Interaction between two essential,conserved bacterial proteins YeaZ and glycoprotease as a potential antibacterial target in multi-drug-resistant Staphylococcus aureus

Abstract

Protein–protein interactions among highly conserved and essential proteins can serve as new targets for antibacterial therapies. One protein–protein interaction between two widely conserved and essential bacterial proteins, YeaZ and its paralog, a putative glycoprotease, is being looked into for its antimicrobial drug potential. These two proteins possess tandem functions, including repression of the branched-chain amino acids biosynthesis and induction of a tRNA modification important in enhancing translation fidelity through anticodon–codon base pairing. Heterodimer formation between these two proteins is essential for Staphylococcus aureus, and other bacterial species including Escherichia coli and Salmonella typhimurium. Such YeaZ–glycoprotease interaction could thus be a target for antimicrobial drugs designed for multi-drug-resistant S. aureus. In this review, we discuss the function, structure, and interaction between these two proteins and their orthologs in other bacteria.

Introduction

Bacterial infections continue to be a serious public health problem despite advances in preventive vaccines and antimicrobial treatments. While a healthy immune system does prevent serious, and potentially fatal, infections; the primary cause of the growing issue of bacterial infections is the emergence of multi-drug-resistant organisms (MDROs). Staphylococcus aureus is a Gram-positive facultative anaerobic bacterial pathogen that presents a leading cause of hospital-acquired infections (HAIs), especially among immunocompromised patients. ¹ This bacterium has acquired resistance to many commonly used antibiotics through lateral gene transfer, random mutations, and microbial biofilms. For example, methicillin-resistant S. aureus (MRSA) is an MDRO that is tolerant to methicillin and all penicillin-derived antibiotics. ² Methicillin normally inhibits cell-wall biosynthesis but becomes inactivated by MRSA due to the mecA gene encoding a unique penicillin-binding protein (PBD) that binds to methicillin and prevents it from reaching its target protein. ² S. aureus strains that have become resistant to vancomycin are termed vancomycin-resistant S. aureus (VRSA). Together, MRSA and VRSA highlight the need to develop new antimicrobial strategies to target MDROs.

HAIs are the primary cause of death within the health-care setting, with S. aureus being a leading cause of HAI. ³ Survival of this pathogen is largely aided by its ability to form biofilms at the site of infection or on medical devices such as catheters.^1,3,4 Biofilms are gatherings of cells protected by a self-produced matrix of extracellular polymeric substances (EPSs), made up of polysaccharides, proteins, lipids, and nucleic acids. Biofilms thwart host immune responses due to the inability of immune cells to effectively reach and recognize their foreign target antigen. ⁵ In fact, antibiotic-resistant S. aureus strains are responsible for more than 60% of skin infections ranging from the treatable impetigo to the potentially lethal necrotizing fasciitis characterized by widespread necrosis of the skin. ⁶ Therefore, it is important to explore new strategies for combating such antibiotic-resistant strains and the diseases they cause.

Within the last 20 years, protein–protein interactions (PPIs) have become a potential drug target that could be disrupted as a strategy to kill resistant organisms. ⁷ One PPI between two widely conserved and essential bacterial proteins, YeaZ and a putative glycoprotease (Gcp), is being looked at for its antimicrobial drug potential because of their tandem functions in repressing branched-chain amino acid (BCAA) biosynthesis and inducing a tRNA modification that enhances translation fidelity through anticodon–codon base pairing. In this review, we discuss the structure, function, and interaction between YeaZ and Gcp in S. aureus and other bacterial species. This can be used to better understand not only what structural moieties between these two proteins are important in their dimerization and bacterial viability but also how they can serve as new antimicrobial targets for debilitating diseases caused by MRSA, VRSA, and biofilm-protected S. aureus.

Bacterial conserved proteins: YeaZ and Gcp

YeaZ (229 amino acids (aa)) is a highly conserved, essential, multifunctional protein of largely unknown function and structure in S. aureus. YeaZ orthologs are found in almost all eubacteria, ⁸ highlighting its conserved nature. In S. aureus and other bacteria (E. coli, S. typhimurium, Thermotoga maritima, Bacillus subtilis, Vibrio parahaemolyticus, and Pyrococcus abyssi) YeaZ interacts directly with another highly conserved and essential putative O-sialoglycoprotein endopeptidase, Gcp (341 aa). Structurally, YeaZ and Gcp are members of the acetate and sugar kinases, 70-kDa heat shock proteins (Hsp70) and Actin (ASKHA) superfamily. Hsp70 proteins are known to assist in protein-folding processes in an adenosine triphosphate (ATP)-dependent manner. ⁹ A conserved metal-binding pocket, with the amino acid motif–HXEXH, is inserted within the Hsp70 actin-like fold (HALF), suggesting these proteins have metal-binding affinity and ATP-dependent protease activity.^8,10–12

The yeaZ and gcp genes are located in the same operon of S. aureus (Figure 1) along with two unidentified genes, sa1857 (460bp) and sa1855 (466bp), that do not directly form dimers with YeaZ or Gcp in the organism. ¹⁰ However, the orthologs of these genes are located in different operons in E. coli (Figure 1).

Figure 1.

The yeaZ operon in S. aureus and its ortholog in E. coli.

YeaZ and Gcp form a heterodimer in S. aureus

Our laboratory began studying Gcp in S. aureus, seeking to better understand what structural moieties are necessary for its binding to YeaZ and for the growth of S. aureus. ¹⁰ A yeast two-hybrid (Y2H) assay was used to study the interaction between YeaZ and Gcp. ¹⁰ Results showed that yeast cells with the Gcp and YeaZ plasmids grew normally on selective media indicating a possible interaction between the two proteins. Co-immunoprecipitation experiments using purified histidine (His)—tagged Gcp and YeaZ fusion proteins showed that YeaZ bound resin was able to retain folded Gcp, whereas control resin without YeaZ could not retain Gcp. ¹⁰ Alanine scanning site-directed mutagenesis, which substitutes a stretch of residues by alanine, was then utilized to determine the enzymatic and functional residues of Gcp. Certain amino acid stretches within the C-terminal region of Gcp were found to be important for cell viability and YeaZ interaction. ¹⁰ Previous work employing bacterial two-hybrid (B2H) system in E. coli also showed that YeaZ (EcYeaZ) could bind to the Gcp ortholog, YgjD, and the Sa1857 ortholog, YjeE, which are both essential for the growth of E. coli. ⁸

YeaZ–Gcp heterodimer regulates t⁶A₃₇ biosynthesis

With the knowledge that Gcp and YeaZ interact in S. aureus, a deeper investigation was initiated to discover the functions of this heterodimer. YeaZ and Gcp are multifunctional proteins. Due to their conserved status, the functions of these proteins in S. aureus were also largely evidenced in other bacteria. In their heterodimer interaction form, these proteins regulate tRNA modifications and BCAA biosynthesis.^13,14 N⁶-threonyl carbamoyl adenosine (t⁶A₃₇) is a universal tRNA modification at adenine position 37 (A₃₇) of codons starting with adenine (ANN codons) of the tRNA anticodon loop. ¹³ The modification consists of a carbonyl group and a threonine attached to the amino group of adenine in position 37 of the tRNA anticodon loop (Figure 2). This modification is important for the selection of start codons, decoding ANN codons, and enhanced translation efficiency by preventing intramolecular base pairing between amino acids U33 and A37.^12,15,16 It is found in more than 70% of tRNAs, specifically for tRNA^Ile, tRNA^Thr, tRNA^Asn, tRNA^Lys, tRNA^Ser, and tRNA^Arg. ¹⁷

Figure 2.

tRNA modification at adenine position 37 of the tRNA anticodon loop. TC-AMP, synthesized by two bacterial families, TsaC and TsaC2, consists of carbonate, threonine, and AMP. This intermediate is added onto the amino group of adenine in position 37 of the anticodon loop (a). Chemical structure of t⁶A₃₇ modification (b).

Nucleotide and metal ion–binding affinities of YeaZ and YgjD are important in understanding the specific roles these proteins play in t⁶A₃₇ modifications. T⁶A₃₇ synthesis is a two-step, ATP-dependent reaction. ¹⁷ First, two bacterial proteins, TsaC and TsaC2 (orthologs Tcs1 and Tcs2 found in Eukarya and Archaea), use L-threonine, bicarbonate, and ATP to synthesize an unstable intermediate called threonylcarbamoyl-AMP (TC-AMP). Another ATP is used to transfer TC-AMP onto position A₃₇ of a tRNA anticodon loop. In most bacteria, this transfer involves YgjD (also called TsaD), YeaZ (also called TsaB), and YjeE (also called TsaE) or their orthologs (Tcs4 in mitochondria; Tcs6, Tcs5, Tcs3, Tcs7, and Tcs8 in Eukarya and Archaea).^12,15,17,18 This raises the possibility that YgjD serves as a partner protein to YeaZ, allowing nucleotide (ATP) binding for this transfer step.^11,19,20 YgjD was shown to be essential for the synthesis of t⁶A₃₇ in E. coli.^21,22 Hydrolysis of ATP in the YeaZ–YgjD–YjeE complex could catalyze this TC-AMP transfer.

YjeE has weak ATPase activity, and in E. coli, the hydrolysis of ATP to adenosine diphosphate (ADP) is observed only when YgjD, YeaZ, and YjeE are in a complex. ¹² The interaction between YeaZ and YgjD in S. typhimurium and E. coli are almost identical. ¹² The YgjD–YeaZ interface creates an atypical ADP-binding site within the N-terminal domain of YeaZ in E. coli. YeaZ underwent a large conformational change between amino acids Cys³⁰–Thr³⁵ upon binding with YgjD in E. coli that results in an alpha-helical loop in the open conformation. In the YeaZ homodimer, this area is in a closed conformation. ¹² This residue is quite conserved in S. aureus (Ser⁴⁰–Ser⁴⁵), offering a potential site important for the YeaZ–Gcp interaction, their regulation of t⁶A₃₇ modification, and subsequently for the viability of this bacterium.

From these data, two models have been proposed for the YgjD–YeaZ–YjeE interaction. Since EcYeaZ was found to cleave YgjD, some researchers believe that upon a stress response signal, YjeE binds to the YeaZ–YgjD heterodimer. YeaZ can then cleave YgjD, activating it and allowing YgjD to perform its function. ATP hydrolysis by YjeE releases it from YeaZ. ⁸ In contrast, others hypothesize that in the YeaZ–YgjD heterodimer, YeaZ cleavage of YgjD inactivates the protein but upon an input signal, YjeE can bind to the YeaZ–YgjD heterodimer, hydrolyze ATP, and release YgjD in its active form before YeaZ cleavage. ⁸ It is not known how these three proteins interact in S. aureus. The YjeE ortholog in S. aureus, Sa1857, is not essential nor does S. aureus YeaZ cleave the YgjD ortholog, Gcp. ¹⁰ Therefore, it is probable that S. aureus and E. coli YeaZ, YgjD, and YjeE orthologs have functional differences in relation to their ability to synthesize t⁶A₃₇ modifications. Nevertheless, high conservation is apparent in that all three orthologous proteins are thought to play a role in similar biosynthetic pathways; however, it is not known whether Gcp and YeaZ’s essential nature is attributable to the t⁶A₃₇ modification.

YeaZ–Gcp heterodimers repress BCAA biosynthesis

BCAAs include isoleucine, leucine, and valine (ILV). These amino acids are important for maintaining protein structure and function. Quantitative polymerase chain reaction (qPCR) analysis showed that a six-fold reduction of YeaZ markedly increased the RNA levels of BCAA operon genes such as ilvD, leuA, and ilvA. In addition, an ilv-promoter-lux reporter system revealed that YeaZ can bind to the ilv promoter. These data demonstrate that YeaZ directly downregulates the ilv–leu operon.^13,14 Addition of Gcp to reaction mixtures containing YeaZ and the ilv promoter had no impact on YeaZ binding to the ilv promoter. Gcp does not have any DNA-binding domains and is proposed to indirectly regulate transcription of this operon, possibly through binding to YeaZ. Ilv–leu operon deletion mutants had no effect on Gcp or YeaZ expression, indicating that the essential nature of these proteins is not attributable to their repression of BCAA biosynthesis. ¹³ It is worthy of note that while Gcp essentiality in S. aureus may not be due to its role in BCAA biosynthesis repression, researchers have discovered that in E. coli, YgjD accounts for the depletion of Amadori-modified proteins (AMPs), which are formed from nonenzymatic glycation reactions and can lead to the formation of harmful advanced glycated end products (AGEs), which are highly stable toxic compounds. Evolutionary conservation of Gcp for this function may, therefore, explain its widespread essentiality in all three domains of life. ²³

YeaZ and Gcp orthologs

YeaZ, YgjD, and YjeE structure

Given the conserved nature of YeaZ, Gcp, and Sa1857, the structures and functions of these protein orthologs are quite similar in other bacterial species including E. coli, S. typhimurium, T. maritima, B. subtilis, V. parahaemolyticus, and P. abyssi).^8,10–12 YeaZ (231 amino acids (aa)) is a truncated version of the E. coli paralog (Figure 1), YgjD (337 aa), with 29% sequence identity in the first 100 amino acids of the protein sequence. ¹² EcYeaZ can cleave YgjD, but S. aureus YeaZ does not possess such an activity.^10,24 YjeE is a small (17 KDa), highly conserved protein that is essential in E. coli and many other Gram-negative bacteria such as S. typhimurium, but it is dispensable in some Gram-positive organisms including S. aureus and B. subtilis.^8,12,25 Studies using x-ray crystallography of the YjeE protein showed that it has a P-loop ATPase that hydrolyzes ATP in Haemophilus influenzae.^26,27 This may be important in the two ATP-dependent reactions of t⁶A₃₇ synthesis. Since YeaZ, YgjD, and YjeE are essential and can interact in E. coli, they may be involved in the same pathway. ⁸ YeaZ appears to be a key component of this protein complex, as it binds both YgjD and YjeE. In addition, a crystal structure study of a conserved Pseudomonas aeruginosa protein called PaYeaZ, which has 49% sequence identity to EcYeaZ showed that this protein is composed of two domains, one of which is said to be the basis for either homodimer assembly or the formation of a protein complex needed for t⁶A₃₇ biosynthesis. ²⁸

In addition to the structural ASKHA family, YeaZ orthologs also belong to a functional family described as the “inactive homologs of metal-binding proteases, putative chaperone” (COG1214). ⁸ The crystal structures of YeaZ from E. coli, S. typhimurium, T. maritima, and V. parahaemolyticus have been published.^11,12,20,24 YeaZ in all these bacterial species adopts a two-lobed structure characteristic of ASKHA superfamily proteins consisting of a β-sheet, α-helix, and a long helical domain.^24,29 Most HALF proteins bind nucleotides together with metal ions like Zn²⁺ or Mg²⁺ for hydrolysis of substrates or phosphate group transfer.^24,29 Nichols notes that YeaZ in S. typhimurium (StYeaZ) and most HALF proteins consist of this two-lobed structure that allows nucleotide binding. ²⁹ StYeaZ and EcYeaZ are closely related orthologs and both lack Zn²⁺ binding sites. Conversely, YgjD in both species was found to have an Mg²⁺ binding site shown to be important in the YgjD–YeaZ–YjeE function of t⁶A₃₇ biosynthesis in these bacterial species.^12,29 Therefore, YgjD may be necessary for YeaZ nucleotide-binding through ATP binding and hydrolysis. All YeaZ and Gcp orthologs have a two-lobed structure. E. coli YeaZ and YgjD both contain Mg²⁺ and Fe³⁺ ions that may be important in nucleotide binding. ¹² In contrast, S. typhimurium YeaZ does not contain any metal ions; however, its YgjD homolog contains an adenosine monophosphate binding pocket next to a cadmium (Cd²⁺) ion. ²⁴ T. maritima ²⁰ and V. parahaemolyticus ¹¹ YeaZ orthologs have no metal ions; their ability to bind nucleotides remains unclear.

Other functions of YeaZ, YgjD, and YjeE orthologs

In E. coli and many other Gram-negative species, YgjD, YeaZ, and YjeE are all essential, and YeaZ interacts with YgjD and YjeE. Researchers made temperature-sensitive (Ts) mutants of YgjD, YeaZ, and YjeE and found that the thr and ilv operons were greatly upregulated in the YgjD (Ts) mutant. ³⁰ It has been revealed that thrL and ilvL genes encode leader peptides, intrinsic terminators of transcription, locate just upstream of the first gene in each of the respective operons. ³⁰ There was no difference in expression of thrL between YgjD (Ts) mutants and wild-type (WT) YgjD strains, indicating that transcriptional initiation of the threonine leader peptide was not affected by the YgjD mutation. The leader peptides of the thr and ilv operons are rich in threonine and isoleucine, which are encoded in ANN codons.

As mentioned before, the t⁶A₃₇ modification is a universal tRNA modification at position 37 of ANN codons of an anticodon loop and helps in decoding these ANN codons. If the yeaZ, ygjD, yjeE genes were transcriptionally active, there would be an increase in t⁶A₃₇ modifications and thus the translation of amino acids like threonine and isoleucine, which are high in concentration in leader peptides of the thr and ilv operons. ³⁰ With these leader peptides, RNA polymerase would not express the downstream genes of the thr and ilv operons, explaining previous accounts of YeaZ–YgjD downregulation of BCAA biosynthesis. ¹⁹ It is, therefore, possible that these three proteins (YeaZ, YgjD, and YjeE) act as indirect regulators of these operons by directly modifying t⁶A₃₇ to decode ANN codons and synthesize leader peptide–associated amino acids, thereby repressing BCAA expression.

Hypotheses of a stress signal to activate the YeaZ–YgjD–YjeE complex may allow for such t⁶A₃₇ synthesis as previously described. ⁸ Indeed, in S. typhimurium, V. parahaemolyticus, and Vibrio harveyi, YeaZ acts as an essential resuscitation-promoting factor from the viable but non-culturable (VBNC) state, highlighting its response to stress-induced signals. Entering a VBNC state can also have important implications on bacterial pathogenesis. In fact, researchers created YeaZ mutants in V. harveyi, which decreased its virulence against zebrafish. ³⁰ The requirement of YeaZ for persistence within and exit from this state presents the protein as a potential drug target.^11,30 There is an evidence in Deinococcus radiodurans that YeaZ repairs interstrand DNA crosslinks formed by the chemotherapeutic agent, mitomycin C (MMC), allowing DNA replication to continue and possibly the cell to exit from the VBNC state. ³¹

Finally, YjeE, YgjD, and YeaZ may play a vital role in cell wall integrity. E. coli YjeE depletion mutants were 1.5–1.75 times wider than WT cells. Many YjeE mutants had irregular protrusions/branches, with alterations in cell wall metabolism. ⁸ YeaZ depletion strains resulted in enlarged cells, but to a lesser extent compared to YjeE-depleted strains. ⁸ The appearance of YeaZ-depleted cells resembled that of a WT E. coli treated with chloramphenicol, a translational inhibitor. This would make sense based on the reasoning that YeaZ plays an integral role in tRNA modifications. YgjD depletion strains had varied morphologies, with 60% of colonies appearing similar to YjeE depletion mutants, and 40% similar to WT cells. ⁸ Interestingly, the YjeE/Sa1857 homolog in B. subtilis, YdiB, is thought to be involved in cell wall biosynthesis based on its conservation in all bacteria except mycoplasma and ureaplasma, which are cell-wall-free species. ²⁶ In addition, YjeE is located next to the amiB gene in E. coli, which is a cell wall amidase. However, disruption of YdiB and YdiC (the ortholog of YeaZ in B. subtilis) had a negligible effect on morphology. ²⁵ In both bacteria, Sa1857 (YdiB and YjeE ortholog in S. aureus) is not essential for survival. In addition, YdiB, YdiC, and the Gcp ortholog, YdiE, are all in the same operon as are Sa1857, YeaZ, and Gcp, respectively, in S. aureus. Therefore, B. subtilis YdiB–YdiC–YdiE interactions could serve as a model for other bacteria including S. aureus.

Conclusion

While YeaZ and Gcp orthologs in E. coli, V. parahaemolyticus, S. Typhimurium, B. subtilis, P. abyssi, and T. maritima, differ in some features, they are largely conserved in terms of their functions and interactions. The conserved nature of YeaZ and Gcp and their tandem role in the t⁶A₃₇ modification and regulation of BCAA synthesis, as well as YeaZ’s role in cell wall biosynthesis, the regulation of the VBNC state, and repairing interstrand DNA cross-links highlight why these proteins can serve as potential targets for new antimicrobial drugs given the continued rise of antibiotic-resistant pathogens. Here, we hypothesize that two different domains of S. aureus YeaZ will be important for its interaction with Gcp and cell growth. A glycine-rich loop (Gly⁷⁴–Gly⁸⁰) associated with nucleotide or polyphosphate binding found to be conserved in YeaZ orthologs in E. coli, S. typhimurium, S. aureus, B. subtilis, and V. parahaemolyticus could be envisaged as an important element in Gcp interaction and S. aureus viability. Second, a conserved ATP-binding site found in a hydrophobic depression formed from YeaZ–YgjD dimerization in E. coli may also be crucial to Gcp interaction and growth in S. aureus (Ser⁴⁰–Ser⁴⁵). Future work shall focus on characterizing these residues of YeaZ for their importance in binding to Gcp and bacterial cell growth. This may be accomplished through the creation of YeaZ or Gcp structural mutants and assaying for their interaction in a bacterial two-hybrid system, as well as complementation experiments to determine if these mutations are critical for bacterial cell viability. Finally, a crystal structure of the YeaZ–Gcp heterodimer in S. aureus will undoubtedly aid in efforts to structurally examine how these proteins interact and function in tandem.