Serum resistance and phase variation of a nasopharyngeal non-typeable Haemophilus influenzae isolate.

Haemophilus influenzae harbours a complex array of factors to resist human complement attack. As non-typeable H. influenzae (NTHi) strains do not possess a capsule, their serum resistance mainly depends on other mechanisms including LOS decoration. In this report, we describe the identification of a highly serum resistant, nasopharyngeal isolate (NTHi23) by screening a collection of 77 clinical isolates. For NTHi23, we defined the MLST sequence type 1133, which matches the profile of a previously published invasive NTHi isolate. A detailed genetic analysis revealed that NTHi23 shares several complement evading mechanisms with invasive disease isolates. These mechanisms include the functional expression of a retrograde phospholipid trafficking system and the presumable decoration of the LOS structure with sialic acid. By screening the NTHi23 population for spontaneous decreased serum resistance, we identified a clone, which was about 103-fold more sensitive to complement-mediated killing. Genome-wide analysis of this isolate revealed a phase variation in the N'-terminal region of lpsA, leading to a truncated version of the glycosyltransferase (LpsA). We further showed that a NTHi23 lpsA mutant exhibits a decreased invasion rate into human alveolar basal epithelial cells. Since only a small proportion of the NTHi23 population expressed the serum sensitive phenotype, resulting from lpsA phase-off, we conclude that the nasopharyngeal environment selected for a population expressing the intact and functional glycosyltransferase.

A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria.

Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.