Abstract/Summary

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.