Dysregulation of Bacterial Proteolytic Machinery
Dysregulation of Bacterial Proteolytic Machinery
Abstract & Commentary
By Joseph F. John, Jr., MD, Chief, Medical Subspecialty Services, Ralph H. Johnson Veterans Administration Medical Center, Professor of Medicine, Medical University of South Carolina, Charleston, SC, is Co-Editor for Infectious Disease Alert.
Dr. John does research for Merck, is a consultant for Cubist, Roche, and bioMerieux, and is on the speaker’s bureau for Pharmacia, GSK, Merck, Bayer, and Wyeth.
Synopsis: Antibiotics useful against human bacterial infections have classically been derived from higher bacteria and fungi. In that tradition, a new class of antibiotics called acyldepsipeptides, have been isolated (then patented) from Streptococcus hawaiiensis. A group predominantly of German scientists from industry and academia have now published the mechanism of action for a group of these acyldepsipeptides (ADEP).
Source: Brotz-Oestehelt H, et al. Dysregulation of Bacterial Proteolytic Machinery By a New Class of Antibiotics. Nat Med. 2005;11:1082-1087.
ADEP I-6 have a complex ring structure. ADEP-I is the primary natural product ("factor A"). ADEP-1 has congeners that have been optimized for antibacterial activity, with the addition of 2 fluorides, to produce a difluoronated phenylalanine side chain. ADEP-2 has MICs for Streptococcus pneumoniae of 0.05 ug/mL, for Streptococcus pyogenes (Group A streptococcus) of 0.05 ug/mL, Enterococcus faecalis of < 0.01 ug/mL, for E. faecium of 0.02 ug/mL, and for Staphylococcus aureus (MRSA) of 0.4 ug/mL. ADEP-4 has an MIC for S. aureus of 0.05 ug/mL. ADEP-3, which is the R epimer of ADEP-2, has MICs for all these bacterial species of > 100 ug/mL.
In lethal S. aureus sepsis, 80% of mice were protected with 12.5 mg/kg dosing. Lower doses (1 mg/kg) were protective for E. faecalis sepsis. In S. pneumoniae sepsis in rats, ADEP performed better than linezolid.
ADEPs act through a complex. likely novel, mechanism. Labeling of precursor DNA, RNA, protein, and fatty acid showed that metabolism of these components was unhindered by ADEP. Microscopic examination of bacteria, immediately after exposure to ADEP, showed distinct filamentous forms, reaching a length of 200 uM, suggesting interruption of routine cell division through a secondary pathway.
Through a set of experiments using a genomic library of E. coli derivatives, some of which had become resistant to ADEP, Brotz-Ostehelt and colleagues found that a caseinolytic protease, designated by the gene ClpP, is necessary for the bactericidal activity of ADEP. Further experiments showed that ClpP specifically interacts with ADEP, independently of ATPases traditionally needed for ClpP to function.
So, what is it about the ClpP-ADEP product that is lethal to bacteria?
In B. subtilis, ClpP requires a Clp-ATPase and ATP to degrade proteins like casein. When exposed to ADEP 1 or ADEP 2, ClpP degrades casein without ATPase. Thus, ADEPs turn a docile ClpP into a proteolytic machine. Using modern proteomic chemistry, Brotz-Ostehelt et al also showed that ClpP was markedly upregulated by ADEP. Further induction by ADEPs was shown for a set of molecules called chaperones (genetic names like ClpC, DnaK, GroEL, and Tig). There were additional novel spots on the proteome pattern after ADEP induction, suggesting a very wide set of interactions for ADEP.
Commentary
Clinicians are increasingly faced with treating resistant bacteria, particularly Gram-positive cocci like staphylococci and enterococci, which are very broadly resistant. Thus, it is crucial that academia and industry continue to work discovering new antibiotics. ADEPs are attractive because they are bactericidal, work through a conserved bacterial mechanism, and have low inhibitory concentrations.
ADEP 1 is also known as "factor A"-the patented product. In the current paper, additional ADEPs 2-6 congeners were derived that showed, in some cases like ADEP 2 and ADEP 4, improved antibacterial activity.
The discovery of highly active ADEPs is encouraging but also daunting in view of the scope and complexity of the work needed to discover and validate new classes of antimicrobials. Modern genomics and proteomics are indispensible for characterizing a gamut of molecules that are altered by the new antimicrobial moieties: for ADEP showing that an intermediate ADEP-bound product triggers a cascade of events leading to cell elongation and death probably through a series of cell regulators that are modified by ClpP-ADEP. The involvement of ADEPs at multiple sites is indeed complex, but the end result is a highly active group of antibacterials.
Resistance is always lurking, however, and it clouds even this novel discovery. ClpP is clearly not indispensable to bacterial cell function since ADEP-resistant mutants arise in vitro at frequencies around 106. At this point, it is concerning that such frequencies of resistance may obviate monotherapy with ADEPs, but only further animal work and clinical trials will answer that question. The use of monotherapy for a lethal systemic Gram-positive murine infections in the current report was highly efficacious.
This work was done under the senior mentoring of Hans-Georg Sahl in Bonn and Harald Labischinski, both of whom have contributed immensely to the field of antibiotic development. Peptides have long been known for their antibacterial potential. With this current work, we now are armed with compounds like the ADEPs that work through novel bacterial cellular mechanisms and offer new hope against the rising tide of resistant Gram-positive bacteria and the ever growing scourge of staphylococcal infections.
ADEP I-6 have a complex ring structure. ADEP-I is the primary natural product (factor A). ADEP-1 has congeners that have been optimized for antibacterial activity, with the addition of 2 fluorides, to produce a difluoronated phenylalanine side chain.Subscribe Now for Access
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