Sialylation of lipooligosaccharides promotes biofilm formation by nontypeable Haemophilus influenzae

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.

FIG. 1.

(A) Biofilm initiation by H. influenzae strains. Comparable numbers of bacteria were inoculated into microtiter dishes, washed, and stained with crystal violet. The density of bacteria remaining within the wells was estimated on the basis of the absorbance of ethanol-solubilized crystal violet at 540 nm (Abs. 540) (see Materials and Methods). P. aeruginosa PAO1 and wfpa60 were included as positive and negative controls, respectively. (B) Biofilm formation by NTHi 2019 in a continuous-flow silicon tubing system. Sterile silicon tubing was seeded with NTHi 2019 (see Materials and Methods) and incubated at 37°C with a continuous flow of supplemented BHI medium for 48 h.

FIG. 2.

SEM analysis of NTHi 2019 biofilms on plastic. Bacteria were grown in biphasic cultures in supplemented BHI (see Materials and Methods). (A) Low-magnification image of bacteria at air-liquid interface; (B) high-magnification image of bacteria, showing multilayered community; (C) bacteria encased in matrix material (denoted by arrow).

FIG. 3.

(A) Comparison of levels of biofilm formation by NTHi 2019 and an isogenic siaB mutant. Bacteria were seeded into microtiter dishes, and biofilm formation was assessed at the times indicated by crystal violet staining (see Fig. 1A and Materials and Methods). OD540, optical density at 540 nm. (B) Comparison of levels of biofilm formation by NTHi 2019 and an isogenic siaB mutant in a continuous-flow silicon tube model system. A continuous flow of supplemented BHI medium was maintained, and the tubes were photographed 48 h postinoculation.

FIG. 4.

Role of sialylation in biofilm formation in biphasic cultures. NTHi 2019 (A) and an isogenic siaB mutant (B) were cultured in supplemented BHI (see Materials and Methods), fixed, and processed for SEM analysis. The panels show dense bacterial communities at the air-liquid interface in each culture after 24 h and are representative of multiple fields of view.

FIG. 5.

Silver stain of NTHi 2019 LOS from planktonic, biofilm, and plate cultures. LOS was prepared from broth, plate, and continuous-flow tube biofilm cultures and examined by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining (see Materials and Methods). Reactivity with MAb 3F11 is an index of asialylated terminal lactosamine and was determined by dot blot analysis.