Neisseria Meningitidis Capsule Switching, Function Of Molecular Chaperones And Stress Resistance And A Novel Method For Making Markerless Mutants

In China,serogroup A strains have long been the most common cause of invasive meningococcal disease,However,recently,Zhejiang CDC published a case report of invasive meningococcal disease caused by a serogroup X ST-7 isolate.In China,ST-7 meningococcal isolates generally belong to serogroup A,and therefore a capsule-switching event was hypothesized.To identify capsule switching breakpoints,the capsule locus and flanking regions of the ST-7 serogroup X isolate and three endemic ST-7 serogroup A isolates were sequenced and compared.To obtain further insight into capsule switching frequency and length of DNA fragments involved,capsule switching assays were performed using genomic DNA containing combinations of antibiotic selection markers at various locations in the capsule locus and flanking regions.Sequence analyses showed that capsule switching has occurred and involved a 8450 bp serogroup X DNA fragment spanning the region from galE to ctrC.Capsule switching assays indicate that capsule switching occurs at a frequency of 6.3 �10^-6 per bacterium per ?g of DNA and predominantly involved DNA fragments of about 8.1-9.6 kb in length.Molecular chaperones are proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures.ROS and low pH released from lysozyme can destroy the normal conformation of protein.So molecular chaperones may play a significant role in the survival of N.meningitidis inside neutrophils.Here we show that the chaperone mutants of N.meningitidis is sensitive to heat,paraquat,H2O2,HClO and acid compared to wild type strain,which indicating chaperone is important for the pathogenesis of N.meningitidis.In the study of chaperones of N.meningitidis,we developed a novel method to make series of markerless deletion mutants,which increased the efficiency of counterselection by using combination of bactericidal antibiotic and bacteriostatic antibiotic.This method could also be used in another meningitis pathogen,Listeria monocytogenes,suggesting that this is a common method in making markerless mutants of both gram positive and gram negative bacteria.