Comparison of loss of serum resistance by defined lipopolysaccharide mutants and an acapsular mutant of uropathogenic Escherichia coli O75:K5

Abstract

In order to determine the importance of the O75 O antigen versus the K5 capsular antigen and the bimodal distribution of lipopolysaccharides (LPSs) in protection from complement-mediated lysis, mutants were made by insertion of a cat or an aphA gene in or in place of genes necessary for the synthesis of LPS and/or the K antigen of an O75(+) K5(+) uropathogenic Escherichia coli strain, GR-12. Mutations were made in the following genes: the rfbD gene (required for the synthesis of TDP-rhamnose), the rfbKM genes (necessary for the synthesis of GDP-mannose), the rol gene (regulating O-antigen length), the kfiC gene (encoding a putative glycosyltransferase), and the kfiC-rfbD genes. The resulting phenotypes were rough (O75(-)), core plus one partial O-antigen subunit, random distribution of O-antigen chain lengths, acapsular (K5(-)), and O75(-) K5(-), respectively. All five mutants and GR-12 were analyzed for survival in 80% serum. The GR-12 parent was resistant, exhibiting a 500% increase in numbers. The rol, rfbKM, rfbD, and kfiC-rfbD mutants were sensitive, experiencing 99%, 99.9%, 99.9%, and at least 99.999% killing, respectively, in the first hour. The kfiC mutant, however, increased in numbers in the first hour but experienced delayed sensitivity, decreasing in viability by 80% in the third hour. Single mutants were complemented with the wild-type gene in trans, showing restoration of the wild-type phenotype and serum resistance. Therefore, the O75 antigen is more important for survival in serum than the K5 antigen, and regulation of the O75 O-antigen chain length is crucial for protection of the bacteria from complement-mediated lysis.

FIG. 1

Structure of the O75 subunit (15). The O75 subunit is made of four sugars: GlcNAc, N-acetylglucosamine; Rha, rhamnose; Gal, galactose; and Man, mannose (side-chain sugar).

FIG. 2

Diagram of construction of chromosomal mutants. Genes deleted or mutated are indicated. Only relevant restriction sites are included: A, SalI; C, ClaI; D, NdeI; E, EcoRV; H, HpaI; K, KpnI; M, MunI; N, NruI; P, PstI; and S, SnaBI. Plasmids are described in Table 2. Crossed lines denote recombination.

FIG. 3

Visualization of LPSs of GR-12, mutants, and complemented mutants. (A) SDS-PAGE. Lanes: 1, GR-12 (wild type); 2, SMB213 (kfiC mutant); 3, SMB122 (rol mutant); 4, SMB122(pRAB10); 5, SMB43 (rfbKM mutant); 6, SMB43(pSMB109); 7, SMB20 (rfbD mutant); 8, SMB20(pGH1610); 9, SMB316 (kfiC-rfbD double mutant). (B) Tricine-SDS buffer system. Lanes: 10 and 13, GR-12; 11, SMB20 (rfbD mutant); 12, SMB43 (rfbKM mutant).

FIG. 4

Analysis of outer membrane protein profiles of GR-12 and mutants. A 5-μl sample of each strain was loaded in each lane. Lanes: 1, protein standard (from top: molecular weight of 94,000 [94K], 67K, 43K, 30K, and 20.1K); 2, GR-12 (wild type); 3, SMB122 (rol mutant); 4, SMB43 (rfbKM mutant); 5, SMB20 (rfbD mutant); 6, SMB213 (kfiC mutant); 7, SMB316 (kfiC-rfbD mutant).

FIG. 5

Serum assays of wild-type strain GR-12 and mutants in 80% serum (A) or 80% heat-inactivated serum (B) with an inoculum of 3 × 10⁵ CFU/ml. The assays were performed for 3 h; samples were taken in duplicate at the start and at each hour. The graphs are a result of at least two different assays for each strain. Results are shown as percent viability. Error bars indicate standard deviations.

FIG. 6

Serum assays (80% serum) of GR-12 and O75⁻, K5⁻, and O75⁻ K5⁻ mutants at an inoculum of 3 × 10⁷ CFU/ml. The assays were performed for 3 h. Samples were taken in duplicate at the start and at each hour. The graph is a result of at least two different assays for each strain. Results are shown as percent viability. Error bars indicate standard deviations.

FIG. 7

Serum assays (80% serum) with 10 mM EGTA plus MgCl₂ of GR-12 and O75⁻, K5⁻, and O75⁻ K5⁻ mutants at an inoculum of 3 × 10⁵ CFU/ml. The assays were performed for 3 h. Samples were taken in duplicate at time zero and at each hour. A control assay was performed with 80% heat-inactivated serum plus EGTA and the double mutant (SMB316). The graph is a result of at least two different assays for each strain. Results are shown as percent viability. Error bars indicate standard deviations.

FIG. 8

Serum assays (80% serum) of GR-12 and each complemented mutant at an inoculum of 3 × 10⁵ CFU/ml. The assays were performed for 3 h. Samples were taken in duplicate at time zero and at each hour. The graph is a result of at least two different assays for each strain. Results are shown as percent viability. Error bars indicate standard deviations.