Strains ofE Research Articles

Bacteria Signal Kidney Damage

Certain strains of E. coli cause inflammation in the kidney and contribute to renal failure. Uhlén et al . identified α-hemolysin as a secreted factor produced by uropathogenic bacteria that induced calcium oscillations in cultured renal proximal tubule cells and renal epithelial cells. Inhibitors of L-type Ca 2+ channels or inhibitors of IP 3 receptors blocked the calcium oscillations. Application of α-hemolysin to renal epothelial cells led to an increase in the secretion of the inflammatory mediators interleukin-6 and interleukin-8. The authors suggest that during early phases of infection, kidney damage arises not by cytolysis, but by the stimulation of inflammatory cytokines by α-hemolysin. Uhlén, P., Laestadlus, Å., Jahnukainen, T., Söderblom, T., Bäckhed, F., Celsi, G., Brismar, H., Normark, S., Aperia, A., and Richter-Dahlfors, A. α-haemolysin of uropathogenic E. coli induces Ca 2+ oscillations in renal epithelial cells. Nature 405 : 694-697. [Online Journal]

Increased expression of periplasmic Cu,Zn superoxide dismutase enhances survival of Escherichia coli invasive strains within nonphagocytic cells.

We have studied the influence of periplasmic Cu,Zn superoxide dismutase on the intracellular survival of Escherichia coli strains able to invade epithelial cells by the expression of the inv gene from Yersinia pseudotuberculosis but unable to multiply intracellularly. Intracellular viability assays, confirmed by electron microscopy observations, showed that invasive strains of E. coli engineered to increase Cu,Zn superoxide dismutase production are much more resistant to intracellular killing than strains containing only the chromosomal sodC copy. However, we have found only a slight difference in survival within HeLa cells between a sodC-null mutant and its isogenic wild-type strain. Such a small difference in survival correlates with the very low expression of this enzyme in the wild-type strain. We have also observed that acid- and oxidative stress-sensitive E. coli HB101(pRI203) is more rapidly killed in epithelial cells than E. coli GC4468(pRI203). The high mortality of E. coli HB101(pRI203), independent of the acidification of the endosome, is abolished by the overexpression of sodC. Our data suggest that oxyradicals are involved in the mechanisms of bacterial killing within epithelial cells and that high-level production of periplasmic Cu,Zn superoxide dismutase provides bacteria with an effective protection against oxidative damage. We propose that Cu,Zn superoxide dismutase could offer an important selective advantage in survival within host cells to bacteria expressing high levels of this enzyme.

Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction

A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426-4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain two FRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomal adhE gene in strains of E. coli K-12 and E. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

Improving DNA damage repair ability ofE. coli to UV-radiosensitivity by DNA-mediated gene transfer of low energy ion beam

Using calcium chloride method of transfer gene as control, a new technique of transferring gene by low energy ion beam has been applied to the study of improving DNA damage repair ability ofE. coli to UV-radiosensitivity. The genome DNA pieces ofDeinococcus radiodurans, as “foreign” genetic materials, were introduced into the UV-radiosensitive strains ofE. coli by implantation of 20 keV Ar+ at doses ranging from 1 × 1015 to 2 × 1015 ions/cm2. Results show that the transfected strains present higher UV-radioresistance than that of un-transfected ones and start ones. The survival rate of transfected strains and their unscheduled DNA synthesis (UDS) ability is increased, indicating that the transfer gene is a success.

Nanomolar levels of dimethylsulfoniopropionate, dimethylsulfonioacetate, and glycine betaine are sufficient to confer osmoprotection to Escherichia coli.

We combined the use of low inoculation titers (300 +/- 100 CFU/ml) and enumeration of culturable cells to measure the osmoprotective potentialities of dimethylsulfoniopropionate (DMSP), dimethylsulfonioacetate (DMSA), and glycine betaine (GB) for salt-stressed cultures of Escherichia coli. Dilute bacterial cultures were grown with osmoprotectant concentrations that encompassed the nanomolar levels of GB and DMSP found in nature and the millimolar levels of osmoprotectants used in standard laboratory osmoprotection bioassays. Nanomolar concentrations of DMSA, DMSP, and GB were sufficient to enhance the salinity tolerance of E. coli cells expressing only the ProU high-affinity general osmoporter. In contrast, nanomolar levels of osmoprotectants were ineffective with a mutant strain (GM50) that expressed only the low-affinity ProP osmoporter. Transport studies showed that DMSA and DMSP, like GB, were taken up via both ProU and ProP. Moreover, ProU displayed higher affinities for the three osmoprotectants than ProP displayed, and ProP, like ProU, displayed much higher affinities for GB and DMSA than for DMSP. Interestingly, ProP did not operate at substrate concentrations of 200 nM or less, whereas ProU operated at concentrations ranging from 1 nM to millimolar levels. Consequently, proU(+) strains of E. coli, but not the proP(+) strain GM50, could also scavenge nanomolar levels of GB, DMSA, and DMSP from oligotrophic seawater. The physiological and ecological implications of these observations are discussed.

Identification of a glycoprotein produced by enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of E. coli to adhere to and invade cultured human intestinal epithelial cells. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant E. coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering E. coli and with pertactin precursor from Bordetella pertussis. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this tib locus open reading frame is proposed to be responsible for TibA modification. These results suggest that TibA glycoprotein acts as an adhesin that may participate in the disease process.

Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria.

We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.

Gut Colonization by an Ice Nucleation Active Bacterium,Erwinia(Pantoea)ananasReduces the Cold Hardiness of Mulberry Pyralid Larvae

To evaluate the suitability of using ice nucleation active (INA) bacteria for the biological control of insect pests, the supercooling point (SCP) of larvae of mulberry pyralid,Glyphodes duplicalis,and silkworm,Bombyx mori,ingesting INA strains ofErwinia(Pantoea)ananasandPseudomonas syringaewas determined. Mean SCP of the guts of silkworm larvae ingesting INA strains ofE. ananasranged from −2.5 to −2.8°C, being 5°C higher than that in control treatments. Similarly, mean SCP of mulberry pyralid larvae ingesting INA strain ofE. ananas,which can grow well in the gut, was −4.7°C at 3 days after treatment, being 6.5°C higher than that in control treatments. On the other hand, mean SCP of the larvae-ingesting INA strain ofP. syringae,which cannot grow in the gut, was −9.0°C at 3 days after treatment, rising by only 2.5°C higher than that in the control treatments. In addition, more than 80% of the larvae of mulberry pyralid ingesting the INA strain ofE. ananasfroze and eventually died when exposed to −6°C for 18 h, while only 36% of the larvae ingesting the INA strain ofP. syringae,or approximately 20% of the control larvae, froze and died. Thus, the gut colonization by INA strains ofE. ananasreduced remarkably the cold hardiness of the insects. These findings suggest that INA strains ofE. ananascould be effective as a potential biological control agent of insect pests.

Inverse relationship between severity of experimental pyelonephritis and nitric oxide production in C3H/HeJ mice.

The contribution of nitric oxide to host resistance to experimental pyelonephritis is not well understood. We examined whether the inhibition of nitric oxide synthesis alters the sensitivity of lipopolysaccharide (LPS) responder (C3H/HeN) and nonresponder (C3H/HeJ) mice to experimental Escherichia coli pyelonephritis. C3H/HeJ and C3H/HeN mice were implanted subcutaneously with minipumps containing an inhibitor of nitric oxide, NG-nitro-L-arginine methyl ester (L-NAME), or a corresponding vehicle. Ascending urinary tract infection by bladder catheterization with two strains of E. coli, an O75 strain bearing Dr fimbriae and an O75 strain bearing P fimbriae, was developed in tested animals. Twenty-four hours following bladder infection, the kidneys of C3H/HeN and C3H/HeJ mice were colonized at a similar rate. However, 5 weeks postinoculation, C3H/HeN mice cleared infection while C3H/HeJ mice showed persistent colonization. Twenty-four hours following infection, C3H/HeN mice treated with L-NAME showed no significant increase of renal tissue infection compared to the saline-treated control group. However, L-NAME-treated C3H/HeJ mice showed an approximately 100-fold increase in E. coli infection rate compared to the saline-treated controls in the Dr+ group but showed no change compared to those in the P+ group. Dissemination of Dr+ E. coli but not P+ E. coli to the liver and uterus was significantly enhanced with L-NAME treatment in C3H/HeJ mice only. Nitric oxide had no direct killing effect on E. coli in vitro. Nitrite production by various organs was found to be significantly lower in C3H/HeJ mice than in C3H/HeN mice. Alteration of nitric oxide and LPS responsiveness was significantly associated with the increased sensitivity of C3H/HeJ mice to experimental Dr+ but not to P+ E. coli pyelonephritis. These findings are consistent with the hypothesis that nitric oxide synthase activity in concert with LPS responsiveness may participate in the antibacterial defense mechanisms of the C3H mouse urinary tract. This phenomenon is strain dependent and possibly related to the invasive properties of E. coli.

Analysis of the genes responsible for the O-antigen synthesis in enterohaemorrhagicEscherichia coliO157

The genes responsible for O157 O-antigen synthesis were cloned from Shiga toxin 1 (Stx1)-producing enterohaemorrhagicEscherichia coliO157:H-. Based on the published sequence of therfbEEcoO157:H7gene, therfbEgene was amplified by PCR and used as the probe. One clone (1-4-1) out of ten agglutinated using the slide agglutination test with O157 antiserum. The DNA sequence of a 31.5 kb fragment in p1-4-1 was determined and found to contain 26 open reading frames (ORFs).These ORFs were organized as two clusters of genes, comprisingorf2toorf16andorf17toorf24regions, which were aligned in opposite directions. The GC contents oforf1,orf12andorf14toorf26were similar to the overall GC content of theE. colichromosome (51%). However,orf2toorf11andorf13showed a much lower GC content of 30.0 to 39.4%. The minimum region essential for O157 O-antigen expression inE.coliK-12 lay betweenorf2andorf13, which corresponded to the region of low GC content inE.coliO157. A colony hybridization test was carried out against reference strains ofE. colirepresenting serogroups O1 to O173 using O157 O-antigen synthesis genes (orf2–orf13) ofE. coliO157 as probes. With the exception oforf12, all probes fromE. coliO157 O-antigen synthesis gene cluster did not hybridize with DNA fromE. coliO1 to O173, except O157 reference strains, under high stringency conditions. These data suggest that the region fromorf2toorf13has originated from a non-E. colispecies with a low GC content.

Conserved organization in the cps gene clusters for expression of Escherichia coli group 1 K antigens: relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae.

Group 1 capsules of Escherichia coli are similar to the capsules produced by strains of Klebsiella spp. in terms of structure, genetics, and patterns of expression. The striking similarities between the capsules of these organisms prompted a more detailed investigation of the cps loci encoding group 1 capsule synthesis. Six strains of K. pneumoniae and 12 strains of E. coli were examined. PCR analysis showed that the clusters in these strains are conserved in their chromosomal locations. A highly conserved block of four genes, orfX-wza-wzb-wzc, was identified in all of the strains. The wza and wzc genes are required for translocation and surface assembly of E. coli K30 antigen. The conservation of these genes points to a common pathway for capsule translocation. A characteristic JUMPstart sequence was identified upstream of each cluster which may function in conjunction with RfaH to inhibit transcriptional termination at a stem-loop structure found immediately downstream of the "translocation-surface assembly" region of the cluster. Interestingly, the sequence upstream of the cps clusters in five E. coli strains and one Klebsiella strain indicated the presence of IS elements. We propose that the IS elements were responsible for the transfer of the cps locus between organisms and that they may continue to mediate recombination between strains.

Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses.

Strains of Escherichia coli O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of E. coli O157 when the efficacy of a novel or mild food preservation treatment is tested.

Differences in thermotolerance of variousEscherichia coliO157:H7 strains in a salami matrix

Three strains ofEscherichia coliO157:H7 (ATCC 43895, Ent C9490 and 380–94) were inoculated into salami and heated in water baths at 50, 55 or 60°C. At intervals between 1 and 360 min, salami samples were removed from the water bath and examined for the presence of survivingE. coliO157:H7. Samples were directly plated onto sorbitol MacConkey (SMAC) agar, and onto tryptone soya agar (TSA) with SMAC overlay. The number of sub-lethally damaged cells in each sample was estimated from the differences between the resultant direct (uninjured cells only) and overlay (total recovery) counts. In samples heated at 50°C, the percentage of cell injury ranged from 71·8–88% for all strains. In samples heated at 55°C the percentage of sub-lethally damaged cells in strains ATCC 43895 and Ent C9490 was significantly higher (P< 0·001) at 97% than that observed in strain 380–94 (64%). Cell injury was not measured at 60°C. There were significant differences between the derived decimal reduction times (D-values) related to the different strains ofE. coliO157:H7, the heat treatment applied and the recovery/enumeration agars used. Significant interstrain differences (P< 0·05) in thermotolerance were noted. Strain Ent C9490 was significantly more heat resistant at 50°C and 60°C (D-values of 116·9 and 2·2 min, respectively), while at 55°C strain 380–94 was more thermotolerant (D-value of 21·9 min). The implications of these findings for the design of studies investigating the heat resistance ofE. coliO157:H7 in fermented meat environments are discussed.

H-NS regulates DNA repair in Shigella.

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30 degrees C and nonfunctional at 37 to 40 degrees C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40 degrees C postirradiation, shows up to 30-fold higher survival than when incubated at 30 degrees C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40 degrees C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40 degrees C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40 degrees C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.

Characterization of a phage specific to hemorrhagic Escherichia coli O157:H7 and disclosure of variations in host outer membrane protein ompC.

Phage AR1, previously known to infectEscherichia coli O157:H7 with high specificity, was further characterized for its genetic properties. The phage DNA sequences including capsid genes and a putative α-glucosyltransferase gene(α-gt) have been deduced. These sequences are conservative but not identical to those of T4 phage. However, a nonessential gene,SegD, organized within the capsid gene cluster of T4 is missing in the corresponding region of AR1 genome, and this characteristic has not been observed among T-even related phages. The difference between AR1 and T4 was further exemplified by their distinct host ranges. Strains ofE. coli O157:H7 collected from different sources were permissive to AR1 but resistant to T4 that normally infects K-12 strains ofE. coli through contact with the outer membrane protein OmpC. Thus, the O157:H7 strains must have a varied OmpC. Indeed, the OmpC sequence of O157:H7 strains was proved to differ from that of K-12 strains by a total of 15 amino acid substitutions and two gaps (a five-residue deletion and a four-residue insertion). The OmpC molecules are relatively conserved across the gram-negative bacteria, and this is the first time OmpC divergence has been found within the sameE. coli species. Since OmpC is located in the outer membrane and its expression is regulated by environmental conditions, alteration of the structure in pathogenic O157:H7 strains may have biological significance.

Distribution of streptomycin-resistant strainsof Erwinia amylovora in Israel and occurrence of blossom blight in the autumn

Following failure in control of fire blight with streptomycin, the distribution of streptomycin-resistant strains ofErwinia amylovora in Israel was surveyed. During 1994–1997 109 pear, apple, loquat and quince orchards were monitored. Streptomycin-resistant strains ofE. amylovora were recovered from flowers and from infected branches collected at 18 locations in the Sharon, Galilee and Golan Heights regions. In the Sharon region all the isolated strains ofE. amylovora were streptomycin-resistant, whereas in the Galilee and Golan Heights, resistant as well as sensitiveE. amylovora strains were recovered at different locations. In the southern coastal plain no resistance could be detected. Streptomycin-resistant strains ofE. amylovora did not hybridize with the DNA probe SMP3, and resistance could not be transferred by mating to a sensitive strain, suggesting that streptomycin resistance in Israel is not plasmid-mediated. Fire blight symptoms were observed, for the first time, on pear blossoms during the autumn of 1994. A high population of 2x 106-6x 107 CFU/flower in the autumn of 1995 and of 1996 was correlated with the appearance of blossom blight symptoms.

Expression of GP63 gene of Leishmania major in Pichia pastoris

We report the bacterial expression and the purification of a monoclonal antibody (mAb) specific for an epitope that coincides with the LDL receptor (LDLr)-binding domain of human apolipoprotein E (apoE). This antibody resembles the LDLr in its primary structure and its specificity for apoE variants. The recombinant Fab (rFab) fragment of mAb 2E8, consisting of the entire light chain and the Fd portion of the heavy chain, was expressed inEscherichia coliand purified to homogeneity. Purification was facilitated by including a five-histidine carboxyl-terminal extension on the Fd chain. A 5- to 10-fold difference in yield of the antibody was observed when the plasmid was expressed in two different strains ofE. coli.Typically 2–6 mg of rFab per liter of culture medium was recovered in the periplasm of the TG1 strain and less than 1 mg was recovered in the periplasm of the XL1-Blue strain. Culture temperatures above 35°C or inclusion of sucrose in the medium reduced rFab yields. The 2E8 rFab was indistinguishable from Fab prepared from 2E8 hybridoma-generated IgG with respect to its affinity and fine specificity. We are using this system to express a panel of 2E8 variant Fabs that will be used as probes to establish the structural features responsible for the binding of apoE to the LDLr.

Direct detection of eae-positive bacteria in human and veterinary colorectal specimens by PCR.

A PCR test based on the amplification of an eae-specific sequence was designed and evaluated for its ability to directly detect homologous sequences in enteropathogenic Escherichia coli and Citrobacter spp. (amplification of eae open reading frame, 178 bp) in sections of the intestines of humans and animals with colonic lesions. Positive PCR results were observed with eae-positive reference strains of E. coli and Citrobacter rodentium (Citrobacter freundii biotype 4280). Known eae-negative reference strains of E. coli and other laboratory strains of enteric bacteria were negative by the amplification test. The sensitivity of the PCR for detection of eae-positive E. coli and C. rodentium was between 1 and 2 CFU. To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were modified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extraneous bacterial DNA from the PCR amplification cocktail without added template. Sections of colon from three pigs experimentally affected with colon lesions due to enteropathogenic (attaching and effacing) E. coli were PCR positive for bacterial eae genome. Sections from control animals were negative. Sections of colon from one of 18 biopsies from confirmed AIDS patients and from 22 of 35 colorectal cancer patients were PCR positive for bacterial eae genome. The PCR test was a simple and quick method of detecting bacterial eae genome in human and veterinary clinical specimens. This method may remove the need for initial culture and detection of the gene by DNA probing from potential associated lesions. The clear relationship of bacteria containing the eae gene with colonic lesions in the pigs and mice indicates that a similar relationship is possible for human patients having similar lesions.

Cloning and molecular analysis of genes encoding two immunodominant antigensofEhrlichia risticii

Ehrlichia risticii, the causative agent of Potomac horse fever, is an obligate intracellular rickettsial organism. To understand the role of 55 and 51 kilodalton immunodominant antigens ofE. risticiiin strain variation, their genes from the 25-D and 90-12 strains were cloned, sequenced, and expressed inE. coli.Sequence analysis revealed that the gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a 10 kDa protein. Homology searches indicated that the 55 kDa antigen and the 10 kDa protein were homologues ofE. coliGroEL and GroES proteins, respectively. There was no nucleotide sequence difference between the genes of the 55 kDa antigen, nor between the entire operons, from both strains ofE. risticii.The sequence-based estimation of the sizes of the putative mature 51 kDa antigens of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectively. The 51 kDa antigens from the 90-12 and 25-D strains shared a 98% identity in their deduced amino acid sequences. The difference in some of the amino acids may be responsible for variation in their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a 2 kDa lower molecular weight region. In Western blots, a 155 kDa protein that appeared to be a trimer product of the 51 kDa antigen was identified. The 55 and 51 kDa antigens were overexpressed inE. coliusing a commercial expression system, pRSET A,B,C (Invitrogen Inc., San Diego, CA, U.S.A.). The purified recombinant proteins cross-reacted with antisera toE. canisandE. sennetsu.

Entamoeba histolyticaHM1:IMSS: Hemoglobin-Degrading Neutral Cysteine Proteases

Serrano-Luna, J. de J., Negrete, E., Reyes, M., and de la Garza, M. 1998.Entamoeba histolyticaHM1:IMSS: Hemoglobin-degrading neutral cysteine proteases.Experimental Parasitology89,71–77.Entamoeba histolyticaHM1:IMSS trophozoites were able to utilize human hemoglobin but not hemin as a sole iron source to growin vitro. Proteases from crude extracts ofE. histolyticadegraded human, porcine, and bovine hemoglobins at pH 7.0. These proteolytic activities were found by electrophoresis in SDS-polyacrylamide gels copolymerized with hemoglobin, with apparent molecular weights of 116, 82, and 21 kDa, the 82-kDa protein being the most active protease against this substrate. The proteases were classified in the cysteine group since the activities were inhibited by l-trans-epoxysuccinylleucylamido (4-guanidino)butane,p-hydroxymercuribenzoate, iodoacetate, andN-ethylmaleimide and activated with dithiothreitol. Other pathogenic strains ofE. histolyticashowed the same pattern of hemoglobinases. These hemoglobin-degrading proteases could be playing an important role in iron acquisition by E. histolytica.