SECRETION OF THE HAEMOPHILUS-INFLUENZAE HMW1 AND HMW2 ADHESINS INVOLVES A PERIPLASMIC INTERMEDIATE AND REQUIRES THE HMWB AND HMWC PROTEINS

Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typable H. influenzae HMW1 and HMW2 non-pilus adhesins mediate at tachment to human epithelial cells, an essential step during colonizat ion. In order to facilitate interaction with host cells, HMW1 and HMW2 are localized on the surface of the organism in a process that involv es cleavage of a 441-amino-acid N-terminal fragment. In the present st udy, we investigated the pathway for the secretion of HMW1 and HMW2, C ell fractionation experiments and cryoimmunoelectron microscopy demons trated that a periplasmic intermediate occurs, suggesting involvement of the Sec machinery, Additional analysis revealed that, ultimately, t he proteins are partially released from the surface of the organism. S tudies with Escherichia coli harbouring plasmid subclones extended ear lier findings and suggested that the secretion of HMW1 requires access ory proteins designated HMW1B and HMW1C, while the secretion of HMW2 r equires proteins called HMW2B and HMW2C. Further analysis established that HMW1B/HMW1C and HMW2B/HMW2C are interchangeable, an observation c onsistent with the high degree of homology between HMW1B and HMW2B and between HMW1C and HMW2C, Additional studies of the hmw1 locus indicat ed that HMW1B is located in the outer membrane and serves to transloca te HMW1 across the outer membrane. In the absence of HMW1B, HMW1 remai ns unprocessed and is degraded in the periplasmic space, at least in p art by the DegP protease, Mutagenesis of an HMW1 N-terminal motif shar ed with other secreted proteins resulted in diminished processing and extracellular release, suggesting interaction of this motif with the H MW1B protein, Continued investigation of the HMW1 and HMW2 adhesins ma y provide general insights into protein secretion and bacterial pathog enesis.