Staphylococcusaureus infection is a severe pathological condition which not only affects theinfected organ, but extensive antimicrobial therapy used to treat such an infectionalso produces detrimental effects on overall health of the infected individual.There are disparate pathophysiological mechanisms associated with acute andchronic S. aureus infection.
The acute infection is associated with moreaggressive and invasive phenotype whereas the chronic infection is associatedwith less aggressive and mild inflammatory phenotype characterised by smallcolony variants (SCV). There are several regulatory factors which play acrucial role in such a dynamic shift in S. aureus phenotype from a greaterinflammatory and shorter intracellular persistence associated acute phase to aless 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 dynamicswitching of bacterial phenotype in acute and chronic S. aureus infection. Toidentify the role of these regulating factors in acute and chronic infection,the investigators designed different mutant models (single, double & triplemutant) and studied the virulence of wild and mutant strains in differentin-vivo and in-vitro infection models.
Wildtype and mutant strains of S. aureus LS1 and SH1000 were used for all theexperiments under the study. Mutant strains of agr, Sig B & sarA were generated by transduction of antibioticresistant cassettes (i.e. RN6911, IK181 and ALC136 respe.) using differentphages (i.e.
11,80 & 85 respe.). Wild type and mutant strains were grownstepwise i.e. wild type and single mutants were grown in first step whereasdouble and triple mutants were grown in second step, the protein fraction were extractedsequentially. Proteomic analysis was performed using mass spectrometry(LTQ-Orbitrap-Velos-Mass Spectrometer) and resulting data was analysed usingappropriate software (i.
e. Rosetta Elucidator 3.3.
01) for the differentialexpression of proteins. As a part of in-vitro studies, primary humanosteoblasts, HUVECs (Human umbilical venous endothelial cells) and humanpolymorphonuclear cells (PMNs) were used as hosts to study the S. aureusinfection patterns. In addition, haemolytic effects of S. aureus infection werestudied using haemolysis assay i.e. incubating different mutant and wild typestrains with extracted human red blood cells. Cytokine fractions were analysedusing cytokine release assay.
Forin-vivo studies, wistar adult rats were used to develop chronic S. aureusinfection (i.e.
osteomyelitis). Quantitative real time PCR was used to studythe differential gene expression in bacterial strains. Appropriate statisticaltests were used to perform statistical analysis of the data (i.
e. one-wayANOVA).agr/serAsingle mutant showed severe reduction in release of virulence factors andcorresponding reduced inflammation and toxicity compared to wild type strains.All mutants (i.e. single, double & triple) showed reduced inflammation anddecreased cytotoxicity except sigB mutant, which showed increased ?-toxin leveland corresponding increased cytotoxic effects on PMNs.
Similar results wereobserved for HUVECs and osteoblasts in-vitro assays, where all double andtriple mutants showed reduced cell activation and inflammation compared to wildtype, whereas sigB mutant showed increased cell activation and inflammation. Thisshowed that in acute phase, agr and sarA are essential to develop an aggressiveand inflammatory phenotype whereas sigB is associated with less aggressivephenotype. To study S. aureus infection pattern in chronic phase, HUVECs andosteoblastswere treated with wild type and mutantbacterial strains and later checked for the presence of bacteria inintracellular compartment after 7-9 days. Single (agr/serA), double and triplemutants were able to localize in intracellular compartment for longer periodbut sigB mutants were totally cleared out from the host cells.
But when sigBmutants were complemented with complete sigB operon, the effects were reversedi.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 whensigB mutants were complemented with complete sigB operon. Gene expressionanalysis of wild type and mutant strain showed agr and sarA upregulation inacute phase and sigB upregulation in chronic phase.
This showed that agr andsarA downregulation and sigB upregulation is essential for long termintracellular persistence of S. aureus in chronic phase. agr/serA mutants couldnot escape phagosomal lysis, thus confirming the essential role of agr and sarAin phagosomal escape. In-vivo studies showed that the single,double and triple mutant strains caused less aggressive phenotype and reducedtoxicity than wild type strain.
Mutant strains either single, double or triple,could not induce severe and chronic infection compared to wild type. Thusinvestigators hypothesized that the combined action of agr, sarA and sigB iscrucial for an acute/chronic S.aureus in-vivo infection.Thus,by using different in-vitro and in-vivo models, study demonstrated theimportance of specific S. aureus regulators in switching from acute to chronicphase infection i.e. it showed that active agr and sarA are crucial for acutetoxicity and severe inflammation whereas sigB is essential for long termintracellular persistence.
Study also highlighted the dynamic interaction ofimportant S. aureus regulators (agr, sarA and sigB) and the necessity of theircombined action to induce severe inflammation and toxicity. More importantly,study also furnished a novel therapeutic marker/target to treat severe chronicS. aureus infection.