Use this URL to cite or link to this record in EThOS:
Title: Investigation of the FetMP-FetABCDEF (Ftr1-P19) iron-uptake system of Campylobacter jejuni
Author: AlSallami, Dhama
ISNI:       0000 0004 7971 9252
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Access from Institution:
C. jejuni is the major cause of human gastroenteritis worldwide (including industrialized nations) inducing mild to acute infection and, in some cases, Guillain-Barré syndrome or reactive arthritis. Infection is largely foodborne, and chickens are considered to be the major source. Chicken gut colonisation by C. jejuni depends upon its iron-acquisition ability, as previously shown for feo mutants (impaired for ferrous iron uptake). In addition to the wellcharacterised FeoAB system, C. jejuni carries a second potential ferrous-iron transporter that is encoded by the Fe-Fur regulated ftr-p19 genes. P19 is a periplasmic, Cu-containing and iron-binding protein thought to deliver iron to Ftr1 (an inner-membrane ferric permease) for high-affinity iron import. Bioinformatic analysis indicated that the ftr1-p19 genes are adjacent to a set of six conserved and Fe-Fur regulated genes (cj1660-5) of unknown function, that are predicted to contribute to Frt1-p19 mediated iron uptake. The cj1660-5 genes encode a predicted ATP-dependent periplasmic-mechanotransducer system of unclear purpose, two membrane anchored periplasmic thioredoxins and a potential inner-membrane-embedded electron-translocation factor. Gene context analysis showed that ftr1-p19-cj1665-like gene clusters are common in bacteria, and are particularly well conserved in Campylobacter, Yersinia and Bifidobacterium species. These observations suggest an important and potentially novel role for the cj1660-5- encoded components in bacterial iron uptake. The aim here was to further explore the functions of the P19 system (in particular the cj1660-5 components) in iron uptake by C. jejuni. The ftr1-p19 and cj1660-5 genes of C. jejuni NCTC 11168 were expressed in E. coli JC32 (an iron-transport-deficient strain), both individually and in combination, using the compatible pBADrha and pBADara vectors. Both the ftr1-p19 and cj1660-5 genes were found to enhance the iron-restricted growth of JC32 (with respect to vector controls) but this iv effect was only observed at acidic pH. Inactivation of ftr1, cj1660 and cj1663 (substitution with a cat cassette) resulted in a significant growth inhibition for all three C. jejuni mutants in Muller-Hinton broth (low iron) at acidic pH, which was reversed with addition of iron or in rich medium. Complementing plasmids carrying ftr1-p19 or cj1660-5 were generated using pMA1 and these elicited a reversal of the iron-restriction phenotype of the mutants. Thus, the results generated here strongly indicate a role for Cj1660-5, as well as Ftr1, in iron uptake in C. jejuni. This finding is consistent with recently published research by another group. Chicken gut (caecum) colonisation experiments with 20-day-old chickens were performed using a highly motile and strongly colonising strain (PT14). The results showed weak colonisation levels at 3 dpi for all three mutants, but at 7 dpi the cj1660 and cj1663 mutants had adapted to show colonisation levels similar to those of the wildtype. The ftr1 mutant retained a lower colonisation level than the wildtype at 7 dpi but had increased levels with respect to those at 3 dpi. It is suggested that the low colonisation levels seen at 3 dpi are due to the reduced growth (extended lag phase) of the mutants under iron restriction (as seen in vitro) and that by 7 dpi the cj1660 and cj1663 mutants have had sufficient time to overcome their growth lag such that a normal colonisation levels are achieved. qRT-PCR was used to test the iron and Fur dependence of the expression of the ftr1-cj1665 cluster, and their cotranscription potential. The results suggest that the genes are indeed expressed as a co-transcript and that they are subject to a high degree of induction (by 100- 2200-fold) under low-iron conditions. This expression effect was strongly dependent on Fur. The Fur-dependent induction under low-iron conditions is consistent with previous reports. However, the fur mutant strain showed downregulation the ftr1-cj1665 genes iron sufficient and deficient conditions suggesting that these genes are subject apoFur activation. This is in contrast to several previous reports that are consistent with an Fe-Fur repression mode of regulation (although Fur is considered to act as an activator in some instances in C. jejuni). v The reason for this difference in Fur control, as found here, is unclear and requires further investigation. In conclusion, this work provides clear evidence that the ftr1-cj1665 gene cluster has a role as an iron transport system under low pH and is therefore required for normal low-iron growth in vitro and is necessary for optimal colonisation of the chicken gut. Further work is necessary to determine precisely how the Cj1660-5 components contribute to iron acquisition, and the role played by the Ftr1-Cj1665 system in iron uptake with respect to that of other iron transporters (particularly FeoAB) in C. jejuni.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral