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Identification of a new site for ferrichrome transport by comparison of the FhuA proteins of Escherichia coli, Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans.

The fhuA genes of Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans were sequenced and compared with the known fhuA sequence of Escherichia coli. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which in E. coli controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and phi80. All the FhuA proteins contained the region in the gating loops required in E. coli for ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of S. paratyphi B which is infected by the E. coli phages, whereas two of the subdomains were deleted in S. typhimurium and P. agglomerans which are resistant to the E. coli phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated E. coli FhuA with regard to transport of ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to phi80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of S. paratyphi B and S. typhimurium displayed S. paratyphi B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions of S. paratyphi B and displayed S. typhimurium FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the S. typhimurium FhuA. The central segment of the S. paratyphi B FhuA flanked on both sides by S. typhimurium FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of ferrichrome and albomycin, identified an additional loop for ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.

Amino Acid Sequence↗

The pathogenicity of strains of Salmonella paratyphi B and Salmonella java.

AIMS: To relate the diseases caused by strains of Salmonella paratyphi B and S. java to pathogenic mechanisms expressed by these bacteria for the purpose of organism discrimination. METHODS AND RESULTS: Epidemiological data relating to cases of disease caused by strains of S. paratyphi B and S. java, isolated over a 10-year period, were analysed with respect to patients' symptoms, particularly those involving enteric fever. Strains of S. paratyphi B and S. java were also examined for a range of known pathogenic mechanisms. Infection with S. paratyphi B involved pyrexia in 12.5% of patients compared with 2.2% of patients infected with S. java. These organisms could not be differentiated based on the pathogenic properties examined. CONCLUSIONS: Strains of S. paratyphi B appear not to be a major cause of enteric fever but primarily a cause of gastroenteritis, in common with S. java. Both organisms express similar pathogenic mechanisms, and strains of S. java are probably d-tartrate utilizing variants of S. paratyphi B. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of S. paratyphi B are very closely related organisms, primarily causing gastroenteritis. From this study it would appear that strains of S. paratyphi B are not a major cause of enteric fever.

Adolescent↗

Utilization of d-tartaric acid by Salmonella paratyphi B and Salmonella java: comparison of anaerobic plate test, lead acetate test and turbidity test.

d-Tartrate dehydrase of Salmonella java is an oxygen-sensitive enzyme active in cultures incubated under the poorly aerated conditions of static culture but not in fully aerated shaken cultures nor on plates incubated aerobically. On plates of d-tartrate minimal agar incubated anaerobically the enzyme or the degradation products of d-tartrate are exported from d-tartrate-positive cells and are available to d-tartrate-negative bacteria. This may give misleading growth results when d-tartrate-positive and d-tartrate-negative strains are tested for growth on the same plate of d-tartrate minimal agar. The lead-acetate test terminated at 24 h, the 24 h turbidity test and the ability to grow on d-tartrate minimal agar within 48 h differentiated 53 S. paratyphi B strains that were negative in each of the three tests from 76 S. java that were positive in each of the tests. An intermediate group of eight strains utilized d-tartrate in Difco bacto-peptone water to give a positive lead acetate reaction at 2 days, were stimulated to a varying degree by d-tartrate in Oxoid peptone water within the same period of incubation and grew poorly on d-tartrate minimal agar. These latter strains may be deficient in a permease controlling uptake of d-tartrate or export of d-tartrate dehydrase. Inability to utilize d-tartrate is unlikely to be the single character accountable for the reputed enhanced pathogenicity of S. paratyphi B when compared with S. java. Indications for the existence of an enzyme, complementary to and mutually exclusive with d-tartrate dehydrase, that has a positive correlation with pathogenicity are discussed.

Anaerobiosis↗

Isolation and characterization of an outer membrane protein of Salmonella paratyphi B: a mitogen and polyclonal activator of human B lymphocytes.

Salmonella paratyphi B (S. paratyphi B) has been previously characterized as a human T-independent polyclonal B cell activator. To define further the nature of the bacterial structure responsible for these properties, we studied the effects of autoclaving and enzyme treatment of S. paratyphi B on its stimulatory capacity. We found that both autoclaving and papain treatment decreased the ability of S. paratyphi B to induce B cell activation, while trypsin treatment did not affect this capacity. Neither type of treatment affected the binding of S. paratyphi B to lymphocytes, suggesting that binding and B cell stimulation are mediated by different structures. The observation that B cell stimulation was significantly reduced by papain treatment led us to attempt to purify membrane proteins so that we could investigate whether they shared the stimulating capacity of S. paratyphi B. A water-insoluble, 43-45,000 mol. wt. protein, rich in aspartic acid, glutamine, glycine, alanine and leucine, similar in mol. wt. and physicochemical chemical properties to the porins of other gram negative bacteria, was isolated and designated as outer membrane protein (OMP). This protein was equally efficient to S. paratyphi B in inducing T-independent B cell activation. By performing time-course studies of [3H]-thymidine incorporation we observed a burst of mitogenic activity after stimulation of PBL or purified B cells with both S. paratyphi B and OMP peaking at 48-96 hr of culture (compared to 96-120 hr for the PWM proliferation peak), and with a magnitude of roughly 10% of that observed after PWM stimulation. Given the fact that the proportion of B lymphocytes in PBL is 4-12%, it appears likely that the proliferation burst seen with S. paratyphi B and OMP corresponds to a mitogenic effect mainly restricted to the B cell population.

Amino Acids↗

Genotypic typing and phylogenetic analysis of Salmonella paratyphi B and S. java with IS200.

Salmonella paratyphi B and Salmonella java are biovars of common serotype 1,4,[5],12:b:1,2 which respectively cause human paratyphoid fever and gastroenteritis. In order to define genotypes and phylogenetic relationships in this group, we examined representative strains for restriction fragment length polymorphisms (RFLPs) in and around the 16S ribosomal RNA (rrn) genes, and the five to eleven insertion sites of the Salmonella-specific DNA insertion sequence IS200. One of four 16S rrn profiles was predominant, and was shared by the majority of strains, irrespective of their designation as S. paratyphi B or S. java. On the other hand, thirteen unique IS200 profiles were found and this technique was able to distinguish, for the first time, distinct genotypes for S. paratyphi B and S. java. One of the S. paratyphi B profiles, Spj-IP1.0, represented a globally-distributed clone. Greater diversity was detected within IS200 profiles of S. java than within those of S. paratyphi B. IS200 profiles described a phylogenetic complex in which strains of both biovars could be placed. They constituted reproducible molecular fingerprints, which could be compared in a band-matching database suitable for molecular epidemiological typing.

Bacterial Typing Techniques↗

Polyclonal activation of human peripheral blood B lymphocytes by formaldehyde-fixed Salmonella paratyphi B. I. Immunoglobulin production without DNA synthesis.

A "new" polyclonal activator of human peripheral blood B cells, formaldehyde-fixed Salmonella paratyphi B, is described. This bacterium does not stimulate cell proliferation as measured by incorporation of tritiated thymidine but does stimulate a subpopulation of B cells to secrete large amounts of IgM, IgG, and IgA in 7-day cell cultures. The immunoglobulins (Ig) produced by cells responding to S. paratyphi B are not specific antibodies against the bacterial antigens. In comparison with other B cell activators (pokeweed mitogen, Staphylococcus aureus Cowan I, and lipopolysaccharide), S. paratyphi B stimulation produced greater amounts of IgM but less IgG than pokeweed mitogen (PWM) or S. aureus Cowan I; lipopolysaccharide failed to stimulate significant Ig production on day 7 in most cases. In addition, the response to S. paratyphi apparently did not require T cell collaboration. These results suggest that the B cell subpopulation(s) responding to S. paratyphi B may be more differentiated B cells than those responding to either PWM or S. aureus Cowan I. Peripheral blood mononuclear cells from five patients with common variable immunodeficiency without evidence of abnormal suppressor T cells or monocytes failed to respond to S. paratyphi B, whereas cells from two of the same patients responded well to S. aureus Cowan I and partially to PWM. Thus, S. paratyphi B appears to be superior to other B cell activators for studies of B cell function in normal and abnormal states.

B-Lymphocytes↗

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella paratyphi B.

The genomic cleavage map of Salmonella paratyphi B was determined through digestion with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 19 XbaI sites, 10 BlnI sites, and 7 CeuI sites. The fragments were arranged in order through excision of fragments from the gel, redigestion with a second enzyme, end labelling with 32P, and reelectrophoresis. Tn10 transposons inserted in 61 different genes of S. typhimurium LT2 were transduced by use of bacteriophage P22 into S. paratyphi B. The locations of Tn10 insertions on the chromosome of S. paratyphi B were determined by use of XbaI and BlnI sites in Tn10, revealing the positions of genes with Tn10 insertions in S. paratyphi B. All seven CeuI sites (in rrl genes for 23S rRNA) and most of the XbaI and BlnI sites in rrn genes for Glt-tRNA are conserved, but only about half of the XbaI and BlnI sites outside rrn genes are conserved. Gene order is identical in the 68 genes that we could compare between S. paratyphi B and S. typhimurium LT2, and the lengths of intervals between the genes are often the same, but there are several instances of differences in interval lengths, indicating that insertions or deletions of DNA have occurred during the evolutionary divergence of these bacteria.

Bacteriophage P22↗

Polyclonal activation of human peripheral blood B lymphocytes by formaldehyde-fixed Salmonella paratyphi B. II. Heterogeneity of B lymphocytes.

B-cell 'activation' in cultures stimulated with pokeweed mitogen (PWM), Staphylococcus aureus strain Cowan I, or formaldehyde-fixed Salmonella paratyphi B (SPB) was evaluated by enumeration of cells secreting immunoglobulin (Ig) and by quantitation of Ig released into culture supernatants. A dissociation between these two values was found after day 6 in cultures activated with PWM or SPB, suggesting that Ig-secreting cells (ISC) are heterogeneous in terms of Ig secretion rate. Generation of ISC in cultures activated with PWM or SPB was partially inhibited by hydroxyurea, but Ig levels in culture supernatants were not affected. These results indicate that there are at least two subpopulations of ISC in stimulated peripheral blood lymphocyte cultures, one sensitive to, and the other resistant to, hydroxyurea. The hydroxyurea-resistant subpopulation appeared to be more mature and to release most all of the Ig detected in culture supernatants. Furthermore, time-course studies of ISC numbers and Ig levels showed that each ISC in SPB-stimulated cultures (but not in PWM-stimulated cultures) was more active in Ig synthesis and secretion after day 8 than before day 6, indicating that after day 8 most of the ISC in cultures activated with SPB were hydroxyurea-resistant. These studies suggest that SPB is another useful polyclonal B-cell 'activator' for studies of human B-cell differentiation and function, and that SPB defines two distinct subsets of B cells.

B-Lymphocytes↗