Mutation of the conserved calcium-binding motif in Neisseria gonorrhoeae PilC1 impacts adhesion but not piliation.

Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae.

Passively released heme from hemoglobin and myoglobin is a potential source of nutrient iron for Bordetella bronchiseptica.

Colonization by Bordetella bronchiseptica results in a variety of inflammatory respiratory infections, including canine kennel cough, porcine atrophic rhinitis, and a whooping cough-like disease in humans. For successful colonization, B. bronchiseptica must acquire iron (Fe) from the infected host. A vast amount of Fe within the host is sequestered within heme, a metalloporphyrin which is coordinately bound in hemoglobin and myoglobin. Utilization of hemoglobin and myoglobin as sources of nutrient Fe by B. bronchiseptica requires expression of BhuR, an outer membrane protein. We hypothesize that hemin is acquired by B. bronchiseptica in a BhuR-dependent manner after spontaneous loss of the metalloporphyrin from hemoglobin and/or myoglobin. Sequestration experiments demonstrated that direct contact with hemoglobin or myoglobin was not required to support growth of B. bronchiseptica in an Fe-limiting environment. Mutant myoglobins, each exhibiting a different affinity for heme, were employed to demonstrate that the rate of growth of B. bronchiseptica was directly correlated with the rate at which heme was lost from the hemoprotein. Finally, Escherichia coli cells expressing recombinant BhuR had the capacity to remove hemin from solution. Collectively, these experiments provided strong experimental support for the model that BhuR is a hemin receptor and B. bronchiseptica likely acquires heme during infection after passive loss of the metalloporphyrin from hemoglobin and/or myoglobin. These results also suggest that spontaneous hemin loss by hemoglobin and myoglobin may be a common mechanism by which many pathogenic bacteria acquire heme and heme-bound Fe.