Haemophilus responses to nutritional immunity: epigenetic and morphological contribution to biofilm architecture, invasion, persistence and disease severity.

In an effort to suppress microbial outgrowth, the host sequesters essential nutrients in a process termed nutritional immunity. However, inflammatory responses to bacterial insult can restore nutritional resources. Given that nutrient availability modulates virulence factor production and biofilm formation by other bacterial species, we hypothesized that fluctuations in heme-iron availability, particularly at privileged sites, would similarly influence Haemophilus biofilm formation and pathogenesis. Thus, we cultured Haemophilus through sequential heme-iron deplete and heme-iron replete media to determine the effect of transient depletion of internal stores of heme-iron on multiple pathogenic phenotypes. We observed that prior heme-iron restriction potentiates biofilm changes for at least 72 hours that include increased peak height and architectural complexity as compared to biofilms initiated from heme-iron replete bacteria, suggesting a mechanism for epigenetic responses that participate in the changes observed. Additionally, in a co-infection model for human otitis media, heme-iron restricted Haemophilus, although accounting for only 10% of the inoculum (90% heme-iron replete), represented up to 99% of the organisms recovered at 4 days. These data indicate that fluctuations in heme-iron availability promote a survival advantage during disease. Filamentation mediated by a SulA-related ortholog was required for optimal biofilm peak height and persistence during experimental otitis media. Moreover, severity of disease in response to heme-iron restricted Haemophilus was reduced as evidenced by lack of mucosal destruction, decreased erythema, hemorrhagic foci and vasodilatation. Transient restriction of heme-iron also promoted productive invasion events leading to the development of intracellular bacterial communities. Taken together, these data suggest that nutritional immunity, may, in fact, foster long-term phenotypic changes that better equip bacteria for survival at infectious sites.

The Haemophilus influenzae Sap transporter mediates bacterium-epithelial cell homeostasis.

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 SapA-deficient 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.

SapF-mediated heme-iron utilization enhances persistence and coordinates biofilm architecture of Haemophilus.

Non-typeable Haemophilus influenzae (NTHI) is a common commensal bacterium that resides in the human upper respiratory tract of healthy individuals. NTHI is also a known causative agent of multiple diseases including sinusitis, otitis media, as well as exacerbates disease severity of patients with cystic fibrosis and chronic obstructive pulmonary disease. We have previously shown that the Sap transporter mediates resistance to host antimicrobial peptides (AMPs) and import of the iron-containing compound heme. Here, we analyzed the contribution of the Sap structural ATPase protein, SapF, in these essential functions. In contrast to SapD, SapF was dispensable for NTHI survival when exposed to AMPs in vitro. SapF was responsible for heme utilization and recovery of depleted internal heme-iron stores. Further, a loss of SapF resulted in morphological plasticity and enhanced community development and biofilm architecture, suggesting the potential role of heme-iron availability in coordinating the complexity of NTHI biofilm architecture. SapF was required for colonization of the nasopharynx and acute infection of the middle ear, as SapF deficiency correlated with a statistically significant decrease in NTHI persistence in vivo. These data suggest that SapF is required for proper heme utilization which directly impacts NTHI survival. Thus, these studies further support a role for the Sap complex in the transport of multiple substrates and further defines substrate specificity for the two ATPase subunits. Given the multiple essential functions provided by the Sap transporter, this complex could prove to be an effective therapeutic target for the treatment of NTHI diseases.

Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus.

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides.

Heme utilization by nontypeable Haemophilus influenzae is essential and dependent on Sap transporter function.

Bacterial strategies of innate immune evasion and essential metabolic functions are critical for commensal-host homeostasis. Previously, we showed that Sap translocator function is necessary for nontypeable Haemophilus influenzae (NTHI) behaviors that mediate diseases of the human airway. Antimicrobial peptide (AP) lethality is limited by binding mediated by the Sap complex. SapA shares homology with the dipeptide-binding protein (DppA) and the heme-binding lipoprotein (HbpA), both of which have previously been shown to bind the iron-containing compound heme, whose acquisition is essential for Haemophilus survival. Computational modeling revealed conserved SapA residues, similarly modeled to mediate heme binding in HbpA. Here, we directly demonstrate that SapA bound heme and was essential for heme utilization by iron-starved NTHI. Further, the Sap translocator permease mediated heme transport into the bacterial cytoplasm, thus defining a heretofore unknown mechanism of intracytoplasmic membrane heme transport in Haemophilus. Since we demonstrate multiple ligand specificity for the SapA-binding protein, we tested whether APs would compete with heme for SapA binding. We showed that human ß-defensins 2 and 3, human cathelicidin LL-37, human neutrophil protein 1, and melittin displaced heme bound to SapA, thus supporting a hierarchy wherein immune evasion supercedes even the needed iron acquisition functions of the Sap system.