages (Fields et al, 1989; Miller et al, 1989a; Groisman et al, 1989). Transposon mutants have also been tested for altered virulence in live animal models of infection (Miller et al, 1989b). This approach has the advantage that genes can be identified which are important during different stages of infection, but is severely limited by the need to test a wide range of mutants individually for alterations to virulence. Miller et al (1989b) used groups of 8 to 10 mice and infected orally 95 separate groups with a different mutant thereby using between 760 and 950 mice. Because of the extremely large numbers of animals required, comprehensive screening of a bacterial genome for virulence genes has not been feasible.
Recently a genetic system (in vivo expression technology [IVET]) was described which positively selects for Salmonella genes that are specifically induced during infection (Mahan et al, 1993). The technique will identify genes that are expressed at a particular stage in the infection process. However, it will not identify virulence genes that are regulated posttranscriptionally, and more importantly, will not provide information on whether the gene(s) which have been identified are actually required for, or contribute to, the infection process.
Lee & Falkow (1994)
Methods EnymoL
236, 531-545 describe a method of identifying factors influencing the invasion of Salmonella into mammalian cells in vitro by isolating hyperinvasive mutants.
Walsh and Cepko (1992)
Science
255, 434-440 describe a method of tracking the spatial location of cerebral cortical progenitor cells during the development of the cerebral cortex in the rat. The Walsh and Cepko method uses a tag that contains a unique nucleic acid sequence and the lacZ gene but there is no indication that useful mutants or genes could be detected by their method.
WO 94/26933 and Smith et al (1995)
Proc. Natl. Acad. Sci. USA
92, 6479-6483 describe methods aimed at the identification of the functional regions of a known gene, or at least of a DNA molecule for which some sequence information is available.
Groisman et al (1993)
Proc. Natl. Acad. Sci. USA
90, 1033-1037 describes the molecular, functional and evolutionary analysis of sequences specific to Salmonella.
Some virulence genes are already known for pathogenic microorganisms such as
Escherichia coli, Salmonella typhimurium, Salmonella typhi, Vibrio cholerae, Clostridium botulinum, Yersinia pestis, Shigella flexneri
and
Listeria monocytogenes
but in all cases only a relatively small number of the total have been identified.
The disease which
Salmonella typhimurium
causes in mice provides a good experimental model of typhoid fever (Carter & Collins, 1974). Approximately forty two genes affecting Salnonella virulence have been identified to date (Groisman & Ochman, 1994). These represent approximately one third of the total number of predicted virulence genes (Groisman and Saier, 1990).
The object of the present invention is to identify genes involved in the adaptation of a microorganism to its environment, particularly to identify further virulence genes in pathogenic microorganisms, with increased efficiency. A further object is to reduce the number of experimental animals used in identifying virulence genes. Still further objects of the invention provide vaccines, and methods for screening for drugs which reduce virulence.
SUMMARY OF THE INVENTION
A first aspect of the invention provides a method for identifying a microorganism having a reduced adaptation to a particular environment comprising the steps of:
(1) providing a plurality of microorganisms each of which is independently mutated by the insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence so that each mutant contains a different marker sequence, or clones of the said microorganism;
(2) providing individually a stored sample of each mutant produced by step (1) and providing individually stored nucleic acid comprising the unique marker sequence from each individual mutant;
(3) introducing a plurality of mutants produced by step (1) into the said particular environment and allowing those microorganisms which are able to do so to grow in the said environment;
(4) retrieving microorganisms from the said environment or a selected part thereof and isolating the nucleic acid from the retrieved microorganisms;
(5) comparing any marker sequences in the nucleic acid isolated in step (4) to the unique marker sequence of each individual mutant stored as in step (2); and
(6) selecting an individual mutant which does not contain any of the marker sequences as isolated in step (4).
Thus, the method uses negative selection to identify microorganisms with reduced capacity to proliferate in the environment.
A microorganism can live in a number of different environments and it is known that particular genes and their products allow the microorganism to adapt to a particular environment. For example, in order for a pathogenic microorganism, such as a pathogenic bacterium or pathogenic fungus, to survive in its host the product of one or more virulence genes is required. Thus, in a preferred embodiment of the invention a gene of a microorganism which allows the microorganism to adapt to a particular environment is a virulence gene.
Conveniently, the particular environment is a differentiated multicellular organism such as a plant or animal. Many bacteria and fungi are known to infect plants and they are able to survive within the plant and cause disease because of the presence of and expression from virulence genes. Suitable microorganisms when the particular environment is a plant include the bacteria
Agrobacterium tumefaciens
which forms tumours (galls) particularly in grape;
Erwinia amylovara; Pseudomonas solanacearum
which causes wilt in a wide range of plants;
Rhizobium leguminosarum
which causes disease in beans;
Xanthomonas campestris
p.v.
citri
which causes canker in citrus fruits; and include the fungi
Magnaporthe grisea
which causes rice blast disease; Fusarium spp. which cause a variety of plant diseases; Erisyphe spp.;
Colletotrichum gloeosporiodes; Gaeumannomyces graminis
which causes root and crown diseases in cereals and grasses; Glomus spp., Laccaria spp.;
Leptosphaeria maculans; Phoma tracheiphila;
Phytophthora spp.,
Pyrenophora teres; Verticillium alboatrum
and
V. dahliae;
and
Mycosphaerella musicola
and
M. fijiensis.
As described in more detail below, when the microorganism is a fungus a haploid phase to its life cycle is required.
Similarly, many microorganisms including bacteria, fungi, protozoa and trypanosomes are known to infect animals, particularly mammals including humans. Survival of the microorganism within the animal and the ability of the microorganism to cause disease is due in large part to the presence of and expression from virulence genes. Suitable bacteria include Bordetella spp. particularly
B. pertussi

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