Hemolytic-uremic syndrome following urinary tract infection with enterohemorrhagic Escherichia coli: case report and review.

A 6-week-old child with acute urinary tract infection caused by Shiga toxin-producing Escherichia coli (STEC) O5:H-developed hemolytic-uremic syndrome (HUS). Molecular and phenotypic analysis of the urinary isolate indicated that it lacked uropathic properties and that it was probably of intestinal origin. Nevertheless, the patient did not experience a diarrheal prodrome, nor was STEC or Shiga toxin detected in his feces at any time. Examination of the patient's serum pointed to recent infection with E. coli O5, with no evidence of exposure to E. coli O157, O111, or O26. A review of 13 previously reported cases of HUS associated with acute urinary tract infection indicated that this was the first case of nondiarrheal HUS in which infection with the most common STEC serogroups was specifically excluded. This case illustrates the need to investigate patients with nondiarrheal HUS for infection with STEC.

Contribution of YopB to virulence of Yersinia enterocolitica.

The 70-kb virulence plasmid, pYV, of Yersinia enterocolitica encodes a number of secreted proteins (Yops) which are essential for virulence. YopD, the 33-kDa product of the lcrGVHyopBD operon, appears to be involved in delivering YopE and YopH (the Yersinia protein tyrosine phosphatase) into target cells. These proteins then act in concert to cause cytotoxicity in host cells. Previously, we reported that bacteria carrying transposon insertions in yopD are not cytotoxic for macrophages, show impaired tyrosine phosphatase activity in host cells, and are avirulent for mice (E. L. Hartland, S. P. Green, W. A. Phillips, and R. M. Robins-Browne, Infect. Immun. 62:4445-4453, 1994). trans complementation of yopD mutants of Y. enterocolitica with the yopD gene restores all these properties. In this study, we show that polar mutations in proximal genes of the lcrGVHyopBD operon also abrogated bacterial virulence and the capacity to induce cytotoxicity in mouse bone marrow-derived macrophages and HEp-2 epithelial cells. Moreover, trans complementation of a yopBD mutant with the yopD gene alone was not sufficient to restore the ability of the bacteria to cause cytotoxicity. Further work showed that YopB was required for cytotoxicity, dephosphorylation of host proteins, and virulence for mice. These findings indicate that YopB and YopD may serve a related function in Y. enterocolitica and that they may act together to deliver intracellularly acting Yops to their respective targets in host cells.

Serological response of sheep to plasmid-encoded proteins of Yersinia species following natural infection with Y. enterocolitica and Y. pseudotuberculosis.

A prospective study of the serological response to natural infection with Yersinia enterocolitica and Y. pseudotuberculosis was performed in an experimental flock of sheep. A preliminary investigation with immunoblotting techniques showed that lambs infected with virulent Yersinia spp. produced antibodies to several yersinia outer-membrane proteins (yops) encoded by a virulence plasmid (pYV) of Y. enterocolitica or Y. pseudotuberculosis. Thereafter, an enzyme immunoassay (EIA) was developed to measure antibodies to yops. Criteria for interpreting the EIA were established by examining sera from a negative control population of lambs which had not been infected with Yersinia spp. since birth. Test samples comprised 25 pairs of pre- and post-infection sera from animals with bacteriologically proven infections with Yersinia spp. The results showed that infection of lambs with pYV-bearing strains of Y. enterocolitica or Y. pseudotuberculosis invariably evoked a significant antibody response to yops, even though all the infections were subclinical. No animal infected with so-called "environmental", pYV-negative Yersinia spp. seroconverted to yops. EIA with yops as antigen provided a sensitive and specific means to diagnose subclinical infection of lambs with virulent Yersinia spp.

Assessment of enterotoxin production by Yersinia enterocolitica and identification of a novel heat-stable enterotoxin produced by a noninvasive Y. enterocolitica strain isolated from clinical material.

Twenty-eight clinical isolates of Yersinia enterocolitica were investigated for their abilities to produce heat-stable enterotoxin (YST). All 21 invasive strains (serogroup O3 biotype 4) carried the previously described gene for YST (yst), with toxin detectable in culture supernatants from 20 strains. One of seven noninvasive, biotype 1A strains also had enterotoxic activity, despite failure to hybridize with a probe for yst. The toxin produced by this noninvasive (serogroup O6) strain resembled YST in terms of molecular size, heat stability, and solubility in methanol. It differed from YST, however, with respect to regulation of its production by temperature and its mechanism of action, which did not appear to involve cyclic GMP.

Yersinia enterocolitica isolated from two cohorts of young children in Santiago, Chile: incidence of and lack of correlation between illness and proposed virulence factors.

Yersinia enterocolitica was isolated from children in two cohorts in Santiago, Chile. In a cohort containing a cross section of children aged 0 to 4 years, Y. enterocolitica was isolated from stool samples of 1.1% of children with diarrhea and 0.2% of age-matched control children. In a subgroup of this cohort from which weekly stool samples were obtained from all children irrespective of clinical status, 6% of children had asymptomatic Yersinia infections. In a birth cohort (with a greater representation of children less than 1 year of age and a significantly higher rate of diarrhea), Y. enterocolitica was isolated from 1.9% of children with diarrhea and 0.6% of controls (P = 0.05). Biogroup 1A strains (which lacked traditional phenotypic and molecular markers for pathogenicity) were isolated from seven children with diarrhea but from no control children in the birth cohort (P = 0.02). All other isolates, including all isolates from asymptomatic children, were "pathogenic" strains in biogroup 4, serogroup O3; no association between these isolates and occurrence of disease was found. Y. enterocolitica is found among young children in Santiago, with asymptomatic infections not uncommon occurrences. However, questions about the association between previously described virulence factors and diarrheal illness remain.

Pathogenicity of Yersinia kristensenii for mice.

Forty-seven strains of Yersinia kristensenii from widely differing sources, representing all known O serogroups of this species, were investigated for virulence with a variety of animal and in vitro assays. Twenty-four (51%) of the isolates were lethal for mice pretreated with iron dextran. Mouse-lethal strains occurred predominantly within O serogroups O:11, O:12,25, and O:16. Virulent Y. kristensenii strains generally did not express the virulence-associated phenotype (Ca2+ dependence and binding of Congo red and crystal violet) which characterizes virulent strains of Y. enterocolitica, nor did they carry the Yersinia virulence plasmid. Although all strains hybridized with a DNA probe derived from the inv (invasin) gene of Y. enterocolitica, none was able to invade HEp-2 epithelial cell culture. Y. kristensenii strains were virulent only when inoculated parenterally into iron-loaded mice. Animals infected in this way succumbed rapidly to infection, generally within 24 h. This finding suggested that the pathogenicity of these bacteria may be attributable to a secreted toxin, but a search for such a substance and for other in vitro correlates of pathogenicity was unsuccessful. These observations indicate that some strains of Y. kristensenii kill mice by a mechanism not previously recognized in yersiniae.

Na+/H+ exchange involvement in colony-stimulating factor-1-stimulated macrophage proliferation. Evidence for a requirement during late G1 of the cell cycle but not for early growth factor responses.

Na+/H+ exchange activation by growth factors is proposed to be an important early signal for mitogenesis; however, little is known of its duration and requirement during later stages of the cell cycle. Macrophage-specific colony factor (CSF-1) rapidly activates murine bone marrow-derived macrophage Na+/H+ exchange, resulting in stimulation of Na+,K(+)-ATPase activity. The response to CSF-1 is maintained for at least 24 h. Inhibition of Na+/H+ exchange with 5-N,N-dimethylamiloride prevents CSF-1-stimulated DNA synthesis and cell growth. This is unlikely to be due to cytoplasmic acidosis, but more likely reflects a requirement for Na+/H+ exchange-mediated Na+ influx. DMA addition even up to 8 h after the growth factors suppresses S-phase progression. Na+/H+ exchange appears not to be involved in the induction of other early growth factor responses (c-fos and c-myc mRNA induction and general RNA and protein synthesis). We propose that growth factor-stimulated Na+/H+ exchange late in G1 of the cell cycle is required for S-phase progression but not for certain early growth factor responses.

Inhibition of the signaling pathways for macrophage proliferation by cyclic AMP. Lack of effect on early responses to colony stimulating factor-1.

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in quiescent murine bone marrow-derived macrophages (BMM). CSF-1 action has been shown to involve activation of the CSF-1 receptor kinase. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (PMA), is itself weakly mitogenic and synergises with CSF-1 for stimulation of BMM DNA synthesis suggesting a possible role for protein kinase C in the stimulation of BMM DNA synthesis. In this report we show that several agents which raise intracellular cAMP (8-bromoadenosine 3':5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine, cholera toxin, and prostaglandin E2) reversibly inhibit DNA synthesis in BMM induced by CSF-1, granulocyte macrophage-colony stimulating factor, interleukin-3, and PMA. The suppressive action of cAMP elevation on the proliferative response to CSF-1 can be manifested even late in the G1 phase of the cell cycle. Several CSF-1-stimulated earlier responses, viz. protein synthesis, Na+/H+ exchange, Na+,K(+)-ATPase and c-myc-mRNA expression, were not inhibited thus showing a striking difference from some other cellular systems involving growth factor-mediated responses. c-fos-mRNA levels were raised and stabilized by the cAMP-elevating agents, and this modulation was not altered by CSF-1. Thus, the signaling pathways in the macrophages involving tyrosine kinase and protein kinase C activation are associated with increased proliferation while those involving elevation of cAMP (and presumably activation of cAMP-dependent protein kinases) appear to have an inhibitory effect.

Activation and proliferation signals in murine macrophages: relationships among c-fos and c-myc expression, phosphoinositide hydrolysis, superoxide formation, and DNA synthesis.

Murine bone marrow-derived macrophages (BMM) undergo DNA synthesis in response to growth factors such as colony stimulating factor-1 (CSF-1) and granulocyte-macrophage CSF (GM-CSF). These macrophages can also be "activated," but without subsequent DNA synthesis, by a number of other agents, including lipopolysaccharide (LPS), concanavalin A, zymosan, formyl-methionyl-leucyl-phenylalanine (FMLP), and the Ca2+ ionophore, A23187. When BMM are treated with a range of stimuli, there is some, although not perfect, correlation between transient elevations in both c-myc mRNA and c-fos mRNA levels and increases in DNA synthesis. However, enhanced DNA synthesis and oncogene expression are readily dissociated from rises in inositol phosphates and, by implication, phospholipase C-mediated hydrolysis of phosphatidyl inositol 4,5-bisphosphate. Superoxide formation in BMM can also be dissociated from the other responses and does not necessarily depend on protein kinase C activation.