Sun Nyunt Wai

The Iron-Binding Protein Dps Confers Hydrogen Peroxide Stress Resistance to Campylobacter jejuni

We identified and characterized the iron-binding protein Dps from Campylobacter jejuni. Electron microscopic analysis of this protein revealed a spherical structure of 8.5 nm in diameter, with an electron-dense core similar to those of other proteins of the Dps (DNA-binding protein from starved cells) family. Cloning and sequencing of the Dps-encoding gene (dps) revealed that a 450-bp open reading frame (ORF) encoded a protein of 150 amino acids with a calculated molecular mass of 17,332 Da. Amino acid sequence comparison indicated a high similarity between C. jejuni Dps and other Dps family proteins. In C. jejuni Dps, there are iron-binding motifs, as reported in other Dps family proteins. C. jejuni Dps bound up to 40 atoms of iron per monomer, whereas it did not appear to bind DNA. An isogenic dps-deficient mutant was more vulnerable to hydrogen peroxide than its parental strain, as judged by growth inhibition tests. The iron chelator Desferal restored the resistance of the Dps-deficient mutant to hydrogen peroxide, suggesting that this iron-binding protein prevented generation of hydroxyl radicals via the Fenton reaction. Dps was constitutively expressed during both exponential and stationary phase, and no induction was observed when the cells were exposed to H(2)O(2) or grown under iron-supplemented or iron-restricted conditions. On the basis of these data, we propose that this iron-binding protein in C. jejuni plays an important role in protection against hydrogen peroxide stress by sequestering intracellular free iron and is expressed constitutively to cope with the harmful effect of hydrogen peroxide stress on this microaerophilic organism without delay.

The Release of Outer Membrane Vesicles from the Strains of Enterotoxigenic Escherichia coli

The clinically isolated heat labile enterotoxin (LT)‐producing strains of Escherichia coli can be separated into two groups, namely spontaneous LT‐releasing strain and non‐spontaneous LT‐releasing strain, based on their phenotypes of spontaneous release of LT into the culture medium. The phenotype of spontaneous LT release was observed to correlate closely with the phenotype of the release of numerous small vesicles into the culture medium. Both morphological and biological examinations of the vesicle showed that the vesicle was released from the outer membrane. It can, therefore, be assumed that LT may be released from the cell at the time the vesicles form.

Construction of a ferritin-deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress

The ferritin‐encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin‐deficient mutant was constructed. The growth of ferritin‐deficient strain SNA1 was clearly inhibited under iron deprivation. The ferritin‐deficient mutant was more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron‐mediated oxidative stress.

Characterization of a Pore-Forming Cytotoxin Expressed by Salmonella enterica Serovars Typhi and Paratyphi A

ABSTRACT Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene that has been characterized so far only in Escherichia coli. Using DNA sequence analysis and PCR, we established that clyA is conserved in the human-specific typhoid Salmonella enterica serovars Typhi and Paratyphi A and that the entire clyA gene locus is absent in many other S. enterica serovars, including Typhimurium. The gene products, designated ClyASTy and ClyASPa, show ≥90% amino acid identity to E. coli cytolysin A, ClyAEC, and they are immunogenically related. The Salmonella proteins showed a pore-forming activity and are hence functional homologues to ClyAEC. The chromosomal clyASTy gene locus was expressed at detectable levels in the serovar Typhi strains S2369/96 and S1112/97. Furthermore, in the serovar Typhi vaccine strain Ty21a, expression of clyASTy reached phenotypic levels, as detected on blood agar plates. The hemolytic phenotype was abolished by the introduction of an in-frame deletion in the clyASTy chromosomal locus of Ty21a. Transcomplementation of the mutant with a cloned clyASTy gene restored the hemolytic phenotype. To our knowledge, Ty21a is the first reported phenotypically hemolytic Salmonella strain in which the genetic determinant has been identified.

Cytocidal and Apoptotic Effects of the ClyA Protein from Escherichia coli on Primary and Cultured Monocytes and Macrophages

ABSTRACT Cytolysin A (ClyA) is a newly discovered cytolytic protein ofEscherichia coli K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.

Purification and characterization of ferritin from Campylobacter jejuni

We purified an iron-containing protein fromCampylobacter jejuni using ultracentrifugation and ionexchange chromatography. Electron microscopy of this protein revealed circular particles with a diameter of 11.5 nm and a central core with a diameter of 5.5 nm. The protein was composed of a single peptide of 21 kDa and did not serologically cross-react with horse spleen ferritin. The UV-visible spectrum of the protein showed no absorption peaks in the visible region, indicating that little or no heme is bound. The ratio of Fe:phosphate ofC. jejuni ferritin was 1.5∶1. From these morphological and chemical examinations, we concluded that theC. jejuni purified protein is a ferritin of the same class as that ofHelicobacter pylori andBacteroides fragilis and differs from the heme-containing bacterioferritin ofEscherichia coli. The 30 N-terminal amino acids were sequenced and were found to resemble the sequences of other ferritins strongly (H. pylori ferritin, 73% identity;B. fragilis ferritin, 50% identity;E. coli gene-165 product, 50% identity), and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 26% identity;Azotobacter vinelandii, 26% identity; horse spleen ferritin 30% identity). Proteins that cross-reacted with antiserum against the ferritin ofC. jejuni were found in otherCampylobacter species and inH. pylori, but not inVibrio, E. coli, orPseudomonas aeruginosa.

Type 1 fimbriation and its phase switching in diarrheagenic Escherichia coli strains.

ABSTRACT Type 1 fimbriae can be expressed by most Escherichia coli strains and mediate mannose-sensitive (MS) adherence to mammalian epithelial cells. However, the role of type 1 fimbriae in enteric pathogenesis has been unclear. Expression of type 1 fimbriae inE. coli is phase variable and is associated with the inversion of a short DNA element (fim switch). Forty-six strains of diarrheagenic E. coli were examined for the expression of type 1 fimbriae. Only four of these strains were originally type 1 fimbriated. Seventeen strains, originally nonfimbriated, expressed type 1 fimbriae in association with off-to-on inversion of the fim switch, after serial passages in static culture. The switching frequencies of these strains, from fimbriate to nonfimbriate, were greater than that of the laboratory strain E. coli K-12. None of the 16 strains of serovar O157:H7 or O157:H− expressed type 1 fimbriae after serial passages in static culture. The nucleotide sequence analysis of thefim switch region revealed that all of the O157:H7 and O157:H− strains had a 16-bp deletion in the invertible element, and the fim switch was locked in the “off” orientation. The results suggest that expression of type 1 fimbriae may be regulated differently in different E. coli pathogens causing enteric infections.

Electron Microscopy of the Major Outer Membrane Protein of Campylobacter jejuni

The surfaces of the disrupted‐cell surfaces of the Campylobacter jejuni strains FUM158432 and M1 were examined using the negative‐staining technique and electron microscopy. The surfaces of the whole cells and the outer membranes were covered with small dark dots which, in some areas, were arranged in hexagonal patterns. The hexagonal arrangement was more clearly seen in extracted outer membrane. The size of each structure was measured based on a center‐to‐center distance with the adjacent structure, and was determined to be 9.9±0.9 nm. A profile of the proteins in the outer membrane by SDS‐PAGE, performed in 0.1% SDS and at 100 C, showed 42 kDa proteins to comprise the major outer membrane protein of this bacterium. Digestion of the outer membrane materials with proteinase reduced this protein band in the SDS‐PAGE, and the amount of dark dots on the electron micrograph indicated the structure to be the major outer membrane protein (porin) of this bacterium. The power spectrogram of a computer‐assisted Fourier transformation of the hexagonally arranged porin proteins suggests that the porin has a trimeric structure rather than a monomeric one.

The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion‐exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star‐shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N‐terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross‐reactive with the antiserum for the 60‐kDa HSP of Yersinia enterocolitica. Production of the 62‐kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62‐kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.

A Structural Analysis of the Regularly Arranged Porin on the Outer Membrane of Campylobacter jejuni Based on Correlation Averaging

A negatively stained electron micrograph of regularly arranged porin proteins of Campylobacter jejuni on the isolated outer membrane of bacteria was analyzed in detail by the correlation averaging method using a computer‐assisted program. The results showed that the porin of C. jejuni had a trimeric structure separated by about 10.4±0.15 nm. In addition, the pores in the trimers were also separated by about 4.3 ±0.1 nm.