Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-05-20
2003-02-04
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C536S023100, C536S024300, C536S024320, C536S024330, C435S320100
Reexamination Certificate
active
06514746
ABSTRACT:
BACKGROUND
This invention relates to the field of antibacterial treatments and to targets for antibacterial agents. In particular, it relates to genes essential for survival of a bacterial strain in vitro or in vivo.
The following background information is not admitted to be prior art to the pending claims, but is provided only to aid the understanding of the reader.
Despite the development of numerous antibacterial agents, bacterial infections continue as a major, and currently increasing, medical problem. Prior to the 1980s, bacterial infections in developed countries could be readily treated with available antibiotics. However, during the 1980s and 1990s, antibiotic resistant bacterial strains emerged and have become a major therapeutic problem. There are, in fact, strains resistant to essentially all of the commonly used antibacterial agents, which have been observed in the clinical setting, notably including strains of
Staphylococcus aureus
(
S. aureus
) The consequences of the increase in resistant strains include higher morbidity and mortality, longer patient hospitalization, and an increase in treatment costs (B. Murray, 1994, New Engl.
J. Med
. 330:1229-1230). Therefore, there is a pressing need for the development of new antibacterial agents which are not significantly affected by the existing bacterial resistance mechanisms.
Such development of new antibacterial agents can proceed by a variety of methods, but generally fall into at least two categories. The first is the traditional approach of screening for antibacterial agents without concern for the specific target.
The second approach involves the identification of new targets, and the subsequent screening of compounds to find antibacterial agents affecting those targets. Such screening can involve any of a variety of methods, including screening for inhibitors of the expression of a gene, or of the product of a gene, or of a pathway requiring that product. However, generally the actual target is a protein, the inhibition of which prevents the growth or pathogenesis of the bacterium. Such protein targets can be identified by identifying genes encoding proteins essential for bacterial growth.
SUMMARY
Each pathogenic bacterial species expresses a number of different genes which are essential for growth of the bacteria in vitro or in vivo in an infection, and which are useful targets for antibacterial agents. This invention concerns the identification and use of particular essential genes and bacterial strains expressing mutant forms of those genes in the identification, characterization, and evaluation of antibacterial agents. Thus, this invention also provides methods of treating bacterial infections in mammals by administering an antibacterial agent active against such a gene, and the pharmaceutical compositions effective for such treatment.
For the
Staphylococcus aureus
essential genes identified in this invention, the essential nature of the genes was determined by the isolation of growth conditional mutants of
S. aureus
, in this case temperature sensitive mutants (ts mutants). Each gene was then identified by isolating recombinant bacteria derived from the growth conditional mutant strains, which would grow under non-permissive conditions but which were not revertants. These recombinant bacteria contained DNA inserts derived from the normal (i.e., wild-type)
S. aureus
chromosome which encoded non-mutant products which replaced the function of the products of the mutated genes. The fact that a clone having such a recombinant insert can complement the mutant gene product under non-permissive conditions implies that the insert contains essentially a complete gene, since it produces functional product.
The Staphylococcal genes described herein have been completely sequenced and analyzed. These two genes are named espA (SEQ ID NO. 2), encoding EspA, a polypeptide of the response regulator family (SEQ ID NO. 4), and espB (SEQ ID NO. 3), encoding EspB, a polypeptide of the histidine kinase family (SEQ ID NO. 5), respectively. These can be considered to be two separate genes or together to comprise one dicistronic gene.
In a first aspect, this invention provides a method of screening for an antibacterial agent by determining whether a test compound is active against one of the identified bacterial genes. These genes have been identified as essential genes by the isolation of a growth conditional mutant strain, and the complementation in recombinant strains of each of the genes, by expression from artificially-inserted DNA sequences carrying genes identified by the specified sequences of SEQ ID NO. 1-3.
In a particular embodiment the method is performed by providing a bacterial strain having a mutant form of a gene selected from the group of genes corresponding to SEQ. ID. NO. 1-3′ or a mutant gene homologous to one of those genes. The mutant form of the gene confers a growth conditional phenotype, e.g., a temperature-sensitive phenotype, on the bacterial strain having that mutant form. A comparison bacterial strain having a normal form of the gene is also provided and the two strains of bacteria are each contacted with a test compound, preferably under semi-permissive growth conditions. The growth of the two strains in the presence of the test compound is then compared; a reduction in the growth of the bacterial strain having the mutant form compared to the growth of the bacterial strain having the normal form of the gene indicates that the test compound is active against the particular gene.
The term “antibacterial agent” refers to both naturally occurring antibiotics produced by microorganisms to suppress the growth of other microorganisms, and agents synthesized or modified in the laboratory which have either bactericidal or bacteriostatic activity, e.g., &bgr;-lactam antibacterial agents, glycopeptides, macrolides, quinolones, tetracyclines, and aminoglycosides. In general, if an antibacterial agent is bacteriostatic, it means that the agent essentially stops bacterial cell growth (but does not kill the bacteria); if the agent is bacteriocidal, it means that the agent kills the bacterial cells (and may stop growth before killing the bacteria).
The term “active against” in the context of compounds, agents, or compositions having antibacterial activity indicates that the compound exerts an effect on a particular bacterial target or targets which is deleterious to the in vitro and/or in vivo growth of a bacterium having that target or targets. In particular, a compound active against a bacterial gene exerts an action on a target which affects an expression product of that gene. This does not necessarily mean that the compound acts directly on the expression product of the gene, but instead indicates that the compound affects the expression product in a deleterious manner. Thus, the direct target of the compound may be, for example, at an upstream component which reduces transcription from the gene, resulting in a lower level of expression. Likewise, the compound may affect the level of translation of a polypeptide expression product, or may act on a downstream component of a biochemical pathway in which the expression product of the gene has a major biological role. Consequently, such a compound can be said to be active against the bacterial gene, against the bacterial gene product, or against the related component either upstream or downstream of that gene or expression product. While the term “active against” encompasses a range of potential activities, it also implies some degree of specificity of target. Therefore, for example, a general protease is not “active against” a particular bacterial gene which produces a polypeptide product. In contrast, a compound which specifically inhibits a particular enzyme is active against that enzyme and against the bacterial gene which codes for that enzyme.
The term “in vivo” in the context of a bacterial infection refers to the host infection environment, as distinguished, for example, from growth of the bacteria in an artificial culture medium (e.g., in vitro
Benton Bret
Malouin Francois
Martin Patrick K.
Schmid Molly B.
Sun Dongxu
Bingham & McCutchen LLP
Epps Janet L.
Essential Therapeutics, Inc.
McGarry Sean
Rose Bernard F.
LandOfFree
Staphylococcus aureus histidine protein kinase essential genes does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Staphylococcus aureus histidine protein kinase essential genes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Staphylococcus aureus histidine protein kinase essential genes will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3180440