Expression of the Moraxella catarrhalis UspA1 protein undergoes phase variation and is regulated at the transcriptional level.

The UspA1 protein of Moraxella catarrhalis has been shown to function as an adhesin that mediates adherence to human epithelial cell lines in vitro (E. R. Lafontaine, L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen, J. Bacteriol. 182:1364-1373, 2000). In the present study, cell lysates prepared from individual colonies of several M. catarrhalis wild-type strains were analyzed by Western blot analysis using monoclonal antibodies (MAbs) specific for the UspA1 protein. Expression of UspA1 was shown to exhibit phase variation that was correlated with both adherence ability in vitro and the number of guanine (G) residues contained within a homopolymeric [poly(G)]tract located upstream of the uspA1 open reading frame (ORF). Nucleotide sequence analysis revealed that isolates expressing relatively high levels of UspA1 had 10 G residues in their uspA1 poly(G)tracts, whereas isolates that expressed much lower levels of UspA1 had 9 G residues. This poly(G) tract was located 30 nucleotides (nt) upstream of the uspA1 ORF and 168 nt downstream of the uspA1 transcriptional start site. Primer extension experiments, RNA slot blot analysis, and cat reporter constructs were used to demonstrate that M. catarrhalis isolates with 10 G residues in their uspA1 poly(G) tracts expressed two-to threefold more uspA1 mRNA than did isolates which had 9 G residues in their poly(G)tracts. Northern hybridization analysis revealed that an intact uspA1 mRNA was readily detectable in RNA from M. catarrhalis isolates that had 10 G residues in their uspA1 poly(G) tracts, whereas no full-length uspA1 mRNA was observed in isolates whose poly(G)tracts contained 9 G residues. M. catarrhalis strain O35E uspA1 genes that contained wild-type and mutated poly(G) tracts were expressed in Haemophilus influenzae to demonstrate that the length and composition of the poly(G)tract affected expression of UspA1.

RegA, iron, and growth phase regulate expression of the Pseudomonas aeruginosa tol-oprL gene cluster.

The tol-oprL region in Pseudomonas aeruginosa appears to be involved in pyocin uptake and required for cell viability. The complete nucleotide sequences of the tolQRA and oprL genes as well as the incomplete sequences of tolB and orf2 have been previously reported. In addition, the sequence of a P. aeruginosa iron-regulated gene (pig6) has been described and found to share homology with an open reading frame located upstream of the Escherichia coli tolQRA genes (U. A. Ochsner and M. L. Vasil, Proc. Natl. Acad. Sci. USA 93:4409-4414, 1996). In this study, we cloned the remainder of the P. aeruginosa tol-oprL gene cluster and determined its nucleotide sequence. This cluster was found to consist of seven genes in the order orf1 tolQ tolR tolA tolB oprL orf2. Transcriptional analysis of this gene cluster was performed by detecting the presence of mRNAs spanning adjacent genes as well as by using a promoterless lacZ reporter gene fused to each of the seven genes contained in the tol-oprL locus. The results show that there are three major transcriptional units or operons in this region, orf1-tolQRA, tolB, and oprL-orf2, in contrast to the E. coli tol-pal region, where there are only two operons, orf1-tolQRA and tolB-pal-orf2. Analysis of gene expression indicated that the tol-oprL genes of P. aeruginosa are both iron and growth phase modulated. The first operon, orf1-tolQRA, is iron regulated throughout growth, but iron-regulated expression of tolB and oprL fusions occurs only in late log phase. The expression of the three operons was significantly less repressed by iron in fur mutants than in the wild-type strain, suggesting the involvement of Fur in the iron regulation of all three operons. RegA is a positive yet nonessential regulator of tol-oprL expression.

The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro.

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.

Characterization of the Moraxella catarrhalis uspA1 and uspA2 genes and their encoded products.

The uspA1 and uspA2 genes of M. catarrhalis O35E encode two different surface-exposed proteins which were previously shown to share a 140-amino-acid region with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367-4377, 1997). The N-terminal amino acid sequences of the mature forms of both UspA1 and UspA2 from strain O35E were determined after enzymatic treatment to remove the N-terminal pyroglutamyl residue that had blocked Edman degradation. Mass spectrometric analysis indicated that the molecular mass of UspA1 from M. catarrhalis O35E was 83,500 +/- 116 Da. Nucleotide sequence analysis of the uspA1 and uspA2 genes from three other M. catarrhalis strains (TTA24, ATCC 25238, and V1171) revealed that the encoded protein products were very similar to those from strain O35E. Western blot analysis was used to confirm that each of these three strains of M. catarrhalis expressed both UspA1 and UspA2 proteins. Several different and repetitive amino acid motifs were present in both UspA1 and UspA2 from these four strains, and some of these were predicted to form coiled coils. Linear DNA templates were used in an in vitro transcription-translation system to determine the sizes of the monomeric forms of the UspA1 and UspA2 proteins from strains O35E and TTA24.

Effects of iron and temperature on expression of the Pseudomonas aeruginosa tolQRA genes: role of the ferric uptake regulator.

The tolQRA genes have been recently identified in Pseudomonas aeruginosa PAO. In this study, we examined the effect of iron and temperature on tolQRA expression. A promoterless lacZ gene was introduced downstream of plasmid-encoded tolQ and tolA, and expression was monitored by measuring beta-galactosidase activity of cultures. Addition of 25 microM FeCl3 to the culture medium reduced tolQRA expression by 50 to 60% in PAO but by only 25% in the fur mutant PAO A4. Northern hybridization analysis revealed that iron regulation occurs at the level of transcription and involves the P. aeruginosa ferric uptake regulator (Fur). Primer extension analysis was used to identify the proposed transcriptional start site of tolA. Although a putative Fur box was identified 20 bp upstream of the proposed start site, purified Fur did not bind to the tolA or tolQR promoter regions in an in vitro gel retardation assay. Therefore, iron regulation of the tol genes appears to involve an intermediate regulatory gene. Expression of tolQR and tolA was optimal at 37 degrees C and was reduced by 40 to 50% when cultures were grown at either 42 or 25 degreesC. Growth in high-iron medium at 25 degrees C further reduced tolQR and tolA expression.

Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis 035E.

The UspA surface antigen of Moraxella catarrhalis was recently shown to be comprised of two different proteins (UspA1 and UspA2) which share an internal region containing 140 amino acids with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367-4377, 1997). Isogenic uspA1, uspA2, and uspA1 uspA2 mutants were tested in a number of in vitro systems to determine what effect these mutations, either individually or together, might exert on the phenotype of M. catarrhalis 035E. Monoclonal antibodies specific for UspA1 or UspA2 were used in an indirect antibody accessibility assay to prove that both of these proteins were expressed on the surface of M. catarrhalis. All three mutants grew in vitro at the same rate and did not exhibit autoagglutination or hemagglutination properties that were detectably different from those of the wild-type parent strain. When tested for the ability to adhere to human epithelial cells, the wild-type parent strain and the uspA2 mutant readily attached to Chang conjunctival cells. In contrast, the uspA1 mutant and the uspA1 uspA2 double mutant both attached to these epithelial cells at a level nearly 2 orders of magnitude lower than that obtained with the wild-type parent strain, a result which suggested that expression of UspA1 by M. catarrhalis is essential for attachment to these epithelial cells. Both the wild-type parent strain and the uspA1 mutant were resistant to the bactericidal activity of normal human serum, whereas the uspA2 mutant and the uspA1 uspA2 double mutant were readily killed by this serum. This latter result indicated that the presence of UspA2 is essential for expression of serum resistance by M. catarrhalis.

Identification and characterization of the tolQRA genes of Pseudomonas aeruginosa.

The tolQ, tolR, and tolA genes from Pseudomonas aeruginosa PAO were cloned using degenerate oligonucleotide PCR primers designed based on conserved transmembrane regions of Escherichia coli TolQ and TolR and E. coli and Pseudomonas putida ExbB and ExbD. The resulting PCR product was used as a probe to isolate a 6.5-kb DNA fragment containing P. aeruginosa tolQ, tolR, and tolA. The nucleotide sequence of a 2.9-kb DNA fragment containing the tolQ, tolR, and tolA genes was determined. The DNA sequence predicts TolQ to be a 25,250-Da protein exhibiting 53% identity to E. coli TolQ. TolR is predicted to be a 15,788-Da protein, sharing 38% identity with the E. coli TolR protein. The P. aeruginosa tolA sequence predicts a 37,813-Da protein with 27% identity to the E. coli TolA. The P. aeruginosa TolQRA proteins were expressed in E. coli minicells. Analysis of plasmid-encoded tolQ::lacZ and tolA::lacZ promoter fusions in E. coli indicated that these genes are expressed at different levels, suggesting transcription from different promoters. Transcriptional analysis of the tol genes in P. aeruginosa revealed that the tolQ and tolR genes are cotranscribed as an approximately 1.5-kb transcript and that tolA is transcribed from its own promoter as an approximately 1.2-kb transcript. The P. aeruginosa Tol proteins were functionally unable to complement E. coli tol mutants, although P. aeruginosa TolQ was able to complement the iron-limited growth of an E. coli exbB mutant. Introduction of the tolQRA genes in the tol-like mutant PAO 1652 restored pyocin AR41 killing, indicating that the Tol proteins are involved in the uptake of pyocin AR41 in P. aeruginosa. Attempts to inactivate the chromosomal copy of the tolA or tolQ gene in the parent strain PAO proved to be unsuccessful, and we propose that inactivation of these genes in P. aeruginosa results in a lethal phenotype.

Motor function in the excluded esophagus and its implications in the management of patients with unresectable carcinoma of the esophagus.

Palliative exclusion of the esophagus by use of transposed stomach was performed in a 63-year-old woman with unresectable cervical esophageal cancer. Twelve weeks after this operation, motor function of the excluded esophagus was assessed. All voluntary swallows produced a motor response in the esophageal body. Eighty-five per cent of the contractions were peristaltic and generated mean pressures of 28 mmHg. Fifteen per cent of the deglutitions were followed by nonpropulsive waves with mean pressures of 24 mmHg. Spontaneous tertiary activity occurred at a rate of 2.5 contractions per minute with an amplitude of 16 mmHg. Motor function in the excluded esophagus persists after bypass of the organ. This suggests that the excluded esophagus should be decompressed after surgery to prevent "blowout" of its closed ends.

Effects of total fundoplication on function of the esophagus after myotomy for achalasia.

Twelve patients underwent distal esophageal myotomy for achalasia. After denuding the esophageal mucosa over 50 percent of its circumference, a short (2 cm) total fundoplication was performed over a size 56 mercury bougie. Clinical evaluation showed marked symptomatic improvement. Obstructive symptoms are minimal, and no reflux symptoms were noted. Manometric documentation showed a significant decrease in resting esophageal and lower esophageal sphincter pressure. Contraction pressure was also lowered, and peristalsis returned in 36 percent of the waves in the proximal esophagus. Radiologic and scanning documentation revealed slow emptying without evidence of significant reflux. Endoscopic evaluation revealed no esophagitis after 19 months' follow-up.