Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-07
2004-03-02
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024100, C536S024500
Reexamination Certificate
active
06699662
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to inhibitors of Staphylococcus SarA protein function involved in the expression of staphylococcal virulence factors and the use of these inhibitors to treat and prevent staphylococcal infections in subjects. Particularly, the inhibitors act to interfere with the binding of the SarA protein to its binding site(s). The selection of specific inhibitors of the SarA protein is made possible as a result of the identification of the binding sites of SarA protein on at least a portion of the agr (accessory gene regultor) gene, a gene that like the sar (staphylococcal accessory regulator) gene, plays a role in the virulence of Staphylococcus.
There is a great and urgent need among infectious-disease specialists, who have begun seeing one of their worst nightmares come true. They may be losing their last line of defense against the dangerous pathogen
Staphylococcus aureus
(
S. aureus
), which causes infections ranging from skin abscesses to such life-threatening conditions as pneumonia, endocarditis, septicemia, and toxic shock syndrome. Roughly one-third of the strains currently isolated from patients who acquire
S. aureus
infections while hospitalized are resistant to all antibiotics but one, vancomycin and now resistance to that antibiotic is cropping up. The present invention provides a new approach to combating
S. aureus
that may sidestep the organism's ability to develop resistance.
Despite intensive research efforts over the past 50 years, Staphylococcus, particularly
Staphylococcus aureus
, remains a serious threat to human health. In fact, recent reports describe clinical isolates with reduced susceptibility to vancomycin. Therefore,
S. aureus
represents a bigger threat to human health now, than at any time since the pre-antibiotic era.
Staphylococcus is an opportunisitic bacteria that takes advantage of immunocompromised subjects and may become pathogenic in these subjects. There are approximately thirty-two species of Staphylococcus with only three consistently causing human disease.
S. aureus
is clearly the most prominent disease causing species, followed by
S. epidermidis
, and in a distant third is
S. saprophyticus. S. epidermidis
is becoming more prominent as a disease causing species because it causes infections of in-dwelling medical devices. As a result, researchers are looking more carefully at
S. epidermidis
, and as a result of this research, have found homologs of both the sar and agr genes in
S. epidermidis
. Fluckiger, U., et al. (1998). Otto,M., et al. (1998), respectively.
S. aureus
can cause a diverse array of diseases ranging from relatively superficial infections of the skin (boils) to infections of the eye (endopthalmitis) to life threatening osteomyelitis, endocarditis and toxic shock syndrome (reviewed by Projan and Novick, 1997).
S. aureus
is armed with a large battery of virulence factors that enable it to colonize a human host and cause a variety of disease states (reviewed by Projan and Novick, 1997). Nosocomial infections are of particular concern for two reasons. The first is that the majority of life-threatening infections arise in the hospital environment. For example, while the frequency of
S. aureus
infections incurred during orthopedic or cardiac implant surgery is steady, the overall number of infections has risen dramatically in the past decade. This is largely due to the increase in the frequency of these procedures.
S. aureus
has an amazing capacity to colonize in-dwelling prosthetic devices. The second reason for increased concern of
S. aureus
infections is that strains of methicillin-resistant
Staphylococcus aureus
(MRSA) are endemic in hospitals. Moreover, strains with some resistance to vancomycin emerged in the United States in 1997 (Tenover et al., 1998; Smith et al., 1999; Sieradzki et al., 1999). Therefore, the need for new, effective treatments for this drug resistant pathogen is urgent.
The variety of virulence factors expressed by
S. aureus
contribute to a highly efficient system for survival. Early in the infection, surface proteins are predominantly expressed. Protein A and the adhesins (e.g., collagen, fibronectin) are representative surface proteins that solve two problems for the
S. aureus
cell. First, they bind to extracellular matrix components and anchor the cell to the host tissue. Second, they provide a host protein camouflage which helps the infecting cell elude the host's immune system. The nascent colony increases in size until a critical number of cells is achieved (quorum) and a switch is thrown to re-organize the expression of virulence factors from surface proteins to exoproteins. These latter factors contribute to sequestration of the colony within a protective biofilm and enzymatic degradation of host tissue with an army of digestive enzymes, such as nucleases, lipases and proteases, which eventually result in an abscess. These enzymes accomplish two important functions or the bacterium: (1) allowing space for growth of the colony by getting rid of host tissue and (2) digested host tissue is assimilated by the bacterial cells for growth. Deep-seated abscesses, such as those found in staphylococcal ostemyelitis and endocarditis, often require surgical intervention to remediate the disease. It is important to note that this phenotypic switching process can be largely recapitulated in the laboratory environment, with surface protein expression occurring in the early log phase of a culture's growth and exoprotein expression occurring late in log and into the stationary phases of growth.
The potency of this pathogen can be attributed to the coordinated, temporally-regulated expression of a wide array of virulence factors. Early in infection expression of surface proteins predominates, e.g., the collagen and fibronectin adhesins and protein A. The surface proteins allow the organism to attach to host tissues and evade the immune system. However, when the concentration of
S. aureus
cells at the site of infection becomes high, surface protein expression is reduced and exoprotein expression increases. The temporal regulation of surface proteins and exoproteins can be recapitulated in laboratory culture growth models, where early log phase growth represents an early infection and stationary phase represents late infection. Using this model system and classical genetics, two major pleiotropically-acting regulatory loci that govern temporal expression of surface proteins and exoproteins have been identified: agr, for accessory gene regulator (Recsei et al., 1986; Morfeldt et al., 1988; Peng et al., 1988) and sar, for staphylococcal accessory gene regulator (Cheung et al., 1992; Cheung and Projan, 1994). Mutations in these loci result in aberrant regulation of most virulence factors (e.g., lipase, coagulase, &agr;-toxin, adhesins, etc), which is reflected in diminished virulence in animal models of staphylococcal disease (Projan and Novick, 1997).
A scheme depicting the agr locus and its encoded proteins is shown in FIG.
1
. Divergent promoters (P2 and P3), separated by approximately 180 bp, are responsible for transcription of the agrBDCA operon and RNAIII/hld operon (Morfeldt et al., 1996). The four Agr proteins combine to make a quorum-sensing system that is homologous to many two-component signal transduction systems found in prokaryotic organisms (Ji et al., 1997). AgrB is a cell membrane-bound transporter/processor of the AgrD peptide. AgrD is a 46 amino acid peptide that is cleaved to an octapeptide pheromone, exported by AgrB, and specifically recognized by (Ji et al., 1997) the AgrC membrane-bound receptor. The AgrD octapeptide pheromone allows an
S. aureus
cell to signal its presence to other cells in the growing colony. As the colony grows, the concentration of pheromone (Agr D) increases and reaches a particular level. AgrC, also an integral membrane protein, is activated by pheromone binding. AgrC is thought to be a kinase that acts on AgrA by initiating a signal transduction
Hurlburt Barry K.
Rechtin Tammy M.
Smeltzer Mark S.
Fredman Jeffrey
University of Arkansas
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