Analysis of lipooligosaccharide biosynthesis in the Neisseriaceae

Abstract

Neisserial lipooligosaccharide (LOS) contains three oligosaccharide chains, termed the alpha, beta, and gamma chains. We used Southern hybridization experiments on DNA isolated from various Neisseria spp. to determine if strains considered to be nonpathogenic possessed DNA sequences homologous with genes involved in the biosynthesis of these oligosaccharide chains. The presence or absence of specific genes was compared to the LOS profiles expressed by each strain, as characterized by their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and their reactivities with various LOS-specific monoclonal antibodies. A great deal of heterogeneity was seen with respect to the presence of genes encoding glycosyltransferases in Neisseria. All pathogenic species were found to possess DNA sequences homologous with the lgt gene cluster, a group of genes needed for the synthesis of the alpha chain. Some of these genes were also found to be present in strains considered to be nonpathogenic, such as Neisseria lactamica, N. subflava, and N. sicca. Some nonpathogenic Neisseria spp. were able to express high-molecular-mass LOS structures, even though they lacked the DNA sequences homologous with rfaF, a gene whose product must act before gonococcal and meningococcal LOS can be elongated. Using a PCR amplification strategy, in combination with DNA sequencing, we demonstrated that N. subflava 44 possessed lgtA, lgtB, and lgtE genes. The predicted amino acid sequence encoded by each of these genes suggested that they encoded functional proteins; however, structural analysis of LOS isolated from this strain indicated that the bulk of its LOS was not modified by these gene products. This suggests the existence of an additional regulatory mechanism that is responsible for the limited expression of these genes in this strain.

FIG. 1

SDS-PAGE profiles of LOS isolated from various Neisseria strains. Lanes: 1, N. cinerea 32864; 2, N. cinerea 32165; 3; N. lactamica 5841; 4, N. sicca 342; 5, N. sicca 4318; 6, N. flavescens 4322; 7, N. flavescens 4323; 8, N. flavescens ATCC 13120; 9, N. gonorrhoeae F62; 10, N. gonorrhoeae FA5100; 11, N. subflava 44; 12, N. subflava 52; 13, N. subflava 4324; 14, N. subflava 4325; 15, N. subflava 4327.

FIG. 2

Southern hybridization to test for the presence of rfaF DNA in various Neisseria strains. Lanes: 1, N. subflava 52; 2, N. subflava 4325; 3, N. subflava 4324; 4, N. subflava 44; 5, N. subflava 4327; 6, N. flavescens ATCC 13120; 7, N. flavescens 4323; 8, N. flavescens 4322; 9, N. sicca 4318; 10, N. sicca 342; 11, N. lactamica 5841; 12, N. meningitidis 89I; 13, N. cinerea 32165; 14, N. cinerea 32824; 15, N. gonorrhoeae FA19; 16, N. gonorrhoeae WR302; 17, N. gonorrhoeae PID2; 18, N. gonorrhoeae F62.

FIG. 3

Diagram of mechanisms of recombination between N. gonorrhoeae chromosome and plasmid DNA. (A) Description of the introduction of lgtA_NS into the chromosome of F62 ΔlgtA. (B) Description of the introduction of lgtB_NS and lgtE_NS into the chromosome of F62 ΔlgtA ΔlgtE. Black box in lgtA, location of the deletion, if present.

FIG. 4

SDS-PAGE analysis of LOS isolated from various N. gonorrhoeae transformation mutants with pNS44lgtABE or pNS44lgtABΔE. The plasmid pNS44lgtABΔE gene was introduced into F62 ΔlgtA, and the plasmid pNS44lgtABE gene was introduced into F62 ΔlgtA ΔlgtE, by spot transformation. Transformants were isolated based on their changes in reactivity with MAb 2-1-L8, and LOS samples were isolated and analyzed on Tris-Tricine gel. Lanes: 1, N. gonorrhoeae F62; 2, N. gonorrhoeae F62 ΔlgtA transformed with pNS44lgtABΔE; 3, N. gonorrhoeae F62 ΔlgtA; 4, N. gonorrhoeae F62 ΔlgtA ΔlgtE transformed with pNS44lgtABE; 5, N. gonorrhoeae F62 ΔlgtA ΔlgtE.