Sialic Acid metabolism and systemic pasteurellosis

Abstract

Pasteurella multocida subsp. multocida is a commensal and opportunistic pathogen of food animals, wildlife, and pets and a zoonotic cause of human infection arising from contacts with these animals. Here, an investigation of multiple serotype A strains demonstrated the occurrence of membrane sialyltransferase. Although P. multocida lacks the genes for the two earliest steps in de novo sialic acid synthesis, adding sialic acid to the growth medium resulted in uptake, activation, and subsequent transfer of sialic acid to a membrane acceptor resembling lipooligosaccharide. Two candidate-activating enzymes with homology to Escherichia coli cytidine 5'-monophospho-N-acetylneuraminate synthetase were overproduced as histidine-tagged polypeptides. The synthetase encoded by pm0187 was at least 37 times more active than the pm1710 gene product, suggesting pm0187 encodes the primary sialic acid cytidylyltransferase in P. multocida. A sialate aldolase (pm1715) mutant unable to initiate dissimilation of internalized sialic acid was not attenuated in the CD-1 mouse model of systemic pasteurellosis, indicating that the nutritional function of sialate catabolism is not required for systemic disease. In contrast, the attenuation of a sialate uptake-deficient mutant supports the essential role in pathogenesis of a sialylation mechanism that is dependent on an environmental (host) supply of sialic acid. The combined results provide the first direct evidence of sialylation by a precursor scavenging mechanism in pasteurellae and of a potential tripartite ATP-independent periplasmic sialate transporter in any species.

FIG. 1.

Genetic organization of sialocatabolic systems in selected gram-negative bacteria. On the basis of the known functions encoded by the nan genes in E. coli (A) (nanR, transcriptional regulator; nanA, sialate lyase; nanT, sialate transporter; nanE, ManNAc 6-phosphate epimerase; nanK, ManNAc kinase, and yhcH, function unknown [27, 40, 51]), homologous genes in H. influenzae (B) and P. multocida (C) were assigned equivalent functions as indicated by the color-coded ORFs (large arrows). Note that the function of pm1710 is unknown, but is presumed to encode a cytidyltransferase, whereas HI0141 and HI0140 encode glucosamine deaminase and N-acetylglucosamine deacetylase, respectively. Bent arrows indicate known or predicted promoters. The functions of HI0148 and pm1707 also are unknown. Open triangles indicate the insertion of the kanamycin-resistant transposon described in reference . Arrows underneath triangles indicate transcriptional direction of the insertionally inactivated genes. Note that an insertion in the same transcriptional orientation as the inactivated gene is nonpolar (14).

FIG. 2.

Model of sialometabolism in P. multocida. Free sialic acid (Neu5Ac_out) may be produced by endogenous sialidase (NanH or NanB) glycolytic activity (scissors) against the sialic acid attached (Neu5Ac_bound) to sialoglycoconjugates (SGC) or endogenous Neu5Ac bound to LOS. Question marks indicate no evidence was obtained for release of cell-bound Neu5Ac by endogenous sialiases. Reactions marked by a cross do not occur in P. multocida, either because the gene products are absent (NeuB and NeuC), or unlikely to carry out the orthologous function (NanT). Evidence for Neu5Ac uptake, activation, sialyltransfer, and sialate dissimilation is summarized in the text. OM, outer membrane. IM, inner membrane; PEP, phosphoenolpyruvate; Pyr, pyruvate.

FIG. 3.

Complementation of EV5 (neuA). EV5 is an E. coli K-12/K1 hybrid strain with a mutation in neuA. This gene normally encodes CMP-sialic acid synthetase, which catalyzes a necessary step in polysialic acid (K1 antigen) synthesis (55). To detect complementation of the neuA defect in EV5, bacteria expressing basal amounts of pm0187 or pm1710 are cross-streaked against K1-specific bacteriophage painted (arrows) down the center of the plate. Strains expressing the K1 capsule are sensitive to the lytic action of the bacteriophage, whereas those not expressing the capsule are resistant and grow beyond the phage streak. (A) Complementation by pSX1001 (pm0187); (B and C) lack of complementation by two independent EV5 transformants harboring the two independent pSX1000 constructs (pm1710). (D) EV5 transformed with pUC18.

FIG. 4.

Biochemical detection of CMP-sialic acid synthetase. (A) Approximately 125 μg of soluble protein prepared from strain Pm70 grown in BHI was incubated with CTP and radiolabeled Neu5Ac for 1 h at 37°C prior to paper chromatography in solvent system I (lane 3). Labeled Neu5Ac (lane 1) or CMP-Neu5Ac (lane 2) standards are shown for comparison. Relative migration (R_f) of the standards and products are given by the value shown for each spot. The R_f (0.83) of ManNAc was previously determined (51). (B) Thin-layer chromatographic analysis of CMP-sialic acid synthetase (45). Labeled Neu5Ac standard (spotted) is shown in lane 1; samples (10 μl) in all other lanes were applied as streaks prior to chromatography and autoradiographic detection. Lane 2 shows quantitative conversion of input Neu5Ac to CMP-Neu5Ac in 1 h in the presence of 30 μg of soluble protein prepared from E. coli BL21(DE3) Star (induced with IPTG) harboring pSX1001. Lane 3 shows partial conversion by a tenfold dilution of the extract, while lane 4 indicates no conversion in the presence of 30 μg of protein that was boiled for 5 min prior to assay. Lanes 5 to 7 show the identical sequence of reactions for an extract prepared from E. coli harboring pSX1000 (construct tested in Fig. 3C).

FIG. 5.

Relative Neu5Ac activation by pm0187 or pm1710 gene products. Aliquots containing 14, 28, 56, or 113 μg of protein from an induced soluble extract of strain BL21(DE3) Star harboring pSX1001 (pm0187) were incubated with CTP and radiolabeled Neu5Ac for 30 min at 37°C. The nucleotide sugar produced by the reaction (inset) was quantified and expressed as picomoles of CMP-Neu5Ac under the defined assay conditions (•). Aliquots containing 40, 59, 159, or 318 μg of protein were assayed in the same manner (○) with an extract of the induced strain harboring pSX1000 (pm1710). Values in parentheses indicate the specific activities of each enzyme preparation determined from the linear portion of the curves, indicating at least 37 times greater activity in the extract containing the pm0187 gene product. Extracts contained similar amounts of overproduced polypeptides as judged after SDS-PAGE and staining with Coomassie blue.

FIG. 6.

Biochemical characterization of P. multocida aldolase (pm1715) mutants. Approximately 100 μg of soluble protein from Pm70A (lane 1), Pm70 (lane 2), TF5 (lane 3), TF5E (lane 4), or TF5A (lane 5) were incubated for 1 h with radiolabeled Neu5Ac (lane 6) in a total volume of 30 μl prior to fractionation of reaction products or substrate by paper chromatography in solvent system II. Lane 7 shows the migration of radiolabeled ManNAc standard. Note the nearly quantitative conversion of the labeled Neu5Ac to ManNAc by the two wild-type extracts and that of TF5E, as well as the absence of detectable aldolase activity in extracts from the two mutants with defects in pm1715.

FIG. 7.

Direct demonstration of precursor scavenging. Pm70 or Pm70A were cultured in HTM and exposed to radiolabeled Neu5Ac as described in Materials and Methods. Harvested cells were fractionated into soluble and insoluble (total membrane) samples. The soluble samples from Pm70A (lane 1) or Pm70 (lane 2) were subjected to paper chromatography and the radiolabel in the extracts visualized by autoradiography. Lanes 3 and 4 show free ManNAc and Neu5Ac standards, respectively. The position of CMP-Neu5Ac was inferred from its migration relative to the two standards.

FIG. 8.

Detection of endogenous acceptor under in vivo growth conditions. Coomassie blue-stained and fluorographically enhanced soluble (lanes 2 and 3) and membrane (lanes 4 to 7) proteins from Pm70 (lanes 2, 4, and 6) or Pm70A (lanes 3, 5, and 7). Lanes 6 and 7 contain twice the protein content as lanes 4 and 5. Lane 1, molecular weight markers with the weight of the lowest marker given at left. (B) Autoradiogram of panel A. The boxes indicate the weak incorporation of label into the low-molecular-weight material. Note the expected absence of label from the soluble fraction, which contains the low-molecular-weight precursors shown in Fig. 5, and the high-molecular-weight material that did not enter the gel, which is indicative of its identity with peptidoglycan as described in the text.