Nontypeable Haemophilus influenzae (NTHi) are Gram‐negative pathogens that contribute to a variety of diseases, including acute otitis media and chronic obstructive pulmonary disease. As NTHi have an absolute requirement for heme during aerobic growth, these bacteria have to scavenge heme from their human hosts. These heme sources can range from free heme to heme bound to proteins, such as hemoglobin. To test the impact of heme structural factors on heme acquisition by NTHi, we prepared a series of heme sources that systematically vary in heme exposure and covalent binding of heme to peptide/protein and tested the ability of NTHi to use these sources to support growth. Results from this study suggest that NTHi can utilize protein‐associated heme only if it is noncovalently attached to the protein.

Nontypable Haemophilus influenzae (NTHi) causes otitis media (ear infections) and other respiratory illnesses. Currently, NTHi's outer membrane protein P6 is a leading vaccine candidate for the pathogenic bacteria. Previous work in our lab has demonstrated that P6 is not a transmembrane protein. However, P6 is also known to interact with peptidoglycan inside the cell. In light of other studies performed in our lab (flow cytometry and confocal microscopy), which demonstrate P6's surface exposure, we conclude that P6 exists in two orientations in the outer membrane of NTHi. To quantify the two populations of inside‐facing and outside‐facing P6, we utilized a biotinylating reagent (NHS‐LC‐LC‐ biotin) to isolate surface exposed proteins from internally localized proteins. Standard protein detection and immunoblotting techniques were utilized to quantify the two populations of P6. We conclude that the predominant population of P6 is located on the inside of NTHi (>;75%). Further elucidation of the dual orientation of P6 and other lipoproteins is certainly important for both the fields of bacteriology and vaccine development. This study was funded by the Rochester Institute of Technology and NIH NIDCD RO1 08671 (to MEP).

Heme is an iron‐containing porphyrin ring that some iron‐dependent bacteria are able to use as their iron source. c‐type heme is differentiated from other hemes (b, d, etc.) by covalent attachment of the heme to the protein chain via two thioether bonds to Cysteines. Covalent attachment of the heme group is energetically expensive, suggesting that there is a biological benefit to the attachment. One proposed advantage, supported by our studies, is that the covalent attachment stops iron‐dependent bacteria, such as Nontypable Haemophilus influenzae (NTHi), from stealing heme. In our study, we evaluated the role of c heme attachment by creating single‐ and double‐mutant cytochrome c proteins, which lacked one or both of the covalent attachments to the heme group, respectively. We then grew NTHi in the presence of wild‐type (two covalent heme attachments), single‐mutant (one covalent attachment) and double‐mutant (no covalent attachments) protein, as well as appropriate controls, to examine the effect of covalent heme attachment on NTHi growth. Results from our studies demonstrated that the covalent linkages do prevent NTHi from scavenging heme from wild‐type and single‐mutant cytochrome c, while non‐covalently attached heme (in double‐mutant cytochrome c) is readily scavenged and used as an iron source. This study was funded by the Rochester Institute of Technology.