the haemophilus influenzae sap transporter mediates bacterium

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  • The Haemophilus influenzae Sap Transporter Mediates Bacterium-Epithelial Cell Homeostasis

    Forrest K. Raffel, Blake R. Szelestey, Wandy L. Beatty, and Kevin M. Mason

    The Research Institute at Nationwide Childrens Hospital and The Ohio State University College of Medicine, Department of Pediatrics, Center for Microbial Pathogenesis,Columbus, Ohio, USA

    Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharynx and a causative agent ofotitis media and other diseases of the upper and lower human airway. During colonization within the host, NTHI must acquireessential nutrients and evade immune attack. We previously demonstrated that the NTHI Sap transporter, an inner membraneprotein complex, mediates resistance to antimicrobial peptides and is required for heme homeostasis. We hypothesized that Saptransporter functions are critical for NTHI interaction with the host epithelium and establishment of colonization. Thus, wecocultured the parent or the sapA mutant on polarized epithelial cells grown at an air-liquid interface, as a physiological modelof 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 par-ent strain. Upon internalization, the sapA mutant appeared free in the cytoplasm, whereas the parent strain was primarily foundin endosomes, indicating differential subcellular trafficking. Additionally, we observed reduced inflammatory cytokine produc-tion by the epithelium in response to the sapA mutant strain compared to the parental strain. Furthermore, chinchilla middleears challenged with the sapA mutant demonstrated a decrease in disease severity compared to ears challenged with the parentalstrain. Collectively, our data suggest that NTHI senses host environmental cues via Sap transporter function to mediate interac-tion with host epithelial cells. Epithelial cell invasion and modulation of host inflammatory cytokine responses may promoteNTHI colonization and access to essential nutrients.

    Nontypeable Haemophilus influenzae (NTHI) is a commensalinhabitant of the human nasopharynx, yet it can cause op-portunistic infections in compromised upper and lower respira-tory tracts. As such, NTHI is a leading cause of otitis media, sinus-itis, and community-acquired pneumonia and is also commonlyassociated with exacerbations of chronic obstructive pulmonarydisease and cystic fibrosis (17). Commensal and opportunisticcolonization of the host requires NTHI to overcome a myriad ofhost defense mechanisms, such as production of bactericidal pro-teins, physical mucociliary clearance, and sequestration of essen-tial nutrients (815). NTHI strains have developed several suc-cessful strategies to evade host immunity and equip themselves forsurvival in vivo, including production of IgA1 protease, expressionof aggregative adhesins, formation of biofilms, and modificationof surface lipooligosaccharide (LOS) (1628). Gram-negativeouter membrane components such as LOS can be potent stimu-lators of the host inflammatory response and serve as bindingtargets for immunoprotective antibodies, complement proteins,and antimicrobial peptides (21, 29, 30). Modification of LOS pro-vides a first line of defense for NTHI and, as such, a mechanism toevade the host innate immune system. For instance, addition ofsialic acid to LOS prevents complement deposition, and phospho-rylcholine (ChoP) modification inhibits the binding of immuno-protective antibodies, further serving to mask bacterial surfacecharge and minimize antimicrobial peptide binding (17, 3032).In addition, LOS modifications induce changes in host epithelialresponses. ChoP-decorated LOS facilitates attachment to and in-vasion of the epithelium and signaling through the platelet-acti-vating factor receptor (PAFr). ChoP activation of the PAFr signal-ing cascade downregulates host expression of Toll-like receptor 2(TLR2), TLR4, and TLR9 and promotes NTHI invasion of hostepithelial cells (20, 21, 33, 34). Traditionally, NTHI has been cat-

    egorized as an extracellular pathogen; however, invasion of hostepithelial cells can offer a temporary or long-term respite from thehost immune response and can counter active nutrient depletionby the host. The process of active nutrient depletion, termed nu-tritional immunity, is a host mechanism to inhibit microbialgrowth by sequestering essential nutrients (i.e., iron, zinc, andmanganese) (9, 11, 12, 14, 15). Recalcitrance to antibiotic therapy,persistence in the presence of protective antibodies, and culture-negative clinical status suggest that biofilm formation and devel-opment of bacterial reservoirs within host cells may contribute tothe chronic nature of NTHI infections (1, 4, 3540).

    Bacterial mechanisms to acquire essential nutrients and evadeinnate immune responses are essential for NTHI survival as acommensal in the nasopharynx and as a pathogen at privilegedsites in the host. We have previously demonstrated an essentialrole for the Sap transporter, a multifunctional inner membraneABC transport complex, in resistance to antimicrobial peptidekilling and the transport of the essential heme iron (4144). An-timicrobial peptides are transported into the bacterial cytoplasm

    Received 5 September 2012 Returned for modification 26 September 2012Accepted 7 October 2012

    Published ahead of print 15 October 2012

    Editor: A. J. Bumler

    Address correspondence to Kevin M. Mason,

    Supplemental material for this article may be found at

    Copyright 2013, American Society for Microbiology. All Rights Reserved.


    January 2013 Volume 81 Number 1 Infection and Immunity p. 4354 43

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  • in a Sap-dependent manner and are subsequently degraded bycytoplasmic peptidase activity (45). Similarly, the Sap transporterfunctions in the uptake of heme iron. First bound by the SapAperiplasmic binding protein, heme iron is delivered for transportacross the cytoplasmic membrane through the SapBC permeasecomplex (44). In addition to the multifunctional roles of Saptransporter function in innate immune resistance and nutrientacquisition, Sap transporter function influences NTHI biofilm de-velopment and architecture. NTHI deficient in the SapF ATPaseprotein developed a more robust biofilm than that of the parentalstrain, coincident with morphological plasticity of NTHI, includ-ing increased chain length and filament production within thebiofilm architecture (46). We have further demonstrated thatneutralization of host antimicrobial peptides restores virulence tothe sapA mutant in vivo, suggesting an essential role for Sap-de-pendent antimicrobial peptide resistance during the acute phaseof disease in the host (45). Sap transporter function thus serves tomaintain NTHI heme iron homeostasis and persistence in thehost, providing mechanisms to resist antimicrobial peptides, aidin nutrient acquisition, and influence NTHI biofilm formation.

    Due to these essential roles, we investigated the consequence ofloss of Sap transporter functions on colonization of epithelial cells.We determined that NTHI deficient in SapA was less adherent toepithelial cells yet was associated with membrane ruffling and ep-ithelial cell destruction. In addition, we observed that SapA-defi-cient NTHI was more invasive and had a decreased immune-stim-ulatory effect on host epithelium compared to host responsesinduced by the parental strain. Collectively, these data support animportant role for Sap transporter function in NTHI interactionwith host epithelial cells. We propose that NTHI utilizes the Saptransporter to sense microenvironmental cues such as heme ironlimitation and antimicrobial peptide production to modulate thehost environment, gain access to essential nutrients, and evade theinnate immune system. Additionally, these data suggest thatNTHI residence in the epithelial cytoplasm may function as abacterial reservoir during chronic infections.

    MATERIALS AND METHODSBacterial strains and growth conditions. The parental NTHI strain 86-028NP::rpsLA128G is a streptomycin-resistant strain constructed as previ-ously described (47). Construction of a strain with an unmarked, nonpo-lar deletion of the sapA gene was performed by the recombineeringstrategy as previously described (46, 47). Briefly, primers 5=-AAGTGCGATGGTATTTTGACGAA-3= and 5=-ACGAGTAATATGATCCGCCTTTGT-3= were used to amplify sapA and 1 kb of the flanking DNA both 5= and3= to sapA. The subsequent amplicon was ligated into the pGEM-T Easyvector (pFR001) and transformed into Escherichia coli strain DY380. Inparallel, primers, 5= TAATATGCCTTACAATTTGACACATAATTTATCACAATGCATTTGTTATG 3= and 5= CAGAATATGGCGAAGAACCGACCAGAACATTAGTGTTTCTCCTGAATAAA 3=, each containing 50 bpof DNA homologous to the 5= and 3= ends of the sapA gene, were used toamplify the spec-rpsL cassette from pRSM2832 (47). This amplicon wasthen electroporated into strain E. coli DY380/pFR001 to form strainDY380/pFR002, in which the sapA gene in pFR001 has been replaced bythe cassette. The plasmid pFR002 was then used to transform NTHI 86-028NP::rpsLA128G, and transformants were selected by growth on specti-nomycin-containing Chocolate II agar plates. To generate a nonpolardeletion mutant, the sapA mutant was transformed with plasmidpRSM2947 and grown at 32C, and FLP expression was induced usinganhydrotetracycline. The cells were cured of the plasmid by growth at37C. Green fluorescent protein (GFP)-expressing


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