Tyrosine Phosphorylation as a Widespread RegulatoryMechanism in Prokaryotes

Landon J. Getz,a Cameron S. Runte,a Jan K. Rainey,b,d Nikhil A. Thomasa,c

aDepartment of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, CanadabDepartment of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, CanadacDepartment of Medicine, Division of Infectious Diseases, Dalhousie University, Halifax, Nova Scotia, CanadadDepartment of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada

ABSTRACT Phosphorylation events modify bacterial and archaeal proteomes, impart-ing cells with rapid and reversible responses to specific environmental stimuli or niches.Phosphorylated proteins are generally modified at one or more serine, threonine, or ty-rosine residues. Within the last ten years, increasing numbers of global phosphopro-teomic surveys of prokaryote species have revealed an abundance of tyrosine-phosphorylated proteins. In some cases, novel phosphorylation-dependent regulatoryparadigms for cell division, gene transcription, and protein translation have been identi-fied, suggesting that a wide scope of prokaryotic physiology remains to be character-ized. Recent observations of bacterial proteins with putative phosphotyrosine bindingpockets or Src homology 2 (SH2)-like domains suggest the presence of phosphotyrosine-dependent protein interaction networks. Here in this minireview, we focus on protein ty-rosine phosphorylation, a posttranslational modification once thought to be rare in pro-karyotes but which has emerged as an important regulatory facet in microbial biology.

KEYWORDS SH2 domain, cell biology, pathogenesis, phosphorylation, proteinchaperone, protein tyrosine binding domain, proteomics, transcriptional regulation,tyrosine kinase

Recent phosphoproteomic data from some bacterial species contrast with earlyreports that detected very low levels of tyrosine-phosphorylated proteins (�1%)

within bacterial proteomes (1, 2). Tyrosine phosphorylation of a small set of bacterialproteins was first linked to specialized cellular events, primarily with the synthesis andexport of polysaccharides associated with lipopolysaccharide (LPS) and capsule biosyn-thesis (3–5). In contrast, eukaryotic tyrosine phosphorylation has long been considereda major tenet in signal transduction mechanisms, with a diverse array of tyrosinekinases and protein substrates (6). However, within the last 10 years, detailed bacterialand archaeal phosphoproteomic studies have resulted in a fresh view that comparesclosely to the eukaryotic condition, where tyrosine phosphorylation is a central para-digm. Improved phosphotyrosine peptide enrichment techniques combined withhighly sensitive mass spectrometry now reveal that tyrosine phosphorylation is indeedwidespread across archaeal and bacterial proteomes (7–13).

CELLULAR FACTORS CONTRIBUTING TO TYROSINE PHOSPHORYLATION

Within a cellular context, several factors are required for a phosphate group to becovalently linked to tyrosine. In brief, a kinase acts to mediate transfer of a phosphategroup (PO4) to a protein substrate that has an accessible hydroxyl group on a tyrosineresidue.

Tyrosine kinase activity. The topic of bacterial tyrosine kinases (BY-kinases) hasbeen extensively reviewed by many groups (14–16), highlighting the diverse aspects of

Citation Getz LJ, Runte CS, Rainey JK, ThomasNA. 2019. Tyrosine phosphorylation as awidespread regulatory mechanism inprokaryotes. J Bacteriol 201:e00205-19. https://doi.org/10.1128/JB.00205-19.

Editor William Margolin, McGovern MedicalSchool

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Nikhil A. Thomas,n.thomas@dal.ca.

Accepted manuscript posted online 1 July2019Published

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bacterial tyrosine kinase biology. Notably, most BY-kinases characterized to date reveala structurally distinct class of proteins from the well-characterized “Hanks-type” eu-karyotic tyrosine kinases (14). Furthermore, BY-kinases are also distinct from so-called“eukaryotic” serine/threonine kinases, which are more prevalent in bacterial genomes(17, 18). BY-kinases have two transmembrane domains and typical Walker A and B ATPbinding motifs in their cytoplasmic catalytic domain. The ATP binding feature of theseproteins supports their eventual autophosphorylation, most often observed on variousclustered C-terminal tyrosine residues (19–21).

The cell surface-exposed feature of BY-kinases is thought to facilitate extracellularsignal integration, leading to cytoplasmic kinase activity and diverse physiologicalresponses (Fig. 1). In the case of Firmicutes, the function of the membrane andcytoplasmic protein domains has been separated into two separate proteins (22–24).Here, a membrane-located protein modulator interacts with a separate cytoplasmicprotein that carries out kinase activity. It is not exactly clear why this bipartite arrange-ment has occurred, although separating the functions potentially allows for an expan-sion of cellular responses. For example, in Bacillus subtilis, TkmA, SalA, and MinD act asindependent membrane association partners for the BY-kinase PtkA, which can thendissociate and target various cytoplasmic proteins for phosphorylation (25).

Another group of phosphorylating enzymes are known as dual-specificity proteinkinases (DSPK) that catalyze the transfer of a phosphate group to serine, threonine, or

FIG 1 Selected phosphotyrosine-regulated processes in Gram-positive and Gram-negative bacterial cells. The two large boxes indicatea representative bacterial cell and the dashed line represents a cell division site (septum). Arrows indicate the pathway direction ofthe cellular process. Interrupted lines (in 3 and 5) indicate transcriptional inhibition. (1) Wzc autophosphorylation increases copoly-merase activity in E. coli. (2) Tyrosine phosphorylation of DnaK by PtkA during heat shock enhances maintenance of misfolded proteinsin Bacillus subtilis. (3) Tyrosine phosphorylation of SalA promotes DNA binding to prevent scoC transcription in B. subtilis. (4) S.pneumoniae CspD requires CpsC to be recruited to cell division site, leading to its autophosphorylation, which supports capsuleproduction. (5) DNA binding and transcriptional repressor activity of Cra is blocked by its tyrosine phosphorylation, leading to LEE-1gene expression in enterohemorrhagic E. coli. (6) Tyrosine phosphorylation of SSB enhances SSB affinity for DNA in B. subtilis. (7)Phosphorylation of CesT on Y152 results in enhanced effector secretion via the type III secretion system in enteropathogenic E. coli.

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tyrosine. DSPK have been identified in Bacillus spp., Salmonella enterica serovar Typhi-murium, Chlamydophila pneumoniae, and Mycobacterium tuberculosis (11, 26–28). WhileDSPK are common in yeasts, their specific roles in bacteria are modestly understooddue to few characterization studies. Interestingly, Escherichia coli with genetic deletionsfor its two known BY-kinases (Etk and Wzc) was still capable of directing tyrosinephosphorylation for a substantial amount of its cellular proteins (7), suggesting thatnovel tyrosine kinases remain to be identified or perhaps that known serine/threoninekinases exhibit a dual specificity that includes tyrosine phosphorylation.

Substrate and site specificity. Kinase substrate specificity is thought to bedependent on the amino acid sequence surrounding the phosphorylation site (29).We are only beginning to understand the nature of bacterial phosphosite motifs,yet differences and some similarities with eukaryotic phosphosites are apparent (7,8). As more examples of tyrosine-phosphorylated peptides are identified throughmass spectrometry-based analyses, conserved motifs will be determined. Phos-photyrosine-specific motifs have recently been proposed for E. coli and Shigella flexneri;however, only two motifs are conserved across both species, YXXXK and YXXK (7, 8). Itshould be noted that these two motifs are not readily observed in other bacteria withvalidated phosphotyrosine proteins, suggesting that other mechanistic or temporaldeterminants could be involved in specific bacterial lineages. Continued work to furtherinvestigate the putative motifs and other possible determinants will be valuable forcharacterizing kinase-substrate specificity. Lastly, cocrystallization studies are requiredto elucidate structural details about how specific tyrosine kinases interact with con-served tyrosine motifs. These and other challenging approaches are needed to betterunderstand mechanistic aspects of tyrosine phosphorylation.

FUNCTIONAL CONSEQUENCES OF TYROSINE PHOSPHORYLATION IN BACTERIA

Beyond the prototypic role in capsule and lipopolysaccharide (LPS) biogenesis,tyrosine phosphorylation in prokaryotic organisms has been functionally linked toregulatory roles in nutrient sensing, protein localization, stress responses, transcrip-tional regulation, and virulence. Many examples were initially discovered in Gram-positive bacteria, primarily in Bacillus subtilis (reviewed by Mijakovic and Deutscher[30]), with roles for tyrosine phosphorylation recently reported in Gram-negative bac-teria.

Capsular and extracellular polysaccharide biogenesis. Some of the first discov-eries for the role of tyrosine phosphorylation in bacteria were based on studies ofcapsular polysaccharide biogenesis. The role of tyrosine phosphorylation in capsuleformation has been a very challenging area of study, and the exact mechanismsinvolved remain elusive. Multiple lines of evidence clearly indicate that autophosphor-ylation of the BY-kinase is required. In an apparent dichotomy, dephosphorylation ofthe requisite BY-kinase, often performed by a cognate phosphatase, is also required forcapsule biogenesis. Therefore, it appears likely that a dynamic, multistep processinvolving reversible tyrosine phosphorylation contributes to capsule biogenesis. Manycomplex models with additional protein partners have been proposed, and the readeris directed to those reports for specific details (21, 31).

Transcriptional regulation. Phosphotyrosine modification of many different tran-scription regulators has been shown to impact DNA binding and, correspondingly, toaffect gene expression. Here, tyrosine phosphorylation and dephosphorylation act as areversible switch for activating or silencing gene expression, allowing a cell to rapidlyrespond to environmental conditions. The examples identified to date highlightphosphorylation-mediated steric hindrance and protein conformational changes asmechanisms of action.

(i) SalA. In vitro studies using the B. subtilis transcriptional repressor SalA revealedthat efficient binding to specific DNA was ATP dependent (32). Further protein inter-action studies showed that SalA interacted with the BY-kinase PtkA, raising the possi-bility that it is subject to tyrosine phosphorylation. Indeed, mass spectrometric datarevealed that SalA was tyrosine phosphorylated at residue 327 by PtkA. Interestingly,

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this region of SalA is not involved in DNA binding; however, when it is tyrosinephosphorylated, it supports an enhanced ability of the amino-terminal region of SalAto bind ATP, thus resulting in higher binding affinity to DNA (32). Therefore, tyrosinephosphorylation of SalA results in its conformational change, which increases itsfunctionality as a DNA binding transcriptional repressor (Fig. 1).

(ii) Cra. The Cra transcriptional regulator is a member of the LacI/GalR repressorfamily, and within enterohemorrhagic E. coli (EHEC), it negatively regulates the firstgenetic operon within the locus of enterocyte effacement (LEE) pathogenicity island(33). An EHEC phosphotyrosine proteomic study identified Cra Y47 as a phosphosite (7),raising the possibility that Cra modification could impact gene expression. The cognatetyrosine kinase for Cra is not known, and therefore an approach to incorporate anonhydrolyzable phosphotyrosine structural analog into Cra (in a site-specific manner)was employed (34). Specifically, p-carboxymethylphenylalanine (pCmF) was incorpo-rated into Cra at residue 47, using a heterologous expression system, to study the roleof Cra Y47 phosphorylation. The purified Cra protein (Cra-pCmF47) was used for in vitroDNA binding assays, which revealed reduced Cra-pCmF47 binding to the LEE1 operonpromoter region compared to that of wild-type Cra or phosphodeficient Cra (Y47F) (Fig.1). These data suggest that tyrosine phosphorylation of Cra sterically hinders andreduces its DNA binding capacity, which permits LEE1 gene expression, leading to EHECvirulence (35).

STRESS RESPONSES INVOLVING TYROSINE PHOSPHORYLATIONSingle-stranded DNA binding proteins and other DNA binding proteins. Single-

stranded DNA binding protein(s) (SSB) is found in all organisms and serves to modulateDNA repair, recombination, and replication. In eukaryotes, SSB and other DNA bindingproteins are known to be phosphorylated on serine and threonine residues (36–38),whereas studies on bacterial SSBs have revealed phosphorylation on tyrosine residues(39). Specifically, a Bacillus subtilis protein kinase, YwqB, was shown to phosphorylateSSB, and phosphorylation increased the DNA binding efficiency of SSB by 200-fold (Fig.1). Furthermore, the data showed that the tyrosine phosphorylation status of SSBdecreases during RecA-dependent DNA repair. It was postulated that DNA bound bySSB would inhibit the nucleation of RecA. Indeed, during the DNA damage response,stimulated by mitomycin, SSB tyrosine phosphorylation status declined, and in a ΔywqDmutant the cells survived at a lower rate than that of wild-type cells. It is likely that SSBis dephosphorylated during the DNA damage response, modulating its binding to DNAand allowing RecA-dependent DNA repair to occur. In another paradigm of DNAdamage responses with Deinococcus radiodurans, phosphorylation of RecA at Tyr77 andadditionally at Thr318 modifies the activity of RecA by increasing its affinity fordouble-stranded DNA (dsDNA) (40). It is unclear whether this occurs for RecA homo-logues in other bacteria, although the above examples of tyrosine phosphorylation ofDNA binding proteins involved in DNA repair and recombination mechanisms warranta fresh look at the dynamic nature of stress responses in bacteria. Future studies thatassess phosphoproteomes under stress-inducing conditions will likely provide newdiscoveries.

DnaK modification upon heat shock. DnaK is a chaperone protein associated withheat shock responses, and it works in concert with its cochaperones, DnaJ and GrpE,among other proteins, to maintain misfolded or denatured proteins (41). Mass spec-trometry analyses with various PtkA-deficient and PtpA-deficient strains (kinase andphosphatase, respectively) demonstrated that B. subtilis DnaK was phosphorylated ontwo tyrosine residues, Y573 and Y601; however, only Y601 was linked to its ability torespond to heat shock (42). Specifically, it was shown that bacteria expressing DnaKwith a Y601F substitution had diminished survival rates after heat shock, andDnaK(Y601F) was deficient for protein interactions with its cochaperones, DnaJ andGrpE. Compared to DnaK sequences in other bacteria, it was noted that some speciescontain a phenylalanine residue at position 601. The authors posit that some bacteria

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may have mutated the phenylalanine at position 601 to tyrosine, resulting in a morerobust and PtkA kinase-inducible regulation mediated by DnaK.

Cell cycle regulation. Capsule production in various Streptococcus pneumoniaeserotypes is linked to the tyrosine phosphorylation state of CpsD (43), although thedetailed mechanism of capsule expression remains elusive and likely includes CpsDdephosphorylation mediated by the phosphatase CpsB (44, 45). A challenging questionrelating to how S. pneumoniae coordinates capsule expression during cell division wasaddressed using mutant strains expressing fluorescently tagged CpsC and CpsD pro-teins (21). CpsD was shown to localize to the cell division site in a CpsC-dependentmanner. Furthermore, localization at the cell division site triggered CpsD autophos-phorylation and recruitment of CpsH, forming a component of the capsule polymerasemachinery. Bacteria expressing nonphosphorylatable CpsD variants were further shownto be deficient for capsule production at the cell division site, thereby linking tyrosinephosphorylation to a temporal-spatial aspect of cell division. Lastly, elongated cells withputative cell division defects were observed for bacteria expressing nonphosphorylat-able CpsD, leading to a model where CpsD phosphorylation coordinates proper celldivision and daughter cell encapsulation (21). It will be interesting to learn whether thisis a widespread mechanism for other capsule-expressing bacteria, including Gram-negative bacteria that contain a single BY-kinase protein structure.

ARCHAEAL PROTEIN TYROSINE PHOSPHORYLATION

Archaeal genomes typically encode multiple Hanks-type protein serine/threoninekinases, in addition to a variety of serine/threonine phosphatases (46). There are noknown tyrosine kinases in the Archaea and there do not appear to be any BY-kinasehomologues encoded in archaeal genomes (47). In contrast, specific protein tyrosinephosphatases (PTP) have been identified and characterized (reviewed by Kennelly [46]),suggesting that tyrosine-phosphorylated proteins (i.e., cellular substrates) likely exist. Insupport of that view, a phosphoproteomic study in the archaeon Sulfolobus solfataricusrevealed extensive protein phosphorylation, with the Ser/Thr/Tyr ratio skewed heavilytoward phosphotyrosine (25.8/20.6/53.6%) (12). This dramatic skew has not beenobserved in other archaeal phosphoproteomes to date and is surprising, given theapparent absence of tyrosine kinase homologues in archaeal genomes. For example, astudy in Halobacterium salinarum revealed a stark absence of phosphotyrosine proteins,with serine and threonine modifications being well represented (13). It is not clear whythe significant skew toward pTyr was observed in the Sulfolobus study, althoughdifferences in phosphopeptide enrichment and mass spectrometry methodologiesmight explain the disparity. The kinase or kinases involved with these modificationshave not been functionally identified, and this remains an open set of questions for thefield.

Some of the extensive S. solfataricus phosphotyrosine modifications were observedon early enzymes required for gluconeogenesis, pentose and hexose metabolism, andthe tricarboxylic acid (TCA) cycle (12). This suggests that many of these enzymes mayrequire tyrosine phosphorylation to regulate specific biochemical reactions in thesepathways. Notably, this pattern of tyrosine phosphorylation in proteins linked to sugarmetabolism and the TCA cycle compares very closely to the finding for the phospho-tyrosine proteome of E. coli (7). This very intriguing similarity between bacterial andarchaeal phosphoproteomes will need to be followed up with mechanistic studies toaddress whether tyrosine phosphorylation impacts protein function toward carbohy-drate and cellular metabolism.

BACTERIAL PATHOGENESIS AND TYROSINE PHOSPHORYLATIONBacterial pathogenesis. LPS and capsule formation are well understood as cell

surface polymers contributing to virulence; therefore, for many bacteria, their respec-tive syntheses broadly associate tyrosine phosphorylation with virulence. Extendingbeyond that corollary, many proteomic and directed studies have reported reproduc-ible site-specific tyrosine phosphorylation of proteins associated with bacterial patho-

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genesis. Some of the modified proteins are encoded from pathogenicity islands (PAI) orvirulence plasmids and are associated with specialized secretion systems.

T3SS and tyrosine phosphorylation. Independent studies have reported that avariety of type III secretion system (T3SS)-associated proteins are modified by tyrosinephosphorylation (7, 8, 27, 48). In the cases of EHEC and Shigella, multiple data setsidentified more than 10 different phosphotyrosine peptides, suggesting a role for thismodification in T3SS-mediated virulence.

In EHEC, the T3SS multicargo chaperone CesT displayed tyrosine phosphorylation onresidue Y152 or Y153 (7). Importantly, each independent modification was later linkedto different virulence-associated outcomes within a related enteropathogenic Esche-richia coli (EPEC) strain (48). CesT comprises 156 amino acids, so the targeted adjacent152 or 153 residues are very close to the C terminus. Bacteria expressing nonphos-phorylatable forms of CesT were severely attenuated for T3SS-mediated intestinalcolonization in a mouse infection model, implicating CesT tyrosine phosphorylation asan important event leading to enteric pathogenesis (48). Furthermore, the CesT 152tyrosine residue was strictly required for the protein expression of the type III effectorNleA, revealing an unexpected site-specific regulation activity for a T3SS chaperoneprotein (further discussed below in context of pTyr binding).

In Shigella flexneri, VirB acts as a master transcriptional regulator of select T3SSgenes. A phosphoproteomic study identified the transcriptional regulator VirB astyrosine phosphorylated on residues Y100 and Y113 (8). Putative phosphomimeticsubstitutions (tyrosine to glutamic acid) at each corresponding residue resulted inreduced T3SS secretion levels, whereas phosphoablative substitutions had no effect.This led the authors to suggest that VirB tyrosine phosphorylation acts to limit VirBactivity toward T3SS genes (8). A Shigella infection plaque assay on HeLa cells revealeda deficiency for bacteria expressing VirB Y100E and no difference for bacteria express-ing VirB Y113E. Additional experiments will be required to determine if VirB tyrosinephosphorylation impacts DNA binding or another mechanistic aspect of T3SS transcrip-tional regulation.

There are other examples of tyrosine-phosphorylated T3SS proteins in EHEC, Shigellaspp., and Chlamydia spp. that include conserved apparatus components and translo-cator proteins (7, 8, 27). Currently, it is unclear what functional roles tyrosine phos-phorylation plays in T3SS function, although mechanisms of host sensing, secretoryactivation, and substrate hierarchy are all unresolved questions in the field (49).Tyrosine phosphorylation could represent a posttranslational reversible mechanism toregulate T3SS function, and thus further investigations will ideally address this andother questions. Notably, threonine phosphorylation serves to activate type six secre-tion in Pseudomonas aeruginosa and Agrobacterium tumefaciens (50, 51), so a paradigm,albeit an analogous comparison, exists for regulatory protein phosphorylation andsecretion system function.

Mycobacterial tyrosine phosphorylation. Several serine/threonine kinase en-zymes (Pkn family, PknA-K) in Mycobacterium tuberculosis that play a functional role incell growth (52) have been identified, with some associated with virulence (53).Notably, members of the Pkn family have been identified as phosphorylated ontyrosine, despite M. tuberculosis lacking traditional BY-kinases (11). Specifically, PknB,PknD, PknE, PknF, and PknG were shown to catalyze Tyr phosphorylation of proteinsubstrates. Therefore, given these findings, these enzymes should be considered DSPK.These Pkn enzymes likely organize into a network for activation of one another, andprotein phosphorylation status has been demonstrated to correspond to variousgrowth conditions. PknA and PknB are both essential for M. tuberculosis replication,with PknB implicated as a key factor in latency regulation. In the case of PknG, severalstudies have linked it to regulating amino acid metabolism under nutrient-limitingconditions and latency (54, 55). Moreover, M. tuberculosis pknG mutants are defectivefor survival in macrophages (56, 57) and have been shown to exhibit reduced virulencein mouse and guinea pig infection models (54, 55). Therefore, it appears that members

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of the Pkn family of enzymes are involved in metabolic functions and, in the context ofinfection, at least PknG contributes to virulence. There remains the broader question ofwhether protein tyrosine phosphorylation mediated by Pkn family members contrib-utes to virulence.

pTyr BINDING POCKETS AND PUTATIVE SH2-LIKE DOMAINS IN BACTERIA

In the case of eukaryotes, it is well established that phosphotyrosine modificationcreates high-affinity binding sites for protein interactions, often supporting regulatorysignaling cascades (6). Proteins that bind phosphotyrosine typically have structuralpolypeptide folds, and common examples include the Src homology 2 (SH2) domainand the protein tyrosine binding (PTB) domain (58). While the current evidence forbacterial pTyr binding domains is scarce, we hypothesize, based on the large amountof phosphotyrosine-modified proteins in bacteria, that prokaryotes have proteins thatcontextually bind phosphotyrosine, akin to the eukaryotic paradigm.

A putative pTyr binding pocket within CsrA for CesT. The carbon storage regulatorA protein (CsrA) is a well-studied RNA binding protein involved in posttranscriptiongene regulation in many bacteria (59). It has been reported that the EPEC type IIIsecretion chaperone CesT competitively interacts with CsrA, displacing it from nleAmRNA, thus resulting in type III effector NleA translation to support EPEC pathogenesis(60). A CsrA-CesT cocrystal structure revealed that CesT tyrosine 152 participates inweak hydrogen-bonding interactions with a helical domain of CsrA that typicallyparticipates in RNA binding interactions (61). Our studies in EPEC have shown that CesTY152 is strictly conserved (invariant) and subject to phosphorylation (48). Moreover,mutation of CesT residue 152 to a nonphosphorylatable phenylalanine strictly abro-gated NleA effector expression, suggesting a possible role for O-linked phosphorylationin this mechanism. Molecular dynamic simulation modeling with tyrosine phosphory-lation of CesT Y152 reveals PO3-mediated tripartite H-bonding coordinated by acontiguous “pocket” encompassed by His43, Val42, and Ser41 of CsrA (Fig. 2). Intrigu-ingly, this exact region is where CsrA binds contextual mRNA “GGA” motifs (59, 62);thus, perhaps, this reveals molecular competition for this localized binding region.While additional work is required to elucidate the CesT-CsrA binding mechanism, it isnotable that eukaryotic pTyr binding pockets tend to contain His and Arg residues thatprovide accessible hydrogen atoms for coordinating H-bonding with protein domainscontaining phosphate groups (58). Lastly, in the case of EHEC and K-12 E. coli, multipleproteomic data sets demonstrated that CsrA was tyrosine phosphorylated at residue 49,although a functional link was not investigated (7). Given that CsrA is a global regulatorof gene expression in many bacteria, we speculate that proteins with appropriatecontextual tyrosine phosphorylation modifications could participate in high-affinityCsrA binding interactions with profound regulatory consequences.

FIG 2 Molecular dynamic simulation of a CsrA-CesT pY152 interaction over a 125-ns period. Relevanttime frames were extracted to show polar contacts between the pY152 and neighboring CsrA proteindomain. The cocrystal within coordinate file PDB entry 5Z38 for the CesT-CsrA dimer of dimers was usedfor the analyses. Note the tripartite polar contacts formed by CesT pY152 and CsrA residues H43, Val42,and Ser41.

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Src homology 2 (SH2) phosphotyrosine binding domains in bacterial species.Eukaryotic proteins containing SH2 protein domains exhibit high binding affinity forspecific phosphotyrosine peptide motifs (63). Such SH2 domains typically encompass100 amino acids and are identifiable by a characteristic structural protein fold. SH2domain-mediated binding to a phosphotyrosine in a given polypeptide is dependenton contextually positioned positively charged amino acids within the SH2 domain thatprovide a binding pocket to coordinate phosphotyrosine binding via electrostaticinteractions (64). Furthermore, neighboring amino acids that surround the modifiedtyrosine within the polypeptide may also interact with allosteric SH2 domain aminoacids, thereby providing specificity for these protein-protein interactions. In most cases,bioinformatic searches of bacterial genomes do not identify SH2 domain-encodinggenes. In the case of Legionella species, putative SH2-like domains had been detectedin a few early studies, although their functional relevance was elusive (65, 66). Given thepredominantly intracellular lifestyle of Legionella bacteria within protozoan hosts, it washypothesized that SH2 domain-encoding genes were acquired through horizontal genetransfer events. With many more genome sequences available, bioinformatic searchesof multiple Legionella genomes have revealed 93 SH2 domains within 84 proteins,indicating that the presence of these proteins is not rare and perhaps has biologicalconsequences (67). Notably, some of these Legionella SH2 domain proteins are trans-located into host cells via the type IV secretion system (T4SS), raising the intriguingpossibility of a role in subversion of pTyr-dependent host signaling cascades. In vitrostudies with selected Legionella SH2 domain proteins revealed high binding affinity toa variety of synthetic pTyr-containing peptides with amino acid sequences correspond-ing to known eukaryotic protein targets (67). Interestingly, the Legionella SH2 domainsbound to some pTyr peptides with affinities that exceeded that of the cognateeukaryotic SH2 domain partner. These observations suggested that the Legionella SH2domain proteins exhibit high binding affinities for phosphotyrosine with relaxedspecificity for the overall peptide architecture. Remarkably, one Legionella SH2 proteinwas found to exhibit higher binding affinity to phosphotyrosine than a rationallydesigned (i.e., recombinantly engineered) SH2 domain pTyr “superbinder” (68). Crys-tallography experiments revealed the spatial conformation of a peptide-associated pTyrside chain within the Legionella SH2 domain binding pocket as a major factor contrib-uting to greater binding affinity. Therefore, certain Legionella SH2 domain proteinsmake extensive electrostatic interactions with pTyr, which collectively contribute tohigh binding affinity. The detailed functional and infection relevance of these LegionellaSH2 domain-pTyr interactions remains elusive; however, their discovery and character-ization open a new direction for host-pathogen interaction biology.

KINASE INHIBITORS AS NOVEL ANTIMICROBIALS—ADDRESSING A NEED TOCOMBAT DRUG RESISTANCE

In mammalian cells, a central dogma is that kinase-mediated protein phosphoryla-tion contributes to regulated cell signaling. Over 30% of a cell’s proteins can bemodified at any given time (6, 69, 70). Critically, dysregulated kinase activity is associ-ated with many diseases, including cancer, and for that reason kinases are widelyrecognized as being “druggable” targets for cancer treatments. In fact, over 20 tyrosinekinase inhibitor drugs, mostly large antibody complexes, are approved for clinical use,and many more are in the development pipeline (71, 72). Coincidently, phosphorylationstudies are continually being pursued to investigate aggressive human cancers (38,73–76), with a goal of improved health care outcomes and novel kinase inhibitortherapies. We and others reason that similar approaches are needed to elucidatetyrosine kinase and phosphorylation aspects of bacterial physiology, with a long-termgoal of developing novel bacterial kinase inhibitors (14, 77).

Currently, there are no specific inhibitors that block the function of BY-kinases.BY-kinases are expected to have ATPase activity; thus, it is surprising that known ATPaseinhibitors are ineffective against this family of enzymes. This might be due to theslightly different architecture of the Walker box motifs in BY-kinases from that of

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canonical ATPases (78), although this has not been investigated. Nonetheless, it isworth exploring strategies to inhibit bacterial tyrosine phosphorylation, perhaps lead-ing to novel antimicrobials. The fact that most BY-kinases are multidomain membrane-associated proteins poses a challenge for large inhibitory antibodies to penetrate thickcell envelopes and hydrophobic barriers. A solution might be found by performingsmall-molecule drug library screening. As with all drug library screens, robust bacterialphenotypes are required, and there is generally a strong preference to target aconserved mechanism for broad-spectrum bacterial inhibition. Therefore, this high-lights the need to explore further phosphoproteomic studies with clinically relevantpathogens, toward a goal to identify biologically and structurally conserved tyrosinephosphorylation substrates across diverse bacterial species.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

As phosphotyrosine proteomic studies continue to be reported, the field has severalchallenges ahead to address a widespread yet modestly understood aspect of prokary-otic biology.

A critical set of tasks is to decipher shared and conserved phosphotyrosine mech-anisms among species. As most proteomic experiments typically represent a singlegrowth condition or time point, they are essentially snapshots of highly dynamicreversible processes, and hence might only embody part of the picture. Additionally,the issue of reproducibility between experiments and sorting through false positives inphosphotyrosine data sets is yet another hurdle. Lastly, attributing functional mecha-nisms to tyrosine phosphorylation can be very challenging, especially if the cognatekinase and phosphatase are not known for the given substrate. The use of putativephosphomimetic substitutions (e.g., glutamic acid, aspartic acid) can be informative torestore negatively charged electrostatic interactions, although these residues do notaccurately recapitulate the spatial and ionic bonding features uniquely presented byphosphotyrosine. Some researchers have employed innovative synthetic approaches tobetter mimic phosphotyrosine in proteins (34) or to generate de novo pTyr-containingproteins with engineered E. coli expression strains (79, 80). These and other approacheswill be useful for future work addressing phosphotyrosine-dependent mechanisms.

The development of quality bench-side reagents to isolate and detect tyrosinephosphorylation (prior to mass spectrometry) is beneficial when studying proteinphosphorylation. The use of commercially available antiphosphotyrosine antibodieshas been a successful approach; however, these antibodies are imperfect, as theirbinding to targets is often amino acid sequence (context) specific, thus introducingunintended bias in some studies. Modifications to established approaches include theuse of a “Phos-tag” compound in standard SDS-PAGE to impart protein mobilizationdifferences (81) or the use of phospho-specific protein stains that detect down to 1 to10 ng of a given phosphoprotein (82). While not phosphotyrosine specific, thesedetection strategies are cost-effective approaches for most laboratories and are thusvaluable to assess workflow and sample content prior to more laborious mass spec-trometry analyses. A recent development has been the recombinant engineeringand/or cloning of so-called SH2 domain superbinder protein domains (83). It has beenshown that these and other superbinders bind to phosphotyrosine with high affinityand reduced contextual sequence bias (67, 83).

After efficient phosphoprotein and phosphopeptide enrichment, the next majoradvance for the field is quantitative and time-resolved phosphoproteomics, which willhelp to address temporal aspects of microbial growth, cell signaling, and bacterialinfection. Phosphotyrosine-dependent cell physiology occurring on a defined timescale can only be elucidated with time course-based experiments, ideally coupled toquantitative detection of phosphoprotein species. Isobaric peptide labeling approachessuch as isobaric tag for relative and absolute quantitation (iTRAQ) and tandem masstags (TMT) can be applied to experimental workflows prior to mass spectrometry,allowing for quantitative differential measurements of distinct peptides in enrichedsamples (84). Another approach is stable isotope labeling by amino acids in cell culture

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(SILAC), which allows for relative quantification of proteins within a sample (85). SILAChas an advantage of labeling proteins in situ; however, it requires auxotrophs foranalyses and has a limited labeling capacity based on the amino acid makeup of a givenprotein.

The inherently reversible nature of tyrosine phosphorylation (mediated by kinasesand phosphatases) makes quantitative phosphoproteomics a difficult area to study.Furthermore, steady-state levels of most bacterial phosphoproteins are typically low,especially in bacterial infection samples where mammalian or plant proteins dominatethe sample’s protein content. Therefore, innovative quantitative approaches combinedwith phosphoprotein enrichment techniques will likely be required to elucidate howphosphotyrosine protein modifications globally contribute to prokaryotic cell physiol-ogy and regulatory events.

Tyrosine phosphorylation has emerged as a widespread occurrence in bacteria andarchaea that in some cases rivals the high levels seen in mammalian systems. Asprokaryotic tyrosine phosphorylation has long been understudied, it remains modestlyunderstood. Preliminary studies suggest that other novel enzymes with tyrosine kinaseactivity exist, suggesting that there remain many phosphotyrosine-dependent cellularmechanisms to discover. Further studies will redefine views around prokaryotic phys-iology and bacterial pathogenesis. We envision the development of bacterial tyrosinekinase inhibitors as novel approaches to limit bacterial growth during infections or tobe used as an adjunct antibiotic therapy in the treatment of challenging bacterialinfections.

ACKNOWLEDGMENTSResearch in the Thomas laboratory is supported by an operating grant (RGPIN/

05807-2019) from the Natural Sciences and Engineering Research Council of Canada(NSERC) and the Dalhousie Medical Research Foundation (DMRF). Research in theRainey laboratory is supported by NSERC operating grant RGPIN/05907-2017.

The funders had no role in interpretation or the decision to submit the work forpublication.

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