REVIEW Hicks and Mullholland, Microbiology 2018;164:1471–1480 DOI 10.1099/mic.0.000728

Cysteine biosynthesis in species

Joanna L. Hicks* and Claire V. Mullholland

Abstract

The principal mechanism of reducing sulfur into organic compounds is via the synthesis of L-cysteine. Cysteine is used for protein and glutathione synthesis, as well as being the primary sulfur source for a variety of other molecules, such as biotin, coenzyme A, lipoic acid and more. Glutathione and other cysteine derivatives are important for protection against the oxidative stress that pathogenic such as and encounter during infection. With the alarming rise of antibiotic-resistant strains of N. gonorrhoeae, the development of inhibitors for the future treatment of this disease is critical, and targeting cysteine biosynthesis enzymes could be a promising approach for this. Little is known about the transport of sulfate and thiosulfate and subsequent sulfate reduction and incorporation into cysteine in Neisseria species. In this review we investigate cysteine biosynthesis within Neisseria species and examine the differences between species and with other bacteria. Neisseria species exhibit different arrangements of cysteine biosynthesis genes and have slight differences in how they assimilate sulfate and synthesize cysteine, while, most interestingly, N. gonorrhoeae by virtue of a genome deletion, lacks the ability to reduce sulfate to bisulfide for incorporation into cysteine, and as such uses the thiosulfate uptake pathway for the synthesis of cysteine.

INTRODUCTION deletion of cysteine synthase in Salmonella typhimurium Sulfur becomes accessible to animals as bacteria and plants caused a 500-fold increase in sensitivity to ciprofloxacin [7]. incorporate inorganic sulfur into cysteine. Cysteine biosyn- The Neisseria species include the human pathogens Neisse- thesis is the primary pathway for the incorporation of sulfur ria meningitidis (meningococci) and Neisseria gonorrohoeae into a variety of cellular constituents, including methionine, (gonococci), as well as other non-pathogenic environmental thiamine, biotin, coenzyme A and more. Cysteine itself species, such as Neisseria mucosa, Neisseria flava, Neisseria plays an important role in protein molecules in Fe–S clus- sicca, etc. Meningococci are facultative commensals that col- ters, catalysis, protein folding and the formation of disulfide onize the nasopharynx and for reasons that are not fully bonds. In micro-organisms, cysteine plays an important understood occasionally enter the bloodstream or central role in protection from oxidative stress via reducing systems nervous system and cause life-threatening septicaemia or such as thioredoxin and glutathione. In bacteria, cysteine meningitis, respectively. Despite the availability of antibiot- biosynthesis is carried out almost exclusively via the con- ics for prophylactic or therapeutic treatment and the avail- served reductive sulfate assimilation pathway, which succes- ability of immunization against specific serogroups of sively reduces sulfate to bisulfide for the production of meningococcal meningitis, it remains the leading cause of cysteine using an activated form of serine. bacterial meningitis worldwide [8]. Meningococcal disease is associated with high morbidity and mortality in children Due to the important cellular roles of cysteine and the and young people, with effects such as limb loss, hearing absence of mammalian biosynthetic pathways, the enzymes loss, cognitive dysfunction, visual impairment, seizure dis- involved in sulfur metabolism and specifically cysteine bio- orders and developmental delays [9]. Gonococci are obligate synthesis are targets for the design of novel antibiotics human pathogens and are the causative agent of the sexually [1–5]. For example, the inactivation of cysteine and methio- transmitted infection gonorrhoea, which causes an esti- nine biosynthetic enzymes in Mycobacterium tuberculosis mated 78 million cases annually worldwide [10]. N. gonor- significantly reduces persistence and virulence during the rhoeae colonize and invade the epithelium of the urogenital chronic stage of infection in mice models [6]. Furthermore, tract to cause localized inflammation, and in rare cases

Received 11 July 2018; Accepted 13 September 2018 Author affiliation: School of Science, University of Waikato, Gate 8 Hillcrest Road, Hamilton, 3216, New Zealand. *Correspondence: Joanna L. Hicks, [email protected] Keywords: cysteine; cysteine biosynthesis; sulfate reduction; Neisseria; gonorrhoea; meningitis. Abbreviations: ABC, ATP-binding cassette; APS, adenosine 5¢phosphosulfate; HMM, hidden Markov model; MFS, major facilitator superfamily; OAS, O-acetylserine; OASS, O-acetylserine sulfhydrylase; PAP, phosphoadenosylphosphate; PAPS, phosphoadenosylphosphosulfate; SAT, serine acetyltransferase; sbp, sulfate-binding protein.

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disseminate and cause more serious disease, such as pelvic when using the Basic Local Alignment Search Tool (BLAST) inflammatory disease, ectopic pregnancy, infertility and to interrogate the entire genome (Fig. 1). It is, however, arthritis. Gonorrhoea is now a global health problem due to present in other N. gonorrhoeae strains, such as SK29344, the increasingly high number of strains that are resistant to SK22871, ATL_2011_01_17, 1291, MS11 and all frontline antibiotics. Since the introduction of antibiotic ATL_2011_01_21. The CysA ATP-binding subunit is pres- treatment for gonorrhoea, resistance has emerged to ent in all Neisseria, with the protein from N. gonorrhoeae sulphonamides, penicillins, tetracyclines, fluoroquinolones, being 97 % identical to that from N. meningitidis. The cysA early-generation cephlasporins and, more recently, and cysW genes appear to be in a bicistronic operon, extended-spectrum cephlasporins, including ceftriaxone, whereas cysU and the sbp appear individually in Neisseria and macrolides, including azithromycin (the main current genomes (Fig. 1). Several studies have found an upregula- treatments) [10]. Therefore, the identification of new or tion of the cysAW operon and cysU (cysT) sulfate uptake alternative effective treatment regimens is critical for future genes upon the adhesion of meningococci to epithelial cells treatment of this disease. [16–18]. Gonococci and meningococci differ in the way they obtain SULFATE REDUCTION sulfur for growth. These differences could reflect the differ- ent pathogeneses of these two organisms, with gonococci Once imported into the cell, sulfate is successively reduced being an obligate human pathogen and meningococci being to bisulfide via a series of enzymatic steps, as shown in an ‘accidental’ pathogen, and their different environmental Fig. 2. The first is catalyzed by ATP sulfhydrylase (CysDN). requirements for growth. Targeting cysteine biosynthesis in This enzyme activates sulfate in an ATP-dependent reaction these pathogens could be a promising avenue for new anti- to form adenosine 5’-phosphosulfate (APS) and pyrophos- microbials to combat gonorrhoea and meningococcal phate. ATP sulfhydrylase typically consists of two subunits, meningitis. CysD, the catalytic subunit, and CysN, the GTP regulatory subunit. In Neisseria species these subunits are encoded by TRANSPORT OF SULFATE separate cysD and cysN genes (Fig. 3), but in some bacteria such as Bacillus subtilis the two subunits are fused [19]. In There are three sulfate transport systems in bacteria, as most bacteria the next step is catalyzed by APS kinase described in [11]. Two belong to the ATP-binding cassette (CysC) to form phosphoadenosylphosphosulfate (PAPS) in (ABC) superfamily and the other belongs to the major facili- an ATP-dependent reaction. In some bacteria, including the tator superfamily (MFS) sulfate transporters. Neisseria spe- Mycobacteria and Pseudomonads, CysC, the APS kinase, is cies have a sulfate transport system belonging to one of the fused to CysN. PAPS is subsequently reduced to sulphite ABC superfamilies. This sulfate transporter has a periplas- and phosphoadneosylphosphate (PAP) by PAPS reductase mic sulfate-binding protein (sbp), the permeases CysT (now (CysH). However, in Neisseria a single enzyme, APS reduc- annotated as CysU to avoid confusion with the tRNA-Cys tase (also termed CysH), directly reduces APS to sulphite gene) and CysW, and the ATP-binding subunit CysA [12] [20] (Fig. 2). Curiously, enzymes with both PAPS and APS (Fig. 1). This system has the ability to take up sulfite and reductase activities are present in B. subtilis, Pseudomonas thiosulfate [13]. An additional ABC transporter with the aeruginosa and M. tuberculosis, for example, CysH can periplasmic binding protein CysP replacing sbp is more spe- complement a cysC mutant and vice versa [21–23]. The final cific for thiosulfate uptake. Using sequence similarity step in the reaction is the reduction of sulfite to bisulfide by searches, we found no homologues of CysP or the MFS sul- the multi-subunit enzyme sulfite reductase (CysJI) (Fig. 2). fate transporters in N. meningitidis, N. gonorrhoeae or any The Escherichia coli sulfite reductase is a complex enzyme, other Neisseria genomes. Although there is an annotated composed of two proteins, CysJ and CysI, in a a8ß4 struc- cysP gene in three recently sequenced N. gonorrhoeae strains ture. The flavoprotein CysJ (a8) contains four FAD and four [14], closer inspection via homology searches revealed that FMN cofactors and NADPH–cytochrome c reductase activ- it is actually an inorganic phosphate transporter protein. As ity. CysJ accepts electrons from NADPH and transfers them such, we hypothesize that the sbp ABC transporter is the to CysI, which reduces sulphite to sulfide [13]. CysI is a hae- only sulfate transport system in Neisseria (Fig. 1). moprotein that contains a [4Fe–4S] cluster and a siroheme. An uroporphyrinogen methyltransferase encoded by cysG The single ABC transporter identified in Neisseria is for the catalyzes the synthesis of siroheme (for incorporation into uptake of sulfate and presumably thiosulfate, given the abil- CysI) from uroporphyrinogen III [13]. CysJI is present in ity of all Neisseria species to grow on thiosulfate as a sole Neisseria species, along with CysG, although in some bacter- source of sulfur [15]. The ABC transporter for the uptake of ia such as B. subtilis uroporphyrinogen methyltransferase sulfate consists of the periplasmic binding protein sbp, activity is encoded by ylnD and ylnF, which encode the which is present in all Neisseria species, although the pro- C-terminal and N-terminal domains, respectively [24]. tein from N. gonorrhoeae is truncated at the N-terminal end compared to the N. meningitidis sbp (Fig. 1). The CysU and The genes encoding the enzymes for the reduction of sulfate CysW permeases are present in most Neisseria, including to sulfide are organized differently in different species of N. gonorrhoeae, although we could not identify the CysU bacteria. For example, in E. coli there are three transcrip- subunit in the genome of N. gonorrhoeae FA 1090, even tional units: cysDNC for the reduction of sulfate to PAPS,

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Fig. 1. The sulfate/thiosulfate ABC transporter and its genomic context in Neisseria species. The sulfate-binding protein (sbp) imports both sulfate and thiosulfate, as CysP (for the import of thiosulfate) is not present in Neisseria species. CysU and CysW are permeases in the membrane and CysA is the transporter protein. The lower panel shows the genomic context of the ABC transporter genes. cysU and sbp are encoded individually, whereas cysA and cysW are in an operon. sbp genes (green arrows) from N. gonorrhoeae strains are truncated at the 5’ end compared to other Neisseria species. cysU is present in all Neisseria strains, but absent from N. gonorrhoeae FA1090, as shown by the dotted arrow.

cysJIH for the reduction of PAPS to bisulfide and cysG the pseudogenes due to the presence of stop codons within their uroporphyrinogen methyltransferase [13]. In B. subtilis a coding sequences (Fig. 3). large cysHPylnBCDEF operon is present with the cysJI Most N. meningitidis and N. flava strains can grow on sul- operon found elsewhere [25, 26]. This led us to investigate fate as a source of sulfur, whereas N. gonorrhoeae lacks this the genomic context of genes for the reduction of sulfate in ability [15, 30]. The loss of the sulfate reduction pathway in Neisseria species, specifically N. meningitidis and N. gonor- N. gonorrhoeae through gene deletions, truncations and the rhoeae. N. meningitidis has all the genes necessary for sulfate insertion of stop codons fits with the inability of the bacte- reduction in the cysGHDNJI operon, whereas N. gonor- rium to grow on sulfate as a unique source of sulfur [15]. rhoeae has an unusual cysGNJI operon. In the N. meningiti- However, the need for cysteine in the culture media of dis serogroup B strain MC58 genome the cysGHDNJI N. gonorrhoeae can be satisfied by thiosulfate [15], suggest- operon appears twice in a ~32 kb region that appears to ing that the sulfate sbp ABC transporter discussed above is have been duplicated [27]. In other serogroup B strains such also transporting thiosulfate into the cell. When N. gonor- as H44/76 this region occurs only once [28]; we also interro- rhoeae is grown in the presence of 2 mM thiosulfate (or gated other N. meningitidis strains from the same and dif- more) as the sole source of sulfur, sulfite accumulates in the ferent clades [29], but could only identify one copy of the culture medium [15]. The cleavage of thiosulfate produces cysGHDNJI operon. The N. meningitidis genome shares sulfur and sulfite, with the sulfur originating from the sul- 90 % homology at the nucleotide level to the N. gonorrhoeae fane moiety being incorporated directly into cysteine. If the genome, and genomic comparisons allowed us to look bacterium lacks the ability to reduce sulfite to sulfur (Fig. 2), closely at the unusual cysGNJI operon arrangement and, sulfite will accumulate in the culture media, as seen in together with homology searches, determine whether the N. gonorrhoeae, reaffirming the inability of N. gonorrhoeae missing cysH and cysD genes from N. gonorrhoeae are to successively reduce sulfate to sulfide. located elsewhere in the genome. We found a deletion of 3500 bp between the cysG and cysN genes that removes cysH CYSTEINE SYNTHASE and cysD and truncates both the cysG and cysN genes Although the pathway of cysteine biosynthesis has not been (Fig. 3), as also seen by Rusniok et al. [20]. In addition, a studied in Neisseria, the fact that all strains are able to use closer inspection of the cysJI genes revealed that they are thiosulfate as a sole source of sulfate suggests that a

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Fig. 2. The sulfate transport and reduction pathway in Neisseria species. The sulfate reduction pathway absent in N. gonorrhoeae is shown in red. All Neisseria species have the sulfate-binding protein (sbp) for the transport of sulfate and thiosulfate. Serine is activated by serine acetyltransferease (SAT) to form O-acetylserine (OAS), which is converted to cysteine or S-sulfocysteine by O-acetylserine sulfhydrylase (OASS). Differences between Neisseria and other bacterial species are shown by two dotted lines; adenosine phosphosul- fate (APS) reductase directly reduces APS to sulfite and only the OASS-A isoform (CysK) is present in Neisseria species. functional serine acetyltransferase (SAT, CysE) and O-ace- cysteine. The crystal structure of SAT from Haemophilus tylserine sulfhydrylase (OASS, CysK/CysM) are present. influenzae in complex with cysteine and also with CoA [32] demonstrates CoA binding in the active site as expected, but SAT catalyzes the first step in the two-step reaction that cysteine also binds in the active site to compete with serine makes L-cysteine from L-serine. The side-chain carboxyl of and inhibit its activity. L-serine undergoes a CoA-dependent acetylation to form O- acetylserine. The second step is then catalyzed by OASS, Mammals synthesize cysteine from methionine and as such which catalyzes the ß-replacement of acetate with sulfide to lack OASS, making it a potential target for antimicrobials. convert O-acetylserine to L-cysteine. SAT and OASS form a OASS is a pyridoxal 5’-phosphate-dependent enzyme bienzyme complex termed cysteine synthase. The SAT– belonging to the cysteine synthase superfamily [13]. Most OASS interaction is highly conserved across bacterial spe- bacteria have two isoforms of O-acetylserine sulfhydrylase, cies. Most enzyme complexes form to channel substrates termed A and B. OASS-A, as discussed earlier, forms a tight from one enzyme to another. However, the cysteine syn- complex with SAT, whereas OASS-B does not interact with thase complex is unusual in that the C-terminal tail of SAT SAT [33, 34]. such as E. coli, S. typhimurium inserts into the active site of OASS, competing with the and H. influenzae have both A and B isoforms of OASS. binding of O-acetylserine [31]. In the complex one SAT OASS-A (CysK) is expressed under aerobic conditions, hexamer binds to two OASS dimers, the formation of which whereas OASS-B (CysM) is expressed under anaerobic con- is disrupted by O-acetylserine [31]. In addition to regulation ditions [35]. The OASS-A and OASS-B isoforms display by complex formation, SAT is feedback-inhibited by subtle but significant structural differences, leading to

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Fig. 3. The sulfate reduction operon from N. meningitidis compared to N. gonorrhoeae. Dotted lines denote the 3.5 kb fragment deleted from the N. gonorrhoeae genome that truncates cysG and cysN and deletes cysH and cysD. Stop codons are shown by dashed lines in cysJ and cysI pseudogenes in N. gonorrhoeae.

differences in their preferred substrates [4], with OASS-B classified as being important for the meningococcal coloni- preferring thiosulfate and OASS-A preferring bisulfide. zation of epithelial cells [41]. Neisseria species appear to have only one OASS isoform, In other organisms, cysteine, as a major reducing agent, annotated as CysK (OASS-A), and multiple searches of the plays an important role in survival, mainly due to protection genomes of species belonging to Neisseria found no OASS- against oxidative stress. N. gonorrhoeae lacks any glutathi- B isoforms. We modelled the structures of the SAT (CysE) one importers [42] and, as such, we hypothesize that cyste- and OASS (CysK) proteins from N. gonorrhoeae using the ine plays a vital role in overcoming oxidative stress that the Phyre2 modelling server [36]. The structures of the SAT bacterium encounters upon infection. (Fig. 4a) and OASS-A (Fig. 4d) enzymes were modelled with 100 % confidence and 93 and 99 % coverage, respec- tively. We then overlaid the model structures with SAT and REGULATION OASS-A structures from enzymes not used in the modelling The cysteine regulon of other is upreg- process. There is good conservation of structure for both ulated in vitro in the presence of nitric oxide [43], under enzymes, even though there is little sequence homology, as oxidative stress [40] and in vivo during infection or long- shown by the hidden Markov model (HMM) [37] for each term survival [6, 44]. Upon infection, pathogenic Neisseria (Fig. 4). Although there is little sequence homology, we see encounter unique conditions, including low pH, oxidative the conservation of key amino acid residues in the SAT and stress and varying iron and nutrient concentrations. The OASS-A proteins from Neisseria species (Fig. 4), confirming cysteine regulon is composed of the sulfate/thiosulfate that these are in fact functional enzymes. As shown in the transporter genes, the sulfate reduction pathway genes and HMM for OASS, there is conservation of amino acid resi- the cysteine synthase genes, and is regulated by the tran- dues in the active site. These conserved amino acids also scription factor CysB. The cysteine regulon is important for interact with a peptide inhibitor based on the C-terminal meningococci host cell colonization [41], and the sulfate tail of SAT in H. influenzae (Fig. 4f) [5]. Neisseria species reduction operon cysDGHIJN is essential for meningococcal have the ability to grow on thiosulfate as the sole source of growth [45]. Within meningococci, the genes of the cysteine sulfate, which suggests that the OASS-A (CysK) protein is regulon show upregulation upon host cell colonization, yet functioning as a dual-function enzyme using both sulfide no expression differences in cysB expression have been and thiosulfate to make cysteine. Although this is unusual, observed [41, 45]. However, CysB is highly expressed and as most bacteria have two isoforms of OASS, there is evi- classified as an essential gene in N. gonorrhoeae [38]. The dence that CysK and CysM are bifunctional in S. typhimu- transcriptome of N. gonorrhoeae during infection of the rium.A cysK/cysM double mutant in S. typhimurium lower genital tract in women (in vivo) has been compared to required cysteine for growth, whereas strains lacking either the growth of N. gonorrhoeae in chemically defined medium cysK or cysM grew in the absence of cysteine [11]. (in vitro) [46]. Of significant note was a decrease in the expression of CysB in two of the three subjects sequenced SAT is classified as an essential gene in N. gonorrhoeae, and this encourages further investigation as to the role of whereas OASS-A is not [38]. SAT is the limiting step in the CysB in activating the cysteine regulon during infection. production of cysteine in N. gonorrhoeae; it is feedback- inhibited by cysteine and represents the rate-limiting step of CysB is a transcription factor belonging to the helix-turn- the pathway, which presumably defines why it is essential. helix family that regulates the cysteine regulon [11, 47–50]. OASS-A (CysK) is not essential in other bacteria, but when CysB is a homotetramer with an N-terminal DNA-binding it is knocked out the bacteria show significantly decreased motif and has been shown to activate the transcription of fitness and increased susceptibility to antibiotics [7, 39, 40]. cysP, cysJIH and cysK promoters in the presence of N-acetyl- SAT is not classified as essential in N. meningitidis, but in a serine in S. typhimurium [48, 50]. It also binds to its own transposon mutagenesis screen exhibited a severe growth promoter to negatively regulate its activity. Based on the defect [41]. OASS-A is also non-essential but has been characterization of the S. typhimurium CysB protein, we

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Fig. 4. Enzymes of the cysteine synthase complex. Model of the N. gonorrhoeae serine acetyltransferase (SAT, CysE) enzyme (a) as predicted with 100 % confidence and 93 % coverage by Phyre2 using the PDB file 1T3D (SAT from E. coli) as a template (56 % sequence identity). Overlay of the model with PDB file 3GVD (SAT from Yersinia pestis) shows the conservation of key structural features (b). The height of the amino acids in the Pfam hidden Markov model (HMM) [37] for the SATase domain shows the conservation of key active site amino acids present in Neisseria SAT enzymes (c) [also shown as sticks in (a)]. Model of the N. gonorrhoeae O-acetylserine sulfhy- drylase A isoform (OASS-A, CysK) enzyme (d) as predicted with 100 % confidence and 99 % coverage by Phyre2 using the PDB file 4AEC (OASS-A from Arabidopsis thaliana) as a template (55 % identity). Overlay of the model with PDB file 4HO1 (OASS from H. influen- zae) shows the conservation of key structural features (e). The active site of OASS-A from H. influenzae (PDB file 31QG) binds peptides corresponding to the C-terminus of SAT to inhibit its activity (f). The height of the amino acids in the Pfam HMM [37] for OASS (g) shows the conservation of key amino acids [shown as sticks in (d)] in the active site required for activity and also for binding of the peptide inhibitor.

identified the inverted repeat sequence CCGTTG-N17- which in turn activates transcription [49]. Sulfide and thio- CAACGG 35 nucleotides upstream of the cysB transcrip- sulfate compete with N-acetylserine for binding to CysB and tional start site in both N. gonorrhoeae and N. meningitidis. as such act as anti-inducers [48]. We could not identify the We suggest that CysB binds to this sequence to regulate its putative CysB-binding sequence upstream of other genes in own activity by occluding the RNA polymerase-binding site, the cysteine regulon, presumably as CysB-binding sites gen- thereby repressing transcription. In this situation, binding erally show poor sequence homology. CysB positively regu- of N-acetylserine would reduce CysB binding to DNA, lates genes within the cysteine regulon (with the exception

Downloaded from www.microbiologyresearch.org by IP: 54.70.40.111476 On: Thu, 03 Jan 2019 06:23:36 Hicks and Mullholland, Microbiology 2018;164:1471–1480 of CysB itself). When N-acetylserine is present a conforma- Cysteine biosynthesis has also been implicated in the devel- tional change is thought to occur in CysB that allows the opment of antibiotic resistance [7, 40]. Antibiotic-induced protein to interact with DNA at ‘activation sites’ in the pro- oxidative stress is a well-known phenomenon and interest- moter DNA and it is hypothesized that this recruits RNA ingly bacteria with impaired cysteine biosynthesis exhibit polymerase. reduced antibiotic resistance. Altogether, this suggests that inhibitors of cysteine biosynthesis could provide new anti- microbials for inhibiting growth or the ability of pathogenic INHIBITORS OF CYSTEINE BIOSYNTHESIS Neisseria to infect and invade, or enhance the efficacy of In the last 6 years, microbial cysteine biosynthesis has been existing antibiotics and help to combat the increase in investigated as a potential drug target, as the repression of resistance. cysteine synthesis leads to a reduction in the fitness of the SAT is a key enzyme in cysteine biosynthesis and, given that bacterium and reduced pathogenesis [2–4, 51]. Amino acid it is essential in N. gonorrhoeae, represents a promising biosynthesis is an exploitable target for new antimicrobials, drug target. There have been few attempts to discover inhib- as most pathogens spend a part of their life cycle in itors of SAT, with the main attempt being virtual screening extremely challenging environments, such as macrophages using the crystal structure of E. coli SAT and the National in the case of M. tuberculosis or the gastric mucosa in the Cancer Institute chemical database [40]. Of the 11 mole- case of S. typhimurium [52–54]. Survival and growth cules with the highest scores, 3 compounds were evaluated depend on the ability of the bacteria to metabolize in this biologically, and the best had an IC50 value of 72 µM and extreme environment. Pathogenic Neisseria inhabit mucosal could serve as a platform for the development of a more surfaces and, as such, are subjected to oxidative stress. Glu- potent inhibitor. tathione is one of the first-line defences against oxidative OASS is the other key enzyme in cysteine biosynthesis and stress [42]; it scavenges free radicals and restores oxidized is the last enzyme in the pathway (Fig. 2). In many patho- molecules by donating hydrogen. High concentrations of genic organisms there are two or more OASS isoforms, glutathione (>15 mM) are present in N. gonorrhoeae [42], which activated under different conditions. As Neisseria presumably for defence against oxidative stress. Cysteine or appear to only have one OASS isoform, efficient inhibition cystine are required as a precursor for the biosynthesis of of this enzyme would drastically reduce cysteine biosynthe- glutathione. In accordance with the role of cysteine in com- sis and the fitness of the organism, leading to an increased bating oxidative stress, upon cysteine depletion the gene susceptibility to antibiotics [7, 39, 40, 56]. expression profile in N. meningitidis resembles that encoun- As discussed earlier, the C-terminus of SAT interacts with tered during oxidative stress [55]. Designing inhibitors to OASS-A to regulate its activity. Peptide inhibitors were cysteine biosynthetic genes from N. gonorrhoeae or N. men- designed based on this sequence and screened in silico for ingitidis could be a promising avenue for future antimicro- binding to OASS [5]. Two pentapeptides, MNWNI and bials. In addition, mammals synthesize cysteine from MBYDI, were hits in the screen and were subsequently crys- methionine and, as such, lack SAT and OASS. tallized with OASS from H. influenzae [5] (Fig. 4f). These

Fig. 5. The cystine and cysteine ABC transporter operons from N. meningitidis and N. gonorrhoeae. The complete cystine ABC trans- porter operon is present in both Neisseria species (top). The cysteine ABC transporter operon is present in N. gonorrhoeae but absent from the N. meningitidis genome (bottom).

Downloaded from www.microbiologyresearch.org by IP: 54.70.40.111477 On: Thu, 03 Jan 2019 06:23:36 Hicks and Mullholland, Microbiology 2018;164:1471–1480 studies determined the important structural requirements stress, and in these oxidizing environments cysteine is for the design of pentapeptide inhibitors of OASS, in that most likely in the cystine form, which both species have the three C-terminal amino acids determine the affinity of the transporters for. the peptide for OASS and the first two positions are import- ant for dictating binding to OASS. CONCLUSION Synthetic peptides and docking studies found peptides that Sulfur is found in many important biomolecules, such as bound tighter than the native peptide and their derivatives. the amino acids cysteine and methionine, the cofactors A carboxylic acid, a small hydrophobic chain at C2 and a CoA, Fe–S clusters and biotin, and the reducing agents cyclopropane linker grant tight packing of the structure and cysteine, glutathione and thioredoxin. Plants and bacteria are essential for synthetic peptides to inhibit OASS by lock- incorporate inorganic sulfur into cysteine, which then ing the enzyme in a closed conformation [57]. becomes available to higher organisms. Cysteine is essen- There is conservation of key amino acids in the active site of tial to all organisms, but cysteine biosynthesis is especially the OASS enzyme from Neisseria species (Fig. 4d) and, important to pathogenic bacteria to overcome the oxida- although the sequence identity is low, the structural conser- tive stress encountered upon infection. The pathogenic vation of these enzymes is high (Fig. 4e). The five C-termi- Neisseria N. meningitidis and N. gonorrhoeae have the nal amino acids of SAT from N. gonorrhoeae form a tail ability to synthesize cysteine, although as outlined in this (Fig. 4d) and have the sequence IDFMI. The conserved iso- review, N. gonorrhoeae has the lost the ability to reduce leucine at the end of the tail is important for peptide binding sulfate to sulfide and, as such, relies on thiosulfate as its and inhibition. We therefore suggest that what is known sole source of sulfate for cysteine biosynthesis. N. gonor- about peptide inhibitors of OASS from other organisms can rhoeae colonize mucosal surfaces in the urogenital tract be applied to the homologues in Neisseria species. To date, and, as such, is exposed to oxidative stress throughout no cysteine biosynthesis enzymes from any Neisseria species most of its in vivo life cycle. It encounters oxidative stress have been characterized, yet inhibitor design relies on the from the host immune system and from commensal lacto- characterization of the key enzymes in this pathway. Inhibi- bacilli that colonize the female urogenital tract and gener- tion of cysteine biosynthesis could be a promising avenue ate oxidants. To protect itself against oxidative stress, for the design of new antimicrobials for the treatment of N. gonorrohoeae has evolved unique defence strategies gonorrhoea and meningococcal disease. This is especially [42], one of which is elevated levels of glutathione [59]. promising for N. gonorrhoeae, given that it has a limited Glutathione synthesis relies on cysteine as a precursor. cysteine biosynthesis pathway, as the ability to reduce sul- Disruption of the cysteine biosynthesis pathway in N. gon- fate has been lost and the SAT appears to be essential. orrhoeae could be an effective mechanism for reducing virulence, reducing the incidence of persistent bacteria and increasing susceptibility to antibiotics. Neisseria species TRANSPORT OF CYSTEINE AND CYSTINE appear to only have a single OASS gene, OASS-A (CysK), Under oxidizing conditions, two cysteine molecules dimer- and not the two isoforms (OASS-A/CysK and OASS-B/ ize by the formation of a disulphide bond to form cystine. CysM) seen in other bacteria. OASS-A from other organ- In most bacteria cystine is imported as a source of cyste- isms is inhibited by peptides that mimic the C-terminus of ine. In N. gonorrhoeae two ABC transporter operons for SAT (CysE), which binds to OASS-A, reducing its activity. the uptake of cystine and cysteine (ngo0372-0374 and This is a promising therapeutic strategy for Neisseria spe- ngo2011-2014, respectively) have been identified [58]. The cies, as in other bacteria, such as S. typhimurium and extracellular solute receptors (NGO0372 and NGO2014) M. tuberculosis, impaired cysteine biosynthesis has been for each transporter were crystallized in an ‘open’ ligand- linked to reduced antibiotic resistance and an increase in free form and a ‘closed’ form bound to L-cystine and L- susceptibility to a broader range of antibiotics [40, 60, 61]. cysteine, respectively. N. meningitidis has an operon with In addition, SAT (CysE) is essential in N. gonorrhoeae 97 % identity to the ngo0372-0374 cystine ABC transporter [38] and is a requirement for growth in N. meningitidis operon from N. gonorrhoeae, so they presumably transport [41] and, as such, also represents an attractive target for cystine in the same manner. Interestingly, the ngo2011- inhibitor design. Targeting cysteine biosynthesis in the 2014 operon encoding the ABC transporter for the import Neisseria species is a promising avenue for the design of of cysteine is absent from N. meningitidis but present in antimicrobial drugs against gonococcal and meningococcal most other Neisseria species (Fig. 5). This suggests the disease. operon has only been deleted in the N. meningitidis species and, as such, meningococci do not require the ability to import cysteine or have an alternative transporter for the Funding information import of cysteine. The extracellular concentration of cys- Funded by a SIF grant to JH from the University of Waikato. teine that these organisms encounter is unknown, but pre- Acknowledgements sumably in the intracellular environments concentrations We would like to thank Professor Vic Arcus for helpful discussions are low. In addition, the Neisseria pathogens N. gonor- about this paper and Dr Erica Prentice for helpful discussions and rhoeae and N. meningitidis often encounter oxidative proofreading of the manuscript.

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Conflicts of interest between sequence and function in the human pathogen Neisseria The authors declare that there are no conflicts of interest. meningitidis. Genome Biol 2009;10:R110. 21. Berndt C, Lillig CH, Wollenberg M, Bill E, Mansilla MC et al. Char- References acterization and reconstitution of a 4Fe-4S adenylyl sulfate/phos- 1. Campanini B, Pieroni M, Raboni S, Bettati S, Benoni R et al. Inhibi- phoadenylyl sulfate reductase from Bacillus subtilis. J Biol Chem tors of the sulfur assimilation pathway in bacterial pathogens as 2004;279:7850–7855. enhancers of antibiotic therapy. Curr Med Chem 2015;22:187–213. 22. Bick JA, Dennis JJ, Zylstra GJ, Nowack J, Leustek T. Identification 2. Poyraz O, Jeankumar VU, Saxena S, Schnell R, Haraldsson M of a new class of 5’-adenylylsulfate (APS) reductases from sul- et al. Structure-guided design of novel thiazolidine inhibitors of O- fate-assimilating bacteria. J Bacteriol 2000;182:135–142. acetyl serine sulfhydrylase from Mycobacterium tuberculosis. 23. Williams SJ, Senaratne RH, Mougous JD, Riley LW, Bertozzi CR. J Med Chem 2013;56:6457–6466. 5’-adenosinephosphosulfate lies at a metabolic branch point in 3. Jean Kumar VU, Poyraz Ö, Saxena S, Schnell R, Yogeeswari P mycobacteria. J Biol Chem 2002;277:32606–32615. et al. Discovery of novel inhibitors targeting the Mycobacterium 24. Johansson P, Hederstedt L. Organization of genes for tetrapyrrole tuberculosis O-acetylserine sulfhydrylase (CysK1) using virtual biosynthesis in gram–positive bacteria. Microbiology 1999;145: – high-throughput screening. Bioorg Med Chem Lett 2013;23:1182 529–538. 1186. 25. Guillouard I, Auger S, Hullo MF, Chetouani F, Danchin A et al. et al. 4. Spyrakis F, Singh R, Cozzini P, Campanini B, Salsi E Iso- Identification of Bacillus subtilis CysL, a regulator of the cysJI zyme-specific ligands for O-acetylserine sulfhydrylase, a novel operon, which encodes sulfite reductase. J Bacteriol 2002;184: antibiotic target. PLoS One 2013;8:e77558. 4681–4689. et al. 5. Salsi E, Bayden AS, Spyrakis F, Amadasi A, Campanini B 26. Mansilla MC, de Mendoza D. The Bacillus subtilis cysP gene enco- Design of O-acetylserine sulfhydrylase inhibitors by mimicking des a novel sulphate permease related to the inorganic phosphate nature. J Med Chem 2010;53:345–356. transporter (Pit) family. Microbiology 2000;146:815–821. 6. Fontan P, Aris V, Ghanny S, Soteropoulos P, Smith I. Global tran- 27. Tettelin H, Saunders NJ, Heidelberg J, Jeffries AC, Nelson KE scriptional profile of Mycobacterium tuberculosis during THP-1 et al. Complete genome sequence of Neisseria meningitidis human macrophage infection. Infect Immun 2008;76:717–725. serogroup B strain MC58. Science 2000;287:1809–1815. 7. Turnbull AL, Surette MG. L-Cysteine is required for induced anti- ’ biotic resistance in actively swarming Salmonella enterica serovar 28. Piet JR, Huis In t Veld RA, van Schaik BD, van Kampen AH, Baas et al. Typhimurium. Microbiology 2008;154:3410–3419. F Genome sequence of Neisseria meningitidis serogroup B strain H44/76. J Bacteriol 2011;193:2371–2372. 8. Sharip A, Sorvillo F, Redelings MD, Mascola L, Wise M et al. Pop- ulation-based analysis of meningococcal disease mortality in the 29. Budroni S, Siena E, Dunning Hotopp JC, Seib KL, Serruto United States: 1990–2002. Pediatr Infect Dis J 2006;25:191–194. D et al. Neisseria meningitidis is structured in clades associ- ated with restriction modification systems that modulate 9. Rosenstein NE, Perkins BA, Stephens DS, Popovic T, Hughes JM. homologous recombination. Proc Natl Acad Sci USA 2011;108: Meningococcal disease. N Engl J Med 2001;344:1378–1388. 4494–4499. 10. Wi T, Lahra MM, Ndowa F, Bala M, Dillon JR et al. Antimicrobial 30. Port JL, Devoe IW, Archibald FS. Sulphur acquisition by Neisseria resistance in Neisseria gonorrhoeae: global surveillance and a call meningitidis. Can J Microbiol 1984;30:1453–1457. for international collaborative action. PLoS Med 2017;14: e1002344. 31. Droux M, Ruffet ML, Douce R, Job D. Interactions between serine  acetyltransferase and O-acetylserine (thiol) lyase in higher 11. Guedon E, Martin-Verstraete I. Cysteine metabolism and its regu- – lation in bacteria. In: Wendisch VF (editor). Amino Acid Biosynthesis plants structural and kinetic properties of the free and bound – ~ Pathways, Regulation and Metabolic Engineering. Berlin, Heidel- enzymes. Eur J Biochem 1998;255:235 245. berg: Springer Berlin Heidelberg; 2007. pp. 195–218. 32. Olsen LR, Huang B, Vetting MW, Roderick SL. Structure of serine 12. Kertesz MA. Bacterial transporters for sulfate and organosulfur acetyltransferase in complexes with CoA and its cysteine feed- – compounds. Res Microbiol 2001;152:279–290. back inhibitor. Biochemistry 2004;43:6013 6019. 13. Kredich NM. Biosynthesis of cysteine. EcoSal Plus 2008;3. 33. Becker MA, Tomkins GM. Pleiotrophy in a cysteine-requiring mutant of Samonella typhimurium resulting from altered protein- 14. Abrams AJ, Trees DL, Nicholas RA. Complete genome sequences protein interaction. J Biol Chem 1969;244:6023–6030. of three Neisseria gonorrhoeae laboratory reference strains, deter- mined using pacbio single-molecule real-time technology. Genome 34. Campanini B, Speroni F, Salsi E, Cook PF, Roderick SL et al. Inter- Announc 2015;3:e01052-15. action of serine acetyltransferase with O-acetylserine sulfhydry- lase active site: evidence from fluorescence spectroscopy. Protein 15. Le Faou A. Sulphur nutrition and metabolism in various species of – Neisseria. Ann Microbiol 1984;135B:3–11. Sci 2005;14:2115 2124. 16. Joseph B, Schneiker-Bekel S, Schramm-Glück A, Blom J, Claus H 35. Tai CH, Nalabolu SR, Jacobson TM, Minter DE, Cook PF. Kinetic et al. Comparative genome biology of a serogroup B carriage and mechanisms of the A and B isozymes of O-acetylserine sulfhydry- disease strain supports a polygenic nature of meningococcal viru- lase from Salmonella typhimurium LT-2 using the natural and – lence. J Bacteriol 2010;192:5363–5377. alternative reactants. Biochemistry 1993;32:6433 6442. 17. Grifantini R, Bartolini E, Muzzi A, Draghi M, Frigimelica E et al. 36. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Gene expression profile in Neisseria meningitidis and Neisseria lac- Phyre2 web portal for protein modeling, prediction and analysis. tamica upon host-cell contact: from basic research to vaccine Nat Protoc 2015;10:845–858. development. Ann N Y Acad Sci 2002;975:202–216. 37. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J et al. The 18. Dietrich G, Kurz S, Hübner C, Aepinus C, Theiss S et al. Transcrip- Pfam protein families database: towards a more sustainable tome analysis of Neisseria meningitidis during infection. J Bacteriol future. Nucleic Acids Res 2016;44:D279–D285. 2003;185:155–164. 38. Remmele CW, Xian Y, Albrecht M, Faulstich M, Fraunholz M et al. 19. Mansilla MC, de Mendoza D. L-cysteine biosynthesis in Bacillus Transcriptional landscape and essential genes of Neisseria gonor- subtilis: identification, sequencing, and functional characterization rhoeae. Nucleic Acids Res 2014;42:10579–10595. of the gene coding for phosphoadenylylsulfate sulfotransferase. 39. Senaratne RH, de Silva AD, Williams SJ, Mougous JD, Reader JR J Bacteriol 1997;179:976–981. et al. 5’-Adenosinephosphosulphate reductase (CysH) protects 20. Rusniok C, Vallenet D, Floquet S, Ewles H, Mouze-Soulama C Mycobacterium tuberculosis against free radicals during chronic et al. NeMeSys: a biological resource for narrowing the gap infection phase in mice. Mol Microbiol 2006;59:1744–1753.

Downloaded from www.microbiologyresearch.org by IP: 54.70.40.111479 On: Thu, 03 Jan 2019 06:23:36 Hicks and Mullholland, Microbiology 2018;164:1471–1480

40. Turnbull AL, Surette MG. Cysteine biosynthesis, oxidative stress regulation by cysB protein and N-acetyl-L-serine. J Bacteriol 1989; and antibiotic resistance in Salmonella typhimurium. Res Microbiol 171:130–140. – 2010;161:643 650. 51. Nagpal I, Raj I, Subbarao N, Gourinath S. Virtual screening, identi- 41. Capel E, Zomer AL, Nussbaumer T, Bole C, Izac B et al. Comprehen- fication and in vitro testing of novel inhibitors of O-acetyl-L-serine sive identification of meningococcal genes and small noncoding sulfhydrylase of Entamoeba histolytica. PLoS One 2012;7:e30305. RNAs required for host cell colonization. MBio 2016;7:e01173-16. 52. Brown SA, Palmer KL, Whiteley M. Revisiting the host as a growth 42. Seib KL, Wu HJ, Kidd SP, Apicella MA, Jennings MP et al. medium. Nat Rev Microbiol 2008;6:657–666. Defenses against oxidative stress in Neisseria gonorrhoeae: a sys- 53. Roop RM, Gaines JM, Anderson ES, Caswell CC, Martin DW. Sur- tem tailored for a challenging environment. Microbiol Mol Biol Rev – vival of the fittest: how Brucella strains adapt to their intracellular 2006;70:344 361. niche in the host. Med Microbiol Immunol 2009;198:221–238. et al. 43. Santi-Rocca J, Smith S, Weber C, Pineda E, Hon CC Endo- 54. McClean CM, Tobin DM. Macrophage form, function, and pheno- plasmic reticulum stress-sensing mechanism is activated in Ent- type in mycobacterial infection: lessons from tuberculosis and amoeba histolytica upon treatment with nitric oxide. PLoS One other diseases. Pathog Dis 2016;74:ftw068. 2012;7:e31777. 55. van de Waterbeemd B, Zomer G, van den Ijssel J, van Keulen L, 44. Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA et al. Tran- Eppink MH et al. Cysteine depletion causes oxidative stress and scriptional adaptation of Mycobacterium tuberculosis within macro- triggers outer membrane vesicle release by Neisseria meningitidis; phages: insights into the phagosomal environment. J Exp Med implications for vaccine development. PLoS One 2013;8:e54314. 2003;198:693. 56. Lestrate P, Delrue RM, Danese I, Didembourg C, Taminiau B et al. 45. Mendum TA, Newcombe J, Mannan AA, Kierzek AM, McFadden J. Identification and characterization of in vivo attenuated mutants of Interrogation of global mutagenesis data with a genome scale Brucella melitensis. Mol Microbiol 2000;38:543–551. model of Neisseria meningitidis to assess gene fitness in vitro and in sera. Genome Biol 2011;12:R127. 57. Bruno A, Amori L, Costantino G. Computational insights into the et al. mechanism of inhibition of oass-a by a small molecule inhibitor. 46. McClure R, Nudel K, Massari P, Tjaden B, Su X The gonococ- – cal transcriptome during infection of the lower genital tract in Mol Inform 2013;32:447 457. women. PLoS One 2015;10:e0133982. 58. Bulut H, Moniot S, Licht A, Scheffel F, Gathmann S et al. Crystal 47. Hryniewicz MM, Kredich NM. Stoichiometry of binding of CysB to structures of two solute receptors for L-cystine and L-cysteine, respectively, of the human pathogen Neisseria gonorrhoeae. J Mol the cysJIH, cysK, and cysP promoter regions of Salmonella typhi- – murium. J Bacteriol 1994;176:3673–3682. Biol 2012;415:560 572. 59. Archibald FS, Duong MN. Superoxide dismutase and oxygen toxic- 48. Hryniewicz MM, Kredich NM. The cysP promoter of Salmonella – typhimurium: characterization of two binding sites for CysB pro- ity defenses in the genus Neisseria. Infect Immun 1986;51:631 tein, studies of in vivo transcription initiation, and demonstration 641. of the anti-inducer effects of thiosulfate. J Bacteriol 1991;173: 60. Rawat M, Newton GL, Ko M, Martinez GJ, Fahey RC et al. Myco- 5876–5886. thiol-deficient Mycobacterium smegmatis mutants are hypersensi- 49. Ostrowski J, Kredich NM. Negative autoregulation of cysB in Sal- tive to alkylating agents, free radicals, and antibiotics. Antimicrob – monella typhimurium: in vitro interactions of CysB protein with the Agents Chemother 2002;46:3348 3355. cysB promoter. J Bacteriol 1991;173:2212–2218. 61. Sareen D, Newton GL, Fahey RC, Buchmeier NA. Mycothiol is 50. Ostrowski J, Kredich NM. Molecular characterization of the cysJIH essential for growth of Mycobacterium tuberculosis Erdman. promoters of Salmonella typhimurium and Escherichia coli: J Bacteriol 2003;185:6736–6740.

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