Identification of a 3-deoxy-D-manno-octulosonic acid biosynthetic operon in Moraxella catarrhalis and analysis of a KdsA-deficient isogenic mutant

Abstract

Lipooligosaccharide (LOS), a predominant surface-exposed component of the outer membrane, has been implicated as a virulence factor in the pathogenesis of Moraxella catarrhalis infections. However, the critical steps involved in the biosynthesis and assembly of M. catarrhalis LOS currently remain undefined. In this study, we used random transposon mutagenesis to identify a 3-deoxy-D-manno-octulosonic acid (KDO) biosynthetic operon in M. catarrhalis with the gene order pyrG-kdsA-eno. The lipid A-KDO molecule serves as the acceptor onto which a variety of glycosyl transferases sequentially add the core and branch oligosaccharide extensions for the LOS molecule. KdsA, the KDO-8-phosphate synthase, catalyzes the first step of KDO biosynthesis and is an essential enzyme in gram-negative enteric bacteria for maintenance of bacterial viability. We report the construction of an isogenic M. catarrhalis kdsA mutant in strain 7169 by allelic exchange. Our data indicate that an LOS molecule consisting only of lipid A and lacking KDO glycosylation is sufficient to sustain M. catarrhalis survival in vitro. In addition, comparative growth and susceptibility assays were performed to assess the sensitivity of 7169kdsA11 compared to that of the parental strain. The results of these studies demonstrate that the native LOS molecule is an important factor in maintaining the integrity of the outer membrane and suggest that LOS is a critical component involved in the ability of M. catarrhalis to resist the bactericidal activity of human sera.

FIG. 1.

Genetic organization of the M. catarrhalis LOS biosynthesis gene cluster containing pyrG-kdsA-eno. The large arrows represent the direction of transcription, and the site of the TN insertion identified in MCTN38 is denoted (EZ::TN IS). The short, numbered arrows indicate the relative annealing positions of the oligonucleotide primers used in PCR (A) and RT-PCR (B) analyses.

FIG. 2.

Detection of the pyrG-kdsA-eno operon by RT-PCR analysis using total RNA isolated from strain 7169 (top panel) or 7169kdsA11 (bottom panel). Refer to Fig. 1 and its legend for the annealing position and polarity of each gene-specific primer. The RT-PCRs were performed using the following nucleic acid templates: lanes 1, strain 7169 chromosomal DNA; lanes 2, total RNA isolated from 7169; lanes 6, strain 7169kdsA11 chromosomal DNA; lanes 7, total RNA purified from 7169kdsA11. Reaction sets contained the following primers: a, 381 and 382; b, 383 and 384; c, 385 and 386; d, 387 and 388; e, 389 and 390. The results for one representative set of control reactions (performed using primers 385 and 386) are depicted on the agarose gels as follows: lanes 3 and 8, RT-PCRs using purified total RNA as the nucleic acid template but without activation of the RT; lanes 4 and 9, RT-PCR with no added nucleic acid template; lanes 5 and 10, RT-PCR using chromosomal DNA as the template. Molecular size standards (lanes M) are depicted in 100-bp increments.

FIG. 3.

A silver-stained SDS-PAGE gel (A) and an MAb4G5- probed immunoblot (B) depicting the LOS profiles of M. catarrhalis 7169 (lanes 1) and 7169kdsA11 (lanes 2). Molecular size standards are shown in kilodaltons.

FIG. 4.

Linear negative-ion MALDI-MS spectrum of O-deacylated LOS from wild-type strain 7169. All ion values are represented as average (avg.) masses. In the high-mass region above m/z 2500, six deprotonated molecular ion species, (M-H)⁻, were identified at m/z 2633.2, 2756.4, 2795.3, 2879.6, 2918.5, and 3042.1. At lower mass levels, several prompt fragments were observed that were generated by cleavage at the labile KDO-lipid A glycosidic linkage yielding two oligosaccharide (OS) fragments at m/z 1737.1 and 1898.6 and three di-N-acyl lipid A (lipid A′) fragments at m/z 895.9, 1019.0, and 1142.5, respectively. The two OS fragments differed by the mass of a single hexose (Hex; ΔM = 162 Da) and the three lipid A′ species differed by the mass of one or two PEAs (ΔM = 123 Da). The heterogeneity of the OS (two isoforms) and lipid A′ (three isoforms) was consistent with the presence of the six LOS glycoforms differing in the total number of hexose residues (8 or 9) and/or the number of PEAs (0, 1, or 2) that modified one or more of the two phosphates (P) on lipid A′. The calculated average masses for the intact molecular ions and their OS and lipid A′ fragments are shown in the boxed inset structure.

FIG. 5.

Reflectron negative-ion MALDI-MS of lipid A isolated from the M. catarrhalis 7169kdsA11 mutant. Results for all ions are represented as deprotonated (M-H)⁻ exact masses. The upper right portion of the figure shows the structure of the major species at m/z 1781.95. Abbreviations: C12:0-OH, 3-hydroxy-dodecanoyl acyl chain; C12:0, dodecanoyl acyl chain; C10:0, decanoyl acyl chain. *, sodiated species.

FIG. 6.

(A) In vitro growth of the parental 7169 (▪) and the mutant 7169kdsA11 (○) strains was monitored spectrophotometrically at 1.5-h intervals. (B) The corresponding OMP profiles of 7169 (lane 1) and 7169kdsA11 (lane 2) were evaluated by SDS-PAGE analysis and Coomassie brilliant blue staining. Molecular size standards are shown in kilodaltons.

FIG. 7.

Bactericidal effects of NHS on strains 7169 (black bars) and 7169kdsA11 (gray bars). The comparative serum sensitivity microscale assay was performed as described in Materials and Methods. Bacterial recovery is expressed as log CFU per milliliter, and the data represent the averages of the results of three independent assays.