Transcription And Regulation Mechanism Of Capsule Genes Instreptococcus Pneumoniae

Streptococcus pneumoniae(pneumococcus)is a commensal in the upper respiratory tract and also a major human pathogen of acute pneumonia,meningitis,otitis media,and septicemia.Pneumococcal infections are globally responsible for at least one million of deaths every year.The capsular polysaccharide(CPS)is the major virulence factor of S.pneumoniae,enabling the bacteria to evade phagocytic killing by the intrinsic negative charge of CPS.S.pneumoniae produces at least 97 antigenically different types of capsule,each of which is synthesized by the capsule gene locus.The genetic and biochemical principles of the CPS synthesis have been extensively documented.While the type-37 capsule is synthesized by a single polysaccharide polymerase gene(tts),the production of all the other known CPSs is realized by different sets of multiple cps genes in the same locus of the pneumococcal chromosome.Except for the type-3 cps genes,the gene clusters of the other 95 CPS types consist of the common and type-specific cps genes.It is known that these cps genes are transcribed from the promoter upstream of cpsA,the first gene in the cps gene clusters.It has also been obserbed that the production level of the pneumococcal capsule is altered by multiple conditions.However,the transcription of the cps locus is poorly understood at the present time.The goal of this dissertation project is to determine how the cps genes of S.pneumoniae are transcribed and transcriptionally regulated.We first characterized the transcriptional features of the cps locus in the type-2 virulent strain D39.The initial analysis revealed that the cps genes are cotranscribed from a major transcription start site at the 25th(G)upstream of cps2A(the first gene of cps locus).The primier extension,for the first time,revealed that the 17 genes in the cps locus are co-transcribed as an operon.Using unmarked chromosomal truncations and a luciferase-based transcriptional reporter,we showed that the promoter of the cps operon consists of four functional modules: core promoter and three enhancer sequences(putative transposable insertion element-IE,repeat unit of the pneumococcus-RUP,and a spacing sequence-SS).The importance of these promoter modules in the transcription of the cps operon was functionally confirmed by significant reduction of the knockout mutants in capsule production and virulence in a mouse systemic infection model.We subsequently assessed the sequence feature of the cps promoter region in 225 clinical pneumococcal isolates.The result revealed entensive modular and sequence variations in the promoter region of these isolates,which are characterized by postional shuffling of the IE and RUP module,as well as mosaic combinations of the enhancer modules with nucleotide polymorphisms.These sequence variations appear to be a result of horizontal DNA exchange mediated by the natural competence.The promoter replacement experiments showed remarkable functional impact of the strain-to-strain variations in the cps promoter on the levels of the cps gene mRNA,capsule production,and virulence.These results strongly suggest that sequence varionations in the cps promoter represent a novel adaptation mechanism of S.pneumoniae by diversifying the capsule thickness among the strains,in addition to the well-known capsule type switch.In summary,this study,for the first time,discovered four functional modules in the cps promoter of S.pneumoniae.All of these promoter modules are required for the full transcription of the cps operon and capsule production.Sequence and modular variations in the cps promoter among the clinical isolates can significantly influence the transcription of the cps genes and capsule production.The insightful information derived from this project has substantially enhaced our understanding of the molecular mechanisms governing the formation of the pneumococcal capsule and thereby the pathogenesis of this important human pathogen.