Regulation of expression of type 3 fimbriae in Klebsiella pneumoniae CG43
4.2.5 CRP and LRP probably play a role for the PhgS/PhgM-independent
expression of type 3 fimbriae
The expression of a certain genes promoted by two separate condition-controlled promoters is not uncommon in bacteria (Heroven et al., 2004). The identification of PhgS/PhgM-dependent and PhgS/PhgM-independent promoters implies different transcripts are produced. In order to map the transcription start site of mrkA, RT-PCR analysis using different pairs of primers were carried out. As shown in Fig. 13, the transcripts extended to 5’-end of pA5 or pA6 were detected and confirmed by
Southern blotting hybridization using the PCR product amplified with primer pair pA2 and pA3 as a probe. No transcript was obtained while the primer pA5 replaced with pA4 indicating that the transcription start site of mrkA could be assigned to the sequence between pA4 and pA5. Analysis of the sequences upstream of pA4 revealed a conserved LRP and CRP binding elements, 5’-YAGHAWATTWTDCTR-3’ (Y=C or T, H≠G, W=A or T, D≠C, R=A or G) (Cui et al., 1995) and 5’-TGTGA -N6-TCATC-3’ (Kolb et al., 1993), respectively. We speculate that other than the regulation of PhgS/PhgM, LRP or CRP also plays a role on the expression of type 3 fimbriae.
CRP, cAMP receptor protein, has been reported to function both aspositive and negative effector to influence many gene expression (Botsford and Harman, 1992).
LRP, leucine-responsive regulatory protein, regulates the expression of more than 40 genes and proteins in E. coli (Calvo and Matthews, 1994). To determine if CRP and LRP play a regulatory role on the expression of type 3 fimbriae, RT-PCR is being employed to analyze the effect of addition of glucose or leucine on the expression of type 1 and type 3 fimbriae. It is probably that the PhgS/PhgM-independent promoter PL-mrkA is under control by CRP or/ and LRP, which had been shown to affect the expression of type 1 fimbriae or P pili (Baga et al., 1985; Kelly et al., 2006) The hypothesis is that, in the presence of certain level of glucose or leucine, CRP or LRP
determines the cross-regulation of the expression of type 1 and type 3 fimbriae. While in the presence of a lot of PhgS and PhgM regulatory proteins, PL-mrkA is under a negative control and PhgS and PhgM play a major role to positively regulate the expression of type 3 fimbriae. However, the possibility remains to be verified.
Overall, a cross-talk regulation was demonstrated between the expression of type 1 fimbriae and type 3 fimbriae. In addition, PhgS/PhgM appeared to be able to regulate positively the activity of PS-mrkA, which is probably leading to the expression of type 3 fimbriae. On the other hand, the deletion of either phgS or phgM had no effect on the activity of PL-mrkA, PfimB orB PfimE. In addition, a PhgS/PhgM-independent regulation was also observed indicating a complicated regulatory mechanism is involved for the expression of type 3 fimbriae.
Chapter V
Summary
The incidence of Klebsiella infection is found to increase in the recent years.
Antibiotics are usually used to cure K. pneumoniae infections. However, misuse of antibiotics caused the nosocomial Klebsiella infections even more severe because of the emergence of muti-drug resistance strains. Therefore, to find new targets for drug intervention to the muti-drug resistance strains is important.
Attachment of pathogens to their host is a prerequisite step of infection and adhesin is one of the major virulence factors involved in K. pneumoniae infections.
Expression of type 3 fimbriae has been correlated with several Klebsiella infections.
However, little is known about the roles of the fimbriae in K. pneumoniae pathogenesis. In part I, to rule out the possibility that other factors resided in K.
pneumoniae interfere with the activity of type 3 fimbriae, an E. coli type 3 fimbriae
display system was established. By using this display system, we have demonstrated that the proper growth of the filament and fimbrial morphology were MrkD adhesin dependent. The changes of fimbrial morphology in turn affected the fimbrial activity as assessed by collagen binding, cell adherence, and biofilm formation. In addition, E.
coli JM109[pmrkABCDV3F] was found to have the highest level of fimbrial activity and the adhesion to HCT-8 cells was probably due to the interaction of RGD sequence on MrkDV3 with integrin. Overall, MrkD adhesin appeared to play a major role in determining the activity of type 3 fimbriae and hence the MrkD is a potent drug
target.
Since only one study (Allen et al., 1991) of a possible role to stabilize type 3 fimbriae the function, how MrkF stables the type 3 fimbirae and the other function roles of MrkF is not known. As determined previously using the recombinant E. coli display system, MrkF was shown to be able to serve as an initiator for the growth of the filament and likely a regulator for the length and the activity of the fimbriae. The demonstration of an operon structure further supported MrkF is a component of the fimbriae. The possibility is investigated by construction of a mrkF deletion mutant and the fimbrial morphology and activity of the mutant will be compared to that of wild-type bacteria. The result provide us the information of how MrkF maintains the stability of type 3 fimbirae.
It is generally believed that coordinate expression of multiple fimbriae is required for the establishment of a systemic infection. In part II, a cross regulation of the expression of type 1 and type 3 fimbriae was demonstrated. Both PhgS/PhgM-dependent and PhgS/PhgM-independent promoters of type 3 fimbriae were identified, indicating a complex regulatory system is involved for the expression of type 3 fimbriae. The promoter for fim operon transcription is carried on a 314-bp invertible DNA and is catalyzed by FimB and FimE sitespecific recombinases (Abraham et al., 1985). In addition to the inversion of the DNA by recombinases,
LRP protein also acts to determine the expression of type 1 fimbriae (Kelly et al., 2006). Although the CRP has not been found to affect the expression of type 1 fimbrial, CRP-depedent expression of P fimbriae has been reported (Baga et al., 1985). The presence of consensus binding elements of LRP and CRP in the putative promoter PL-mrk suggested the involvement of the two regulators in modulating the expression of type 3 fimbriae.
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呂文鈴，國立清華大學碩士論文，克雷白氏肺炎菌 CG43 第三型纖毛黏附因子的 鑑定，中華民國九十年六月。
陳美甄，國立交通大學碩士論文，克雷白氏肺炎菌 CG43 第三型纖毛黏附蛋白特 性，中華民國九十一年六月。
廖心瑋，國立交通大學碩士論文，克雷白氏肺炎菌 CG43 第三型纖毛組成蛋白 MrkD 與 MrkF 之功能探討，中華民國九十五年六月。
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Table 1. Bacterial strains and plasmids used in this study
Strains or plasmids Descriptions Reference or source
K. pneumoniae
VHm1~VHm17 Clinical isolates of K. pneumoniae,
from the Veteran General Hospital, Taipei. This study
CG43 Clinical isolate of K2 serotype (Chang et al., 1996)
CG43S3 CG43, Smr (Lai et al., 2003)
CG43S3mrkA- CG43S3 with deletion in mrkA gene (呂文鈴，民國九十年)
CG43S3phgS- CG43S3 with deletion in phgS gene This study
CG43S3phgM- CG43S3 with deletion in phgM gene This study
CG43S3lacZ- CG43S3 with deletion in lacZ gene (Lin et al., 2006)
CG43S3lacZ-phgfS- CG43S3lacZ- with deletion in phgS gene This study
CG43S3lacZ-phgfS- CG43S3lacZ- with deletion in phgS gene This study