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.

Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media

“…More detailed analysis by scanning electron microscopy showed dense multilayered bacterial communities (Fig. 5C) that are reminiscent of those observed by our group and others in vitro (12,38,43,48).…”

“…Moreover, prior work has clearly shown that NeuAc promotes biofilm formation and bacterial persistence in animal models of NTHi disease (4,18,43). Our previous studies did not show any biofilm phenotype associated with loss of PCho, at least in terms of initial attachment or numbers of viable bacteria found within biofilms (55).…”

“…LOS confers resistance to host killing (8,9,37) and is also the primary target of the Toll-like receptor 4 pathway that mediates protection against H. influenzae in the airways (47). It has been established that NTHi strains that express NeuAc-LOS forms comprise a greater proportion of biofilm communities than of planktonic cultures, and that mutations eliminating these forms decrease biofilm formation and bacterial persistence in animal models of OM (4,12,18,43). More recently, we showed that LOS purified from biofilms has decreased potency as an inflammatory agonist, which correlated with an increase in PCho ϩ LOS forms that were present within biofilms (55).…”

Haemophilus influenzaeForms Biofilms on Airway Epithelia

“…We hypothesized that NTHi biofilm formation on epithelia requires matrix formation and the ability to incorporate sialic acid into the biofilm, as suggested by previous studies (6,7). To test this hypothesis, NTHi 2019siaA, 2019siaB, and 2019wecA were cocultured on airway epithelia.…”

“…Haemophilus influenzae (NTHi) has recently been shown to form biofilms in vitro and in otitis media (OM) (6)(7)(8)(9)(10); however, there is no information on NTHi biofilm formation in the lung or in CF.…”