A point mutation in the active site of Legionella pneumophila O-acetyltransferase results in modified lipopolysaccharide but does not influence virulence.

The majority of clinical isolates of Legionella pneumophila serogroup 1 produce lipopolysaccharide (LPS) that reacts with monoclonal antibody (MAb) 3/1. By using a negative cell sorting method, we isolated a spontaneous LPS mutant from L. pneumophila serogroup 1 strain Corby that lost reactivity with this MAb. The mutant contained a single nucleotide exchange in position 169 of the lag-1 gene that encodes an O-acetyltransferase that is responsible for O-acetylation of the L. pneumophila O-repeat unit (legionaminic acid). This mutation resulted in a single amino acid exchange in a highly conserved motif present in many O-acetyltransferase-like proteins. RT-PCR analysis revealed that the mutant lag-1 gene was transcribed, but the resulting protein lacked O-acetyltransferase activity. Chemical analysis of the mutant LPS revealed that it lacked 8-O-acetyl groups in legionaminic acid. In addition, the mutant failed to produce high-molecular-weight long-chain O-polysaccharide. Complementation of the mutant with the wild-type lag-1 gene restored reactivity with MAb 3/1 and the chemical structure of the wild-type LPS. Strain Corby and its MAb 3/1-negative mutant were indistinguishable in their serum resistance characteristics, and in uptake and intracellular multiplication in Acanthamoeba castellanii and macrophages.

Complex O-acetylation in Legionella pneumophila serogroup 1 lipopolysaccharide. Evidence for two genes involved in 8-O-acetylation of legionaminic acid.

A putative gene encoding an O-acetyl transferase, lag-1, is involved in biosynthesis of the O-polysaccharide (polylegionaminic acid) in some Legionella pneumophila serogroup 1 strains. To study the effect of the presence and absence of the gene on the O-polysaccharide O-acetylation, lag-1 from strain Philadelphia 1 was expressed in trans in the naturally lag-1-negative OLDA strain RC1, and immunoblot analysis revealed that the lag-1-encoded O-acetyl transferase is active. O-Polysaccharides of different size were prepared from the lipopolysaccharides of wild-type and transformant strains by mild acid degradation followed by gel-permeation chromatography. Using NMR spectroscopy and MALDI-TOF mass spectrometry, it was found that O-acetylation of the first three legionaminic acid residues next to the core occurs in the short-chain O-polysaccharide (<10 sugars) from both strains. Hence, there is another O-acetyl transferase encoded by a gene different from lag-1. In the longer-chain O-polysaccharide, a legionaminic acid residue proximal to the core is N-methylated and could be further 8-O-acetylated in the lag-1-dependent manner. Only strains expressing a functional lag-1 gene were recognized in Western blot analysis by monoclonal antibody 3/1 requiring 8-O-acetylated polylegionaminic acid for binding. The highly O-acetylated outer core region of the lipopolysaccharide is involved in the epitope of another serogroup 1-specific monoclonal antibody termed LPS-1. The O-acetylation pattern of the L. pneumophila serogroup 1 core oligosaccharide was revised using MALDI-TOF mass spectrometry. lag-1-independent O-acetylation of the core and short-chain O-polysaccharide was found to be a common feature of L. pneumophila serogroup 1 strains. The biological importance of conserved lag-1-independent and variable lag-1-dependent O-acetylation is discussed.

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

Cloning and functional characterization of a 30 kb gene locus required for lipopolysaccharide biosynthesis in Legionella pneumophila.

The spontaneous Legionella pneumophila lipopolysaccharide (LPS) mutant 137, which did not bind the LPS-specific mAb 2625, was complemented with a genomic library from the parental wild-type strain. Transformants were screened for reconstitution of the wild-type LPS phenotype, able to bind mAb 2625. By this strategy, a 32,661 bp region comprising 30 open reading frames (Orfs) was identified. Orfs with significant homologies to genes encoding enzymes required for LPS or capsule biosynthesis of Gram-negative bacteria were located on the gene locus. The mutation of strain 137 could be assigned to a deletion of a cytosine residue in Orf 8. The protein encoded by Orf 8 exhibited homology to bacterial methyl-transferases. The L. pneumophila LPS gene locus included genes with deduced products likely to be involved in LPS core oligosaccharide biosynthesis (rmlA-D, rhamnosyl-transferases, acetyl-transferase) as well as LPS O-chain biosynthesis and translocation (mnaA, neuB, neuA, wecA, wzt, wzm). The neuA (Orf 25) and neuB (Orf 24) gene products were functionally characterized by complementation of the capsule negative E. coli K1 mutants EV5 and EV24, respectively. By introduction of the L. pneumophila neuA gene into E. coli EV5 and the neuB gene into EV24, expression of the K1 polysialic acid capsule could be restored. We, therefore, conclude that the biosynthesis pathway of legionaminic acid, the structural unit of the L. pneumophila Sg1 O-antigen, might be similar to the biosynthesis of sialic acid. Southern blot analysis indicated the entire gene locus to be present in L. pneumophila serogroup (Sg)1 strains, whereas only parts of the DNA stretch hybridized to DNA from Sg2 to Sg14 strains.

The siaA gene involved in capsule polysaccharide biosynthesis of Neisseria meningitidis B codes for N-acylglucosamine-6-phosphate 2-epimerase activity.

The capsule polysaccharide of Neisseria meningitidis serogroup B is composed of a homopolymer of alpha-2-->8 linked N-acetyl-neuraminic acid (sialic acid). The enzymes required for sialic acid biosynthesis and polymerization are encoded in region A of the capsule gene complex. We here describe the enzymatic activity of the siaA gene product as determined by biochemical analysis. siaA was overexpressed in Escherichia coli and the SiaA protein was purified to homogeneity. Enzymatic assays revealed that SiaA did not accept N-acetyl-glucosamine as substrate, but only N-acetyl-glucosamine-6-phosphate (EC 5.1.3.9). SiaA catalyzes the isomerization of N-acetyl-glucosamine-6-phosphate to form N-acetyl-mannosamine-6-phosphate. This reaction represents the first step in capsule biosynthesis of N. meningitidis B.

Systemic infection of an immunocompromised patient with Methylobacterium zatmanii.

We describe the identification of Methylobacterium zatmanii as the causative agent of bacteremia and fever in an immunocompromised patient. The patient, a 60-year-old man, had a 5-month history of acute myeloid leukemia and had been on chemotherapy throughout this period. Seven days after the onset of neutropenia, the patient developed fever. The combination of ciprofloxacin, co-trimoxazole, imipenem, amikacin, and vancomycin led to a complete defervescence. On subculture from six positive blood cultures, the organism grew only on buffered charcoal yeast extract agar and not on standard agars. Identification by universal PCR and subsequent sequence analysis of the amplified 16S rRNA gene segment was achieved. This identification by molecular biology techniques was confirmed by conventional biochemical tests. To our knowledge, this is the first description of M. zatmanii isolated from patient material.

Phase-variable expression of lipopolysaccharide contributes to the virulence of legionella pneumophila.

With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625-positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement components, whereas the parental strain RC1 was almost serum resistant. Moreover, mutant 811 was not able to replicate intracellularly in macrophage-like cell line HL-60. In the guinea pig animal model, mutant 811 exhibited significantly reduced ability to replicate. Among recovered bacteria, mAb 2625-positive revertants were increased by fourfold. The relevance of LPS phase switch for pathogenesis of Legionella infection was further corroborated by the observation that 5% of the bacteria recovered from the lungs of guinea pigs infected with the wild-type strain RC1 were negative for mAb 2625 binding. These findings strongly indicate that under in vivo conditions switching between two LPS phenotypes occurs and may promote adaptation and replication of L. pneumophila. This is the first description of phase-variable expression of Legionella LPS.

The central, surface-exposed region of the flagellar hook protein FlgE of Campylobacter jejuni shows hypervariability among strains.

In a previous study, we observed that monoclonal antibodies raised against the hook protein FlgE of Campylobacter jejuni LIO 36, isolate 5226, bound exclusively to this strain. The aim of this study was to elucidate the molecular basis for these binding specificities. The hook protein-encoding gene flgE of C. jejuni was cloned in Escherichia coli and sequenced. The flgE genes of four additional C. jejuni strains were amplified by PCR and also sequenced. Comparison of the deduced amino acid sequences revealed a high degree of variability in the central parts of the FlgE proteins among the strains, including variable and hypervariable domains. These findings may indicate a selective pressure of C. jejuni hosts, forcing the bacteria to generate variations in surface-exposed antigenic determinants.

Monoclonal antibody against species-specific epitope of Pseudomonas aeruginosa Hsp60 protein cross-reacts with Pseudomonas stutzeri and other Pseudomonas species.

In a previous study, we determined the epitope of the Pseudomonas aeruginosa Hsp60 heat shock protein which is recognized by the specific monoclonal antibody (MAb) 2528. Subsequent investigations revealed a weak cross-reactivity of MAb 2528 with P. stutzeri, P. alcaligenes, P. mendocina and P. pseudoalcaligenes. To elucidate the molecular structure for these cross-reactions, we cloned the P. stutzeri hsp60 gene in Escherichia coli and determined the nucleotide sequence of the gene. In addition, the hsp60 gene of further Pseudomonas species was amplified and sequenced and amino acid substitutions within the epitope recognized by MAb 2528 were determined. The decapeptide QADIEARVLQ is unique to the P. aeruginosa Hsp60 protein, and cross-reaction of MAb 2528 reflects the phylogenetic relationship of Pseudomonas species as P. aeruginosa and all four cross-reacting species constitute a DNA homology group within the rRNA group I of the family Pseudomonadaceae, which belong to the gamma-subclass of the Proteobacteria.

Development of an amplification and hybridization assay for the specific and sensitive detection of Mycoplasma fermentans DNA.

A polymerase-chain-reaction-based detection system for Mycoplasma fermentans was established. The highly conserved tuf gene, which encodes elongation factor Tu of prokaryotes, served as target sequence for the PCR. With two PCR oligodeoxynucleotides, which were selected from M. fermentans specific sequences of the tuf gene, we amplified a 850 base pair DNA fragment. Via the biotin-moiety of one primer the PCR fragments were immobilized on streptavidin-coated microtitre plates. After alkaline denaturation a digoxigenin-labelled M. fermentans specific DNA probe was hybridized to the single stranded immobilized PCR fragment. Detection was performed by addition of an alkaline phosphatase conjugated anti-digoxigenin antibody. 4-methyl-umbelliferyl-phosphate was used as a fluorogenic substrate. Amplification of 10 fg chromosomal target DNA was detected by this 'DNA enzyme immuno assay (DEIA)' technique, corresponding to seven genome copies. Our study supports the presumption that the tuf gene proves to be a suitable target sequence for the PCR based detection of any bacterial species. Furthermore, hybridization of PCR fragments with radio-labelled DNA probes should no longer be necessary, because a very sensitive non-radioactive test system can easily be established with the 'DEIA' technique.

Detection of Mycoplasma pneumoniae by polymerase chain reaction and nonradioactive hybridization in microtiter plates.

In order to improve the diagnosis of a Mycoplasma pneumoniae infection, we developed a polymerase chain reaction (PCR)-based assay. The gene encoding elongation factor Tu (tuf) was selected as the target sequence. Oligonucleotides derived from variable stretches of the tuf gene were able to prime the amplification of a 950-bp fragment exclusively when M. pneumoniae DNA was used as the template. The sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal M. pneumoniae-specific oligonucleotide. The use of three to four genome copies for PCR was sufficient for obtaining a hybridization signal. In addition, we substituted radioactive filter hybridization with a microtiter plate assay. Via a biotin moiety of one PCR primer, the amplification products were immobilized on streptavidin-coated microtiter plates. Subsequent hybridization with a digoxigenin-labeled oligonucleotide resulted in the same sensitivity and specificity as those obtained by filter hybridization. Clinical application of the assay was performed on 102 throat swab specimens from patients with respiratory tract infections. Of 21 culture-positive samples, 19 were confirmed to be positive in the PCR-based assay (sensitivity, 90%). Furthermore, 14 of 19 seropositive but culture-negative samples gave a positive hybridization signal. Of 62 culture-negative and seronegative specimens, 60 gave a negative result in our assay (specificity, 97%). Of the 33 samples that were positive in our PCR-based assay, 5 samples initially gave false-negative results because of the presence of inhibitory substances in those specimens. Inhibition of Taq polymerase in these five cases was prevented by an additional step of phenol extraction and subsequent ethanol precipitation.

Glycation of serum albumin and its role in renal protein excretion and the development of diabetic nephropathy.

The present study was designed to investigate whether microalbuminuria at the onset of diabetic nephropathy might be partially due to the glycation of serum albumin. It is postulated elsewhere (Ghiggeri et al., Proc. Eur. Dial. Transplant. Assoc. 21 (1984) 633-636) that the glycation of serum albumin and the subsequent cationization may induce microalbuminuria. To investigate whether a relationship exists between the amount of glycated albumin in its cationized form and the development, and progression of diabetic nephropathy, the urinary excretion of glycated albumin was studied in diabetic patients. The diabetic patients (type I and II diabetes) were divided into groups according to their albumin excretion rates: group I diabetics had a normal albumin excretion (n = 30, x = 4.2 mg/12 h); group II diabetes displayed microalbuminuria (n = 17, x = 38.6 mg/12 h); group III diabetics displayed macroalbuminuria (n = 21, x = 582.5 mg/12 h). The fraction of glycated albumin in serum (Glyco Gel Test Kit) was 0.032 in group I, 0.042 in group II, and 0.038 in group III, all these values were significantly higher than the value for the controls (0.014%; n = 17, 2 alpha = 0.001) as measured with the Glyco Gel Test Kit. The concentration of glycated albumin in the urine of the controls and group I was below the detection limit. Urine in group II contained only a glycated albumin fraction of 0.0002 of total albumin, and the fraction for group III was 0.0008. Isoelectric focussing (IEF) and chromato-focussing revealed native albumin with an isoelectric point of 4.7-4.9, and anionic glycated albumin with a pI of 3.0-4.2.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence and expression in Escherichia coli of a Mycoplasma hominis gene encoding elongation factor Tu.

We describe the molecular cloning and the complete nucleotide (nt) sequence of a Mycoplasma hominis gene common to a broad range of Mycoplasma species, as defined by hybridization analysis with the cloned gene. Production of M. hominis protein in Escherichia coli was assayed by use of a monoclonal antibody. The nt sequence analysis revealed a 1194-bp open reading frame that could encode a 43 516-Da protein. Computer-aided sequence comparison indicated that the gene codes for elongation factor Tu.