DISCUSSION
Our findings suggest that there are differences in virulence gene
carriage between S. aureus cultured from control subjects and
patients with CRSsNP or CRSwNP. In the case of CRSwNP isolated S.
aureus these differences may translate into the ability to localise and
replicate intracellularly, thus conferring a bacterial survival
advantage.
Phenol soluble modulins such as delta haemolysin gene (hld ) are
specific to S. aureus bacteria and induce neutrophil and
erythrocyte lysis.23 We noted its absence in 50% of
CRSwNP isolates but presence in all other isolates. Deletion ofhld has been shown to nearly abolish transcription of the
accessory gene regulator (agrA ) transcription and downstream
virulence genes including alpha toxin, beta haemolysin and enterotoxin B
and gamma haemolysin.24 Given that mutations in the
accessory gene regulator locus have been shown to create senescent
bacteria known as small colony variants with reduced virulence gene
expression and superior intracellular translocation in epithelial cells,
the absence of hld may allow for similar pathogenic behaviour
thus establishing an intracellular source of
infection.25-27
Immune modulators including aureolysin which have been shown to cleave
the C3 protein of the classical and alternative complement pathways were
well preserved within the groups. Staphylococcal complement inhibitor
(scn) was less frequently carried by the CRSwNP strain. Complement
lowers the threshold for B-cell activation and antibody formation; hence
the absence of staphylococcal complement inhibitor could lead to greater
antibody formation towards S. aureus as seen in
CRSwNP.3, 28
Exfoliative virulence factors involved in pore formation in host cells
including bi-component gamma haemolysin components (hlgA, hlgB
HlgC ) were well conserved throughout all isolate groups. However,
leukocidin E/D (LukE/D ), which is not under control of the
accessory gene regulator locus, was carried by 40% of control and 75%
of CRSwNP isolates but no CRSsNP isolates. Both toxins have been shown
to create pores within target neutrophils and erythrocytes while
leukocidin E/D has been shown to induce calcium channel activation in
neutrophils independent of its pore forming function leading to cell
death.29 Furthermore, leukocidin E/D can deplete the
adaptive immune response by lysing polymorphic neutrophils leading to
failure of local immunity and was essential for bacterial growth and
seeding in a mouse model of bacteraemia.30 Often found
in invasive S. aureus disease, serine proteases (splA, splB and
splE) have been shown to cleave the mucin 16 glycoprotein which forms a
mucosal barrier. There carriage was abundant in the CRSwNP group (75%)
but not seen in the CRSsNP group and only in 40% of the control
group.31 Furthermore, serine proteases (splA, splB)
and leukocidins E/D (lukE/D ) were absent in presence of the
enterotoxin gene cluster and may suggest different phenotypic behaviours
between these strains. This data would be in keeping with the findings
of Nepal et al who recently demonstrated greater number of CRSwNP
isolates carrying the Sa3int prophage which contains lukE/D genes, when
compared with controls and CRSsNP patients.17
Superantigens including the enterotoxin gene cluster sei, sem, sen, seo
seu were carried by 60% of control isolates, 25% of CRSwNP isolates
and only minimally present in a CRSsNP isolates. These enterotoxins are
associated with long term colonisation in the nasal airways, cystic
fibrosis lung and atopic dermatitis wounds, and were found at a similar
carriage frequency in our control group.32,33Furthermore, they have been shown to stimulate T cell proliferation, but
conversely act like decoy targets for antibodies protecting S.
aureus from attack.33
As greater numbers of intracellular bacteria have been observed within
CRSwNP than CRSsNP tissue, we selected a strain from the control group
and CRS group which represented the virulence gene pattern
observed.7 When investigating intracellular survival,
we confirmed that a representative CRSwNP S. aureus strain
carrying serine proteases (splA, splB) and leukocidins E/D without the
enterotoxin gene cluster was better able to internalise, survive and
replicate within the LAD2 cell line than a representative control strain
carrying the enterotoxin gene cluster with absent serine protease (splA,
splB) and leukocidinE/D genes. Confocal microscopy confirmed the
intracellular localisation of the bacteria and demonstrated a large
number of apoptotic mast cells with a number of live bacteria residing
within them when compared to the control group. Hence the CRSwNPS. aureus phenotype may further support its own survival by using
dead cell bodies to protect itself from the effects of antibiotics and
the immune system.
There may be a survival advantage for S. aureus in a CRSwNP
environment to shed the hld gene and enterotoxin gene cluster
allowing a more senescent phenotype with reduced virulence factor
production and increased intracellular survival. Furthermore, the
carriage of LukE/D may well promote the Th2 environment as this
has been shown to skew the immune response, depleting memory T
lymphocytes thereby reducing the number of IL-17 and IFN-gamma producing
cells, and impairing bacterial clearance.13,34 The
CRSsNP group S. aureus genomes did not carry lukE/D orsplA/B genes, with only one isolate demonstrating minimal gene
carriage from the enterotoxin gene cluster. This may lead to lower
levels of IgE raised towards S. aureus enterotoxins, and could
explain why CRSsNP patients have lower serum IgE levels towards S.
aureus enterotoxins than CRSwNP patients.
Sequencing large numbers of virulence factors from genomic samples is
likely to demonstrate significant heterogeneity which makes statistical
significance testing difficult without large cohorts. Nevertheless, our
findings complement existing sequencing data and further our
understanding of mechanisms of enhanced pathogenicity for S.
aureus in CRS.