Characterization of the nucleotide-binding domain NsrF from the BceAB-type ABC-transporter NsrFP from the human pathogen Streptococcus agalactiae

Abstract

Treatment of bacterial infections is a great challenge of our era due to the various resistance mechanisms against antibiotics. Antimicrobial peptides are considered to be potential novel compound as antibiotic treatment. However, some bacteria, especially many human pathogens, are inherently resistant to these compounds, due to the expression of BceAB-type ABC transporters. This rather new transporter family is not very well studied. Here, we report the first full characterization of the nucleotide binding domain of a BceAB type transporter from Streptococcus agalactiae, namely SaNsrF of the transporter SaNsrFP, which confers resistance against nisin and gallidermin. We determined the NTP hydrolysis kinetics and used molecular modeling and simulations in combination with small angle X-ray scattering to obtain structural models of the SaNsrF monomer and dimer. The fact that the SaNsrFH202A variant displayed no ATPase activity was rationalized in terms of changes of the structural dynamics of the dimeric interface. Kinetic data show a clear preference for ATP as a substrate, and the prediction of binding modes allowed us to explain this selectivity over other NTPs.

Self‐immunity to antibacterial peptides by ABC transporters

“…The domain organisation of these ABC transporters is completely different in comparison with the LanT‐type or LanFEG‐type transporters [155]. The BceB protein (TMD) is predicted to have 10 TM helices and an additional large extracellular subdomain (ECD) between TM helix 7 and TM helix 8 of roughly 200 amino acids in size, where the BceA protein is the NBD [156] (Table 1). Interestingly, the topology of the C‐terminal four TM helices and the ECD is similar to MacB [157].…”

New insights into the resistance mechanism for the BceAB-type transporter SaNsrFP

“…We included two controls where the strains were transformed with (I) an empty plasmid ( L. lactis NZ9000Cm) (II) a plasmid containing a variant of the nsrfp gene ( L. lactis NZ9000 Sa NsrF H202A P) 34 . This NZ9000 Sa NsrF H202A P strain is used since the transporter carries a mutation in the H-loop, a highly conserved region of ABC transporters, and as a result is not able to hydrolyze ATP 47 . This mutation causes loss of ATP hydrolysis and stabilization of the closed conformation 53 .…”

“…This mutation causes loss of ATP hydrolysis and stabilization of the closed conformation 53 . Although the substrate still binds to it as the transporter, it cannot be translocated because the required energy cannot be provided 34 , 47 . The growth of L. lactis NZ9000Cm, L. lactis NZ9000 Sa NsrFP, L. lactis NZ9000 Sa NsrF H202A P and L. lactis NZ9000NisT was monitored online over a time period of 500 min (Fig.…”

“…Resistance at a low level against nisin (2.6-fold) was shown in Khosa et al, in which an inactive variant of the protease Sa NSR ( Sa NSR S236A ) was produced 32 . Even though, it was demonstrated that NZ9000 Sa NsrF H202A P shows no ATPase activity in-vitro 47 , it is known that it is difficult to compare in-vitro with in-vivo data since it cannot be excluded that other processes in the bacterial cell might lead to such a residual bacitracin resistance for Sa NsrF H202A P. On the other hand, the residual resistance is only observed with bacitracin not with zinc-bacitracin. It has been shown that bacitracin shows a higher attraction to the membranes in the presence of zinc and the target most probably due to the observation that it is forced into an amphiphile conformation 20 .…”

“…In previous work, it was shown that Sa NsrFP is able to confer resistance against nisin without its TCS 34 . As a control for our study, we also analyzed the ATPase-deficient mutant of the ABC transporter that showed no ATPase function in-vitro 47 .…”