Felicia
Ikolo
ab,
Meng
Zhang
a,
Dean J.
Harrington
c,
Carl
Robinson
d,
Andrew S.
Waller
d,
Iain C.
Sutcliffe
*a and
Gary W.
Black
a
aDepartment of Applied Sciences, Faculty of Health & Life Sciences, University of Northumbria at Newcastle, Newcastle upon Tyne, NE1 8ST, UK. E-mail: iain.sutcliffe@northumbria.ac.uk; Fax: +44 (0)191 227 3519; Tel: +44 (0)191 227 4071
bDepartment of Biochemistry, School of Medicine, St. George's University, True Blue, St. George's, Grenada
cDivision of Biomedical Science, School of Life Sciences, University of Bradford, West Yorkshire, BD7 1DP, UK
dCentre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, UK
First published on 8th October 2015
Peptidyl-prolyl isomerase (PPIase) lipoproteins have been shown to influence the virulence of a number of Gram-positive bacterial human and animal pathogens, most likely through facilitating the folding of cell envelope and secreted virulence factors. Here, we used a proteomic approach to demonstrate that the Streptococcus equi PPIase SEQ0694 alters the production of multiple secreted proteins, including at least two putative virulence factors (FNE and IdeE2). We demonstrate also that, despite some unusual sequence features, recombinant SEQ0694 and its central parvulin domain are functional PPIases. These data add to our knowledge of the mechanisms by which lipoprotein PPIases contribute to the virulence of streptococcal pathogens.
Several lipoprotein PPIases have been shown to have significant roles in bacterial physiology, notably PrsA in Bacillus subtilis.9 Moreover, in some pathogens PPIases have been shown to affect virulence,7 including PrsA of Bacillus anthracis,10Enterococcus faecalis EF0685 and EF1534,11Listeria monocytogenes PrsA2,12,13Streptococcus pneumoniae SlrA and PpmA14 and Streptococcus pyogenes PrsA.15 Some of these PPIase belong to the cyclophilin subfamily (e.g. S. pneumoniae SlrA; E. faecalis EF1534) but many belong to the parvulin subfamily,16 including the members of PrsA family that appear to be ubiquitous in Firmicute genomes.
Streptococcus equi is the causative agent of the widespread equine disease Strangles.17,18 We have previously shown that the PrsA homologue of S. equi (UniProt: C0M9L5, originally denoted PrtM) plays a significant role in S. equi virulence, both in an air interface tissue culture model, a mouse model and, most significantly, in the equine host.19 PrtM is here referred to as SEQ0694, based on its annotation in the S. equi genome.17
To further investigate the role of SEQ0694 we have here characterised the recombinant protein as a functional PPIase and used a proteomic approach to demonstrate that SEQ0694 likely influences the folding and activity of multiple secreted proteins of S. equi, including at least two putative virulence factors.
In addition to rSEQ0694, the section of the seq0694 ORF encoding the predicted parvulin domain of SEQ0694 (amino acids 148–242, ESI,† Fig. S1; rSEQ0694parv) was amplified using primer pair TGCCATAGACTACTCAGGTCACTACTCTAGACAATG (forward, NdeI site underlined) and TGCCATAG
TTAGGCTTTTTTGGTTACCTTAACA (reverse, XhoI site underlined), cloned, expressed and the protein purified as described above, except that 5 kDa, 6 mL cut-off concentrator units (Viva Science) were used.
The concentration of both purified proteins was determined using the Bradford Assay.
Reported kinetic data are given as the mean value of triplicate measurements for every condition. To ascertain if these data reflected true Michaelis–Menten kinetics, a Lineweaver–Burk plot was constructed and used to determine value of Km (calculated by reciprocalising the X intercept in the Line-weaver-Burk plot). The specificity constant (M s) was determined by dividing Kcat by Kmα.
Protein sequence alignments were performed using Clustal Omega24 (http://www.ebi.ac.uk/Tools/msa/clustalo/).
To confirm in vitro PPIase activity of SEQ0694, we produced full-length SEQ0694 as a recombinant protein, rSEQ0694 (ESI,† Fig. S2), for assay using a standard protease-coupled PPIase assay in which the rate of cis to trans isomerisation of a tetrapeptide substrate is measured through selective and colourigenic chymotrypsin hydrolysis of the trans isomer.12,21 In addition we produced the central parvulin domain of SEQ0694 as a recombinant protein, rSEQ0694parv. Both recombinant proteins were assayed against three tetrapeptide substrates varying in the amino acid preceding the critical proline residue. Whereas no activity could be detected using tetrapeptide substrates containing lysine–proline or alanine–proline bonds (data not shown), both rSEQ0694 and rSEQ0694parv were found to exhibit PPIase activity using Suc-Ala-Phe-Pro-Phe-pNA as substrate (Fig. 1). However, both recombinant proteins exhibited notably lower activities than the calf thymus cyclophilin used as a positive control.
Recombinant protein stability to chymotrypsin under the assay conditions was assessed. Significant cleavage of rSEQ0694parv by chymotrypsin was observed (ESI,† Fig. S3), whereas rSEQ0694 remained relatively stable for up to 5 min. This meant that although rSEQ0694parv showed an apparently faster rate of reaction compared with rSEQ0694 (Fig. 1), enzyme kinetics could only be determined for the latter (Fig. 2). A KcatKm−1 of 5.84 × 106/M s for rSEQ0694 was calculated from triplicate PPIase assays, suggesting that rSEQ0694 is a moderately active PPIase compared to other members of the parvulin family, with a similar activity to E. coli PpiC (Table 1). This activity was somewhat surprising as sequence alignments indicate that several amino acids considered functionally significant in parvulins31–35 are not conserved in rSEQ0694 (Fig. 3). However, a candidate Asp (D187) which might fulfil the role of the critical conserved Asp/Cys could be identified in rSEQ0694 (Fig. 3). Although a role of this Asp/Cys as a catalytic nucleophile is not yet fully resolved,36 its conservation in rSEQ0694 is likely to be significant. Moreover, the conserved residues in bacterial PrsA proteins identified by Jakob et al.26 are well conserved in SEQ0694 (ESI,† Fig. S1).
![]() | ||
Fig. 2 Kinetic analysis of rSEQ0694. The Kcat for rSEQ0694 was determined to be 583.75 s−1 and the Km 100 μM. Calculated Kcat/Km is 5.84 × 106 M−1 s−1. |
Parvulin | Substratea | K cat/Km/M s | Ref. |
---|---|---|---|
a Data from protease-coupled assays where substrate is a colourigenic tetrapeptide Succ-Ala-X-Pro-Phe-pNA in which X is the amino acid indicated in the Table. b Estimation from Fig. 1 in Heikkinen et al.33. c Subsequently Weininger et al.49 have reported that PpiD is inactive as a PPIase using modified substrates in a protease-free assay. d Data from a protease-free assay using the tetrapeptide Succ-Ala-Ala-Pro-Phe-2,4-difluroanilide as substrate. | |||
rSEQ0694 | Phe | 5.8 × 106 | This study |
rSEQ0694 | Lys | Inactive | This study |
rSEQ0694 | Ala | Inactive | This study |
B. subtilis PrsA | Lys | 1.5 × 104 | 27 and 33 |
B. subtilis PrsA | Ala | 0.6 × 104b | 33 |
B. subtilis PrsA | Glu | 0.8 × 104b | 33 |
S. aureus PrsA | Lys | 0.5 × 104b | 33 |
S. aureus PrsA | Ala | 1.7 × 104b | 33 |
S. aureus PrsA | Glu | 3.3 × 104 | 33 |
E. coli PpiC (Par10) | Leu | 1.3 × 107 | 46 |
E. coli PpiC (Par10) | Ser | 3.7 × 105 | 47 |
E. coli PpiD (Par68) | Ala | 1.1 × 109c | 48 |
E. coli PpiD (Par68) | Glu | 3.4 × 109c | 48 |
E. coli PpiD (Par68) | Leu | 2.3 × 109c | 48 |
Human Pin4 (Par14) | Arg | 3.9 × 103 | 46 |
L. lactis PpmA | Ala | Inactived | 29 |
S. pneumoniae PpmA | Ala, Phe, Gly, Val, Leu, Gln, Glu | Inactive | 14 |
![]() | ||
Fig. 3 Sequence alignment of SEQ0694 with representative members of the parvulin family. Alignment produced with Clustal Omega. The signal peptide sequences of the Firmicutes proteins have been removed so that each sequence starts from the lipidated cysteine at the N-terminus of the mature protein. Key active site residues of the characterised parvulins are highlighted in yellow. For the longer bacterial sequences, the region aligning with the short E. coli PpiC sequence corresponds to the central parvulin domain. Realignment of the gapping in the central parvulin domain region in SEQ0694 could bring D187 into alignment with the critical D/C residue present in the characterised parvulins. The position of the region deleted in the S. equi mutant strain ΔprtM138–21319 is shown in bold. Abbreviations and UniProt accession codes for the sequences are: Bsu_PRSA (Q81U45); B. subtilis PrsA (P24327); Eco_PpiC, E. coli PpiC/Par10 (P0A9L5); Hsa_Par14, Homo sapiens Pin4 (Q9Y237); LMO_PrsA2, L. monocytogenes PrsA2 (Q71XE6); Sau_PrsA, Staphylococcus aureus PrsA (A6QI23); and SEQ0694, S. equi PrsA (C0M9L5). |
Master 2D PAGE gels from 6 matched gel pairs (ESI,† Fig. S4) were analysed for differential protein expression and significant spots identified by mass spectrometry (Tables 2 and 3). Of the detectable total cell proteins, 12 differentially expressed proteins in 10 spots were identified (Table 2). The changes were primarily in cytoplasmic enzymes (e.g. enolase) which, because the proteins fold in the cytoplasm, may reflect general responses to stress due to lack of fully functional SEQ0694 (see below). Four of these proteins were also detected in the cell-free supernatant proteins (Table 3). In the cell-free supernatant proteomes, 13 proteins in 17 spots were found to be differentially expressed. As expected, the majority of these are proteins predicted to be either secreted or cell envelope localised and because of this could be plausible substrates for SEQ0694 (Table 3). As multiple proteins were found to be absent from the cell-free supernatant proteome of the mutant strain ΔprtM138–213, we hypothesise that SEQ0694 is likely to influence folding and secretion of multiple substrates rather than a specific substrate. Interestingly, two previously reported virulence factors of S. equi were notably absent from the cell-free supernatant proteome of the ΔprtM138–213 mutant: the truncated fibronectin-binding protein FNE40–42 and IgG endopeptidase IdeE2.43 FNE is noted to be misannotated as a pseudogene in the strain 4047 genome17 due to a misplaced start methionine. Our data therefore confirm the expression of FNE by strain 4047. SEQ0882, a putative DNase virulence factor homologous to S. pyogenes DNAse44 was also absent from the cell-free supernatant proteome of the ΔprtM138–213 mutant.
Spot #a | Protein identifiedb | Scorec | Matched peptidesd | % covere | Predicted functionf | Signal peptide |
---|---|---|---|---|---|---|
a Spot marked in ESI, Fig. S4. WT spots are upregulated or only detected in the wild type strain 4047, Prt spots were only detected in the ΔprtM138–213 mutant proteome. b As annotated in Holden et al.17 c Mascot score. d Number of non-redundant peptides identified for each protein. e Percent amino acid coverage of entire protein. f As determined from Uniprot annotation, BlastP and PFAM analysis. | ||||||
WT2201 | SEQ0898 | 1229 | 17 | 54 | Enolase (PF00113,PF03952) | No |
WT2201 | SEQ1657 | 117 | 3 | 8 | Cyclophilin PPIase (PF00160) | Lipoprotein |
WT2201 | SEQ0210 | 91 | 2 | 26 | 10 kDa chaperonin GroES (PF00166) | No |
WT3201 | SEQ1366 | 206 | 5 | 14 | Xaa-His dipeptidase (PF01546) | No |
WT3601 | SEQ0434 | 158 | 3 | 14 | Mannose-6-phosphate isomerase (PF01238) | No |
WT4001 | SEQ0408 | 318 | 6 | 68 | 30S ribosomal protein S6 (PF01250) | No |
WT4204 | SEQ1025 | 188 | 3 | 25 | Asp23 domain protein (PF03780) | No |
WT5302 | SEQ1354 | 184 | 3 | 23 | Purine nucleoside phosphorylase (PF01048) | No |
WT5504 | SEQ0046 | 293 | 6 | 30 | Alcohol dehydrogenase (PF00107,PF08240) | No |
WT6201 | SEQ1418 | 163 | 4 | 26 | Putative dTDP-4-keto-6-deoxyglucose-3,5-epimerase (PF00908) | No |
WT6501 | SEQ1011 | 408 | 6 | 22 | 6-Phosphofructokinase (PF00365) | No |
Prt9401 | SEQ1642 | 103 | 3 | 23 | Ribosome-recycling factor (PF01765) | No |
Spot #a | Protein identifiedb | Scorec | Matched peptidesd | % covere | Predicted functionf | Signal peptide |
---|---|---|---|---|---|---|
a Spot marked in ESI, Fig. S4. b As annotated in Holden et al.17 c Mascot score. d Number of non-redundant peptides identified for each protein. e Percent amino acid coverage of entire protein. f As determined from Uniprot annotation, BlastP and PFAM analysis. | ||||||
WT1002 | SEQ0210 | 174 | 4 | 57 | 10 kDa chaperonin GroES (PF00166) | No |
WT1401 | SEQ1821 | 334 | 4 | 38 | PepSY (PF03413) protease inhibitor domain lipoprotein | Lipoprotein |
WT1402 | SEQ1177 | 198 | 5 | 22 | Domain of Unknown Function (PF06207/DUF1002) | Present |
WT2101 | SEQ1800 | 119 | 2 | 30 | Unknown function, no conserved domains. Restricted distribution within streptococci; spot position shifted compared to mutant Prt1103 | Present |
WT2202 | SEQ1025 | 146 | 3 | 20 | Asp23 domain protein (PF03780) | No |
WT2202 | FNE | 72 | 2 | 6 | Truncated fibronectin binding protein (PF08341) | Present |
WT2401 | SEQ1177 | 526 | 8 | 36 | Domain of unknown function (PF06207/DUF1002) | Present |
WT3301 | SEQ1657 | 409 | 6 | 35 | Cyclophilin type PPIase (PF00160) | Lipoprotein |
WT7301 | SEQ0882 | 519 | 7 | 39 | DNA/RNA non-specific endonuclease | Present |
WT7301 | FNE | 361 | 6 | 26 | Truncated fibronectin binding protein (PF08341) | Present |
WT8401 | SEQ0938 | 331 | 6 | 19 | IdeE2 Mac family protein (PF09028) | Present |
WT8501 | SEQ0938 | 204 | 4 | 11 | IdeE2 Mac family protein (PF09028) | Present |
WT9202 | FNE | 221 | 5 | 13 | Truncated fibronectin binding protein (PF08341) | Present |
WT9202 | SEQ0882 | 93 | 3 | 14 | DNA/RNA non-specific endonuclease | Present |
WT9403 | SEQ0520 | 556 | 10 | 41 | Hydrolase/esterase (PF07859) | Present |
Prt0301 | SEQ1171 | 165 | 4 | 25 | Sortase A (PF04203) | Signal anchor |
Prt1103 | SEQ1800 | 133 | 3 | 36 | Unknown function, no conserved domains. Restricted distribution within streptococci; position shifted compared to mutant WT2101. | Present |
Prt1202 | SEQ1919 | 221 | 3 | 6 | OppA olipopeptide binding lipoprotein (PF00496) | Lipoprotein |
Prt2101 | SEQ0408 | 139 | 2 | 26 | 30S ribosomal protein S6 (PF01250) | No |
Prt2301 | SEQ1919 | 86 | 3 | 6 | OppA olipopeptide binding lipoprotein (PF00496) | Lipoprotein |
Cumulatively, these proteomic changes likely explain, at least in part, the attenuation of the ΔprtM138–213 mutant.19 However, as the ΔprtM138–213 mutant should still express a N + C domain fusion protein (lacking most of the parvulin domain), it may be that more dramatic proteome changes would be evident in an seq0694 null mutant, since a L. monocytogenes PrsA N + C construct partly complemented the proteome defect of a full prsA deletion12 and an N + C fusion construct of B. subtilis PrsA partially restored secretion of an AmyQ reporter protein (although it did not restore viability to PrsA-depleted cells27). In B. subtilis, the N and C domain is notable in driving dimerization of PrsA and, although lacking primary sequence homology, has structural similarity to other ‘foldases’ such as trigger factor.26 Without structural characterisation of the N + C fusion encoded by the S. equi ΔprtM138–213 mutant we cannot speculate whether this construct is likely to have a native-like conformation and functionality. However, it is notable that the sequence deletion removes not only the majority of the parvulin domain of SEQ0694 but also a conserved lysine of the Firmicutes PrsA protein N-domains. It is worth reemphasising that the partial deletion in the S. equi ΔprtM138–213 mutant is sufficient to cause significant attenuation of virulence in the natural host.19
It was interesting to note that SEQ1657, a cyclophilin PPIase lipoprotein (orthologous to S. pneumoniae SlrA14 and L. lactis PpiA29) was up-regulated in both the total cell and secreted proteins of the parental strain. Likewise, it was observed that the SEQ1171 sortase is up-regulated in the mutant strain, perhaps suggesting a need to remodel protein localisation within the mutant cell envelope.
As the proteomic data suggested a range of protein functions are likely to be perturbed in strain ΔprtM138–213, including stress responses, we performed several physiological tests. Although the mutant strain grows normally in nutrient rich broth, we observed pleiotropic changes including increased sensitivity to salt stress (ESI,† Table S1) and increased sensitivity to various antibiotics with diverse cellular targets (ESI,† Fig. S5). Increased sensitivity to salt stress has previously been observed in a prsA mutant of E. faecalis11 and a prsaA2 mutant of L. monocytogenes.30 A range of findings have been observed regarding antibiotic susceptibilities of other prsA mutants. Similar to our findings, a prsaA2 mutant of L. monocytogenes displayed increased sensitivity to bacitracin, penicillin and vancomycin but not gentamicin30 and a mutant in Staphylococcus aureus prsA showed increased sensitivity to vancomycin.45 However, a prsA mutant of E. faecalis was unaffected in its sensitivity to ampicillin and norflaxin,11 in contrast to our findings. Cumulatively, our data suggest a general perturbation in cell envelope function in the ΔprtM138–213 mutant, which likely reflects multiple changes in the extracytoplasmic proteome of the mutant (consistent with our proteomic data). This is conclusion is consistent with the pleiotropic effects of PrsA mutation in other Firmicutes.11,28,30,45
pNa | Paranitroaniline |
PPIase | Peptidyl-prolyl isomerase |
rSEQ0694 | Recombinant N-terminally His-tagged mature SEQ0694 |
rSEQ0694parv | Recombinant N-terminally His-tagged parvulin domain of SEQ0694 |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5mb00543d |
This journal is © The Royal Society of Chemistry 2015 |