The Diverse Functional Roles of Elongation Factor Tu (EF-Tu) in Microbial Pathogenesis

1Elongation Factor Thermo Unstable (EF-Tu) is one the most abundant proteins found in bacteria, comprising up to 6% of the total protein expressed in Escherichia coli and as high as 10% of the total protein expressed in the genome reduced pathogen Mycoplasma pneumoniae. The primary, canonical function of EF-Tu is to transport aminoacylated tRNAs to the ribosome. Ef-Tu has been a therapeutic target for antibiotics (elfamycins) since the 1970s. However, current issues with elfamycins’ poor pharmacokinetics and solubility has prevented their commercialization as therapeutic agents.

2Diverse functions have been ascribed to EF-Tu many of which include important virulence traits in Gram positive and Gram-negative pathogenic bacteria. To effect alternate virulence-associated functions, including adhesion to host extracellular matrix components, EF-Tu must gain access and be retained on the extracellular surface. This poses a challenge as signal secretion motifs are absent in this highly structured protein, and motifs required for binding diverse host cell surface receptor and matrix molecules must evolve without jeopardizing structural constraints needed to execute canonical function as a G protein. Here we refer to secondary functions as “moonlighting” functions. The concept of protein moonlighting is well established in eukaryotes, and is rapidly gaining traction in prokaryotes indicating that it is an ancient and evolutionally conserved phenomenon. Although EF-Tu executes various functions in eukaryotes, a review of the moonlighting roles of EF-Tu in bacteria is lacking. Therefore, this review has a focus to discuss the ever-expanding moonlighting roles of EF-Tu in prokaryotes, and how these roles relate to pathogenesis.

3Elongation factors (Table 1) in bacteria (e.g., EF-Tu also known as EF1A) and in eukaryotes (e.g., the eukaryotic Elongation Factor 1 Complex [eEF1A]) all have the same primary and critical function to shuttle aminoacylated tRNAs to the ribosome during protein translation. A codon–anticodon system ensures that the correct amino acid is added to the growing protein chain, a process that consumes guanosine triphosphate (GTP) prior to releasing the elongation factor from the aminoacyl tRNA. However, bacteria and eukaryotes differ in the mechanism by which they recharge the elongation factor/guanosine diphosphate (GDP) complex. This recharging function is executed by the Elongation Factor Thermo stable (EF-Ts) in prokaryotes and by eukaryotic Elongation Factor 1B (eEF1B) in eukaryotes (Figure 1).

4EF-Tu is comprised of three domains known as domains i, ii and iii which have evolved a high degree of molecular flexibility. To perform its canonical function, EF-Tu must form a functional binding pocket for an aminoacyl-tRNA, and to achieve this, domain i must become aligned more closely to domains ii and iii (i.e., they must move by around 90°). The extent of intramolecular movement needed to accommodate the aminoacyl-tRNA is about one third of the protein’s total diameter, indicating how significant this conformational change is. Once the incoming aminoacyl-tRNA has docked with the mRNA, GTPase activity induces a reverse conformational change enabling the release of EF-Tu from the ribosome. The structural and functional constraints needed to execute these critical molecular interactions ensure that key domains within EF-Tu evolve slowly compared to molecules that perform their functions on the cell surface, where they face constant challenge from the host’s immunological defenses and undergo diversifying selection. As such, EF-Tu is considered to be an ancient molecule that is comprised of domains that are highly conserved in phylogenetically diverse prokaryotes. This sequence conservation extends to EF-Tu homologs in eukaryotes, which have also evolved a similar overall protein synthesis pathway.

5In bacteria, EF-Tu is encoded by the tuf gene. tuf has a highly conserved genomic location and amino acid sequence, and has been used in the construction of phylogenetic trees for species discrimination. Amongst different bacterial species the EF-Tu sequences have less than 30% sequence divergence. Low G + C Gram positive bacteria carry only a single copy of tuf. In contrast, many enteric bacteria have two copies (tufA and tufB) while three tuf-like genes have been identified in Streptomyces ramocissimus. In species with two copies of the gene, the two genes differ by less than 1.4%, based on nucleotide comparison. In some bacteria with two copies of tuf, deletion of one copy of the gene is not lethal to the bacterium. It has been postulated that a second copy of this gene (which is mainly conserved in Gram negative bacteria) evolved before the branching of eubacteria. The cause for the intermittent presence of the second copy of tuf within eubacteria has been debated. It has been proposed that the second copy arose by lateral gene transfer, at least within Enterococci, whilst others argue that lateral gene transfer is unlikely in translation factors and attribute the discontinuous observation of a second tuf gene to the theory that it had been randomly lost in some lineages.

6Eukaryotes have two isoforms of EF-Tu known as eEF1A1 and eEF1A2 (Table 1), with each sharing 96% amino acid similarity. Both isoforms are also highly expressed representing 1–11% of the total protein expressed. Some cells express just one of the eEF1A isoforms, while both are expressed after muscle trauma and in some tumor cell types. The number of genes encoding eEF1A varies widely within eukaryotes, from ten in maize to four in rice.

7There is now overwhelming evidence that proteins with canonical functions in the bacterial cytosol also perform important tasks on the bacterial cell surface. EF-Tu features prominently in many of these studies. Many moonlighting proteins are ancient, highly expressed enzymes, or are proteins that are perform essential roles in glycolysis, respiration and respond to stress. There is evidence that only a subset of cytosolic proteins can traffic onto the cell because other highly expressed cytosolic proteins are not observed on the cell surface or in extracellular secretions. Mass spectrometry studies have been instrumental in revealing the identities of surface accessible proteins that are not predicted to reside on the cell surface that have canonical functions in the bacterial cytosol. The presence of surface-associated moonlighting proteins has been confirmed using florescence and electron microscopy. It is notable that purified, soluble moonlighting protein fails to associate with the surface when exogenously incubated with bacterial cells suggesting that posttranslational modification(s) that occur in the host bacteria and/or passage through the cell membrane may be important events in a proteins ability to moonlight on the cell surface. Another unusual feature of protein moonlighting is that not all strains belonging to the same species present moonlighting proteins on their cell surface. For example, only a subset of pathogenic E. coli express surface GAPDH which binds host molecules. Finally, it is now known that moonlighting proteins are processed on the surface of bacterial pathogens. Processing is expected to increase protein disorder and alter function compared to the full length proteoform. Here we present key studies that describe the salient features that define the diverse moonlighting functions of EF-Tu related to pathogenesis (Figure 2 and Table 2).

8EF-Tu was first described as having a moonlighting function on the cell surface of M. pneumoniae. As EF-Tu has now been found on the surface of a wide range of prokaryotes (Table 3), the potential mechanisms behind its extracellular locale shall be summarized here.

9Typically, newly synthesized proteins destined for the cell surface possess either signal peptides or signal motifs that are recognized by transport machinery and translocated through the cytoplasmic membrane. However, conventionally cytosolic proteins lacking signal sequences, like EF-Tu, have also been identified extracellularly. These proteins, termed non-classically secreted proteins, can be differentiated from other cytosolic proteins by assessing properties such as amino acid composition and structurally disordered regions. Indeed, EF-Tu was one protein used to construct the feature-based non-classically secreted protein prediction software SecretomeP. However, the actual mechanisms behind non-classical protein secretion remain a topic for debate. While there are translocation systems that do not require signal peptides, such as the Holin–Antiholin system, ABC transporters, and a type seven secretion system in Gram-positive bacteria, these only account for a small portion of non-classically secreted proteins. Therefore, the presence of cytosolic proteins in extracellular locations is often linked with cell lysis.

10There is evidence supporting cytosolic protein excretion through cell lysis, and there is evidence supporting excretion through alternative secretion pathways (reviewed in ). In addition to specific secretion pathways, cytosolic protein excretion can also occur through compromised membrane integrity, translational and osmotic stress, the protein’s biochemical and structural properties, and via membrane vesicles (MVs) (Outer Membrane Vesicles [OMVs] in Gram-negative bacteria) (reviewed in ).

11Multiple mechanism may contribute to the surface location of EF-Tu; however, there are substantial reports of MVs carrying EF-Tu. Bacterial MVs are nanoparticles produced through processes such as membrane blebbing and endolysin-triggered cell death and contain various membrane and cytosolic proteins, as well as lipopolysaccharides, peptidoglycan and DNA. MVs are involved in diverse biological processes, including virulence, biofilm development, quorum sensing, horizontal gene transfer, and exportation of cellular components. EF-Tu is present within MVs derived from Gram-positive bacteria including Listeria monocytogenes, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pnuemoniae, and Streptococcus pyogenes; Gram-negative bacteria including Acinetobacter baumannii, Bacteroides fragilis, Cronobacter sakazakii, Escherichia coli, Francisella novicida, Haemophilus influenzae, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, and Pseudomonas aeruginosa; and in six Mycoplasma species. Indeed, EF-Tu was reported as one of the most abundant protein in some of these studies. Interestingly, several MVs that contain EF-Tu have been reported to increase virulence, modulate immune responses, and offer protection to infection via immunization. As the number of MV-encapsulated proteins varied dramatically within this subset of studies, ranging from 8 in S. agalactiae to 416 in F. novicida, it is not possible to determine the exact role EF-Tu plays in these processes in most instances. However, in the case of S. pneumoniae MVs, which were shown to have high immunogenic capacity and induce protective responses in mice, EF-Tu was one of 15/161 MV proteins that were immunogenic and one of two proteins from which antibodies were generated against in immunized mice.

12Antibodies against EF-Tu have also been detected in a range of natural infections, including those caused by Mycoplasma hyopneumoniae, Chlamydia trachomatis and K. pneumonia. Recombinant EF-Tu (rEF-Tu) from Mycoplasma ovipneumoniae induces an immune response in mice, increasing levels of IgG, TNF-α, IFN-γ, IL-12(p70), IL-4, IL-5, and IL-6. Sera from mice immunized with rEF-Tu also reduced M. ovipneumoniae growth. In Mycoplasma fermentans, EF-Tu interacts specifically with the C-terminal 34 amino acids of CD21 in human B lymphoma cells. CD21 receptors on the B cells enable the complement system to influence B-cell activation and maturation. The implication is that Mycoplasma species are involved in malignancies (reviewed in ) and the interaction between EF-Tu and CD21 may be significant in this regard. EF-Tu from L. monocytogenes is the main activator of host dendritic cells (DC), antigen presenting cells (APCs) that play a key role in host immune modulation. Activation of DCs is achieved when L. monocytogenes interacts with the pattern recognition receptors (PRRs) on the surface of DCs. These PRRs recognize bacterial proteins known as PAMPS (pathogen-associated molecular pattern). In L. monocytogenes EF-Tu was identified as a potent immuno-stimulatory effector in this process indicating that EF-Tu is a candidate for DC maturation-based therapies.

13Recombinant EF-Tu (rEF-Tu) has recently shown promising results as a vaccine candidate against bacterial pathogens. Immunization with rEF-Tu elicited both Th1 and Th2-type responses against Streptococcus suis and anti-rEF-Tu sera reduced viable load detection in porcine blood. Mice immunized with rEF-Tu demonstrated significant protection against lethal challenges with S. pneumoniae and increased cytokine, IgG1 and IgG2a, and CD4+ T-cell production. rEF-Tu mediated protection against S. pneumoniae has also been demonstrated in fish, and partial protection against H. influenzae was achieved in mice.

14Besides stimulating an immune response in mammals (Figure 2A), EF-Tu is also a recognized PAMP in plants. EF-Tu is secreted by via unknown mechanisms in soil dwelling, plant–pathogenic bacteria and is recognized by membrane-associated PRRs found on the extracellular surface of root epithelial cells in different plant species. The interaction between PRRs and PAMPS identifies the bacteria as an infectious threat, triggering a signal transduction cascade, that elicits an innate immune response that includes the production of reactive-oxygen species and programed cell death.

15Both monocots and dicots use EF-Tu to notify their immune system of an infection. There is however, another level of sophistication to this interaction. Different plant species are known to have evolved recognition mechanisms in their respective PRRs that interact with different regions in the EF-Tu molecule. Rice PRRs recognize the amino acids (aa) 175-225 of EF-Tu, termed EFa50, from the plant pathogenic bacteria Acidovorax citrulli (formerly Acidovorax avenae) whereas Arabidopsis thaliana recognizes the first 18 aa of EF-Tu from E. coli (termed elf18). Although elf18 does not illicit a response to this pathogen in rice plants, engineering the Arabidopsis PRR for elf18 into the rice plant enabled rice to recognize elf18 and respond by increasing resistance to bacterial attack. This proof-of-concept experiment demonstrated that PRRs can to be engineered into the genomes of different crop species and may be beneficial to the farming and food production industry. Transfer of PRRs has already been demonstrated on food crops such as tomatoes and wheat. Earlier structural studies of EF-Tu suggested that the first 12 amino acids of EF-Tu are exposed on the surface and the dodecapeptide can act as a competitive inhibitor of the elf18 elicitor. The first 12aa of EF-Tu can also suppress the apoptotic response in plant cells allowing bacterial pathogens sufficient time and nutrient resources to colonize and replicate within plant cells. elf18 and EFa50 do not seem to have similar properties, but more recent structural studies have shown that they do appear to be (at least partially) surface exposed on the EF-Tu molecule. These data indicate that these two PAMPS can interact with PRRs on plant cell surfaces.

16EF-Tu has also been shown to bind selectively to neuropeptide hormone substance P (SP). SP belongs to the tachykinin family of neuropeptides released by nerve and inflammatory cells and is linked to many inflammatory diseases because it binds to the neurokinin 1 receptor (NK-1R) which stimulates pro-inflammatory responses. The inability to trigger NK-1R decreases bacterial clearance and increases death rates in mouse models of infection. SP and/or NK-1R have been linked to disease caused by infectious agents, autoimmune disorders, psychological disturbances, cancer, atopic dermatitis, and cell proliferation. EF-Tu from S. aureus, S. epidermidis, and B. cereus has been shown to bind SP, with an associated increase in virulence and biofilm formation.

17rEF-Tu derived from Lactobacillus johnsonii triggers a pro-inflammatory response in HT29 cells and increased IL-8 secretion in the presence of CD14. IL-8 is known to increase levels of calcium ions within the cell in which it is expressed. In Bacillus subtilis EF-Tu is a calcium binding protein. However, the link between calcium and EF-Tu in prokaryotes remains tenuous and further studies are needed to investigate the voracity of this association and its implication in the inflammatory response.

18Adhesion to host cells and molecules is fundamental to pathogenesis in many bacterial species as it facilitates colonization, invasion, and host immune subversion. In many instances, moonlighting functions for EF-Tu are associated with a role in adherence to a range of host molecules and host cells (Figure 2B). This does not appear to be a trend in specific phylogenetic groups of prokaryotes, as it extends through a range of bacteria. EF-Tu resides on the surface of Francisella tularensis where it binds to the RGG domain of nucleolin on the surface of the human monocytic cell line THP-1. The HB-19 pseudopeptide irreversibly binds to the RGG domain in the C-terminus of nucleolin and effectively blocks attachment of Francisella tularensis. Consistent with these experiments, EF-Tu and a 32 kDa cleavage fragment of EF-Tu were recovered during affinity chromatography pull-down experiments using nucleolin as bait. It is notable that cleavage fragments of EF-Tu have been previously described in the cytoplasm and membrane fraction of L. monocytogenes and more recently on the extracellular surfaces of S. aureus, Mycoplasma hyopneumoniae and M. pneumoniae. Despite this finding, data describing the cleavage products in L. monocytogenes has not been reported but is an interesting observation nonetheless that warrants further investigation.

19Fibronectin (Fn) is a key component of the extracellular matrix. It is a glycoprotein that binds to integrins embedded in eukaryote cell membranes and provides support and anchors cells to substrata. Many bacterial pathogens and commensals express adhesins that bind Fn and these interactions can trigger cytoskeletal rearrangements that promote host cell invasion. Interestingly, Fn also plays a role as a signaling molecule so, binding Fn may serve other functions for the bacteria, compounding their infectivity. Indeed, many bacteria have a repertoire of dedicated, secreted adhesins that target Fn.

20EF-Tu localizes to both the outer membrane (OM) and outer membrane vesicles (OMV) of A. baumannii and binds to DsbA, a protein important in protein folding and maturation. In its external location in A. baumannii EF-Tu directly binds Fn. The genome-reduced, human pathogen, M. pneumoniae also displays EF-Tu on its cell surface and plays an important role in interactions with Fn. Anti-EF-Tu antibodies are able to prevent the binding of M. pneumoniae to immobilized Fn demonstrating the specificity of this interaction. Binding of EF-Tu is confined to the C-terminal region of EF-Tu, with two Fn-binding regions being identified at amino acid positions 192-292 and 314-394. The specificity of the Fn binding domain between amino acids 314-394 was confirmed when peptides spanning this region blocked the binding capacity of EF-Tu to Fn by 62%. Notably, EF-Tu from Mycoplasma genitalium, which shares 96% identity with EF-Tu from M. pneumoniae, does not bind Fn. Experimental comparison between the two sequences identified the residues S343, P345 and T357 to be key in the interaction with Fn. However, the Fn-binding A. baumannii EF-Tu does not possess these key binding residues identified in M. pneumoniae (see Supplementary File), suggesting an alternative Fn-binding mechanism. More recently, we have shown that EF-Tu from M. pneumoniae is a multifunctional, adhesive moonlighting protein that can bind fetuin, heparin, actin, as well as to plasminogen, vitronectin, lactoferrin, laminin, and fibrinogen.

21The exogenous addition of soluble Fn is known to promote the ability of Mycobacterium avium subsp. paratuberculosis to attach and invade two epithelial cell lines but the identity of bacterial cell surface receptor(s) for Fn were not known. EF-Tu is a surface exposed cell wall protein in Mycobacterium avium subspecies paratuberculosis (MAP). With the importance of Fn to MAP adhesion and invasion, and as EF-Tu is known to bind Fn in M. pneumoniae and A. baumannii, it was investigated for its role as a Fn-binding protein. Knowledge of the Fn-binding regions of EF-Tu from M. pneumoniae was used to map putative Fn-binding regions in EF-Tu from A. baumannii. Although the two previously identified Fn-binding regions only showed 73 and 69% identity respectively in the EF-Tu homolog from MAP, ELISA assays showed that MAP EF-Tu binds Fn in a dose-dependent manner.

22EF-Tu binds to human intestinal cells and mucins in a pH-dependent manner in the probiotic bacterium, Lactobacillus johnsonii suggesting a role for EF-Tu in gut colonization. At pH 5, EF-Tu derived from L. johnsonii was able to bind to the human colorectal adenocarcinoma cell line HT29 cells, undifferentiated CACO-2 cells and mucins isolated from HT29-MTX cells. The ability of EF-Tu to bind mucins extends to other anatomical locations, including the saliva mucin, MUC7. EF-Tu was identified as one of six proteins to bind this mucin in Streptococcus gordonii, a major oral colonizer.

23When H. pylori is co-cultured with THP-1 cells, expression of EF-Tu is upregulated and secreted and shown to localize to the surface of the human monocytic cell line THP-1 implicating it in host adhesion.

24While there are currently no universal moonlighting motifs, basic residues, both singularly and in clusters, are known to be binding anchors for anionic glycosaminoglycans, are key in plasminogen, DNA and actin-binding, and have been linked with biofilm formation. Such basic residue clusters are found throughout the sequences of EF-Tus with reported moonlighting functions (Figure 3 and Supplementary File).

25A basic residue cluster is located at the N-termini of EF-Tu molecules with known moonlighting functions (Figure 3 and Table 2). This short linear motif (SLiM) has at least three surface exposed basic residues, resides within a region of protein disorder, and possesses predicted protein:protein interaction sites. The arginine and lysine residues in positions 7 and 9 are unconserved in some bacterial species, thus may have arisen from advantageous point mutations (Figure 4).

26Interestingly, the moonlighting EF-Tu from Mycoplasma fermentans has a unique, highly surface exposed, N-terminal extension that is 39 amino acids in length and possess an additional basic residue cluster at 31nKmKgKy38 (see Supplementary File). Whether M. fermentans EF-Tu harbors moonlighting adhesive capabilities is currently unknown and may warrant future investigation.

27Apart from the N-termini, the remaining basic residue clusters observed in EF-Tu molecules that moonlight are at least partially buried, which may impede their ability to bind host molecules. However selective cell surface proteolysis, described in detail in Section “Post-translational Modifications of EF-Tu,” can overcome some of the structural impediments.

28Many bacterial pathogens of medical and veterinary significance have the ability to bind and activate plasminogen, a process that relies on interactions between basic amino acid residues on surface-accessible bacterial adhesins and kringle domains in plasminogen (Figure 2A). EF-Tu is also utilized by bacteria to dampen the host immune response. The ability of EF-Tu to bind to host complement factors and plasminogen has been demonstrated in Pseudomonas aeruginosa, Leptospira sp., Streptococcus pneumoniae M. pneumoniae (plasminogen only) and Acinetobacter baumannii (plasminogen only). The complement system is part of the innate immune system which non-specifically acts to clear the body of infection by lysing bacteria. Complement factors bound on the bacterial surface remain active, and plasminogen can be converted to plasmin in its bound state. This allows the bacteria to regulate and utilize these components for their own gain. By recruiting FH, FH1 and plasminogen the bacteria are able to inactivate C3b via a cleavage event mediated by FH, FH1 and plasmin. The C3b complement factor is an important opsonization trigger that binds and labels bacteria ready for opsonization by the host immune system. By degrading C3b, the bacteria are able to overcome this aspect of the innate immune system. Additionally, the recruitment of plasmin to the bacterial cell surface plays an important role in degrading ECM and facilitating tissue invasion. By binding complement factors such as Factor-H, FHL-1 and CFHR1 the bacterium is able to suppress this process and evade the host innate immune response.

29Bacterial infections can also lead to a reduction in white blood cell (WBC) counts in infected hosts leading to leukopenia. Isolates of K. pneumoniae from patients with leukopenia express higher levels of EF-Tu compared with K. pneumoniae isolates from patients with leucocytosis suggesting that EF-Tu may be a pathogenicity factor in K. pneumonia-based leukopenia. Interestingly, EF-Tu is upregulated in Mycobacterium species that have been phagocytosed by macrophages. The purpose of this upregulation has yet to be determined, but may further support the idea of a role for EF-Tu in bacterial evasion of the host immune system.

30Not only do bacteria have to defend themselves against the host immune response, but often they must defend themselves against each other. Due to the competitive environment in which bacteria live, they have developed toxins that target other bacterial cells. The Type VI secretion system (T6SS) can deliver toxin molecules into the cytosol of competing bacteria inhabiting the same niche. For the T6SS effector molecule in P. aeruginosa to enact its toxic effect, it must interact with EF-Tu prior to delivery into the recipient’s cytoplasm. The effector molecule and EF-Tu directly bind to each other in members of the Pseudomonas genus. These observations suggest that EF-Tu has a wide variety of moonlighting functions relating to pathogenesis in different prokaryote species.

31Some bacteria can change cell shape as a protective strategy against the immune system. By changing shape, bacteria can become less easily engulfed by phagocytes, and can enhance biofilm formation thereby increasing persistence in the host. Moreover, major actin-like cytoskeletal proteins, such as MreB, have a role in virulence. For example, in P. aeruginosa MreB regulates the type IV pili assemblage; in H. pylori MreB regulates the secretion of virulence factors; and in Salmonella enterica serovar Typhimurium a disruption in the mreB gene led to downregulation of genes involved in pathogenicity.

32The hypothesis that EF-Tu may interact with the cytoskeleton in prokaryotes is not a new idea, with the concept being introduced as early as the 1970s when EF-Tu was shown to form filaments. More recently, the formation of amyloid-like filaments by Gallibacterium anatis EF-Tu has been linked to biofilm formation. EF-Tu also interacts with MreB in E. coli. MreB forms helical filaments beneath the cell membrane and is essential for regulating cell shape. As it is well known that eukaryotic EF-Tu (eEF1A) interacts with actin, and influences cell shape, it is conceivable that this moonlighting function also occurs in prokaryotes. In B. subtilis and E. coli, EF-Tu modulates the formation of MreB filaments by binding MreB in a ratio of 1:1 (Figure 2C). One hypothesis suggests a link between EF-Tu cell concentration and cytoskeletal function. By reducing the expression of EF-Tu in the cell, cell shape can be modulated from the typical rod-like appearance to an abnormal cell shape. Alteration of cell shape is due to disruption of the process that places MreB in helical structures beneath the membrane. Further studies investigated whether EF-Tu’s role in the translation mechanism was directly related to the population of EF-Tu molecules that interact with MreB, or whether separate populations of EF-Tu are generated for these alternate roles. Treatment of bacterial cells with kirromycin, which inhibits the release of EF-Tu from the ribosome, failed to interfere with EF-Tu localization in the cytoskeleton or its interactions with MreB filaments. As previous studies have only revealed an interaction between tufB with mreB (not tufA), it is tantalizing to consider whether only one of the tuf genes is responsible for the alternate function of cytoskeletal integrity. This might suggest that evolution of two tuf genes is useful in the delegation of EF-Tu moonlighting roles.

33Proteolytic processing is an irreversible post-translational modification (PTM) that can result in a loss, gain or change of function in a protein, as well as in degradation. Processing may be a mechanism to unlock moonlighting functions that are inherent in the newly created cleavage fragments via a mechanism similar to ectodomain shedding (Figure 2D). Furthermore, cleavage fragments may serve as competitive inhibitors to host immune cells. Host cytokines, chemokines, enzymes, antimicrobial peptides and growth factors all bind ECM components, including heparin and fibronectin, to control immune responses such as leukocyte emigration through tissue. By binding to the same ligands as host effector molecules, bacterial proteins and their cleavage fragments may dampen an immune response. Additionally, cleavage may result in the loss of antigenic epitopes in surface proteins thereby circumventing host immune detection.

34Cell surface EF-Tu is proteolytically processed in M. hyopneumoniae. Cleavage fragments of EF-Tu are retained on the cell surface and recovered during affinity chromatography using different host molecules as bait. The cleavage of EF-Tu has recently been demonstrated to be more widespread than previously appreciated with cleavage sites now also mapped in the M. pneumoniae and S. aureus. Processing generates fragments that are predicted to be more structural disordered and exposes regions of EF-Tu that are normally inaccessible to the aqueous environment. In particular we have shown that novel SLiMs enriched in positive charges are exposed allowing them to bind to a range of host molecules. Processing has so far been described in bacteria that belong to the low G + C Firmicutes. Many protein:protein and protein:nucleic acid interactions require correctly spaced positive charges derived from lysine, arginine and histidine side chains in short regions of peptide sequence. Amino acids with positively charged side chain residues are encoded by A:T rich triplet codons and members of the low G + C Firmicutes are well suited to employ this as a strategy to expand their functional proteome. Single amino acid substitutions caused by single nucleotide polymorphisms (SNPs) have previously been described as pathogenicity-enhancing. Specifically, SNPs in E. coli and S. typhimurium adhesin genes have led to distinctive pathogenicity-enhanced phenotypes. SNPs can provide bacteria a selective advantage, leading to niche expansion and ultimately, novel species. We propose that the accumulation of positively charged residues via SNPs in SLiMs facilitates binding interactions with diverse host molecules. Processing presents a mechanism to release fragments, each with the potential to expose a different repertoire of SLiMS to the aqueous environment compared with the parent molecule, and generate protein multifunctionality.

35Bacterial EF-Tus are also the target for reversible PTMs such as phosphorylation, methylation and acetylation. Phosphorylation of EF-Tu has been identified in E. coli, Thermus thermophiles, B. subtilis, Corynebacterium glutamicum, Streptomyces collinus, Thiobacillus ferrooxidans, S. pneumoniae, M. genitalium, and M. pneumoniae. While phosphorylation of EF-Tu lowers its binding affinity to GTP, subsequently reducing protein synthesis, the PTM also inhibits binding by the antibiotic kirromycin, a specific inhibitor of EF-Tu. Furthermore, the associated decreased bacterial growth facilitated by EF-Tu phosphorylation has been implicated as an acclimation measure to stress conditions during infection. Similarly, demonstrated that EF-Tu methylation in E. coli lowers GTP hydrolysis and suggest a more accurate translation process as a result.

36Lysine acetylation and lysine glutarylation has been described in EF-Tu from Mycobacterium tuberculosis. These modifications can affect protein–protein and protein:nucleic acid interactions and it may be important to the pathogenicity of M. tuberculosis, although this is yet to be determined. Evidence for the role of PTMs in EF-Tu moonlighting functions have been described in P. aeruginosa, where the trimethylation of the lysine at residue 5, allows EF-Tu to structurally mimic phosphorylcholine. This modification means that EF-Tu can specifically bind a platelet-activating receptor, resulting in successful bacterial adhesion.

37EF-Tu has evolved to be a multifunctional protein in a wide variety of pathogenic bacteria. While moonlighting functions vary among microbial species there is a common theme for roles in adherence and in immune regulation. The understanding of how this essential and highly expressed protein evolved moonlighting functions is an active area of research and is likely that more diverse and important roles are yet to be discovered.

38KH and SD conceived and co-wrote the first draft with input from all authors for the final draft. VJ created the figures and contributed significantly to the revised manuscript. All authors contributed to revision, read and approved the submitted version.

39The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.