Tucker C. Roberts

Broad-Spectrum Antibiotic Activity of the Arylomycin Natural Products Is Masked by Natural Target Mutations

Novel classes of broad-spectrum antibiotics are needed to treat multidrug-resistant pathogens. The arylomycin class of natural products inhibits a promising antimicrobial target, type I signal peptidase (SPase), but upon initial characterization appeared to lack whole-cell activity against most pathogens. Here, we show that Staphylococcus epidermidis, which is sensitive to the arylomycins, evolves resistance via mutations in SPase and that analogous mutations are responsible for the natural resistance of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We identify diverse bacteria lacking these mutations and demonstrate that most are sensitive to the arylomycins. The results illustrate that the arylomycins have a broad-spectrum of activity and are viable candidates for development into therapeutics. The results also raise the possibility that naturally occurring resistance may have masked other natural product scaffolds that might be developed into therapeutics.

Secretion of the chlamydial virulence factor CPAF requires the Sec-dependent pathway

The chlamydial protease/proteasome-like activity factor (CPAF) is secreted into the host cytosol to degrade various host factors that benefit chlamydial intracellular survival. Although the full-length CPAF is predicted to contain a putative signal peptide at its N terminus, the secretion pathway of CPAF is still unknown. Here, we have provided experimental evidence that the N-terminal sequence covering the M1–G31 region was cleaved from CPAF during chlamydial infection. The CPAF N-terminal sequence, when expressed in a phoA gene fusion construct, was able to direct the export of the mature PhoA protein across the inner membrane of wild-type Escherichia coli. However, E. coli mutants deficient in SecB failed to support the CPAF signal-peptide-directed secretion of PhoA. Since native PhoA secretion was known to be independent of SecB, this SecB dependence must be rendered by the CPAF leader peptide. Furthermore, lack of SecY function also blocked the CPAF signal-peptide-directed secretion of PhoA. Most importantly, CPAF secretion into the host cell cytosol during chlamydial infection was selectively inhibited by an inhibitor specifically targeting type I signal peptidase but not by a type III secretion-system-specific inhibitor. Together, these observations have demonstrated that the chlamydial virulence factor CPAF relies on Sec-dependent transport for crossing the chlamydial inner membrane, which has provided essential information for further delineating the pathways of CPAF action and understanding chlamydial pathogenic mechanisms.

In Vitro Activities of Arylomycin Natural-Product Antibiotics against Staphylococcus epidermidis and Other Coagulase-Negative Staphylococci

ABSTRACT The arylomycins are a class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), and we have found in diverse bacteria that their activity is limited by the presence of a resistance-conferring Pro residue in SPase that reduces inhibitor binding. We have also demonstrated that Staphylococcus epidermidis, which lacks this Pro residue, is extremely susceptible to the arylomycins. Here, to further explore the potential utility of the arylomycins, we report an analysis of the activity of a synthetic arylomycin derivative, arylomycin C16, against clinical isolates of S. epidermidis and other coagulase-negative staphylococci (CoNS) from distinct geographical locations. Against many important species of CoNS, including S. epidermidis, S. haemolyticus, S. lugdunensis, and S. hominis, we find that arylomycin C16 exhibits activity equal to or greater than that of vancomycin, the antibiotic most commonly used to treat CoNS infections. While the susceptibility was generally correlated with the absence of the previously identified Pro residue, several cases were identified where additional factors also appear to contribute.

Type I Signal Peptidase and Protein Secretion in Staphylococcus epidermidis

Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the β-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationary-phase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.

Synthesis and Biological Characterization of Arylomycin B Antibiotics

Antibiotics are virtually always isolated as families of related compounds, but the evolutionary forces underlying the observed diversity are generally poorly understood, and it is not even clear whether they are all expected to be biologically active. The arylomycin class of antibiotics is comprised of three related families that are differentiated by nitration, glycosylation, and hydroxylation of a conserved core scaffold. Previously, we reported the total synthesis of an A series member, arylomycin A2, as well as the A series derivative arylomycin C16 and showed that both are active against a broader spectrum of bacteria than previously appreciated. We now report the total synthesis of a B series analogue, arylomycin B-C16, and its aromatic amine derivative. While the aromatic amine loses activity against all bacteria tested, the B series compound shows activities that are similar to the A series compounds, except that it also gains activity against the important pathogen Streptococcus agalactiae.