Cation/proton antiporters (CPAs) play a major role in maintaining living cells’ homeostasis. CPAs are commonly divided into two main groups, CPA1 and CPA2, and are further characterized by two main phenotypes: ion selectivity and electrogenicity. However, tracing the evolutionary relationships of these transporters is challenging because of the high diversity within CPAs. Here, we conduct comprehensive evolutionary analysis of 6537 representative CPAs, describing the full complexity of their phylogeny, and revealing a sequence motif that appears to determine central phenotypic characteristics. In contrast to previous suggestions, we show that the CPA1/CPA2 division only partially correlates with electrogenicity. Our analysis further indicates two acidic residues in the binding site that carry the protons in electrogenic CPAs, and a polar residue in the unwound transmembrane helix 4 that determines ion selectivity. A rationally designed triple mutant successfully converted the electrogenic CPA, EcNhaA, to be electroneutral.

Bacteria survive on any surface through the generation of biofilms that provide a protective environment to grow as well as making them drug resistant. Extracellular polymeric matrix is a crucial component in biofilm formation. The presence of biofilms consisting of common opportunistic and nosocomial, drug-resistant pathogens has been reported on medical devices like catheters and prosthetics, leading to many complications. Several approaches are under investigation to combat drug-resistant bacteria. Deployment of bacteriophages is one of the promising approaches to invade biofilm that may expose bacteria to the conditions adverse for their growth. Penetration into these biofilms and their destruction by bacteriophages is brought about due to their small size and ability of their progeny to diffuse through the bacterial cell wall. The other mechanisms employed by phages to infect biofilms may include their relocation through water channels to embedded host cells, replication at local sites followed by infection to the neighboring cells and production of depolymerizing enzymes to decompose viscous biofilm matrix, etc. Various research groups are investigating intricacies involved in phage therapy to mitigate the bacterial infection and biofilm formation. Thus, bacteriophages represent a good control over different biofilms and further understanding of phage-biofilm interaction at molecular level may overcome the clinical challenges in phage therapy. The present review summarizes the comprehensive details on dynamic interaction of phages with bacterial biofilms and the role of phage-derived enzymes – endolysin and depolymerases in extenuating biofilms of clinical and medical concern. The methodology employed was an extensive literature search, using several keywords in important scientific databases, such as Scopus, Web of Science, PubMed, ScienceDirect, etc. The keywords were also used with Boolean operator “And”. More than 250 relevant and recent articles were selected and reviewed to discuss the evidence-based data on the application of phage therapy with recent updates, and related potential challenges. Graphical Abstract:

Na⁺/H⁺ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na⁺, and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na⁺/H⁺ exchanger family and a model system for all related Na⁺/H⁺ exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H⁺ ions that NhaA exchanges for one Na⁺ ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na⁺ and H⁺ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na⁺/H⁺ exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300.