Superbug's 'CPU' Revealed: Researchers Discover Chemical

Clue Directing Staphylococcus Aureus

(Credit: CDC/Janice Haney Carr)

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.

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)
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.

Prediction of Cryptic nonribosomal peptide assembly in Staphylococcus.

(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.
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.
 Isolation of tyrosine-valine dipeptides.

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

A and aureusimine B (phevalin) were determined by mass spectrometry and NMR
experiments
 To verify that the aureusimines are synthesized by the S. aureus NRPS

(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.
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

 Microarray analysis of virulence expression.

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

(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.
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………………..