The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA) suggests that the channel-forming domain contains beta-strands.

Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic ß-strands according to secondary structure predictions (residues 71-110, 158-167, 180-203, and 264-286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71-110 and HlyAΔ264-286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158-167 and HlyAΔ180-203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71-110 and HlyAΔ264-286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71-110, and HlyAΔ264-286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain ß-strands, possibly in addition to α-helical structures.

Immunoproteomic analysis of antibody in lymphocyte supernatant in patients with typhoid fever in Bangladesh.

We have previously shown that an assay based on detection of anti-Salmonella enterica serotype Typhi antibodies in supernatant of lymphocytes harvested from patients presenting with typhoid fever (antibody in lymphocyte supernatant [ALS] assay) can identify 100% of patients with blood culture-confirmed typhoid fever in Bangladesh. In order to define immunodominant proteins within the S. Typhi membrane preparation used as antigen in these prior studies and to identify potential biomarkers unique to S. Typhi bacteremic patients, we probed microarrays containing 2,724 S. Typhi proteins with ALS collected at the time of clinical presentation from 10 Bangladeshis with acute typhoid fever. We identified 62 immunoreactive antigens when evaluating both the IgG and IgA responses. Immune responses to 10 of these antigens discriminated between individuals with acute typhoid infection and healthy control individuals from areas where typhoid infection is endemic, as well as Bangladeshi patients presenting with fever who were subsequently confirmed to have a nontyphoid illness. Using an ALS enzyme-linked immunosorbent assay (ELISA) format and purified antigen, we then confirmed that immune responses against the antigen with the highest immunoreactivity (hemolysin E [HlyE]) correctly identified individuals with acute typhoid or paratyphoid fever in Dhaka, Bangladesh. These observations suggest that purified antigens could be used with ALS and corresponding acute-phase activated B lymphocytes in diagnostic platforms to identify acutely infected patients, even in areas where enteric fever is endemic.

Mutations affecting export and activity of cytolysin A from Escherichia coli.

Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix alphaA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix alphaG, the "beta-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

Characterization of anti-Salmonella enterica serotype Typhi antibody responses in bacteremic Bangladeshi patients by an immunoaffinity proteomics-based technology.

Salmonella enterica serotype Typhi is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide 50 to 90% protection for 2 to 5 years, and available practical diagnostic assays to identify individuals with typhoid fever lack sensitivity and/or specificity. Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines. Here we describe a platform immunoaffinity proteomics-based technology (IPT) that involves the use of columns charged with IgG, IgM, or IgA antibody fractions recovered from humans bacteremic with S. Typhi to capture S. Typhi proteins that were subsequently identified by mass spectrometry. This screening tool identifies immunogenic proteins recognized by antibodies from infected hosts. Using this technology and the plasma of patients with S. Typhi bacteremia in Bangladesh, we identified 57 proteins of S. Typhi, including proteins known to be immunogenic (PagC, HlyE, OmpA, and GroEL) and a number of proteins present in the human-restricted serotypes S. Typhi and S. Paratyphi A but rarely found in broader-host-range Salmonella spp. (HlyE, CdtB, PltA, and STY1364). We categorized identified proteins into a number of major groupings, including those involved in energy metabolism, protein synthesis, iron homeostasis, and biosynthetic and metabolic functions and those predicted to localize to the outer membrane. We assessed systemic and mucosal anti-HlyE responses in S. Typhi-infected patients and detected anti-HlyE responses at the time of clinical presentation in patients but not in controls. These findings could assist in the development of improved diagnostic assays.

ClyA cytolysin from Salmonella: distribution within the genus, regulation of expression by SlyA, and pore-forming characteristics.

Functional homologs of the Escherichia coli cytolysin A (clyA, hlyE, sheA) gene have recently been detected in Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A). In this study, analysis of a collection of Salmonella strains showed that all S. Typhi and S. Paratyphi A strains tested harbor an intact copy of the corresponding clyA variant, i.e. clyA(STy) and clyA(SPaA), respectively. On the other hand, clyA proved to be absent in the S. enterica serovar Paratyphi B and serovar Paratyphi C strains, in various non-typhoid S. enterica subsp. enterica serovars (Typhimurium, Enteritidis, Choleraesuis, Dublin, and Gallinarum), and in S. enterica subsp. arizonae and Salmonella bongori strains. When grown under normal laboratory conditions, the S. Typhi and S. Paratyphi A strains produced only basal amounts of ClyA protein and did not exhibit a clyA-dependent hemolytic phenotype. RT-PCR and immunoblot analyses as well as phenotypic data revealed, however, that the expression of clyA(STy) and clyA(SPaA) can be activated by the Salmonella transcription factor SlyA. In addition, osmotic protection assays and lipid bilayer experiments demonstrated that the hemolytic ClyA(STy) and ClyA(SPaA) proteins are effective pore-forming toxins which, similar to E. coli ClyA, generate large, stable, moderately cation-selective channels in target membranes. Taken together with our recent serological findings which have indicated that S. Typhi and S. Paratyphi A strains produce substantial amounts of ClyA during human infection, these data suggest that ClyA may play a role in S. Typhi and S. Paratyphi A pathogenesis.

Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae.

Cytolysin A (ClyA, HlyE, SheA) is a hemolytic pore-forming toxin found in Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A. In the present study, analysis of several Shigella strains revealed that they harbor only nonfunctional clyA gene copies that have been inactivated either by the integration of insertion sequence (IS) elements (Shigella dysenteriae, Shigella boydii, and Shigella sonnei strains) or by a frameshift mutation (Shigella flexneri). Shigella dysenteriae and S. boydii strains also exhibited IS-associated deletions at the clyA locus. PCR and Southern blot analyses as well as database searches indicated that clyA-related DNA sequences are completely absent in strains belonging to various other genera of the family Enterobacteriaceae. According to these data, ClyA may play a role only for a rather small subset of the enteric bacteria.

Linezolid resistance in Staphylococcus aureus: gene dosage effect, stability, fitness costs, and cross-resistances.

Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol.

Release of latent ClyA cytolysin from Escherichia coli mediated by a bacteriophage-associated putative holin (BlyA) from Borrelia burgdorferi.

Introduction of the Borrelia burgdorferi blyAB locus into Escherichia coli has recently been reported to cause a hemolytic phenotype that is dependent on the E. coli clyA (hlyE, sheA) gene (a cytolysin gene present in many E. coli strains, including E. coli K-12, which is repressed under standard in vitro growth conditions). The blyA gene product has been suggested to be a prophage-encoded holin, but the processes triggered in E. coli by the expression of blyA and/or blyB, which lead to the hemolytic phenotype, remained unclear. Here we show that expression of blyA in E. coli causes damage to the E. coli cell envelope and a clyA-dependent hemolytic phenotype, regardless whether blyB is present or absent. The expression of blyB in E. coli, on the other hand, did not have obvious phenotypic effects. Transcriptional studies demonstrated that the clyA gene is not induced in E. coli cells expressing blyA. Furthermore, protein analyses suggested that the impairment of the E. coli cell envelope by BlyA is responsible for the emergence of the hemolytic activity as it allows latent intracellular ClyA protein, derived from basal-level expression of the clyA gene, to leak into the medium and to lyse erythrocytes. These findings are compatible with the presumption that BlyA functions as a membrane-active holin.

Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus.

Thymidine-auxotrophic small colony variants (SCVs) of Staphylococcus aureus are frequently isolated from the chronically infected airways of patients suffering from cystic fibrosis. To date, little is known regarding the molecular mechanisms leading to the formation of this special phenotype, but the auxotrophism for thymidine suggests that impaired thymidine metabolism might play a major role. Sequence analysis of the thymidylate synthase-encoding thyA gene of six clinical thymidine-auxotrophic S. aureus SCVs revealed that all isolates had mutations within thyA. In five isolates the function of the thymidylate synthase was definitely impaired: three of them showed a truncation of the thyA coding sequence by nonsense or frame-shift mutations, in one further isolate the active site of the enzyme was affected by an internal 12-bp deletion, and another isolate had a 173-bp deletion spanning the 5'-terminal region of thyA and the preceding DNA sequence. The sixth isolate showed two amino acid substitutions within the thyA gene product. To confirm the importance of impaired thymidylate synthase synthesis or activity for the formation of the thymidine-auxotrophic SCV phenotype, we constructed a thyA knock-out mutant of a wild-type S. aureus strain. This mutant showed all characteristics of clinical SCVs, such as slow growth, decreased pigment production, reduced hemolytic activity, auxotrophism for thymidine, resistance to trimethoprim/sulfamethoxazol, and reduced plasma coagulase activity. Complementation of the thyA knock-out mutant with intact thyA in trans nearly restored the normal phenotype. In conclusion, these data confirm at the molecular level that impaired thymidylate synthase function is causative for the formation of the thymidine-auxotrophic SCV phenotype in S. aureus.

Serologic evidence for effective production of cytolysin A in Salmonella enterica serovars typhi and paratyphi A during human infection.

ClyASTy and ClyASPaA are closely related pore-forming cytolysins of Salmonella enterica serovars Typhi and Paratyphi A whose expression is strongly repressed under standard in vitro growth conditions. We show here that human infections by these pathogens cause a specific antibody response to ClyA, indicating effective toxin production during infection.

Compensatory adaptation to the loss of biological fitness associated with acquisition of fusidic acid resistance in Staphylococcus aureus.

Recent studies have shown that individual amino acid exchanges within elongation factor G (EF-G) cause fusidic acid resistance in Staphylococcus aureus. The data from the present study illustrate that the fusidic acid resistance-mediating amino acid substitutions P406L and H457Y are associated with a marked impairment of the biological fitness of S. aureus. In particular, strains producing EF-G derivatives with these mutations showed reduced growth, decreased plasma coagulase activity, and an impaired capability to compete with the isogenic wild-type strain. Second-site mutations within EF-G, such as A67T and S416F, that have been encountered in clinical fusidic acid-resistant isolates containing the amino acid exchanges P406L and H457Y, respectively, were shown not to contribute to resistance. Furthermore, the substitution A67T had no impact on the biological fitness in vitro. The exchange S416F, however, was found to function as a fitness-compensating mutation in S. aureus carrying the substitution H457Y in EF-G. In conclusion, the data presented in this report provide evidence at the molecular level that the deleterious effects of fusidic acid resistance-mediating exchanges within EF-G of S. aureus can be reduced considerably by specific compensating mutations in this target protein. This compensatory adaptation most likely plays a significant role in the stabilization of resistant bacteria within a given population.

Molecular analysis of cytolysin A (ClyA) in pathogenic Escherichia coli strains.

Cytolysin A (ClyA) of Escherichia coli is a pore-forming hemolytic protein encoded by the clyA (hlyE, sheA) gene that was first identified in E. coli K-12. In this study we examined various clinical E. coli isolates with regard to the presence and integrity of clyA. PCR and DNA sequence analyses demonstrated that 19 of 23 tested Shiga toxin-producing E. coli (STEC) strains, all 7 tested enteroinvasive E. coli (EIEC) strains, 6 of 8 enteroaggregative E. coli (EAEC) strains, and 4 of 7 tested enterotoxigenic E. coli (ETEC) strains possess a complete clyA gene. The remaining STEC, EAEC, and ETEC strains and 9 of the 17 tested enteropathogenic E. coli (EPEC) strains were shown to harbor mutant clyA derivatives containing 1-bp frameshift mutations that cause premature termination of the coding sequence. The other eight EPEC strains and all tested uropathogenic and new-born meningitis-associated E. coli strains (n = 14 and 3, respectively) carried only nonfunctional clyA fragments due to the deletion of two sequences of 493 bp and 204 or 217 bp at the clyA locus. Expression of clyA from clinical E. coli isolates proved to be positively controlled by the transcriptional regulator SlyA. Several tested E. coli strains harboring a functional clyA gene produced basal amounts of ClyA when grown under standard laboratory conditions, but most of them showed a clyA-dependent hemolytic phenotype only when SlyA was overexpressed. The presented data indicate that cytolysin A can play a role only for some of the pathogenic E. coli strains.

Molecular analysis of fusidic acid resistance in Staphylococcus aureus.

Fusidic acid is a potent antibiotic against severe Gram-positive infections that interferes with the function of elongation factor G (EF-G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF-G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid-resistant clinical S. aureus strains as well as in 10 fusidic acid-resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF-G. To confirm the importance of observed amino acid exchanges in EF-G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF-G derivatives with the exchanges P406L, H457Y and L461K were constructed by site-directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid-susceptible S. aureus strain RN4220 caused a fusidic acid-resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid-resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF-G for the generation of fusidic acid resistance in S. aureus.

Biological cost of rifampin resistance from the perspective of Staphylococcus aureus.

Resistance determinants that interfere with normal physiological processes in the bacterial cell usually cause a reduction in biological fitness. Fitness assays revealed that 17 of 18 in vitro-selected chromosomal mutations within the rpoB gene accounting for rifampin resistance in Staphylococcus aureus were associated with a reduction in the level of fitness. There was no obvious correlation between the level of resistance to rifampin and the level of fitness loss caused by rpoB mutations. Among 23 clinical rifampin-resistant S. aureus isolates from six countries, only seven different rpoB genotypes could be identified, whereby the mutation 481His-->Asn was present in 21 (91%) of these 23 isolates. The mutation 481His-->Asn, in turn, which confers low-level rifampin resistance on its own, was not shown to be associated with a cost of resistance in vitro. The restriction to distinct mutations that confer rifampin resistance in vivo, as demonstrated here, appears to be determined by the Darwinian fitness of the organisms.

Differential regulation of multiple proteins of Escherichia coli and Salmonella enterica serovar Typhimurium by the transcriptional regulator SlyA.

SlyA is a transcriptional regulator of Escherichia coli, Salmonella enterica, and other bacteria belonging to the ENTEROBACTERIACEAE: The SlyA protein has been shown to be involved in the virulence of S. enterica serovar Typhimurium, but its role in E. coli is unclear. In this study, we employed the proteome technology to analyze the SlyA regulons of enteroinvasive E. coli (EIEC) and Salmonella serovar Typhimurium. In both cases, comparative analysis of the two-dimensional protein maps of a wild-type strain, a SlyA-overproducing derivative, and a corresponding slyA mutant revealed numerous proteins whose expression appeared to be either positively or negatively controlled by SlyA. Twenty of the putative SlyA-induced proteins and 13 of the putative SlyA-repressed proteins of the tested EIEC strain were identified by mass spectrometry. The former proteins included several molecular chaperones (GroEL, GroES, DnaK, GrpE, and CbpA), proteins involved in acid resistance (HdeA, HdeB, and GadA), the "starvation lipoprotein" (Slp), cytolysin ClyA (HlyE or SheA), and several enzymes involved in metabolic pathways, whereas most of the latter proteins proved to be biosynthetic enzymes. Consistently, the resistance of the EIEC slyA mutant to heat and acid stress was impaired compared to that of the wild-type strain. Furthermore, the implication of SlyA in the regulation of several of the identified E. coli proteins was confirmed at the level of transcription with lacZ fusions. Twenty-three of the Salmonella serovar Typhimurium proteins found to be affected by SlyA were also identified by mass spectrometry. With the exception of GroEL these differed from those identified in the EIEC strain and included proteins involved in various processes. The data suggest that gene regulation by SlyA might be crucial for intracellular survival and/or replication of both EIEC and Salmonella serovar Typhimurium in phagocytic host cells.