The Haemophilus influenzae Sap Transporter Mediates Bacterium-Epithelial Cell Homeostasis

Abstract

Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharynx and a causative agent of otitis media and other diseases of the upper and lower human airway. During colonization within the host, NTHI must acquire essential nutrients and evade immune attack. We previously demonstrated that the NTHI Sap transporter, an inner membrane protein complex, mediates resistance to antimicrobial peptides and is required for heme homeostasis. We hypothesized that Sap transporter functions are critical for NTHI interaction with the host epithelium and establishment of colonization. Thus, we cocultured the parent or the sapA mutant on polarized epithelial cells grown at an air-liquid interface, as a physiological model of NTHI colonization, to determine the contribution of the Sap transporter to bacterium-host cell interactions. Although SapAdeficient NTHI was less adherent to epithelial cells, we observed a significant increase in invasive bacteria compared to the parent strain. Upon internalization, the sapA mutant appeared free in the cytoplasm, whereas the parent strain was primarily found in endosomes, indicating differential subcellular trafficking. Additionally, we observed reduced inflammatory cytokine production by the epithelium in response to the sapA mutant strain compared to the parental strain. Furthermore, chinchilla middle ears challenged with the sapA mutant demonstrated a decrease in disease severity compared to ears challenged with the parental strain. Collectively, our data suggest that NTHI senses host environmental cues via Sap transporter function to mediate interaction with host epithelial cells. Epithelial cell invasion and modulation of host inflammatory cytokine responses may promote NTHI colonization and access to essential nutrients.

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment

“…The size of each circle is scaled to the number of nodes contained within. The top 10 significantly overrepresented nodes within each ontological category are listed on the figure, whereas all nodes are listed in supplemental Tables S8 -S13. ent with previous reports (22), in the absence of CK-636 viable internalized NTHI were not observed in NHBE cells following 24 and 48 h of infection. When Arp2/3 function was inhibited, we observed gross morphological changes in actin structure and an increase in the number of extracellular bacteria indicating that bacterial uptake was reduced (Fig.…”

Haemophilus influenzae

“…The pathogen also utilizes urease, found to be more prevalent in disease-causing isolates than commensals, to survive in acid environments [43,44]. Other systems involved in sensing the microenvironment within the host include the multifunctional Sap-transporter, possibly via detection of host molecules as well as heme accessibility [45,46]. As alluded to by its name, Haemophilus lacks the ability to synthesize heme that is essential for its survival.…”

“…We showed that NTHiderived outer membrane vesicles (OMVs) carried functional b-lactamase that protected GAS from amoxicillin-mediated killing. Interestingly, NTHi OMV shedding is seen in several infection models, suggesting that they may play important roles during pathogenesis [22,45].…”

Ferric Uptake Regulator and Its Role in the Pathogenesis of Nontypeable Haemophilus influenzae

“…In addition, NTHiinduced OM middle ear effusions are frequently sanguineous, likely a result of these hallmarks of middle ear inflammation (92; B. Szelestey and K. Mason, unpublished observations). NTHi invasion of host epithelial cells may also provide a mechanism of nutrient acquisition when bacteria are exposed to nutrient-limiting conditions in the host (92). NTHi infection of the middle ear coincides with an influx of polymorphonuclear leukocytes (PMNs).…”