RESUMEN
Western blot analysis of Orientia tsutsugamushi whole-cell lysates with scrub typhus patient sera has identified at least five protein antigens of O. tsutsugamushi with molecular sizes of 22 kDa, 47 kDa, 56 kDa, 58 kDa, and 110 kDa. In this study, sera from serial bleedings of 108 patients were used to study the kinetics and the magnitude of specific antibody responses against the 47-kDa and 56-kDa antigens. Recombinant protein of the conserved 47-kDa antigen (r47b) or a mixture of truncated 56-kDa antigen (r56s) from three prototype strains was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). Our results showed that 76% and 93% of these patients had elevated IgM and IgG against r47b, respectively, and 98% and 100% had elevated IgM and IgG against r56s, respectively. The kinetics of antibody responses against r47b and r56s can be grouped into three patterns. In the first type of response, IgM and IgG against r47b and r56s appeared about the same time. The IgM and IgG titers against r56s were much higher than those against r47b. In the second type of response, induction of IgM appeared to be similar to that in the first type. The major difference to the first type is that the IgG titers against r47b were induced at least 1 week later than those against the r56s. The third type showed strong IgG responses against both r47b and r56s, and low or no IgM responses indicated a secondary infection. This is the first systematic investigation of antibody response kinetics against the conserved 47-kDa antigen versus the variable 56-kDa antigen in scrub typhus patients.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/sangre , Orientia tsutsugamushi/inmunología , Tifus por Ácaros/inmunología , Formación de Anticuerpos , Antígenos Bacterianos/química , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Peso Molecular , Proteínas Recombinantes , Factores de TiempoRESUMEN
Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus. We analyzed the proteome of the virulent Breinl strain of R. prowazekii purified from infected egg yolk sacs. Total proteins from purified R. prowazekii Breinl strain were reduced by dithiothreitol, alkylated by iodoacetic acid and digested with trypsin followed by analysis with an integrated two-dimensional liquid chromatography and mass spectrometry system (2D-LC/MS/MS). A comparison was made using previously analyzed proteome of the Madrid E strain and current analysis of the Breinl strain. For Breinl 251 proteins were identified, representing 30% of the total protein-encoding genes, using a shotgun 2D-LC/MS/MS proteomic approach. This result is identical to that of Madrid E strain. Among the identified proteins, 33 from Breinl and 37 from Madrid E have an unknown function. A methyltransferase, RP028/RP027, whose gene is mutated in the avirulent Madrid E strain but not in the virulent Breinl strain, was only detectable in the Breinl strain, consistent with the genetic mutation in Madrid E. This result suggests the possible relationship between this gene product and the virulence of the strains.
Asunto(s)
Proteómica , Rickettsia prowazekii/metabolismo , Rickettsia prowazekii/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , Datos de Secuencia Molecular , Análisis por Matrices de Proteínas , Rickettsia prowazekii/química , Rickettsia prowazekii/clasificación , Vacunas contra Rickettsia/genética , Vacunas contra Rickettsia/inmunología , Espectrometría de Masas en Tándem , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Virulencia/fisiologíaRESUMEN
Homoserine transsuccinylase catalyzes the succinylation of homoserine in several bacterial species, the first unique step in methionine biosynthesis in these organisms. The enzyme from Escherichia coli is reported to be a dimer and uses a ping-pong catalytic mechanism involving transfer of succinate from succinyl-CoA to an enzyme nucleophile, followed by transfer to homoserine to form O-succinylhomoserine. Site-directed mutagenesis and steady-state kinetics were used to identify three amino acids that participate in catalysis. Mutation of cysteine-142 to serine or alanine eliminated all measurable activity, suggesting this amino acid acts as the catalytic nucleophile. Cysteine nucleophiles are often deprotonated by histidine residues, and histidine-235 was identified as the sole absolutely conserved histidine residue among family members. This residue was mutated to both alanine and asparagine, and no activity was observed with either mutant. Lysine-47 had been previously identified as an essential residue. Mutation of this amino acid to arginine reduced catalytic activity by greater than 90%, while mutation to alanine yielded an enzyme with <1% of wild-type activity. A pH-rate profile of the K47R mutant demonstrated that this amino acid participates in the first half reaction. The data presented here provide the first detailed description of the homoserine transsuccinylase active site and provide a framework for additional mechanistic characterization of this enzyme.