Tamm-Horsfall protein inhibits binding of S- and P-fimbriated Escherichia coli to human renal tubular epithelial cells.

Escherichia coli is the predominant pathogen in urinary tract infections. Fimbriae are one of the major virulence factors of these bacteria, since these protein appendices contribute towards bacterial adhesion to epithelial cells. In clinical E. coli isolates from urinary tract infections, P fimbriae are more frequently present than S fimbriae. However, these two types of fimbriae mediate adhesion to cultured tubular epithelial cells equally well. Tamm-Horsfall protein, which is the most abundant protein in normal human urine, inhibits hemagglutination by E. coli expressing S fimbriae, but does not interfere with hemagglutination by P-fimbriated E. coli. Therefore, it has been speculated that Tamm-Horsfall protein may serve as a clearance factor for S-fimbriated E. coli in human urine. In our experiments, adherence of purified S fimbriae and of S-fimbriated E. coli to tubular epithelial cells was inhibited by Tamm-Horsfall protein, but the protein also decreased binding of P-fimbriated E. coli to approximately the same degree. We found less adherence of both types of fimbriae to a Madin-Darby canine kidney cell line expressing soluble and membrane-bound Tamm-Horsfall protein as compared with the control cell line. In conclusion, our in vitro data suggest that urinary Tamm-Horsfall protein may serve as a clearance factor for E. coli expressing both S and P fimbriae. In the light of these findings, the low clinical relevance of S-fimbriated E. coli for urinary tract infections may be readily explained; however, the predominance of P fimbriae remains unresolved.

Influence of cloned tRNA genes from a uropathogenic Escherichia coli strain on adherence to primary human renal tubular epithelial cells and nephropathogenicity in rats.

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) possesses pathogenicity islands which are incorporated into two tRNA genes, the selC and the leuX gene. The leuX gene influences the expression of different putative virulence factors. We demonstrate an effect of the leuX-specific tRNA on adherence and uropathogenicity.

Diagnosis of ornithosis by cell culture and polymerase chain reaction in a patient with chronic pneumonia.

We report the case of a woman who had pneumonia due to Chlamydia psittaci. A Chlamydia species was determined to be the causative agent of the pneumonia because it was isolated from bronchoalveolar lavage fluid, because it could be detected in lung biopsy specimens by the direct immunofluorescence technique, and because Chlamydia-specific antibodies could be detected by ELISA and microimmunofluorescence. The infectious agent could not be identified at the species level with use of serological techniques, but the isolate was determined to be C. psittaci by PCR with use of species- and genus-specific sequences within the chlamydial lipopolysaccharide biosynthesis gene gseA. The case reported herein exemplifies the problems encountered in diagnosing ornithosis and shows that isolation of the etiologic agent followed by identification of the species by PCR is helpful in diagnosing this rare disease. In addition, the findings in our case show that laboratory personnel who are conducting tests for Chlamydia pneumoniae should be aware of the risk of accidentally isolating highly infectious C. psittaci organisms.

S fimbriae of uropathogenic Escherichia coli bind to primary human renal proximal tubular epithelial cells but do not induce expression of intercellular adhesion molecule 1.

We have recently reported an increase of expression of the intercellular adhesion molecule 1 by renal carcinoma cells in response to S fimbriae of Escherichia coli. Now we demonstrate that E. coli expressing S and P fimbriae strongly binds to human proximal tubular epithelial cells. However, in primary and simian virus 40-transfected renal tubular epithelial cells S fimbriae do not enhance the expression of intercellular adhesion molecule 1.

Relevance of cysteine residues for biosynthesis and antigenicity of human hepatitis B virus e protein.

The mature form of the secretory core protein (HBe protein) of human hepatitis B virus contains four cysteines which are located at amino acid positions -7, 48, 61, and 107 relative to the HBc start methionine. In addition, there is a cysteine, Cys-183, located in the C-terminal domain of the HBe precursor, which is cleaved during HBe maturation. Here, the significance of these cysteines for biosynthesis and antigenicity of the HBe protein was examined. The cysteines at positions -7 and 61 were found to be crucial for HBe biosynthesis. As has already been described, if the Cys at position -7 is mutated, disulfide-linked HBe homodimers which have both HBe antigenicity and HBc antigenicity are expressed. Here we show that these dimers are due to Cys-61-Cys-61 disulfide bridges which are formed only if the Cys at position -7 is not present. In the wild-type protein, this dimerization appears to be inhibited by formation of intramolecular disulfide bridges between the Cys at -7 and one of the internal cysteines. Moreover, Cys-61 is important for HBe biosynthesis in general since mutation of this amino acid results in production of HBe proteins which are either only poorly secreted or possess a different antigenicity.

Molecular basis of the diversity of hepatitis B virus core-gene products.

All hepatitis B viruses examined to date code for at least two different core-gene products which are referred to as the c- and the e-protein. In the case of the human hepatitis B virus, they are known as the HBcAg and the HBeAg. Although these proteins share most of their primary amino acid sequence, they exhibit quite distinct properties. The e-protein is located in the cytoplasm and the nucleus of infected cells and very efficiently assembles into nucleocapsids. By contrast, the e-protein does not form particles. It enters the secretory pathway and is actively secreted by the cells. Here we describe the biosynthetic pathways by which the c- and e-proteins are expressed and summarize recent data from our laboratory showing that the antigenic and biophysical properties which distinguish the HBeAg from the HBcAg are primarily due to the 10 amino acid long portion of the HBeAg leader sequence that remains attached to the HBeAg after cleavage.

A cysteine and a hydrophobic sequence in the noncleaved portion of the pre-C leader peptide determine the biophysical properties of the secretory core protein (HBe protein) of human hepatitis B virus.

The molecular basis of the biophysical and antigenic differences between the cellular core protein (HBc protein) and the secreted core protein (HBe protein) of human hepatitis B virus was examined. The data show that the properties which distinguish the HBe protein from the HBc protein are due mostly to the 10-amino-acid portion of the HBe leader sequence which remains attached to the HBe protein after cleavage. A cysteine located within this region determines the quaternary structure and the antigenicity of the HBe protein. If this cysteine is lacking, the HBe protein, which is predominantly a monomer with only HBe antigenicity, is expressed as a disulfide-linked homodimer showing both HBe and HBc antigenicity. However, dimerization of the HBe protein was found to be neither sufficient nor required for particle formation. In fact, aggregation of the HBe protein was found to be inhibited by the strongly hydrophobic tripeptide Trp-Leu-Trp, which is also located in the noncleaved portion of the signal sequence. If this tripeptide was converted into either Asp-Asn-Asn or Ala-Asp-Leu, the HBe protein assembled into particles, independent of the presence of the cysteine.

The quaternary structure, antigenicity, and aggregational behavior of the secretory core protein of human hepatitis B virus are determined by its signal sequence.

Human hepatitis B virus encodes a secretory core protein, referred to as the HBe protein, whose secretion is mediated by the pre-C signal sequence. Here we examined whether this sequence is important only for translocation of the HBe precursor (the precore protein) or whether it also contributes to the structural and biophysical properties of the mature HBe protein. When a truncated hepatitis B virus precore protein, lacking the basic C-terminal domain which is cleaved from the wild-type protein during its conversion into HBe, was expressed in human hepatoma cells, only a small amount of HBe-like protein was produced. This protein was slightly smaller than the wild-type HBe protein, suggesting that C-terminal cleavage of the precore protein does not occur at the suggested site. When the authentic signal sequence of the precore protein (the pre-C sequence) was replaced by the unrelated signal sequence of an influenza virus hemagglutinin, not only the full-length but also the C-terminally truncated protein was expressed and secreted with high efficiency. Western blot (immunoblot) analyses with nonreducing gels and conformation-specific monoclonal antibodies revealed that the HBe protein secreted under control of the pre-C signal sequence was a monomer with HBe antigenicity, whereas the HBe-like protein secreted under control of the hemagglutinin signal sequence was a disulfide-bridge-linked dimer with both HBe and HBc antigenicity. Electron microscopic examination of gradient-purified particulate core gene products showed that HBe protein secreted under control of the hemagglutinin signal sequence forms core particles, whereas HBe protein secreted under control of the pre-C sequence does not. Thus, the pre-C sequence not only mediates the secretion but also determines the structural and aggregational properties of the HBe protein.