Staphylococcus (SCV). There are several regulatory factors which

aureus infection is a severe pathological condition which not only affects the
infected organ, but extensive antimicrobial therapy used to treat such an infection
also produces detrimental effects on overall health of the infected individual.
There are disparate pathophysiological mechanisms associated with acute and
chronic S. aureus infection. The acute infection is associated with more
aggressive and invasive phenotype whereas the chronic infection is associated
with less aggressive and mild inflammatory phenotype characterised by small
colony variants (SCV). There are several regulatory factors which play a
crucial role in such a dynamic shift in S. aureus phenotype from a greater
inflammatory and shorter intracellular persistence associated acute phase to a
less cytotoxic and longer intracellular persistence associated chronic phase.
The study aimed at finding the specific role of such essential factors (i.e. agr, sig B & sarA) in dynamic
switching of bacterial phenotype in acute and chronic S. aureus infection. To
identify the role of these regulating factors in acute and chronic infection,
the investigators designed different mutant models (single, double & triple
mutant) and studied the virulence of wild and mutant strains in different
in-vivo and in-vitro infection models.

type and mutant strains of S. aureus LS1 and SH1000 were used for all the
experiments under the study. Mutant strains of agr, Sig B & sarA were generated by transduction of antibiotic
resistant cassettes (i.e. RN6911, IK181 and ALC136 respe.) using different
phages (i.e. 11,80 & 85 respe.). Wild type and mutant strains were grown
stepwise i.e. wild type and single mutants were grown in first step whereas
double and triple mutants were grown in second step, the protein fraction were extracted
sequentially. Proteomic analysis was performed using mass spectrometry
(LTQ-Orbitrap-Velos-Mass Spectrometer) and resulting data was analysed using
appropriate software (i.e. Rosetta Elucidator 3.3.01) for the differential
expression of proteins. As a part of in-vitro studies, primary human
osteoblasts, HUVECs (Human umbilical venous endothelial cells) and human
polymorphonuclear cells (PMNs) were used as hosts to study the S. aureus
infection patterns. In addition, haemolytic effects of S. aureus infection were
studied using haemolysis assay i.e. incubating different mutant and wild type
strains with extracted human red blood cells. Cytokine fractions were analysed
using cytokine release assay.  For
in-vivo studies, wistar adult rats were used to develop chronic S. aureus
infection (i.e. osteomyelitis). Quantitative real time PCR was used to study
the differential gene expression in bacterial strains. Appropriate statistical
tests were used to perform statistical analysis of the data (i.e. one-way

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single mutant showed severe reduction in release of virulence factors and
corresponding reduced inflammation and toxicity compared to wild type strains.
All mutants (i.e. single, double & triple) showed reduced inflammation and
decreased cytotoxicity except sigB mutant, which showed increased ?-toxin level
and corresponding increased cytotoxic effects on PMNs. Similar results were
observed for HUVECs and osteoblasts in-vitro assays, where all double and
triple mutants showed reduced cell activation and inflammation compared to wild
type, whereas sigB mutant showed increased cell activation and inflammation. This
showed that in acute phase, agr and sarA are essential to develop an aggressive
and inflammatory phenotype whereas sigB is associated with less aggressive
phenotype. To study S. aureus infection pattern in chronic phase, HUVECs and

were treated with wild type and mutant
bacterial strains and later checked for the presence of bacteria in
intracellular compartment after 7-9 days. Single (agr/serA), double and triple
mutants were able to localize in intracellular compartment for longer period
but sigB mutants were totally cleared out from the host cells. But when sigB
mutants were complemented with complete sigB operon, the effects were reversed
i.e. complemented strains were localized intracellularly for longer time.
Moreover, it has also been observed that sigB mutants were unable to show SCV,
a characteristic of chronic infection. But reverse effects were observed when
sigB mutants were complemented with complete sigB operon. Gene expression
analysis of wild type and mutant strain showed agr and sarA upregulation in
acute phase and sigB upregulation in chronic phase. This showed that agr and
sarA downregulation and sigB upregulation is essential for long term
intracellular persistence of S. aureus in chronic phase. agr/serA mutants could
not escape phagosomal lysis, thus confirming the essential role of agr and sarA
in phagosomal escape.

In-vivo studies showed that the single,
double and triple mutant strains caused less aggressive phenotype and reduced
toxicity than wild type strain. Mutant strains either single, double or triple,
could not induce severe and chronic infection compared to wild type. Thus
investigators hypothesized that the combined action of agr, sarA and sigB is
crucial for an acute/chronic S.aureus in-vivo infection.

by using different in-vitro and in-vivo models, study demonstrated the
importance of specific S. aureus regulators in switching from acute to chronic
phase infection i.e. it showed that active agr and sarA are crucial for acute
toxicity and severe inflammation whereas sigB is essential for long term
intracellular persistence. Study also highlighted the dynamic interaction of
important S. aureus regulators (agr, sarA and sigB) and the necessity of their
combined action to induce severe inflammation and toxicity. More importantly,
study also furnished a novel therapeutic marker/target to treat severe chronic
S. aureus infection.




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