β-lactams are a class of most widely used broad-spectrum antibiotics1, which inhibit bacteria cell growth by targeting transpeptidase, therefore, inhibiting the biosynthesis of peptidoglycan, the main component of bacterial cell wall. Bacteria can develop the ability of resisting β-lactam antibiotics by producing β-lactamase2, the enzyme that cleaves the peptide bonds in β-lactam rings and thereby stopping β-lactams from damaging bacterial cell wall synthesis. β-lactamase can be both constitutive or inducible. Inducible systems are studied by scientist and many mechanism has been proposed on how bacteria sense β-lactams and how they induce β-lactamase synthesis after receiving the signals. Mechanisms vary in bacteria species but in this issue a new mechanism was discovered by Li et al.3, which showed direct sensing of β-lactam antibiotics and inducing β-lactamase production by a Two-component system (TCS).
In a TCS, a histidine kinase (HK) acts as a receptor that senses environmental signals, which leads to the autophosphorylation of HK. The phosphate group is then transferred to a response regulator (RR) which regulates gene expression thus affects protein production. In this study, Li et al. found a TCS regulate system in Vibrio parahaemolyticus that could sense environmental β-lactams directly with HK VbrK and the signal was passed downstream to the RR VbrR, which regulates the expression of β-lactamase.
The latest study carried out by the Li group employed a traditional yet effective method to find out which protein was indispensable in the β-lactamase induction process by genetically knocking out histidine kinase genes and scanned for the viable mutant in carbenicillin-containing LB plate. Vibrio parahaemolyticus, a Gram negative bacteria that could develop β-lactam resistance, was studied in this experiment. To find out where did the phosphorylation took place, histidines on VbrK and possible amino acids on VbrR were substituted with alanines. As a result, H286 and D51 were the phosphorylation sites in the induction pathway. Using DAS Transmembrane prediction server, the VbrK was predicted to have two transmembrane domains: one in 9-16, the other 240-260 (Fig. 1). VbrR with a C-terminal 6xHis tag was expressed, which allowed to track VbrR in vivo. As predicted, both H286 of VbrK and VbrR were inside the membrane.
Having established what were the main components of TCS and where were they located, a chemical-genetic essay was adopted to see the relationship between β-lactam, VbrK, VbrR and blaA (a gene that encoded a class A CARB β-lactamase). RNA-seq and RT-PCR were introduced in the experiment and discovered that β-lactam, VbrK, VbrR and also ATP were essential to blaA expression and β-lactamase activity reacted to carbenicillin in a dose-dependent manner. Also a shift-band in VbrR Phos-tag assay was observed, indicating that VbrK and VbrR formed a functional TCS.
Focusing on how did the extracellular domain of VbrK functioned as a receptor of β-lactams, single substitution of L82 with alanine was performed and it resulted in no production of β-lactamase, which proved that L82 was critical for β-lactam recognition. Prediction of the structure of the extracellualar domain revealed that the amino acids formed a pocket to bind β-lactams.
It is significant for the experiment to find one of the few physiological signals identified for bacterial TCRs, yet more is to be found in future studies. However, this undoubtedly identified a potential target for inhibiting β-lactamase production thus solve β-lactams resistance problem faced in clinical practice. TCS is an inducible system yet to be fully studied, however, identifying β-lactam as the signalling ligand of VbrK gives insight to the structure-activity relationship of VbrK and the mechanism of its autophosphorylation.
Figure 1 Induction of β-lactamase production after carbenicillin (β-lactams) binds to the pocket of HK VbrK
Reference
1. Elander RP (2003) Industrial production of beta-lactam antibiotics. Appl Microbiol Biotechnol 61(5-6):385–392.
2. Chiou J, Li R, Chen S (2015) CARB-17 family of β-lactamases mediates intrinsic resistance to penicillins in Vibrio parahaemolyticus. Antimicrob Agents Chemother 59(6):3593–3595.
3. Li, L., et al. (2016). “Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics.” Proceedings of the National Academy of Sciences 113(6): 1648-1653.