In Situ Biofilm Affinity-Based Protein Profiling Identifies the Streptococcal Hydrolase GbpB as the Target of a Carolacton-Inspired Chemical Probe.

Natural products are important precursors for antibiotic drug design. These chemical scaffolds serve as synthetic inspiration for chemists who leverage their structures to develop novel antibacterials and chemical probes. We have previously studied carolacton, a natural product macrolactone fromSorangium cellulosum, and discovered a simplified derivative, A2, that maintained apparent biofilm inhibitory activity, although the biological target was unknown. Herein, we utilize affinity-based protein profiling (AfBPP) in situ during biofilm formation to identify the protein target using a photoexcitable cross-linking derivative of A2. From these studies, we identified glucan binding protein B (GbpB), a peptidoglycan hydrolase, as the primary target of A2. Further characterization of the interaction between A2 and GbpB, as well as PcsB, a closely related homologue from the more pathogenic S. pneumoniae, revealed binding to the catalytic CHAP (cysteine, histidine, aminopeptidase) domain. To the best of our knowledge, this is the first report of a small-molecule binder of a conserved and essential bacterial CHAP hydrolase, revealing its potential as an antibiotic target. This work also highlights A2 as a useful tool compound for streptococci and as an initial scaffold for the design of more potent CHAP binders.

Structure predictions and functional insights into Amidase_3 domain containing N-acetylmuramyl-L-alanine amidases from Deinococcus indicus DR1.

BACKGROUND: N-acetylmuramyl-L-alanine amidases are cell wall modifying enzymes that cleave the amide bond between the sugar residues and stem peptide in peptidoglycan. Amidases play a vital role in septal cell wall cleavage and help separate daughter cells during cell division. Most amidases are zinc metalloenzymes, and E. coli cells lacking amidases grow as chains with daughter cells attached to each other. In this study, we have characterized two amidase enzymes from Deinococcus indicus DR1. D. indicus DR1 is known for its high arsenic tolerance and unique cell envelope. However, details of their cell wall biogenesis remain largely unexplored. RESULTS: We have characterized two amidases Ami1Di and Ami2Di from D. indicus DR1. Both Ami1Di and Ami2Di suppress cell separation defects in E. coli amidase mutants, suggesting that these enzymes are able to cleave septal cell wall. Ami1Di and Ami2Di proteins possess the Amidase_3 catalytic domain with conserved -GHGG- motif and Zn2+ binding sites. Zn2+- binding in Ami1Di is crucial for amidase activity. AlphaFold2 structures of both Ami1Di and Ami2Di were predicted, and Ami1Di was a closer homolog to AmiA of E. coli. CONCLUSION: Our results indicate that Ami1Di and Ami2Di enzymes can cleave peptidoglycan, and structural prediction studies revealed insights into the activity and regulation of these enzymes in D. indicus DR1.

Biofilm-disrupting effects of phage endolysins LysAm24, LysAp22, LysECD7, and LysSi3: breakdown the matrix.

The ability of most opportunistic bacteria to form biofilms, coupled with antimicrobial resistance, hinder the efforts to control widespread infections, resulting in high risks of negative outcomes and economic costs. Endolysins are promising compounds that efficiently combat bacteria, including multidrug-resistant strains and biofilms, without a low probability of subsequent emergence of stable endolysin-resistant phenotypes. However, the details of antibiofilm effects of these enzymes are poorly understood. To elucidate the interactions of bacteriophage endolysins LysAm24, LysAp22, LysECD7, and LysSi3 with bacterial films formed by Gram-negative species, we estimated their composition and assessed the endolysins' effects on the most abundant exopolymers in vitro. The obtained data suggests a pronounced efficiency of these lysins against biofilms with high (Klebsiella pneumoniae) and low (Acinetobacter baumannii) matrix contents, or dual-species biofilms, resulting in at least a twofold loss of the biomass. These peptidoglycan hydrolases interacted diversely with protective compounds of biofilms such as extracellular DNA and polyanionic carbohydrates, indicating a spectrum of biofilm-disrupting effects for bacteriolytic phage enzymes. Specifically, we detected disruption of acid exopolysaccharides by LysAp22, strong DNA-binding capacity of LysAm24, both of these interactions for LysECD7, and neither of them for LysSi3.

Engineering of chimeric enzymes with expanded tolerance to ionic strength.

Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.