The contribution of competence system to the virulence of Streptococcus pneumoniae

Streptococcus pneumoniae;Competence;virulence

Zhu, Luchang

关键词: Streptococcus pneumoniae;    Competence;    virulence;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46923/Luchang_Zhu.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Competence induction in Streptococcus pneumoniae (pneumococcus) is initiated by the binding of the 17-amino acid competence stimulating peptide (CSP) to its membrane-associated histidine sensor kinase ComD. ComD autophosphorylates and subsequently phosphorylates and activates its cognate response regulator ComE. CSP-ComDE triggers the expression of 24 “early” competence genes and approximately 80 “late” competence genes. Some late competence genes encode effectors for genetic transformation. In recent years, the competence regulon of S. pneumoniae has been shown to cross regulate virulence. For example, Lau et al reported that the loss of function in ComB, an accessory protein to the ComA ABC transporter required for the export of CSP, as well as loss of function in the ComD, attenuate the ability of S. pneumoniae to cause pneumonia and bacteremia in mice. Because these studies suggest that competence regulon is not only essential for genetic transformation, but also for virulence, I sought a strategy to suppress pneumococcal competence development as a mean to reduce horizontal gene transfer as well as virulence. Of all the steps required for the development of competence for genetic transformation, the only one that occurs extracellularlly is the interaction between CSP and ComD. I hypothesized that blocking CSP-ComD interaction could effectively inhibit the induction of competence system. Previous studies have shown that the variable central region of CSP determines its receptor specificity. However, the roles of the conserved amino acid residues on both the N- and C-termini are unknown. I examined if amino acid substitution or deletion of these conserved residues could generate analogs that inhibit competence induction by wild-type CSP. I demonstrate that one of the CSP analogs, CSP1-E1A, competitively inhibits the ability of CSP1 to induce genetic transformation in a concentration dependent manner. I also demonstrate that CSP1-E1A successfully attenuates the expression of two virulence factors, CbpD and LytA during competence induction in a concentration-dependent manner. Inhibition of LytA and CbpD by CSP1-E1A reveals the possibility of using this peptide analog to attenuate pneumococcal infection. I examined whether CSP1-E1A could attenuate the virulence of S. pneumoniae D39 using a mouse model of acute pneumonia. CSP1-E1A significantly reduced the mortality rate as well as the kinetic of death infected mice. These results suggest that CSP1-E1A is able to attenuate virulence mechanisms of pneumococcus regulated by the competence regulon. One important feature of pneumococcal DNA uptake during competence is the degradation of one strand of donor DNA by the pneumococcal membrane nuclease EndA. EndA-mediated DNA degradation and small DNA fragment release were used as indicators of competence development. Interestingly, EndA has been identified as a virulence factor, most probably by degrading the neutrophil extracellular traps (NETs) and facilitate pneumococcal dissemination. I hypothesized that the nucleolytic activity of EndA during competence development is important for virulence. Contrary to my expectations, my experimental results demonstrate that EndA-mediated degradation of extracellular DNA is independent of the competence state of pneumococcus. S. pneumoniae mutants with deletion in genes essential for competence induction and regulation have extracellular nuclease activity comparable to their parental wild-type. In addition, nuclease activity of EndA is independent of LytA-mediated partial cell lysis, which may release the endonuclease into DNA-rich environments. Based on these results, I have proposed the existence of two distinct physiological states of EndA: the “competent state” and the “constitutive state”. During the competent state, a portion of EndA molecules is recruited by the competent apparatus to degrade one strand of the DNA, allowing the uptake of the remaining single stranded DNA for integration. During the “constitutive state”, EndA degrades extracellular DNA randomly. This portion of EndA activity is primarily responsible for the degradation of (NETs, virulence and bacterial dissemination. In addition, my data demonstrate that EndA molecules are secreted into the environment, and secreted EndA contributes substantially to “constitutive state” nucleolytic activity. Finally, the nuclease inhibitor aurintricarboxylic acid attenuates EndA-mediated genetic transformation as well as degradation of NETs. Since EndA is essential for both virulence and genetic transformation, it may serve as an interesting drug target to attenuate pneumococcal infection and inhibit horizontal gene transfer.A key event of competence development in S. pneumoniae is the expression of pneumococcal alternative sigma factor ComX. ComX initiates the expression of 80 “late” competence genes in pneumococcal cells competent for genetic transformation. I hypothesized that the expression of ComX-regulated genes is beneficial to the fitness of S. pneumoniae during host infection. In this study, I conducted a systematic deletion study on the contribution ComX-regulated late genes to mouse infection. I showed that 13 mutants with deletions of ComX-regulated genes are attenuated in at least one model of mouse infection. For some late competence genes, their specific expression during competence contributes to bacterial fitness. While for some other ComX regulated genes, their constitutive baseline expression is important for bacterial fitness. Finally, inactivating dprA, a gene essential for competence shut off, severely attenuates the virulence of pneumococcus, indicating competence needs to be properly regulated during host infection. Collectively, these experimental data suggest that the competence regulon of S. pneumoniae plays crucial rules in both genetic transformation as well as fitness and survival in the hosts.