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Updated: Jan 19, 2026
Culturing Non-typeable Haemophilus influenzae
Published on: October 30, 2025
Phylogenetic and functional characterisation of the Haemophilus influenzae multidrug efflux pump AcrB
Martijn Zwama1, Akihito Yamaguchi2, Kunihiko Nishino1
11Department of Biomolecular Science and Regulation, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047 Japan.
Abstract:
Multidrug resistance in Gram-negative bacteria can arise by the over-expression of multidrug efflux pumps, which can extrude a wide range of antibiotics. Here we describe the ancestral Haemophilus influenzae efflux pump AcrB (AcrB-Hi). We performed a phylogenetic analysis of hundreds of RND-type transporters. We found that AcrB-Hi is a relatively ancient efflux pump, which nonetheless can export the same range of antibiotics as its evolved colleague from Escherichia coli. AcrB-Hi was not inhibited by the efflux pump inhibitor ABI-PP, and could export bile salts weakly. This points to an environmental adaptation of RND transporters. We also explain the sensitivity of H. influenzae cells to β-lactams and novobiocin by the outer membrane porin OmpP2. This porin counterbalances the AcrB-Hi efflux by leaking the drugs back into the cells. We hypothesise that multidrug recognition by RND-type pumps is not an evolutionarily acquired ability, and has been present since ancient promiscuous transporters.
Insights
The ancient Haemophilus influenzae AcrB efflux pump (AcrB-Hi) exports a broad spectrum of antibiotics, similar to its E. coli counterpart. Its function is counterbalanced by the OmpP2 porin, influencing bacterial drug resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Multidrug resistance in Gram-negative bacteria is often mediated by multidrug efflux pumps.
- These pumps expel various antibiotics, contributing to treatment challenges.
Purpose of the Study:
- To characterize the ancestral Haemophilus influenzae AcrB efflux pump (AcrB-Hi).
- To investigate the evolutionary origins and functional capabilities of RND-type transporters.
Main Methods:
- Phylogenetic analysis of hundreds of RND-type transporters.
- Functional characterization of AcrB-Hi, including antibiotic export and inhibition assays.
- Analysis of the role of the OmpP2 porin in H. influenzae drug sensitivity.
Main Results:
- AcrB-Hi is an ancient RND-type efflux pump with broad antibiotic export capabilities, comparable to the evolved AcrB from Escherichia coli.
- AcrB-Hi was not inhibited by ABI-PP and weakly exported bile salts, suggesting environmental adaptation.
- The OmpP2 porin contributes to H. influenzae sensitivity to beta-lactams and novobiocin by facilitating drug influx, counteracting AcrB-Hi efflux.
Conclusions:
- The broad substrate recognition of RND-type pumps may be an ancient, inherent trait rather than an evolutionarily acquired one.
- The interplay between efflux pumps and porins is crucial in determining the overall drug resistance profile of Gram-negative bacteria.
- Environmental factors likely shaped the adaptive evolution of RND transporters.
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