Superbug's 'CPU' Revealed: Researchers Discover Chemical Clue Directing Staphylococcus Aureus

(Credit: CDC/Janice Haney Carr)

It has an array of virulence factors:

Surface proteins responsible for adhesion and invasion of host tissues (e.g., fibrinogen and fibronectin-binding proteins)

Exoproteins responsible for immune evasion (e.g., chemotaxis-inhibitory protein)

Numerous hemolytic and pore-forming toxins (e.g., hemolysins, leukocidins, and enterotoxins)

S. aureus is a facultatively anaerobic , Gram-positive bacteria

It was discovered in Aberdeen, Scotland in 1880 by the surgeon Sir Alexander Ogston in pus from surgical abscesses.

Each year some 500,000 patients in American hospitals contract a staphylococcal infection.

Early research by Novick and colleagues identified an accessory gene regulator (agr) that controls several virulence factors.

Expression of the agr locus is positively regulated by the agr pheromone, a ribosomally encoded secondary metabolite.

Subsequent genomic sequencing has revealed that homologs of the agr pheromones exist in several Gram-positive cocci, many of which are non-pathogenic.

Although referred to as the “master” regulator of S. aureus virulence, expression of agr is not always detected in vivo, and agr-deficient clinical isolates are known, which raises the possibility that other small molecules factor prominently in the regulation of virulence factor expression.

Outlines

Prediction of Cryptic nonribosomal peptide assembly in Staphylococcus.

• Genome-mining approach

Isolation of tyrosine-valine dipeptides.

• High-performance liquid chromatography (HPLC) and mass

spectroscopy analysis

Microarray analysis of virulence expression.

Role of aureusimines in vivo.

(A) Genetic loci of S. aureus Newman containing the NRPS gene.The NRPS locus

is found in all sequenced S. aureus genomes. The NRPS cluster contains two

open reading frames: ausA (the NRPS gene) and immediately downstream of it

ausB (phosphopantetheinyl transferase). ausB encodes the enzyme (AusB)

predicted to posttranslationally modify AusA with a 4′-phosphopantetheine

prosthetic group.

Prediction of Cryptic nonribosomal peptide assembly in Staphylococcus.

A major class of bacterial secondary metabolites comprises the nonribosomal peptides, which are produced, in microorganisms, by multifunctional enzyme assembly lines known as nonribosomal peptide synthetases (NRPSs)

Antibiotics are the best known nonribosomal peptides produced by soil-dwelling microbes, which use them as weapons.

Penicillin, for example, is not constructed ribosomally but is dependent on an NRPS that uses valine, cysteine, and -aminoadipic acid precursors.

Although penicillin was the first nonribosomal peptide used for S. aureus infections, S. aureus itself has not previously been shown to construct nonribosomal peptides.

NONRIBOSOMAL PEPTIDE SYNTHETASES

Nonribosomal peptides belong to a family of complex natural products built from simple amino acid monomers.

They are synthesized in many bacteria and fungi by large multifunctional proteins called nonribosomal peptide synthetases (NRPS).

A unique feature of NRPS system is the ability to synthesize peptides containing proteinogenic as well as non-proteinogenic amino acids.In many cases these enzymes work in conjunction with polyketides synthases  (PKS) giving hybrid products.

The product of these multifunctional enzymes are very important pharmaceutical compounds like cyclosporin, gramicidin etc.

The minimum set of domains required for an elongation cycle consist of a module with  Adenylation(A), Thiolation(T) or Peptidyl Carrier Protein (PCP), and Condensation(C) domain.

(B) S. aureus NRPS is a dimodular nonribosomal peptide assembly line encoding

a putative cyclic dipeptide. Domains A, C, T, and Re within the S. aureus NRPS

(AusA) are shown as round spheres shaded in yellow. Curved blue lines originate

from the T domain and indicate the phosphopantetheinyl arm that is predicted to

be delivered via action of AusB. Amino acid substrates (valine and tyrosine) were

predicted according to established NRPS codes. Release of a linear valine

tyrosine dipeptide aldehyde and the predicted nonribosomal peptide structure are

shown.

(C) Identification of S. aureus non ribosomal peptides. Structures of aureusimine

A and aureusimine B (phevalin) were determined by mass spectrometry and NMR

experiments

Isolation of tyrosine-valine dipeptides.

(D) Liquid chromatographic separations (HPLC chromatograms) of organic

extracts of S. aureus Newman and S. aureus Newman Del ausA. Aureusimine A

(peak 1) and aureusimine B (phevalin) (peak 2) are present within extracts of S.

aureus Newman but absent in extracts of S.aureus Newman Del ausA strain. ERM,

erythromycin.

To verify that the aureusimines are synthesized by the S. aureus NRPS

Discovery of a nonribosomal peptide unique to S. aureus raises the

possibility for its role as a regulator of S. aureus virulence factor

expression.

• Global microarray analysis

(A) Differential gene expression caused by th presence of aureusimines A and B

in S. aureus in early and late exponential phase growth. Results are presented as

mean fold up-regulation (shades of red) and down regulation (shades of blue) in

three separate experiments (see scale bar).

Microarray analysis of virulence expression.

(B) Aureusimines induce hemolysis. (Left) S. aureus Newman wild-type and

(center) S. aureus Newman Del ausA were grown on 5% sheep blood agar; (right)

S. aureus Newman Del ausA was grown on 5% sheep blood agar supplemented

with 100 mg/mL aureusimines A and B. Zones of clearance around colonies

indicate hemolysis.

Genes encoding for hemolysins were also significantly up-regulated in strains producing aureusimines

Role of aureusimines in vivo.

(A)Weight change(4days after infection) for mice infected with S. aureus Newman

(filled circles) or S. aureus Newman DausA (open circles). Solid bars represent

average weight change. (B to E) CFUs obtained from kidneys, livers, spleens, and

hearts 4 days after infection. Solid bars represent the average log10 CFUs for the

group

Summery

Staphylococcus aureus is a major human pathogen that is resistant to numerous

antibiotics in clinical use.

They found two nonribosomal peptide secondary metabolites—the aureusimines,

made by S. aureus—that are not antibiotics, but function as regulators of

virulence factor expression and are necessary for productive infections.

In vivo mouse models showed that strains of S. aureus unable to produce

aureusimines were attenuated and/or cleared from major organs, including the

spleen, liver, and heart. Targeting aureusimine synthesis may offer novel leads for

anti-infective drugs.

THANKS………………..