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<p>Nontypeable Haemophilus influenzae (NTHi) is a common commensal in the human nasopharynx
that can cause localized respiratory tract diseases such as otitis media, bronchitis,
and pneumonia. NTHi adheres to respiratory epithelial cells, a critical step in the
process of colonization enabled by bacterial surface adhesive structures called adhesins.
One group of NTHi adhesins are autotransporters, proteins that have an N-terminal
signal sequence, a C-terminal β-barrel domain, and an internal passenger domain
with effector function. The goal of this work was to increase our understanding of
two NTHi autotransporters, Hap and Hia.</p><p>Hap is a monomeric autotransporter that
mediates adherence to epithelial cells and extracellular matrix (ECM) proteins. Hap
also self-associates with protein on neighboring bacteria, resulting in bacterial
aggregation and microcolony formation. The Hap passenger domain contains the regions
responsible for adhesive activity. To define the molecular mechanism of Hap adhesive
activity, we crystallized the Hap passenger domain. Characterization of the crystal
structure revealed an N-terminal globular domain and a more ordered, prism-like C-terminal
domain. Interestingly, Hap crystallized as a multimer, suggesting that Hap-Hap interactions
occurred in the passenger domain. Progressive deletions of the β-loops that comprise
the C-terminal region disrupted Hap-Hap interactions and led to a defect in bacterial
settling. To further support that the C-terminal domain was responsible for Hap-Hap
interactions,</p><p>7</p><p>we purified the wild type and truncated passenger domains
and conjugated the proteins to latex beads. By light microscopy we visualized bead
aggregation when the wild type passenger domain was conjugated to the beads, but not
when the truncated passenger domain was conjugated. These results show that the C-terminal
portion of the Hap passenger domain is responsible for Hap-Hap interactions leading
to multimerization. Hap multimerization could be important in microcolony formation
that leads to biofilm formation in vivo.</p><p>The ECM binding domain in located in
the final 511 amino acids of the Hap passenger domain. To pin-point the region of
the ECM protein fibronectin that is recognized by Hap, we spotted small fragments
of fibronectin onto nitrocellulose membranes and incubated the membrane with purified
Hap passenger domain. Far Western analysis using Hap antibody revealed that the smallest
fibronectin region necessary for binding was comprised of the first two type III repeats,
FNIII(1-2). To define the regions of Hap responsible for interaction with fibronectin,
we mutated motifs in the Hap passenger domain that are important for fibronectin binding
in other bacterial proteins. Based on assessment by ELISA, many of the mutations located
between amino acids 525-725 caused reduced bacterial binding to fibronectin. However,
no mutation totally ablated binding, suggesting that a larger Hap region is involved
in fibronectin binding.</p><p>8</p><p>In an additional study, we identified a relationship
between Hap levels in the outer membrane and the expression of lipopolysaccharide
(LPS) biosynthesis enzymes. Through Western and qPCR analysis, we found that mutation
of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB and lsgD genes involved in the synthesis
of LPS oligosaccharide core in H. influenzae strain Rd/HapS243A resulted in loss of
Hap in the bacterial outer membrane and a decrease in hap transcript. In contrast,
the same mutations had no effect on outer membrane localization of H. influenzae P5
and IgA1 protease or levels of the p5 or iga1 transcripts, suggesting a Hap-specific
effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to
the outer membrane and restoration of wild type levels of hap transcript. We speculate
that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate
in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease
in hap transcript. lgtC is one of several phase variable LPS biosynthesis genes. Using
an antibody against the epitope formed in part by the lgtC gene product, we identified
lgtC phase-off bacteria by Western analysis of colony blots. Consistent with our previous
observations, in lgtC phase off bacteria Hap was absent from the outer membrane and
hap transcript was reduced. By analyzing a lgtC/lic2A double mutant, we found that
Hap localization in the outer membrane and hap transcript levels were not related
to LPS size but instead to the functions of the LPS synthesis enzymes themselves.
This relationship could be beneficial to bacteria in vivo as a way to regulate Hap
expression.</p><p>9</p><p>Early models suggested that autotransporters do not require
accessory factors for folding and OM insertion. However, mounting recent evidence
has suggested that the Bam complex is required for OM localization of most β-barrel
proteins, including autotransporters. We studied the role of the Bam complex in OM
localization of the trimeric autotransporter Hia. We expressed Hia in E. coli strains
with mutations in the Bam complex and found that BamA and BamD were needed for Hia
localization, while BamB, BamC, and BamE were not necessary. In further studies, we
mutated the C-terminus of Hia and found that the final and third-to-last amino acids
were the most important for outer membrane localization.</p><p>In summary, this work
provides insights into the regulation and adhesive activity of Hap and the outer membrane
localization of Hia. We have learned important details about these factors that shed
light on aspects of H. influenzae disease and could lead to new antimicrobial therapies.</p>
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