Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-05
2004-02-24
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S252300
Reexamination Certificate
active
06696249
ABSTRACT:
This invention relates to the field of biochemical assays involving regulated expression of reporter genes, and to mutant strains of bacteria useful in biochemical assays. More particularly, it relates to methods of screening for molecules capable of affecting expression and/or activity of type III secretion machinery in gram-negatve bacteria.
Type III secretion machinery is present in numerous gram-negative bacteria (including members of the species Shigella, Salmonella, Yersinia, Eschedchia, Pseudomonas, Xanthomonas, Ralstonia, and Erwinia) that are pathogenic for man, animals, and plants. For example, the Sec-independent type III secretion pathway is involved in secretion of Yersinia anti-host proteins. In Salmonella and Shigella species, it is involved in the process of entry into epithelial cells. It is also implicated in EPEC signal transducing proteins,
Pseudomonas aeruginosa
toxins, and virulence factors of many plant pathogens, as well as in flagellum assembly of bacteria such as
S. typhimurium
and
Bacillus subtilis.
Features of this secretion pathway can include activation of secretion by contact of the bacterium with host cells (Ménard et al., 1994, “The secretion of the
Shigella flexneri
Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD.”,
EMBO J
., 13:5293-5302; Watarai et al., 1995, “Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness.”,
EMBO J
., 14:2461-2470; Zierler and Galan, 1995, “Contact with cultures epithelial cells stimulates secretion of
Salmonella typhimurium
invasion proteins InvJ.”,
Infect. Immun
., 63:4024-4028); that some of the secreted proteins are delivered into the cytoplasm of host cells (Rosqvist et al., 1994, “Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells.”,
EMBO J
., 13:964-972; Sory and Cornelis, 1994, “Translocation of an hybrid YopE-adenylate-cyclase from
Yersinia enterocolitica
into HeLa cells.”,
Mol. Microbiol
., 14:583-594; Wood et al., 1996, “SopE, a secreted protein of
Salmonella dublin
, is translocated into the target eukaryotic cell via a sip-dependent mechanism and promotes bacterial entry.”,
Mol. Microbiol
., 22:327-338; Collazo and Galan, 1997, “The invasion-associated type III system of
Salmonella typhimurium
directe the translocation of Sip proteins into the host cell.”,
Mol. Microbiol
, 24:747-756); and that transcription of genes encoding secreted proteins is controlled by secretion of regulatory proteins (Hughes et al., 1993, “Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator.”,
Science
, 262:1277-1280; Pettersson et al., 1996, “Modulation of virulence factor expression by pathogen target cell contact.”,
Science
, 273:1231-1233).
Based on the observations that (1) the secretion machinery is involved in secretion of factors which are active against the host, and (2) secretion mutants are avirulent, the type III secretion machinery provides an attractive target for the screening of molecules that would prevent or inhibit gram-negative bacteria from secreting their virulence factors. However, the search for molecules capable of inhibiting the secretion mechanism has previously required two conditions to be present. First, the type III secretion machinery must be active. And second, the product of the secretion activity, i.e., the secreted proteins, must be measurable. Unfortunately, the secretion machinery is, at best, only weakly active when bacteria are grown in standard laboratory media, making the search for inhibitor molecules difficult or impossible. In addition, there is no way to easily measure the presence of a protein secreted in the culture medium by the type III secretion machinery. These proteins do not have an easily assayable enzymatic activity and their secretion must be evaluated using ELISA, which is time consuming and expensive.
This invention provides mutant strains of gram-negative bacteria that constitutively secrete proteins via the type III secretion machinery.
This invention also provides methods of identifying molecules that are able to activate or inhibit secretion in wild-type strains of gram-negative bacteria. These methods comprise the steps of:
a) exposing gram-negative bacterial cells to a sample molecule, wherein said bacterial cells contain a reporter gene transcriptionally fused to a promoter of a gene activated or regulated by the type III secretion machinery; and
b) detecting the presence or activity of the product of the reporter gene.
To practice the methods of this invention, genes under transcriptional control of the type III secretion machinery are identified. Transcriptional fusions between the promoters of these genes and a reporter gene, such as the lacZ reporter gene, are constructed and introduced into wild-type gram-negative bacteria and mutants of these bacteria that constitutively secrete proteins via the type III secretion machinery or are deficient for secretion via the type III secretion machinery. The presence (or activity) of the reporter gene product is evaluated under conditions leading to active secretion to demonstrate that the transcriptional activity of these promoters can be used as an indicator of the secretion activity of the type III secretion machinery.
Using Shigella as a model system for the screening of inhibitors of type III secretion, five genes under transcriptional control of the type III secretion machinery have been identified and the promoters of these genes have been used to create transcriptional fusions with the reporter gene, lacZ. &bgr;-galactosidase activity can be induced in recombinant Shigella cells harboring these transcriptional fusion constructs under conditions known to lead to active secretion.
Any gram-negative bacteria containing type III secretion machinery may be used in the methods of this invention. Suitable bacteria include members of the species Shigella, Salmonella, Yersinia, Escherichia, Pseudomonas, Xanthomonas, Ralstonia, and Erwinia. Similarly, any suitable reporter gene may be used to create a transcriptional fusion construct for use in the methods of this invention. Some suitable reporter genes are, for example, lacZ, phoA, luxAB, and gfp. In a preferred method of this invention, the reporter gene is the lacZ gene.
In one method of the invention, the promoter is from a gene selected from the group consisting of virA and the ipaH family of genes, particularly, ipaH9.8, ipaH7.8, ipaH4.5, and ipaH1.4. Promoters such as ipgD, icsB, ipaA, and mxiD are not regulated by the secretion machinery and thus may be used in the methods of this invention as internal controls. Other suitable promoters for use in the methods of this invention may be easily identified following the teachings detailed in this specification.
In a preferred method according to this invention, candidate inhibitor molecules are screened against three strains of bacteria which contain a reporter gene transcriptionally fused to a promoter of a gene regulated by the activity of the type III secretion machinery: a strain in which secretion is regulated, a strain which has a phenotype of constitutive secretion, and a strain which is deficient for secretion.
REFERENCES:
patent: WO 97/40063 (1997-10-01), None
patent: WO 99/45136 (1999-09-01), None
Sasakawa et al (J Bacteriol 1993 vol 175(8) p. 2334-46).*
Hartman et al. (J. Bacteriology 1990 vol. 172(4) pp. 1905-1915).*
Uchiya et al. (Molecular Microbiol. 1995 vol. 17(2) pp. 241-250).*
Demers, B., et al., “Induction of type III secretion inShigella f lexneriis associated with dif ferential control of transcription of genes encoding secreted proteins”, T he EMBO Journal, vol. 17, No. 10, (1998), pp. 2894-2903.
Demers, B., et al., “Expression of secreted proteins is controlled by the activity of the type III secretion machinery ofShigella flexneri”, Abstracts of the Interscience Conference on Antimirobial Agents and Chemotherapy, vol. 37, (1997), pp. 34.
Abdelmounaaïm Allaoui, et al., “Charact
Demers Brigitte
Parsot Claude
Sansonetti Philippe
Finnegan Henderson Farabow Garrett & Dunner
Institut Pasteur
Navarro Mark
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