Site-2 proteases in prokaryotes: regulated intramembrane proteolysis expands to microbial pathogenesis

Microbes and Infection 8 (2006) 1882e1888 www.elsevier.com/locate/micinf

Review

Site-2 proteases in prokaryotes: regulated intramembrane proteolysis expands to microbial pathogenesis Hideki Makinoshima a, Michael S. Glickman a,b,* a

Immunology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA b Division of Infectious Diseases, Memorial Sloan-Kettering Cancer Center, Box 9 New York, NY 10021, USA Available online 27 April 2006

Abstract Regulated intramembrane proteolysis (RIP) is a widely distributed mechanism of signal transduction in which membrane-bound proteases cleave transmembrane domains of substrate proteins. The site-2 protease (S2P) class of RIP metalloproteases is present in most bacterial genomes but is generally of unknown function except for the well-characterized proteases RseP and SpoIVFB. In this review we will discuss the biochemical functions and physiologic roles of S2P proteases in bacteria and highlight recent data implicating S2P family members in hostepathogen interactions. Ó 2006 Elsevier SAS. All rights reserved. Keywords: Regulated intramembrane proteolysis; Site-2 protease; Membrane zinc metalloprotease; Lipid regulation; Mycobacterium tuberculosis; Microbial pathogenesis; Cell membrane

1. Introduction This review will discuss the emerging physiologic and pathogenetic role of the site-2 protease (S2P) class of prokaryotic membrane metalloproteases. This family of proteases is a subset of a group of membrane-bound proteases designated intramembrane cleaving proteases (I-Clips), which mediate the diverse signal transduction mechanisms of regulated intramembrane proteolysis (RIP) [1,2]. In the most general terms, RIP proteases are transmembrane proteins whose proteolytic active sites lie within the predicted transmembrane segments and which cleave substrate proteins within transmembrane segments. A comprehensive classification and listing of RIP proteases is available through the MEROPS database (http://merops.sanger.ac.uk/) [3]. RIP proteases are subdivided into serine, aspartyl, or metalloprotease subfamilies, which are * Corresponding author. Division of Infectious Diseases, Memorial SloanKettering Cancer Center, Box 9 New York, NY 10021, USA. Tel.: þ1 212 639 3191; fax: þ1 646 422 2124. E-mail address: [email protected] (M.S. Glickman). 1286-4579/$ - see front matter Ó 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2006.02.021

readily identifiable by conserved active site residues typical of these protease classes. Well-studied members of the three classes include rhomboid (serine, MEROPS family S54), presenillin (aspartyl, MEROPS family A22), and site-2 protease (metallo, MEROPS family M50). The S2P/M50 group is widely distributed in bacteria and is identified by a characteristic metalloprotease signature, HExxH, where the histidines chelate a zinc ion, and the glutamate is the catalytic residue, and x is any amino acid. While this motif is common to zinc metalloproteases, in M50 family members this HExxH motif lies within a predicted transmembrane segment, as does a more C-terminal aspartic acid residue in the context of an LDG or FDG conserved motif. Despite low overall amino acid sequence identity between M50 family members, these conserved motifs superimposed on hydropathy profiling identify S2P family members that are the subject of this review. Fig. 1 demonstrates the conserved residues and hydropathy profiling characteristic of M50 proteases using Mycobacterium tuberculosis Rv2869c as an example. Over 400 members of the M50 protease family are identifiable in available genome sequences, indicating that signal

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Fig. 1. Conserved sequence motifs of M50 RIP proteases. Panels A and B are hydropathy plots of the M50 RIP protease Rv2869c from M. tuberculosis. Panel A represents each amino acid on a color scale from hydrophobic (red) to hydrophilic (blue) along the length of the amino acid sequence (indicated by numbering above). The expansions show the amino acid sequences of the transmembrane domains containing the conserved HExxH and FDG motifs which are underlined in red. Panel B shows a Kyle and Doolittle plot in which hydrophobic amino acids have a positive score. The region of the Rv2869c PDZ domain is labeled by the purple bar. The hydropathy plots in A and B were created using CLC Protein Workbench software (CLC Bio).

transduction by S2P family members is a broadly distributed mechanism in bacteria [4]. In this review we will discuss the accumulated knowledge about S2P family members in bacteria, including recent evidence from our laboratory that an M. tuberculosis S2P family member is an important virulence determinant of M. tuberculosis through control of cell envelope composition. Due to space limitations, we will not comprehensively review all known information about bacterial S2P family members, but focus on paradigmatic examples. The prokaryotic S2P family members that have been characterized are listed in Table 1 with substrates, physiologic function, and appropriate references. The reader is also directed to several recently published reviews of regulated intramembrane proteolysis in general [1,5,6] and specific RIP pathways [7e9]. 2. The SREBP/S2P model The founding member of the M50 protease clan is mammalian site-2 protease. S2P, together with site-1 protease (S1P), cleaves membrane-bound sterol regulatory element binding

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proteins (SREBPs). SREBPs are membrane-bound transcription factors that activate sterol and fatty acid biosynthetic genes when liberated from the membrane by the combined proteolytic action of S1PeS2P. S2P was identified by cDNA complementation of a mutagenized CHO cell line auxotrophic for cholesterol and unsaturated fatty acids in which site-2 cleavage of SREBP did not occur [10], resulting in membrane retention of SREBP, failure to activate sterol biosynthesis, and lipid auxotrophy. Analysis of the complementing cDNA revealed a predicted protein with an HExxH motif within a hydrophobic segment now known as characteristic of the M50 class of metalloproteases, multiple predicted transmembrane domains, and a central hydrophilic PDZ domain. The SREBP cleavage, which depends on S2P function, occurs between a leucine and cysteine in the first transmembrane segment of the protein [11]. Second-site cleavage of SREBP is not regulated by cholesterol, but depends on prior site-1 cleavage by S1P. Thus, characterization of the mammalian SREBP/S2P pathway demonstrated the following features that enlighten the ongoing characterization of prokaryotic S2P: (1) S2P cleaves a membrane-bound transcriptional regulatory protein within a transmembrane domain; (2) the SREBP pathway transduces information about membrane composition to the nucleus to regulate transcription; (3) activation of the SREBP pathway is the cumulative result of multiple interdependent proteolytic cleavage events. 3. Escherichia coli YaeL/RseP: second-site cleavage of anti-sigma E An extensively studied bacterial S2P family member is YaeL/RseP of E. coli, which is an essential component of the sigma E pathway. In this review we will refer to this protease as RseP. Early studies in E. coli demonstrated that overexpression of outer-membrane porins or heat shock induce sigma E-dependent transcription of genes involved in restoring membrane protein folding or membrane integrity. Sigma E is a soluble cytoplasmic protein but forms a complex with the single pass transmembrane protein anti-sigma factor E (RseA), in which the cytoplasmic N-terminal domain of RseA binds sigma E [12]. As such, rseA is a negative regulator of the sigma E pathway, and E. coli cells lacking rseA display constitutive activation of the sigE pathway. Under conditions of envelope stress, RseA is degraded by the

Table 1 Prokaryotic M50 proteases of known function Organism

M50 protease

Substrates

Function or pathway

Reference

B. subtilis B. subtilis

SpoIVFB YluC

Pro-sigmaK RsiW(anti-sigW)

[15] [45]

Caulobacter crescentus E. coli Enterococcus faecalis M. tuberculosis V. cholerae

MmpA RseP (YaeL) Eep Rv2869c YaeL

PodJ RseA (anti-SigE) cAD1 Pheromone Unknown TcpP

Sporulation Alkaline shock, osmotic shock, cell wall active antibiotics Swarmer to stalked transition Periplasmic stress response Plasmid conjugation Cell envelope lipid composition, virulence Negatively regulates virulence gene expression

[49] [7] [50] [43] [25]

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sequential proteolysis first by the periplasmic protease DegS, and then by the S2P family member RseP. This second cleavage liberates the sigE/RseA complex from the membrane, after which RseA is degraded by ClpXP, finally liberating active SigE [13]. 4. SpoIVFB and control of Bacillus sporulation SpoIVFB of Bacillus subtilis was initially defined as a genetic locus required for sporulation [14], which promoted proteolytic processing of pro-sigmaK to the mature transcription factor sigmaK. SpoIVFB is an M50 protease with the conserved HExxH motif within a transmembrane segment and a C-terminal aspartate/glycine motif. SpoIVFB does not have a predicted PDZ domain [15]. SpoIVFB proteolytic activity for pro-sigmaK is inhibited by the protein BofA, which exists in a multiprotein complex with SpoIVFB [16]. The inhibition of SpoIVFB by BofA depended on a histidine residue in BofA which is postulated to act in trans to bind the catalytic zinc ion in the SpoIVFB HExxH active site, thereby inhibiting SpoIVFB proteolytic activity [17]. This pathway shares multiple features with both the SREBP and sigE pathways. Specifically, SpoIVFB acts to generate an active transcriptional regulator from an inactive membrane-bound precursor. The signals for the ultimate activation of SigK are the result of multiple proteolytic events which signal across cellular compartments. In the Bacillus example, these compartments are the mother cell and forespore, while in E. coli, the compartments are the periplasm and cytoplasm. 5. Positive and negative regulation of bacterial S2P activity: role of the PDZ domain One of the remarkable features of both the SREBP and sigE pathways is the strict order of proteolytic steps mediated by S1P/S2P or DegS/RseP. Theoretically, release of SREBP or RseA from the membrane only requires a single cleavage event near the cytosolic side of the transmembrane domain, yet this cleavage event is always preceded by a first cleavage. Overexpression of RseP does not hyperactivate the sigE pathway [18], and RseP does not degrade RseA in a DegS null strain [18,19]. Many bacterial S2P family members contain a PDZ domain that lies in the central hydrophilic part of the molecule between the HExxH and LDG motifs (see Fig. 1). PDZ domains frequently recognize the C-terminal sequences of proteins [20], but can also recognize lipids or internal protein sequences [21]. One defined initiating signal for SigE pathway activation of E. coli is the recognition of C-terminal residues in unfolded outer-membrane proteins by the PDZ domain of DegS. Outer-membrane porin terminal peptide binding relieves an autoinhibitory effect of the DegS PDZ domain on DegS cleavage of RseA [22]. Similarly, the PDZ domain of RseP is a negative regulator of proteolytic activity. Although RseP lacking its PDZ domain can rescue the viability defect of RseP null mutant, demonstrating that the PDZ domain is not required for RseP proteolytic activity for RseA [23,24], RsePDPDZ degrades intact RseA without prior

DegS periplasmic cleavage and hyperactivates the SigE pathway [23,24]. By analogy to the PDZ domain of DegS, the prediction of these studies is that a specific ligand interacts with the PDZ domain of RseP to stimulate second-site cleavage of RseA. The role of the PDZ domain of other bacterial S2P members has not been examined in detail, but it is likely that the PDZ domains play important regulatory roles in controlling site-2 cleavage of their respective substrates. The protein or other ligands for S2P PDZ domains are an area of significant interest to understand the regulatory signals that control the proteolytic activity and physiologic function of S2P family members. 6. Proteolytic activity of bacterial S2P: substrates and sequence specificity The evidence indicating that the proteins of the M50 family are indeed metalloproteases is strong, but until recently, indirect. Most of the evidence for proteolytic activity is derived from detailed genetic studies. First, the HExxH motif is characteristic of zinc metalloproteases, and mutations in either the histidine or glutamate abolish proteolytic degradation of postulated substrate in vivo. This has been shown for the following proteaseesubstrate pairs: RseP/RseA [18,19], SpoIVFB/ pro-sigmaK [15], and Vibrio YaeL/TcpP [25]. Recently, a series of studies has demonstrated proteolytic activity of each class of RIP proteases. Several members of the rhomboid class of serine proteases [26], the aspartyl RIP protease gamma secretase [27], and RseP [28] have in vitro proteolytic activity when purified from detergent solubilized membranes. Purified RseP will cleave a substrate that contains the RseA transmembrane domain linking mannose binding protein (MBP) to a short periplasmic fragment of RseA [28]. Proteolytic activity was dependent on the conserved histidine motif, substantiating the in vivo results. Surprisingly, RseP also cleaved unrelated transmembrane sequences without sequence conservation with RseA TM both in vivo and in vitro. These findings provide direct evidence that RseP is a protease that can directly cleave the transmembrane domain of its substrate. The apparent nonspecificity of proteolytic activity is of unclear biologic significance but could either signify that RseP has multiple specific substrates or that the in vitro system lacks some factor which determines specificity. There is strong evidence from the mammalian system that M50 proteases can cleave multiple substrates, as S2P has two known substrates, SREBP and ATF6 [29]. In summary, S2P proteases participate in pathways which convey information across compartments via a highly regulated set of proteolytic steps which ultimately culminate in transcriptional regulation. This function has clear relevance to the interaction of microbial pathogens with their hosts and has been examined recently in Vibrio cholerae and M. tuberculosis. 7. Bacterial S2P and microbial pathogenesis The broad distribution of M50 protease family members in bacteria, including many bacterial pathogens, strongly

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suggests a role in microbial pathogenesis. However, until recently, data about the role of S2P family members in microbeehost interactions were limited. Recent work in V. cholerae and M. tuberculosis has provided the first evidence for a role for this protease class in bacterial pathogenesis and expanded the paradigms of S2P-mediated RIP described above. 7.1. V. cholerae In V. cholerae, virulence gene expression is controlled by the transcription factor ToxT. Transcription of toxT is in turn positively regulated by TcpP, a membrane-localized transcriptional regulator that binds the toxT promoter. TcpP is unstable in the absence of a second membrane protein, TcpH, which is also required for the activation of the ToxT promoter [30]. In a genetic screen for Vibrio transposon mutants that restored ToxT activation in a TcpH null strain, these investigators isolated a null mutant of YaeL [25]. In contrast to RseP/YaeL of E. coli, the V. cholerae YaeL mutant is fully viable. Vibrio YaeL constitutively degrades TcpP in a TcpH null background, but in wild-type strains this degradation occurs in conditions which inhibit virulence gene expression [25]. Thus, Vibrio TcpH appears to protect TcpP from YaeL-mediated proteolysis in conditions which activate virulence gene expression. These findings are thematically similar to the RseP/SigE pathway in E. coli, in that a membrane-localized transcriptional regulator is degraded by YaeL, but they differ in important respects. First, Vibrio YaeL is a nonessential gene, emphasizing the divergent roles of even highly conserved bacterial S2P members. In addition, in contrast to YaeL of E. coli, Vibrio YaeL negatively regulates gene expression, because TcpP is active when membrane localized and inactive when liberated from the membrane by RIP. As such, Vibrio YaeL serves to inhibit virulence gene expression under unfavorable environmental conditions, presumably by sensing an environmental signal. The identity of the signal that activates the degradation of TcpP and the mechanism by which this signal is communicated to Vibrio YaeL are unknown. 7.2. M. tuberculosis M. tuberculosis is a highly successful global pathogen whose pathogenic strategies are emerging from genetic studies. Intense investigation into M. tuberculosis before the advent of mycobacterial genetic techniques highlighted the unique chemical constituents and structure of the mycobacterial cell envelope. The mycobacterial envelope is highly hydrophobic due to mycolic acids: a-alkyl, b-hydroxy fatty acids which are over 80 carbons in total length. These lipids are synthesized by a unique lipid biosynthetic pathway utilizing both fatty acid synthase I and a multi-component fatty acid synthase II. Mycolic acid biosynthesis is the target of the first-line antituberculosis drug isoniazid, and components of the FasII system that polymerize the mycolic acid chain are essential for the viability of mycobacterial cells [31,32].

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In addition to the core biosynthetic enzymes of mycolic acid biosynthesis, several other pathways of mycolic acid modification are implicated in mycobacterial pathogenesis in mice. Modification of mycolic acids by cis or trans cyclopropane rings by the S-adenosyl methionine-dependent methyltransferases PcaA and CmaA2, respectively, is nonessential for viability. However, inactivation of pcaA attenuates Mtb through attenuation of Mtb trehalose dimycolate-induced inflammation [33,34], while deletion of cmaA2 enhances Mtb-induced virulence and inflammation [35]. Similarly, Mycobacterium marinum kasB null mutants are viable, but are defective for growth in macrophages and synthesize truncated mycolic acids [36]. These and other [37] studies have substantiated a view that, while core mycolic acid biosynthesis is essential for mycobacterial viability, mycolate modification pathways are important determinants of pathogenesis. In addition to mycolic acids, the mycobacterial cell envelope contains many unique lipids, many of which are now implicated in pathogenesis. Biosynthesis of phthiocerol dimycocerosate (PDIM) is an important determinant of initial replication of M. tuberculosis in mice [38,39], while the structurally related phenolic glycolipid confers hypervirulence to a clinical strain [40]. Taken together, these studies have replaced the view of the mycobacterial cell envelope as an inert barrier with a model in which individual cell envelope lipids are distinct pathogenesis effector molecules that have separable roles in pathogenesis. This emerging model of M. tuberculosis cell envelope lipids as pathogenesis effectors raises the important question of how pathogenic mycobacteria regulate the composition of the cell envelope during infection. In E. coli and B. subtilis, several transcriptional regulatory systems are known that control the level of unsaturation in membrane fatty acids [41] or transcription of FasII components themselves [42]. However, the molecular mechanisms regulating mycobacterial cell envelope composition are unknown. In addition, the unique structure of mycobacterial cell envelope suggests unique mechanisms of regulation. The M. tuberculosis genome encodes three S2P homologs: Rv0359, Rv2625c, and Rv2869c. We will discuss our recent investigation into the role of Rv2869c in regulating M. tuberculosis cell envelope composition. Rv2869c is a member of the M50 protease class and contains multiple predicted transmembrane domains, an N-terminal HExxH motif within the first transmembrane segment, a C-terminal FDG motif within a transmembrane segment, and a central PDZ domain (Fig. 1). 8. Phenotypes of the Rv2869c null mutant Deletion of Rv2869c in M. tuberculosis or its homolog in Mycobacterium bovis BCG Pasteur causes a defect in cording, a colonial and microscopic morphologic marker of virulence in pathogenic mycobacteria [43]. Cording is a polygenic trait which can be affected by multiple perturbations in cell envelope lipids, including mycolic acid structure [33,36,44]. The cording defect of the DRv2869c strain was

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complemented by a wild-type copy of the Rv2869c gene, but not by Rv2869c with a histidine to alanine mutation in the HExxH proteolytic active site. This morphologic phenotype reflected perturbations in mycolic acid biosynthesis due to the Rv2869c null mutation. Direct examination of mycolic acid biosynthesis revealed that Rv2869c was required to maintain synthesis of all three major mycolic acids when grown in detergent-free media, but not in standard media containing Tween-80 [43]. In addition, deletion of Rv2869c affected relative ratios of phosphatidyl inositol mannoside acylation forms. DRv2869c cells lacked wild-type levels of PIM6 but synthesized wild-type levels of PIM2 [43]. These results demonstrated a direct role for an S2P family member in the regulation of prokaryotic lipid biosynthesis and membrane composition. As discussed above, although RseP of E. coli and YluC of Bacillus have well-described roles in transducing membrane stress [45], they do not appear to directly regulate lipid biosynthesis, as is the case for S2P in mammalian cells. Transcriptional profiling of DRv2869c cells revealed a complex transcriptional perturbation of multiple lipid biosynthetic and catabolic genes. Multiple mycolic acid biosynthetic genes were overexpressed in mutant cells. These mycolic acid biosynthetic genes included the condensation enzyme pks13 [46], two desaturases (desA1 and desA3) one of which (desA3) is likely to be involved in mycolic acid biosynthesis [47] and two fabG genes (3-oxoacyl reductase). Thus, this expression profile is consistent with Rv2869c positively and negatively regulating lipid biosynthesis. Interestingly, several of these regulated genes were recently found to be upregulated in M. tuberculosis isolated from human tuberculosis lung lesions [48]. The in vivo consequences of Rv2869c inactivation were assessed in the mouse model of M. tuberculosis infection and found to be severe. Rv2869c mutant titers in the lungs of infected mice were 100-fold lower than wild-type cells at 3 weeks of infection, the end of active M. tuberculosis replication in the mouse model. In the mouse model of M. tuberculosis infection, wild-type bacteria persist at constant titers in the lungs of mice indefinitely. However, DRv2869c cells also failed to persist, declining in titer over 3 to 22 weeks of the infection, such that titers of Rv2869c cells at 22 weeks were 1000e10,000-fold lower than wild-type cells. This decline in bacterial load was accompanied by significant reduction of macroscopic and microscopic pulmonary inflammatory lesions. These data implicated regulated intramembrane proteolysis in the pathogenesis of tuberculosis infection and suggested that the Rv2869c transcriptional regulon includes critical cell envelope determinants of M. tuberculosis in vivo growth and persistence. Several important questions are unanswered about the Rv2869c pathway. First, the proteolytic substrate(s) of Rv2869c is unknown. By analogy to other M50 proteases, Rv2869c may cleave a membrane-bound transcriptional regulator that activates or represses lipid biosynthetic and modification genes. Other members of the Rv2869c pathway may include an upstream protease acting to initiate the

pathway via first-site cleavage analogous to DegS or S1P. Finally, the initiating signals for Rv2869c pathway activation are unknown, but are important to understand, as the Rv2869c pathway may be transducing host-derived membrane stress or lipid modifications across the cell wall to modify transcription in a pattern that is essential for pathogenesis. These questions are an area of active investigation. 9. Conclusions Although the S2P/M50 family of RIP proteases is a widely distributed protein family in bacteria, the physiologic functions of most of these family members are unknown. However, it is already evident that this protein family participates in diverse processes including envelope protein misfolding, sporulation, virulence gene expression, and mycobacterial cell envelope composition. Although these pathways are diverse, they share a common mechanistic theme. All of the studied members of this family use proteolytic activity within the membrane to transfer information across membranes to integrate gene expression with physiologic stresses occurring in another cellular compartment. In the case of the microbial pathogens V. cholerae and M. tuberculosis, YaeL and Rv2869c control important virulence genes. Thus, S2P in these pathogens seem to participate in pathways that sense host-derived signals or stress and modify gene expression accordingly. Although the identity of these host-derived factors is presently unclear, further study of the role of S2P family members in microbial pathogenesis will yield critical insights into the mechanisms by which microbial pathogens sense their environment within the host. Acknowledgements Work described in this review from the Glickman laboratory is supported by the NIH, Burroughs Wellcome Fund, and the Ellison Medical Foundation. References [1] M. Ehrmann, T. Clausen, Proteolysis as a regulatory mechanism, Annu. Rev. Genet. 38 (2004) 709e724. [2] A. Weihofen, B. Martoglio, Intramembrane-cleaving proteases: controlled liberation of proteins and bioactive peptides, Trends Cell Biol. 13 (2003) 71e78. [3] N.D. Rawlings, F.R. Morton, A.J. Barrett, MEROPS: the peptidase database, Nucleic Acids Res. 34 (2006) D270eD272. [4] L.N. Kinch, K. Ginalski, N.V. Grishin, Site-2 protease regulated intramembrane proteolysis: sequence homologs suggest an ancient signaling cascade, Protein Sci. (2005). [5] M.S. Brown, J. Ye, R.B. Rawson, J.L. Goldstein, Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans, Cell 100 (2000) 391e398. [6] M.S. Wolfe, R. Kopan, Intramembrane proteolysis: theme and variations, Science 305 (2004) 1119e1123. [7] B.M. Alba, C.A. Gross, Regulation of the Escherichia coli sigmadependent envelope stress response, Mol. Microbiol. 52 (2004) 613e619.

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