Stoebner JA, Butterton JR, Calderwood SB, Payne SM. Identification of the vibriobactin receptor of Vibrio cholerae. J Bacteriol. 174 (10) :3270-4.Abstract
Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.
Schmitt MP, Payne SM. Genetic analysis of the enterobactin gene cluster in Shigella flexneri. J Bacteriol. 173 (2) :816-25.Abstract
The genes for transport and synthesis of the phenolate siderophore enterobactin are present on the chromosomes of both Ent+ and Ent- clinical isolates of Shigella flexneri. To determine why Ent- S. flexneri isolates fail to express a functional enterobactin system, the structure and expression of enterobactin genes were examined. Several alterations may be responsible for the inability of S. flexneri to express enterobactin. (i) The mRNA levels produced from the entC and fepB genes were not derepressed in low-iron media. (ii) DNA sequence analysis of the entC-fepB intergenic region revealed an 83-bp noncontiguous deletion in the putative fepB leader sequence. The deleted sequences are in a region which would be capable of forming extensive stem-and-loop structures. (iii) An amber codon in the 5' portion of the entC gene was also detected. (iv) An IS1 element, previously mapped to the Ent- S. flexneri enterobactin gene cluster, was found to lie within a potential transcriptional termination sequence in the entF-fepE intergenic region. (v) A mutation responsible for the inactivation of the entF gene was mapped to the entF coding region by using entF hybrid gene fusions. (vi) A comparison of outer membrane profiles from an E. coli strain harboring the cloned fepA gene from either an Ent+ or Ent- Shigella isolate revealed that the Ent- FepA protein is present in the outer membrane but at greatly reduced levels than that of the Ent+ FepA protein. This observation, along with additional studies, suggests that the Ent- FepA may be defective in translation and/or translocation.
Dolence EK, Lin CE, Miller MJ, Payne SM. Synthesis and siderophore activity of albomycin-like peptides derived from N5-acetyl-N5-hydroxy-L-ornithine. J Med Chem. 34 (3) :956-68.Abstract
N5-Acetyl-N5-hydroxy-L-ornithine (1), the key constituent of several microbial siderophores, has been synthesized in 23% yield overall from N-Cbz-L-glutamic acid 1-tert-butyl ester (6) derived from L-glutamic acid. Reduction of 6 to 7 and treatment with N-[(trichloroethoxy)carbonyl]-O-benzylhydroxylamine (8), and diethyl azodicarboxylate and triphenylphosphine followed by deprotection produced the protected N5-acetyl-N5-hydroxy-L-ornithine derivatives 11 and 12 in large quantities (10-20 g). Following alpha-amino and alpha-carboxyl deprotections of 11 and 12, EEDQ [2-ethoxy-N-(ethoxycarbonyl)-1,2-dihydroquinoline] mediated peptide coupling and final deprotection provided amino acid 1 and six albomycin-like peptides (20, 23, 25, 28, 35, and 36). The growth-promoting ability of each was evaluated with the siderophore biosynthesis mutant Shigella flexneri SA240 (SA 100 iucD:Tn5). These results indicate that substantial modification of the framework of peptide-based siderophores can be tolerated by microbial iron-transport systems.
Barghouthi S, Payne SM, Arceneaux JE, Byers BR. Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila. J Bacteriol. 173 (16) :5121-8.Abstract
Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.
Daskaleros PA, Stoebner JA, Payne SM. Iron uptake in Plesiomonas shigelloides: cloning of the genes for the heme-iron uptake system. Infect Immun. 59 (8) :2706-11.Abstract
The iron uptake systems of Plesiomonas shigelloides strains were determined. Siderophore production was not detected by chemical or biological assays, and the strains tested were unable to use enterobactin, aerobactin, or vibriobactin for growth in low-iron media. Both hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources, but neither transferrin nor lactoferrin served as a source of iron. Hemolysin was detected, and the production of hemolysin was iron repressible. DNA sequences encoding hemolysin production and DNA sequences encoding the ability to use heme or hemoglobin as a sole source of iron were cloned from P. shigelloides and expressed in Escherichia coli. The abilities to use heme and hemoglobin as iron sources were closely linked, and the cloned sequences encoded the ability to transport the porphyrin, as well as iron, into the cells.
Dolence EK, Minnick AA, Lin CE, Miller MJ, Payne SM. Synthesis and siderophore and antibacterial activity of N5-acetyl-N5-hydroxy-L-ornithine-derived siderophore-beta-lactam conjugates: iron-transport-mediated drug delivery. J Med Chem. 34 (3) :968-78.Abstract
N5-Acetyl-N5-hydroxy-L-ornithyl-N5-acetyl-N5-hydroxy-L-ornithyl-N5-acety l- N5-hydroxy-L-ornithine, the functionally instrumental component of the albomycins and ferrichromes, has been incorporated as a "carrier" substructure into both carbacephalosporin and oxamazin type beta-lactam antibiotics. The previously synthesized protected version of this tripeptide (14) was coupled with various beta-lactam analogues 17, 19, 24, and 25 to give protected conjugates 21, 22, 26, and 27. Final deprotection by hydrogenolysis provided the deprotected siderophore-beta-lactam antibiotic conjugates 1-4. The growth-promoting ability of each has been evaluated using either the siderophore-deficient mutant Shigella flexneri SA 100 or S. flexneri SA240 (SA 100 iucD:Tn5). Measurement of the growth-promoting activity using two isogenic Escherichia coli strains differing only in the presence or absence of fhuA (hydroxamate ferrichrome receptor) suggests uptake by the hydroxamate iron-transport system. The antibacterial activity of these conjugates has been investigated, and the potential for use of the ferrichrome iron-transport system as a means of drug delivery is discussed.
Field LH, Underwood JL, Payne SM, Berry LJ. Virulence of Campylobacter jejuni for chicken embryos is associated with decreased bloodstream clearance and resistance to phagocytosis. Infect Immun. 59 (4) :1448-56.Abstract
The 11-day-old chicken embryo has been shown to be a useful animal model for comparing the virulence of human isolates of Campylobacter jejuni. Virulence in this system is associated with the ability to invade the chorioallantoic membrane and to survive and proliferate in vivo. In this study, the survival and multiplication of C. jejuni in the embryonic host was investigated. It was possible to enhance the virulence of a relatively avirulent C. jejuni strain by passaging it intravenously through the embryos. The resulting isogenic variants demonstrated enhanced abilities to survive in vivo but were still unable to invade when inoculated onto the chorioallantoic membrane. The bloodstream clearance of C. jejuni was studied, and virulent, but not avirulent, strains persisted and multiplied both in the bloodstream and in embryonic liver. Virulent strains also were cleared significantly more slowly from the bloodstream of adult BALB/c mice after intravenous challenge than were avirulent strains. C. jejuni strains which were cleared slowly in vivo were also ingested slowly in vitro by mouse peritoneal macrophages. Clearance studies in mice pretreated with cobra venom factor demonstrated that opsonization by serum complement was not a prerequisite for clearance of campylobacters from the murine bloodstream.
Headley VL, Payne SM. Differential protein expression by Shigella flexneri in intracellular and extracellular environments. Proc Natl Acad Sci U S A. 87 (11) :4179-83.Abstract
Shigellae were intrinsically radiolabeled with [35S]methionine either extracellularly or while multiplying within infected HeLa cell monolayers. A complex pattern of suppression and induction of proteins was observed. Proteins of approximately 97, 62, 58, 50, 25, and 18 kilodaltons (kDa) were induced in Shigella flexneri isolated from infected monolayers. Proteins of 100, 85, 70, 64, and 55 kDa were suppressed under the same conditions but were seen in cells labeled in the tissue culture medium alone. Protein expression during the stages of attachment, invasion, and intracellular multiplication was examined by pulse-labeling. The 58-kDa protein was induced only during invasion, and the 62- and 25-kDa proteins were induced only during intracellular multiplication. Shift into a minimal medium with ion concentrations and pH mimicking intracellular conditions and endosomal pH resulted in the induction of the 97- and 58-kDa proteins, and reduction of the intracellular-like medium with 2-mercaptoethanol resulted in the induction of the 97-, 50-, and 25-kDa proteins and suppression of the 55-kDa protein. Radioimmunoprecipitations of shigellae grown in vitro and in vivo revealed differential expression of immunogenic proteins. Proteins corresponding in size to IpaB (62 kDa), IpaC (42 kDa), and IpaD (38 kDa) were lost during intracellular multiplication, whereas another protein corresponding to IpaA (80 kDa) was found to increase under the same conditions.
Stugard CE, Daskaleros PA, Payne SM. A 101-kilodalton heme-binding protein associated with congo red binding and virulence of Shigella flexneri and enteroinvasive Escherichia coli strains. Infect Immun. 57 (11) :3534-9.Abstract
The ability of Shigella flexneri to bind Congo red or hemin is associated with virulence. A 101-kilodalton (kDa) protein responsible for this phenotype (Crb+) in S. flexneri was identified by a tetramethylbenzidine staining procedure which detects heme-protein complexes in polyacrylamide gels. Labeling of cell-surface polypeptides with 125I revealed that the 101-kDa heme-binding protein is expressed on the cell surface. Expression of the protein was regulated by growth temperature and was found to be encoded by the large virulence plasmid of S. flexneri. Deletion mutants and a Tn5 insertion mutant which were negative for Congo red binding (Crb-) did not express the 101-kDa protein. Enteroinvasive Escherichia coli strains that were Crb+, and whose plasmids shared homology with the S. flexneri virulence plasmid, also expressed the 101-kDa protein. Expression of the protein in S. flexneri and enteroinvasive E. coli correlated with the presence of a 9.2-kilobase EcoRI fragment of these plasmids.
Payne SM. Iron and virulence in Shigella. Mol Microbiol. 3 (9) :1301-6.Abstract
Iron limitation, a condition encountered within mammalian hosts, induces the synthesis of a number of proteins in pathogenic Shigella species. These include several outer membrane proteins, Shiga toxin, and proteins involved in the biosynthesis and transport of high-affinity iron-binding compounds or siderophores. Although siderophores have been shown to play a major role in the virulence of some bacterial pathogens, these compounds do not appear to be essential for the virulence of Shigella species. Unlike those pathogens which are restricted to the extracellular compartments of the host, the Shigella species invade and multiply within host cells. Alternative iron-acquisition systems, such as the ability to utilize haem-iron, permit growth of the intracellular bacteria. Virulent shigellae also possess a cell-surface haem-binding protein, and synthesis of this protein correlates with infectivity and virulence. This protein does not appear to be involved in iron acquisition. Rather, it may allow the bacteria to coat themselves with haem compounds, thus enhancing their ability to interact with target host cells.
Sharma SK, Miller MJ, Payne SM. Spermexatin and spermexatol: new synthetic spermidine-based siderophore analogues. J Med Chem. 32 (2) :357-67.Abstract
Syntheses of hexanediamine-based dihydroxamate (Hexamate), spermidine-based trihydroxamate (Spermexatins), and spermidine-based mixed siderophore analogues (Spermexatols) are described. Key intermediates include the N-hydroxysuccinimide esters of various hydroxamic acids, e.g., malonohydroxamate, succinohydroxamate, and glutarohydroxamate. These intermediates were synthesized, characterized, and incorporated as the ligating chains on spermidine. Also, mixed iron chelating compounds (Spermexatols) with both catechol and hydroxamic acid side chains were synthesized. The reagent carbobenzoxyimidazole was employed to distinguish between the primary and secondary amino groups of spermidine. The ability of these iron chelators to stimulate microbial growth is also described.
Schmitt MP, Payne SM. Genetics and regulation of enterobactin genes in Shigella flexneri. J Bacteriol. 170 (12) :5579-87.Abstract
Although Shigella flexneri possesses the genes for two siderophore systems, enterobactin and aerobactin, the enterobactin system is only rarely utilized. To investigate the regulation of enterobactin expression in S. flexneri, all of the genes specifically required for synthesis and transport of enterobactin were cloned from both an expressing (Ent+) and a nonexpressing (Ent-) strain. Notable differences between the cloned genes included endonuclease restriction site changes and the presence of an IS1 element in the Ent- DNA. Southern hybridization revealed that this IS1 element, present at the 3' end of the entF gene, is conserved at this location in different strains and serotypes of Ent- S. flexneri. The Ent- cloned genes were tested for their ability to complement the defect in 11 different Escherichia coli enterobactin mutants. The Ent- genes fully complemented nine mutants but failed to complement the entF mutant AN117 and only partially complemented the entE mutant AN93. Whole-cell RNA isolated from E. coli and the Shigella strains was hybridized to 32P-labeled DNA containing the entB gene or a fragment carrying a portion of the entF gene. E. coli and the Ent+ Shigella strains exhibited derepression of transcription of these genes in low-iron media. Transcription in the Ent- strain remained repressed regardless of iron concentration. Expression of the entB and entF genes was also examined in an Ent- Shigella fur mutant. Expression of entF was only partially derepressed and entB remained fully repressed at all iron concentrations, suggesting that factors other than Fur are responsible for the repression of these enterobactin genes in the Ent- Shigella strains.
Payne SM. Iron and virulence in the family Enterobacteriaceae. Crit Rev Microbiol. 16 (2) :81-111.Abstract
The ability of bacterial pathogens to acquire iron in the host is an essential component of the disease process. Pathogenic Enterobacteriaceae spp. may either scavenge host iron sources such as heme or induce high-affinity iron-transport systems to remove iron from host proteins. The ease with which iron is acquired from the host will be at least partially determined by the iron status of the host at the time of infection. In response to infection, mammalian hosts reduce serum iron levels and withhold iron from the invading microorganisms. Thus the competition for iron is an active process which influences the outcome of a host-bacterial interaction.
Stoebner JA, Payne SM. Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae. Infect Immun. 56 (11) :2891-5.Abstract
El Tor and non-O1 strains of Vibrio cholerae were analyzed to determine whether synthesis of secreted hemolysin was influenced by the concentration of iron in the medium. Synthesis of hemolysin was found to be iron regulated in both El Tor and non-O1 isolates. Increased levels of hemolytic activity were detected in supernatants of iron-starved cells. Spontaneous hemolysin-deficient mutants of one non-O1 strain were found to occur at high frequency. These variants also failed to synthesize vibriobactin, the iron transport compound utilized by V. cholerae. Another non-O1 strain was found to synthesize both hemolysin and vibriobactin constitutively. When the cloned Escherichia coli fur gene, encoded on the plasmid pABN203, was introduced into this constitutive strain, normal iron regulation of both hemolysin and vibriobactin was reestablished. The ability of V. cholerae to utilize mammalian iron compounds was determined, and it was found that both hemin and hemoglobin could serve as sole sources of iron.
Daskaleros PA, Payne SM. Congo red binding phenotype is associated with hemin binding and increased infectivity of Shigella flexneri in the HeLa cell model. Infect Immun. 55 (6) :1393-8.Abstract
Wild-type isolates of Shigella flexneri bind the dye Congo red from solid media, thus producing red (Crb+) colonies. Mutants which fail to bind the dye produce white colonies (Crb-) and are avirulent in a variety of systems. In S. flexneri the ability to bind Congo red correlates with the ability to bind hemin and protoporphyrin IX. Binding of hemin by Crb+ S. flexneri was observed both in solid media and in liquid assays. Results of competition experiments suggest that Congo red and hemin bind to the same site on the bacterial cell and are retained on the cell surface. Binding of hemin by Crb+ S. flexneri is independent of hemin transport since both Crb+ and Crb- cells can utilize hemin as a sole source of iron. Both Crb- and Crb- organisms were able to grow in HeLa cell lysates, indicating that the gene(s) that is responsible for Congo red binding does not play a role in the acquisition of intracellular heme iron. By using the HeLa cell invasion system, the effect of hemin prebinding on the invasiveness of Crb+ S. flexneri was determined. Crb+ cells which had prebound hemin exhibited increased invasiveness, indicating a possible role for the crb gene product in the initial stages of invasion by S. flexneri.
Lawlor KM, Daskaleros PA, Robinson RE, Payne SM. Virulence of iron transport mutants of Shigella flexneri and utilization of host iron compounds. Infect Immun. 55 (3) :594-9.Abstract
Mutants of Shigella flexneri defective in aerobactin-mediated iron transport were assayed for virulence in several model systems. A Tn5 insertion mutant was invasive in HeLa cells, lethal in the chicken embryo, and produced keratoconjunctivitis in the guinea pig, indicating little or no loss of ability to invade and multiply intracellularly. Although the mutant failed to grow in low-iron medium in vitro, growth equivalent to that of the wild type was observed in HeLa cell lysates. Thus, there appears to be sufficient available iron inside the HeLa cell to allow growth in the absence of siderophore synthesis. Possible host iron sources were tested, and both the mutant and wild type utilized hemin or hematin as a sole source of iron. Only the wild-type, aerobactin-producing strain could remove iron from transferrin or lactoferrin. Two deletion mutants were also assayed for virulence and were found to be avirulent for the chicken embryo. These deletions encompass flanking sequences as well as the aerobactin genes; therefore, adjacent genes may be required for virulence.
Marolda CL, Valvano MA, Lawlor KM, Payne SM, Crosa JH. Flanking and internal regions of chromosomal genes mediating aerobactin iron uptake systems in enteroinvasive Escherichia coli and Shigella flexneri. J Gen Microbiol. 133 (8) :2269-78.Abstract
We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.
Daskaleros PA, Payne SM. Characterization of Shigella flexneri sequences encoding congo red binding (crb): conservation of multiple crb sequences and role of IS1 in loss of the Crb+ phenotype. Infect Immun. 54 (2) :435-43.Abstract
The ability to bind Congo red (Crb+) is associated with virulence of Shigella flexneri and is encoded by a large, 220-kilobase plasmid. We cloned fragments of this plasmid to isolate the sequences encoding Congo red binding, to determine the degree of conservation of these sequences among S. flexneri strains, and to study the molecular basis for loss of the Crb+ phenotype. At least two separate BamHI fragments cloned into plasmid vectors encode Congo red binding in E. coli or S. flexneri. One Crb+ clone, pTKS2, contains a copy of IS1 adjacent to the crb sequences. IS1 appears to be responsible for deletions leading to loss of Congo red binding in this clone. In addition, this clone was found to integrate into the chromosome at relatively high frequency. Integration resulted in loss of the Crb+ phenotype. A second clone, pTKS15, which has only limited homology to pTKS2, also encodes Congo red binding. The Crb+ phenotype of transformants carrying pTKS15 was detected at 37 degrees C but not at 30 degrees C, and thus it resembles Congo red binding in wild-type S. flexneri. HindIII digests of plasmid DNA from 10 different S. flexneri strains were hybridized to both of these Crb+ clones and to an IS1 probe. More than one fragment hybridized to pTKS2 or pTKS15. In general, the sizes of these fragments were the same in S. flexneri strains of different serotypes, indicating conservation of these sequences. Three of five copies of IS1 were also found on the large S. flexneri plasmids. Two of the copies were on fragments of the same size in each strain. Analysis of Crb- derivatives of the 10 strains indicated that, although IS1 may be closely linked to crb sequences on the 220-kilobase plasmid, it is not responsible for the majority of deletions of this plasmid associated with loss of Congo red binding.
Field LH, Headley VL, Underwood JL, Payne SM, Berry LJ. The chicken embryo as a model for campylobacter invasion: comparative virulence of human isolates of Campylobacter jejuni and Campylobacter coli. Infect Immun. 54 (1) :118-25.Abstract
Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.
Field LH, Headley VL, Payne SM, Berry LJ. Influence of iron on growth, morphology, outer membrane protein composition, and synthesis of siderophores in Campylobacter jejuni. Infect Immun. 54 (1) :126-32.Abstract
Three human isolates of Campylobacter jejuni were grown in a biphasic culture medium with and without the addition of a synthetic chelator to induce iron limitation. Cells grown in low-iron medium exhibited slower growth rates and altered cellular morphology. Increased numbers of longer, more filamentous forms were seen in Gram-stained smears. Three proteins, with apparent Mrs of 82,000, 76,000, and 74,000, were consistently present in the outer membrane of cells grown in low-iron medium. At least one of these proteins (76,000 to 74,000) was exposed on the cell surface. A bioassay was used to look for the production of siderophores by these and other strains of C. jejuni. Seven of 26 strains tested produced detectable amounts of siderophores. Growing strains at 42 degrees C failed to suppress siderophore synthesis or to alter the outer membrane protein profiles of iron-starved cells. The ability of three strains to utilize exogenously supplied siderophores for growth in low-iron medium was also examined. All three strains were able to utilize enterochelin and ferrichrome, but none utilized aerobactin, rhodotorulic acid, or desferrioxamine B. The effect of iron on the virulence of C. jejuni for 11-day-old chicken embryos inoculated via the chorioallantoic membrane was also determined.