Calcium Binding Properties of the Kingella kingae PilC1 and PilC2 Proteins Have Differential Effects on Type IV Pilus-Mediated Adherence and Twitching Motility

Abstract

Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, which is dependent on type IV pili and is influenced by two PilC-like proteins called PilC1 and PilC2. Production of either PilC1 or PilC2 is necessary for K. kingae piliation and bacterial adherence. In this study, we set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional 9-amino-acid calcium-binding (Ca-binding) site with homology to the Pseudomonas aeruginosa PilY1 Ca-binding site and that PilC2 contains a functional 12-amino-acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we showed that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in K. kingae and provide insights into the biology of the PilC-like family of proteins.

Mutation of the Conserved Calcium-Binding Motif in Neisseria gonorrhoeae PilC1 Impacts Adhesion but Not Piliation

“…While previous studies found that elimination of the ability of PilY1 (the P. aeruginosa PilC homolog) to bind calcium abolished both adhesion and twitching by preventing surface piliation in P. aeruginosa (25,36), the attenuated adhesion observed in K. kingae (37) and this study for N. gonorrhoeae was not dependent on preventing the surface expression of pili. We found that while the gonococcal calcium-binding-deficient PilC1 mutant was reduced in its ability to adhere to epithelial cells, this defect could not be attributed to a lack of piliation.…”

“…PilC from N. gonorrhoeae also has this conserved domain, which has been found in other PilC family members as well ( Fig. 1; see Table S1 in the supplemental material) (37). The focus of this study was to observe whether the calcium-binding domain in the N. gonorrhoeae T4P adhesion protein, PilC, also affected piliation in the same manner as that previously seen in P. aeruginosa.…”

Respiratory Colonization by Kingella kingae, Person-to-Person Transmission, and Pathogenesis of Invasive Infection

“…This observation suggests that piliation offers a selective advantage in the colonization process and at the early stages of the infection, but is detrimental to the bacterium for the invasion of deep body tissues. Additional work by the same research group identified two other genes named pilC1 and pilC2 in physically separated chromosomal locations that encode homologs of the Neisseria PilC proteins that are essential for adherence and piliation [16,19]. Kingella kingae PilC1 and PilC2 proteins have a low level of homology to each other, contain calcium-binding sites and are dispensable for pilus asssembly [16,19].…”

“…Additional work by the same research group identified two other genes named pilC1 and pilC2 in physically separated chromosomal locations that encode homologs of the Neisseria PilC proteins that are essential for adherence and piliation [16,19]. Kingella kingae PilC1 and PilC2 proteins have a low level of homology to each other, contain calcium-binding sites and are dispensable for pilus asssembly [16,19]. While the PilC1 site is necessary for twitching motility and adherence, the PilC2 site has only a minor influence on motility and no influence on adherence [19].…”

“…Kingella kingae PilC1 and PilC2 proteins have a low level of homology to each other, contain calcium-binding sites and are dispensable for pilus asssembly [16,19]. While the PilC1 site is necessary for twitching motility and adherence, the PilC2 site has only a minor influence on motility and no influence on adherence [19]. Additional research indicated that a trimeric autotransporter protein called Knh is crucial for a firm adherence of K. kingae to the epithelium [20].…”