Porphyromonas gingivalis galEIs Involved in Lipopolysaccharide O-Antigen Synthesis and Biofilm Formation

Abstract

Porphyromonas gingivalis is a crucial component of complex plaque biofilms that form in the oral cavity, resulting in the progression of periodontal disease. To elucidate the mechanism of periodontal biofilm formation, we analyzed the involvement of several genes related to the synthesis of polysaccharides in P. gingivalis. Gene knockout P. gingivalis mutants were constructed by insertion of an ermF-ermAM cassette; among these mutants, the galE mutant showed some characteristic phenotypes involved in the loss of GalE activity. As expected, the galE mutant accumulated intracellular carbohydrates in the presence of 0.1% galactose and did not grow in the presence of galactose at a concentration greater than 1%, in contrast to the parental strain. Lipopolysaccharide (LPS) analysis indicated that the length of the O-antigen chain of the galE mutant was shorter than that of the wild type. It was also demonstrated that biofilms generated by the galE mutant had an intensity 4.5-fold greater than those of the wild type. Further, the galE mutant was found to be significantly susceptible to some antibiotics in comparison with the wild type. In addition, complementation of the galE mutation led to a partial recovery of the parental phenotypes. We concluded that the galE gene plays a pivotal role in the modification of LPS O antigen and biofilm formation in P. gingivalis and considered that our findings of a relationship between the function of the P. gingivalis galE gene and virulence phenotypes such as biofilm formation may provide clues for understanding the mechanism of pathogenicity in periodontal disease.

Deletion of a 77-Base-Pair Inverted Repeat Element Alters the Synthesis of Surface Polysaccharides in Porphyromonas gingivalis

“…had been reported previously for another strain (21). This effect is not due to a change in the transcription of PG0121 downstream in the operon (Fig.…”

“…We previously reported that deletion of the HU protein PG0121, which is encoded at the 3= end of the capsule locus, has altered polysaccharide content and that a variety of loci are differentially expressed in the PG0121 deletion mutant, including genes in known polysaccharide synthesis loci, including the APS synthesis locus PG1135-PG1142 (25,26). It has also been reported that deletion of PG0106 in P. gingivalis strain 33277, the first gene in the K-antigen operon, yields a strain with an altered LPS profile on silver-stained gels of purified LPS (21). To examine the possibility that the structure of the LPS molecules is also modulated by the 77bpIR element, proteinase K-digested whole-cell lysates were compared for LPS profile by silver ammonia-stained SDS-PAGE gels.…”

“…Genes in the PG0104-PG0121 locus have been shown to be required for K1 capsule synthesis (19,20). Yet PG0106 (a putative UDP-phosphate alpha-N-acetylglucosaminyltransferase) mutant in strain 33277 is also deficient in LPS synthesis (21), indicating that PG0106 and possibly other genes in this locus are involved in synthesizing both K antigen and LPS molecules. Unlike the K-antigen locus, the coding sequence of another polysaccharide synthesis locus (PG1135 to PG1142) is highly conserved in various P. gingivalis strains (19).…”

Metabolic Network Model of a Human Oral Pathogen

“…UDP-galactose is required in multiple steps within the LPS synthesis pathway. This specific mutant has been documented (59) and is known to have a shortened O-antigen chain as a result but was still viable. In light of this experimental validation of our modeling approach, it will be useful to experimentally test the other potential knockouts listed in Table 3.…”

“…The UDPG4E reaction is linked to the galE gene, producing a UDP-glucose 4-epimerase. The galE mutants are known to have modified lipid A molecules (59). The pathway culminates in the production of lps_PG1, lps_PG2, and lps_PG3 molecules, which refer to the PG1690, PG1450, and PG1435 lipid A structures, respectively.…”