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Heterologous protein exposure and secretion optimization in Mycoplasma pneumoniae

Microbial Cell Factories volume 23, Article number: 306 (2024) Cite this article

Abstract

The non-pathogenic Mycoplasma pneumoniae engineered chassis (Mycochassis) has demonstrated the ability to express therapeutic molecules in vitro and to be effective for treatment of lung infectious diseases in in vivo mouse models. However, the expression of heterologous molecules, whether secreted or exposed on the bacterial membrane has not been optimized to ensure sufficient secretion and/or exposure levels to exert a maximum in vivo biological effect. Here, we have improved the currently used secretion signal from MPN142 protein. We found that mutations at P1’ position of the signal peptide cleavage site do not abrogate secretion but affect it. Increasing hydrophobicity and mutations at the C-terminal of the signal peptide increases secretion. We tested different lipoprotein signal peptides as possible N-terminal protein anchoring motifs on the Mpn cell surface. Unexpectedly we found that these peptides exhibit variable retention and secretion rates of the protein, with some sequences behaving as full secretion motifs. This raises the question of the biological role of the lipobox motif traditionally thought to anchor membrane proteins without a helical transmembrane domain. These results altogether represent a step forward in chassis optimization, offering different sequences for secretion or membrane retention, which could be used to improve Mycochassis as a delivery vector, and broadening its therapeutic possibilities.

Background

Mycoplasma pneumoniae (Mpn) is a mild human pathogen and one of the causative microorganisms for community-acquired and hospital-acquired pneumonia [1, 2]. Mpn belongs to Mycoplasma genus and is characterized by a reduced genome of 816 kb encoding about 700 proteins [3]. This genome-reduced bacteria does not have a cell wall, allowing the direct release of any secreted biomolecule into the medium (patent 15/553552 USA), its genome is extraordinarily stable over time and geographic distribution, with no evidence of horizontal gene transfer [4], and it has a unique genetic code that prevent genes from being passed onto other cells [5]. Global analysis of this biological system [6] gave rise to integration of multi-omics to evaluate gene expression, post-transcriptional regulation [7], protein abundance and protein interactions [8,9,10], as well as regulatory networks [10,11,12] and gene essentiality [13], thus becoming a model biological system. Mpn comprehensively empowered the exploration of protein function, as demonstrated by the initial extensive in vivo approach of protein-protein interactions at a global scale [14], elucidating functions of previously uncharacterized proteins.

Although Mpn is a mild pathogen, by removing the few and well-known pathogenicity determinants found in its genome [15], we have obtained a non-pathogenic bacterial chassis (Mycochassis) (patent applications EP 20382288.7 and EP 20382207.7) for embedding gene platforms encoding therapeutic molecules. This engineered bacterium Mycochassis can survive more than 4 days in the murine lung, causes no lesions, and induces a mild inflammatory response when compared to its wild type (WT) counterpart [16]. We have demonstrated that Mycochassis can actively express and secrete complex molecules, such as the human IL-10-swapped dimer with two disulphide bridges [16] or simultaneously secrete three different enzymes to target polysaccharides from Pseudomonas aeruginosa’s biofilm [17] (patent application EP 20382288.7) and enzymes capable of dissolving biofilms caused by gram-positive bacteria [18]. Thus, it is possible to take advantage of Mpn’s natural tropism toward the human respiratory tract [19] and its enrichment in solid lung tumors [20] to develop strains capable of delivering therapeutic molecules locally, with high bioavailability and less systemic adverse effects.

For protein secretion we fused proteins of interest to the MPN142 signal peptide [16,17,18] (patent application US010745450B2, EP16706622NWB1). The mpn142 gene encodes for a 130 kD protein that after proteolytic cleavage originates P90/P40 polypeptides, which are part of the attachment organelle complex [21]. The MPN142 secretion signal has been improved (MPN142(Opt)ss) to minimize secondary structures in the 5’ of the mRNA [18]. To expose proteins in the membrane we fuse them to the transmembrane domain of Mpn adhesin P30 [22, 23]. Having only two sequences to secrete and expose proteins could be limiting for the development of Mycochassis as a therapeutic vector. In the case of the MPN142 signal peptide, the cleavage site is located between amino acids 25 and 26 of the P40 precursor protein (Ala25-Asn26) [24, 25] resulting in proteins starting with an Asn residue which could affect functionality of some heterologous secreted proteins. In the case of using fusions to P30 transmembrane domain, we could be constrained by the distribution in the cell membrane of this adhesin, which is part of the attachment organelle.

The ubiquitous bacterial secretion pathway (Sec) is the most important transport route for proteins that are exported out of the cytosol via a signal peptide. Sec substrates are synthesized with an N-terminal signal peptide responsible for membrane targeting [26,27,28]. The polypeptide is subsequently transported and once it has crossed the membrane, the signal peptide is cleaved by a membrane-bound enzyme, SPase I [26, 28, 29] (Fig. 1A). Secretion signal peptides are a 15–30 amino acid sequence at the N-terminal of a protein with three conserved domains: a 5 to 8-residue-long positively charged N-terminal (n-region), a 8 to 12-residue-long hydrophobic central region (h-region), and a polar 5 to 7-residue-long C-terminal cleavage region (c-region) [26] (Fig. 1B). The c-region contains crucial motifs required for cleavage by signal peptidase [26, 29]. Mpn contains a fully functional Sec complex machinery (mpn210 SecA, mpn396 SecD, mpn068 SecE, mpn242 SecG, mpn184 SecY), the SRP protein (mpn061) and the protein translocase to insert membrane proteins (mpn680, YidC) [30] but no SPase I [31]. However, an enzymatic activity like that of SPase I was experimentally demonstrated by identifying the N-terminal amino acid of product P40 of MPN142 [24].

Lipoproteins are synthesized in the cytoplasm as precursors with a specific N-terminal signal peptide that contains a lipobox motif. Lipoproteins are involved in several processes, many of which are essential [32, 33]. The lipobox motif has a four residue-molecular determinant at the C-terminal of the signal peptide that is unique to bacteria, containing a Cys residue strictly conserved at its 4th position [33] (Fig. 1C). Considering all non-homologous lipoproteins from Mpn (Supp. Figure 1A), its lipobox consensus is [LF][SAVLT][ASL]C (Fig. 1D). Lipoprotein processing involves a diacylglycerol transferase (mpn224, Lgt) and a lipoprotein signal peptidase (mpn293, Lsp) enzyme activities, highly conserved in bacteria. Lipoproteins are modified at the conserved Cys residue by a diacylglycerol moiety that anchors the protein to the membrane, followed by peptide cleavage of the signal peptide [34]. The lipoproteins present in Mpn appear to be a combination of di-acylated and tri-acylated lipoproteins, although an orthologous protein for N-acyl-transferase has not been described [35] (Fig. 1E).

In this work we have determined the specificity of the amino acid at position P1’ of the cleavage site and improved the MPN142(Opt)ss secretion efficiency by mutating different positions at the h- and c-regions. We have analyzed which signal peptides containing a lipobox motif could be used for protein exposure at the Mpn membrane and found that some of them are as efficient as MPN142(Opt)ss for protein secretion. The optimized sequences identified here that favor secretion or exposure could be used in Mycochassis for human therapy.

Fig. 1
figure 1

A Graphical representation of bacterial Sec system highlighting the co-translational (pathway 1) or post-translational (pathway 2) secretion that preferentially occurs, with respective elements involved in each pathway depending on the signal peptides (SP) characteristics and protein final localization. Mpn contains the genes for the proteins involved in both pathways. B General structure of a signal peptide indicating the residue positions relative to the cleavage site, as well as the general biophysical properties of its different regions. C General structure of a lipoprotein signal peptide (liposignal) including the lipobox and cleavage site motif. D Mpn liposignal logo sequence and lipobox consensus [LF][SAVLT][ASL]C for Mpn. E General scheme of diacyl (orange) and triacyl (yellow) lipoprotein generation by membrane anchoring after secretion. The lipoprotein signal peptide is shown as an elongated light blue box. Lgt is the enzyme that adds a diacyl group to the Cys of the lipobox motif. Lsp is the enzyme that cleaves the signal peptide before the Cys residue

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Results

Mutations in MPN142 signal peptide P1’ position of the cleavage site does not abrogate secretion

Optimization of secretion signals is an important goal in bacterial engineering [26, 36]. Also, in some proteins of therapeutic relevance like chemokines [37] the identity of the N-terminal residue is key [38, 39].

Here, to see if we could secrete proteins with an amino acid different from Asn at the N-terminus we assessed the relevance of the amino acid identity at P1’ position (Asn26 in MPN142(Opt)ss) (Fig. 2A) in cleavage efficiency of the MPN142(Opt)ss signal peptide. To do so we constructed 19 variants of the MPN142(Opt)ss generating point mutations in the P1’ position of the cleavage site (Supp. Table 1) and fused them to a luminescence producing enzyme (NanoLuc) [40] (Fig. 2B). The plasmid was assembled and transformed first in E. coli and then in Mpn strain 129 as proof of concept (Fig. 2C). We measured the luminescence and calculated the fold change ratio between the mutants with respect to the WT after normalizing the respective values by the total protein concentration (Fig. 2D). Although none of the mutations fully abolished secretion, only Asn26 substitution for Ala, His, or Thr have a similar secretion capacity as the WT (Fig. 2D). Interestingly hydrophobic (L, M, V) including the aromatics (F, W), and positive charged (K, R) residues are clearly disfavored at this position. This improves the range of amino acids that we could have at the N-terminus of the secreted protein.

Fig. 2
figure 2

A Structure of the cleavage motif within MPN142(Opt)ss fused to NanoLuc. P3: synthetic promoter P3. B General structure of the vector for the MPN(Opt)ss variants fused to NanoLuc sequence. IR-OR: inverted repeated regions; Tnp: Transposase; CmM438: Cloramphenicol antibiotic resistance. C Scheme of workflow applied for Mpn strains generation included in all the experiments. Vectors were constructed by Gibson assembly reaction, cloned in DH5α E. coli strain for further amplification, and used to transform Mpn 129 strain by electroporation and random transposon insertion. D Luminescence from NanoLuc activity was measured in the culture supernatant of Mpn strains expressing each variant of MPN142(Opt)ss fused to NanoLuc protein. Normalization of each condition was performed by determining total protein concentration by BCA assay. Different amino acid characteristics are represented in greyscale colors. Fold change of NanoLuc activities from different variants with respect to the WT sequence was calculated and T-test was performed from 4 independent experiments. We show the standard deviation of the experiments as error bars and the significance of differences in each variant compared to WT sequences as asterisks (p value < 0.03 (*), < 0.002 (**), < 0.0002 (***) and < 0.0001 (****)). The scheme was created with BioRender.com

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Modifications in the C-terminal and in hydrophobic region of MPN142(opt)ss improve the secretion rate

Secretion relies on how efficiently the signal peptide drives protein translocation. In this sense, small modifications to the signal peptide can in some cases significantly improve the secretory expression over the WT sequence [27].

Although there is no obvious SPase I orthologue in Mpn, there must be an enzyme with a similar activity. Thus, we assumed that we could optimize the MPN(Opt)ss by looking into frequencies of cleavage motifs in other bacteria. As a rule, amino acids with small neutral side chains must be placed in the P3 and P1 position of the SPase I cleavage motif of the secretion signal with a preference for Ala at both positions [26]. However, in some bacteria there is a preference for Ser instead of Ala at position P3 and P1, while others have Leu and Ile at P3 and Ser or Ala at P1. There is one archaeon with a deviated motif that has a Val in position P3, as present in humans and yeast. Lastly, Phe at P2 position is also significantly found in bacteria [41, 42]. Although the cleavage motif is just one of the three components of the signal peptide. It is widely known that disruption of the hydrophobic helix by polar or positively charged amino acids reduces or abolishes membrane transportation [26] and that the length and hydrophobicity of the h-region are critical features [26, 43, 44].

Thus, we aimed to investigate whether modifications in the sequence at positions P3-P1 and changes in hydrophobicity could have an impact on the secretion capacity of MPN142(Opt)ss (Fig. 3A) (Supp. Table 1), by doing the same fusion and procedures as shown in Fig. 2. The fold change for the NanoLuc activity was calculated as previously in Fig. 2. In all cases, the NanoLuc signal generated in the culture supernatant from all MPN142(Opt)ss variants was at least around 4 times higher. Mutation of the residues at positions P3 to P1 improved the NanoLuc secretion in all cases, with the best sequence being that with Ser at P3 mutated to Val and Leu at P2 to Ser. Similarly, the variant [K8L, R9F, Y10I] that increases the hydrophobicity of the transmembrane segment improved quite significantly the NanoLuc secretion. Both the replacement of an Arg [45] and the increase of hydrophobicity in the h-region could have a cooperative effect on secretion. The combination of mutations in the h-region, together with the double mutant at positions P3 and P2, have a synergistic effect on secretion improvement. These variants correspond to [K8L, R9F, Y10I, L24F, A25S] and [K8L, R9F, Y10I, S23A] (Fig. 3B) (Supp. Table 2).

Fig. 3
figure 3

A Table with the different variants designed for MPN142(Opt)ss optimization and its WT sequence. B Luminescence corresponding to NanoLuc activity was measured in the culture supernatant of Mpn different strains. In the Y axis we show the fold change in luminescence relative to the WT MPN142(Opt)ss. We show the results of unpaired T-test analysis from 3 independent experiments. Standard deviations are represented in the plot as error bars and asterisks indicate significant differences with respect to the WT sequence (*p < 0.05, **p < 0.002, ***p < 0.0005 and ****p < 0.0001). Normalization was performed determining total protein concentration employing a BCA assay

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Some Mpn lipoprotein signals can display heterologous proteins on the cell surface

When trying to identify natural signal peptide sequences that could promote protein secretion in Mpn we found a significant number of proteins in the supernatant of Mpn whose gene sequences contain a lipobox motif within the signal peptide sequence [46]. There were other lipoproteins mainly found associated with the cells and not in the supernatant (Fig. 4A, Supp. Table 3).

Thus, to identify lipoprotein signal peptides that could be used to expose proteins on the cell surface we selected those signal peptides corresponding to lipoproteins significantly expressed (more than 100 copies per cell) as determined by Mass Spectrometry (MS) [46] and not found significantly enriched in the supernatant of cell culture (Fig. 4A, Supp. Table 3). Since it was possible to divide lipoproteins into secreted or membrane-associated lipoproteins according to MS data [18], we aimed to address if there was any difference in amino acid sequence between these two clusters. First, we performed a phylogenetic tree [47] of all lipoproteins detected by MS to discard duplicated homologues genes (Supp. Figure 1A). From the selected ones, a cut-off limit of secretion ratio of 1 was established to delimit the secreted and non-secreted lipoproteins. Logo plots (Fig. 4B) and multiple alignment of liposignals from the two clusters of lipoproteins (Supp. Figure 1B, C) were performed. We found the main differences between both groups of sequences at − 1 position of the lipobox motif. Although both are Ala-enriched at this position, secreted ones contain also Gly and Tyr, while the retained ones have Ser and Leu. Additionally, one of the main differences involves the presence of more Leu in the helix of the liposignal from less secreted lipoproteins (Fig. 4B).

With these criteria we selected the lipoprotein signal peptides (liposignal) corresponding to MPN052, MPN058, MPN162, MPN200, MPN233, MPN284, MPN288, MPN415 and MPN523 lipoproteins that were mainly found in the cells and not in the supernatant. We included in the liposignals of these proteins four more amino acids after the Cys from the lipobox motif (Supp. Table 3).

The above-selected liposignals were fused to a DNA sequence containing the NanoLuc luciferase enzyme and a HaloTag protein [48]. NanoLuc assay was performed in the supernatant to evaluate the secreted protein, while the anchored protein level was assessed by fluorescence detection using a cell-impermeant HaloTag fluorescent ligand that covalently binds to the enzyme to determine the levels of surface display (Fig. 4C). The NanoLuc substrate can pass the cell membrane and therefore we cannot be sure if the signal coming from the cell corresponds to protein exposed in the membrane or protein in the cytoplasm. The HaloTag fluorescence detection signal cannot be used to determine protein in the supernatant because we cannot wash the free fluorescent ligand. Thus, the strategy relies on both detections to assess the retention and secretion fractions. We included an empty vector (EV) as a negative control, the MPN142(Opt)ss and the transmembrane region of P30 Cytadhesin Mpn (mpn453), as controls for full secretion and full retention, respectively.

We see as expected that the P30-fusion is mainly at the cell surface while the MPN142(Opt)ss is found mainly in the medium (Fig. 4D, E). Regarding the tested lipoprotein signals, we found no significant correlation between the luminescence and fluorescence signals (Fig. 4E). In other words, there are some lipoprotein signals that favor protein secretion while others favor protein retention at the membrane. Surprisingly, we see that some of the signal exhibits a similar secretion capacity as MPN142(Opt)ss even though we selected them from the ones with a lower secretion/retention ratio (MPN288 and MPN523). In fact, we see that these two had the highest ratio between secreted/retained fractions of the chosen liposignals, while MPN233 that shows the highest retention, has also high secretion (Fig. 4E).

To see if these differences in the behavior of the liposignal peptides were independent of the protein fused, we replaced the NanoLuc-HaloTag by a SpyCatcher domain and the cells and supernatants were incubated with SpyTag (Supp. Figure 2 A, see Methods). Comparison between the secreted/retained ratios obtained with the NanoLuc-HaloTag and a SpyCatcher domains show a good correlation (R = 0.63) except for MPN523 (Supp. Figure 2B). This indicates that the secretion/retention capacity is mainly related to the liposignal and is independent of the protein being fused to it.

Fig. 4
figure 4

A Scatter plot of Mpn lipoproteins secreted fraction expressed as the ratio out/in of lipoproteins determined by MS of culture supernatant (secretome), and its corresponding copies per cell calculated from MS data (Supp. Table 3). B Sequence logo plot of liposignal sequences including lipobox N-terminal Cys of mature protein and the next four residues from all, anchored and secreted lipoproteins found in Mpn. C Representative scheme of the display system-detection strategy that involves fusing selected liposignals to reporter enzymes. Retention efficiency can be evaluated by quantifying HaloTag activity on the cell surface and NanoLuc activity in the supernatant of the released fraction. D Bar plots of fluorescence and luminescence quantification in cell surface and supernatant, respectively. Both determinations were performed in different Mpn strains expressing NanoLuc-HaloTag fused to the liposignal candidates. E Scatter plot of different liposignals according to retained and released proportions of the reporter fusion protein. A Pearson’s correlation was performed with a resulting coefficient of 0.2. EV, empty vector. Secretion (yellow) and anchor (purple) controls are above and below the fitted line, respectively

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The modification in length and mutations in the C-terminal of the liposignals increase exposure levels and reduces protein release

We tested the impact of mutations around the lipobox cleavage sequence using the MPN058 signal peptide that did not show any preference for secretion or retention (Fig. 4E). We mutated Cys26 to Ala (MPN058a) and combined this mutation with Thr21 to Val substitution (MPN058va), abrogating the anchoring via the acylated Cys. It has been reported that Asp residue at position + 2 and a Glu at + 3 after the lipobox favors retention while for example Lys at position + 3 favors secretion [49]. To see if this was the case, we made the mutants MPN058de (Ser24 to Asp, Ser25 to Glu) and MPN058kr (Ser24 to Lys, Ser25 to Arg), where the lipobox motif is conserved, and MPN058iy (Thr21 to Ile, Ala22 to Tyr) (Supp. Table 4). We found that all mutants except for MPN058de resulted in a decrease in protein expression. In the case of the MPN058de, we see an increase in protein expression with proportionally more protein retained than in the WT MPN058 (Fig. 5A, B).

In gram-negative bacteria some outer-membrane lipoproteins possess unstructured linkers at their N-terminal that play a crucial role in their targeting to the outer membrane [50]. To assess this possibility in Mpn we chose a lipoprotein that is preferentially found in the supernatant of Mpn cell culture but still present to a small extent in the cell (MPN643) [18, 46]. This way we could test if the secretion to the medium is due to the liposignal or to the cleavage of a sequence after the lipobox motif like in MPN142. The MPN643 liposignal variants were generated by extension of the liposignal peptide using the MPN643 natural sequence: MPN643 (1–22) MPN643 (1–27), MPN643 (1–32), MPN643 (1–3), MPN643 (1–42), MPN643 (1–47), MPN643 (1–52), MPN643 (1–57), and MPN643-linker by adding a flexible linker of 15 residues length (Supp. Table 4). These sequences were fused to the NanoLuc-HaloTag sequences as described above. We found that increasing the length of the MPN643 N-terminal sequence does not have a significant effect in secretion until we reached 52 aa at which point the amount of secreted protein increases (Fig. 5C, D), suggesting a proteolytic cleavage. In the case of a non-natural linker introduction, we also see a relative increase in secretion supporting the idea that a non-structured spacer of enough length after the lipobox could be cleaved by a Mpn membrane protease (Fig. 5C, D). In general, the secretion ratio for longer liposignals is higher, while for intermediate lengths the exposure fraction is predominant over secretion (as it is for MPN643(1-32, 1-37, and 1-42)). Although there is a low correlation (R = -0.2), the modifications show that the more secretion, the less exposure, as opposed to the MPN058 mutants (Fig. 5D). It is possible that modifications in retention/secretion ratios have to do with Lgt saturation, leading to less acylation rates and thus limiting cleavage by Lps [51]. To confirm that differences in the behavior of the different variants were independent of the protein fused, we replaced the NanoLuc-HaloTag by a SpyCatcher domain as indicated above (see Methods) and tested most of the variants. Comparison between the secreted/retained ratios obtained with the NanoLuc-HaloTag and a SpyCatcher domains shows a good correlation (R = 0.67) for the different variants tested (Supp. Figure 2C). This indicates that the effect of the different mutations and linkers on the secretion capacity is related to them and independent on the protein fused to it.

In the SpyCatcher/SpyTag experiment, the cell lysates show processed and unprocessed lipoproteins corresponding to a lower and upper molecular weight, respectively (Supp. Figure 3A). In general, we see that for shorter liposignal lengths there is relatively less processed protein, while this is reversed when increasing length. These results suggest that less processing reduces both surface exposure and secretion capacity. The MPN643 (1–57) shows a close to 100% processing and the supernatant fraction is considerably increased. In E. coli, it has been reported that unprocessed lipoproteins affect bacterial growth [52]. We also see a similar effect, especially in the intermediate length-MPN643 liposignals strains compared to MPN643 (1–57) (Supp. Figure 3B). These results confirmed that negative charges at positions + 2 and + 3 result in an increase in overall exposure level, and that the length and nature of the sequence between the end of the liposignal and the fused protein could affect the anchoring potential of liposignals.

Fig. 5
figure 5

A Bar plots represent relative fluorescence (cell surface) and luminescence (supernatant) quantification for MPN058 mutants. B Scatter plot of pellet (fluorescence) vs. supernatant signal (luminescence) representing the retention/secretion ratio of each liposignal corresponding to MPN058 mutants. C Bar plots show relative fluorescence (cell surface) and luminescence (supernatant) quantification for MPN643 elongated variants. D Scatter plot of pellet (fluorescence) vs. supernatant signal (luminescence) representing the retention/secretion ratio of MPN643 variants. WT MPN142(Opt)ss (yellow) is included as a control of maximum secretion and P30 signal (purple) as a positive control of anchoring. Results were normalized by growth curves of each Mpn strain, measured for 48 h. Early, intermediate and end points were taken as reference. 3 replicates per condition were done for this experiment and standard deviation is represented by error bars

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Discussion

The constant advancement in synthetic biology requires the investigation of new strategies to optimize biological systems in terms of fitness, product synthesis, and exportation. Live therapeutics must offer a real and superior benefit over other available therapies. This includes the chance to locally treat different conditions while avoiding adverse systemic effects and, more importantly, ensuring the therapy’s effectiveness. That involves a potent expression system that guarantees the sustained availability of active molecules within a therapeutic dynamic range.

Current preclinical investigations in this field demonstrate the capacity of bacterial systems to deliver active molecules. Limitations involve the saturation of the expression system, unreachable therapeutic concentrations, and disturbances in bacterial fitness, all of which translate into inefficient live therapy treatments. Efforts are currently underway to tackle these issues [53].

Here, we describe the optimization of secretion and exposure of heterologous molecules in a biological system that has already demonstrated therapeutic capacity in in vivo infection models. We have generated a non-pathogenic bacterial chassis (Mycochassis) based on Mpn that has shown to have therapeutic effects in infection [17, 18] as well as in lung inflammation [16]. Mpn has a significant advantage over other bacteria since it does not have cell wall and therefore secreted or membrane exposed proteins do not need to pass any barrier. We had previously identified two ways of secreting and exposing proteins at the cell membrane of this bacterium. For secretion, we identified the signal peptide sequence of MPN142 which when fused to any protein results in very efficient secretion despite Mpn not having a bona fide SPase I. For protein exposition, fusion to the P30 protein results in efficient protein exposure [23]. However, both methods have their limitations. The use of MPN142(Opt)ss results in all secreted proteins starting with an Asn residue which could limit their biological activity. In addition, if we would like to secrete more than one protein we could saturate the protease involved in MPN142 signal peptide cleavage, as well as the machinery responsible of targeting nascent peptides or already synthesized proteins to the membrane. In the case of P30 transmembrane domain, we are constrained by the distribution in the cell membrane of this adhesin to the terminal organelle.

To solve these issues, we have explored the cleavage efficiency of different amino acids at position P1’ replacing the wild type Asn as well as sequence variations of the C-terminal region and transmembrane domain of the signal peptide of MPN142. We have found that the signal peptide of MPN142 can tolerate other amino acids than Asn, although mainly hydrophobic amino acids (Leu, Met, Val and Trp) and positively charged ones (Lys and Arg) are poorly cleaved. This allows the expression of proteins whose functionality depends on the identity of its N-terminal amino acid (i.e. chemokines [38, 39]). Additionally, by increasing the overall hydrophobicity and length of the hydrophobic region of the MPN142(Opt)ss we could increase protein secretion levels by around four times.

Regarding the possible saturation of the unknown signal peptidase that cleaves MPN142 signal peptide and the identification of membrane exposure signals, we have investigated Mpn lipoproteins. Bacterial lipoproteins contain a lipobox motif with a conserved Cys that is acylated and then the signal sequence is cleaved by a specific peptidase right before that Cys. Theoretically, lipoproteins remain anchored to the cell membrane by the diacyl or triacyl groups linked to the Cys. However, in some bacteria they have been found in the cell medium by lipase cleavage of the acyl groups [54] or in extracellular vesicles [46]. In a previous work we found several Mpn lipoproteins that were significantly enriched in the cell medium and others that were found mostly in the cell and therefore could be used as an alternative to P30 fusion [18]. To assess this, we selected a group of lipoproteins that were significantly expressed in Mpn as shown by MS data analysis [46] and were not enriched in the cell culture medium. These lipoproteins signal peptides despite being found in non-related proteins have some specific amino acids preferences at some positions of the signal peptide and lipobox motif. We fused them to luminescence and fluorescence reporters, and we could see that they exhibited different retained/secreted ratios that were independent of the fused reporter protein. In fact, we showed that the Mpn natural lipoproteins efficiently secreted or retained, have some differences in the liposignals sequence specificity at the transmembrane helical domain. This sequence-dependence is also supported by the fact that changing one or two residues in a liposignal modifies the protein retention capacity. Negatively charged residues positioned at + 2 and a Glu at + 3 positions after the lipobox favors retention as shown in other bacteria [49].

Mycoplasma species synthesize simple phospholipids, comprising primarily of glycerophospholipids like phosphatidylglycerol and cardiolipin. In addition, it incorporates from the medium significant amounts of phosphatidylcholine and sphingomyelin although these lipids are very uncommon in cell-wall containing bacteria, and its growth is dependent on the presence of cholesterol in the medium [55]. This unique membrane composition of Mycoplasma species and the lack of a cell wall could favour the release of lipoproteins as compared to other bacteria.

We also found that having long unstructured regions could result in efficient secretion, probably due to cleavage by proteases present in the membrane of Mpn. Finally, we see that the length of the sequence between the liposignal and the fused protein affects the processing capacity of the Lgt protease, with longer sequences being more efficient. What remains to be explained is why the lipoproteins that we selected because they were enriched in the cell surface instead of in the cell medium, when fused to heterologous proteins could be as effective in secretion as the MPN(Opt)ss sequence. Probably, some of these natural lipoproteins interact with any of the Mpn surface membrane proteins and remain attached, or they could contain folded elements very close to the cleavage site of the Lgt protease decreasing its efficiency.

Thus, it is not feasible to predict modifications that could further improve the retention proportion. Although we could not identify any specific sequence motif that results in efficient membrane exposure, we found several that could be used for efficient secretion comparable to MPN142(OPT)ss (MPN288 and MPN523).

In conclusion, these results represent not only an advance in our understanding of Mpn but also an evolution and potential enhancement of our chassis to express and expose molecules more optimally, thereby expanding the boundaries for future applications.

Materials and methods

Plasmids and oligonucleotides

Plasmids were generated following the Gibson assembly method [56] or ligation by KLD Enzyme Mix (New England Biolabs Cat. M0554S). When required, IDT Incorporation performed oligonucleotides and gene synthesis. Gene amplifications were carried out with Phusion DNA polymerase (Thermo Fisher Scientific Cat. F530L). Mpn does not require a Shine–Dalgarno region at the 5′ end of the mRNA to efficiently translate the transcripts, so the promoter sequence contains the region placed immediately upstream of each of the coding sequences controlled by them. Thus, researchers working with other Mycoplasma species may need to define their own regulatory regions or use a recently reported regulatory region that is functional in all Mycoplasma species. All oligonucleotides employed for plasmid assembly/ligation are available in Supp. Table 5. The correct assembly or ligation of all plasmids were verified by Sanger sequencing (Eurofins Scientific).

Mpn transformations

Mpn cultures were grown to late-exponential phase in 75 cm2 tissue culture flasks for transposon vector transformations. The adherent cell layer was washed three times with chilled electroporation buffer (sucrose 0.2 µM and HEPES 8 mM), scraped off, and resuspended in 500 µL of this buffer at a concentration of approximately 1010 cells mL−1. Next, this cell suspension was passed 10 times through a 25-gauge (25G) syringe needle, and 50 µL aliquots were mixed with the desired DNA plasmid for transformation. 1–2 µg of DNA was added to the mix and the volume was adjusted to 80 µL with electroporation buffer. The mix was transferred into 0.1 cm electro-cuvettes, for a 15 min incubation on ice before being electroporated in a BIO-RAD Gene Pulser Xcell device. The settings employed were 1250 V/25 µF/100 Ω. After the pulse, cells were incubated on ice for 15 min and subsequently harvested by adding 420 µL of Hayflick into the cuvette. Cells were left for recovery and resistance gene expression at 37 °C for 2 h before inoculating 100 µL of the transformation volume into a 25 cm2 flask filled with 5 mL of Hayflick supplemented with the appropriate antibiotic. One week later, the total volume of the transformation was directly inoculated into 75 cm2 flasks containing 15 mL of Hayflick medium with selection antibiotic for 72 h of expansion. Cultures were collected in 2 mL volume of Hayflick medium, disaggregate as previously and frozen at − 80 °C.

Transposon insertion detection by PCR

All Mpn strains were grown in 25 cm2 flasks to confluence. The adherent layer of cells was washed three times with PBS and scraped off in 300 µL of this buffer. This cell suspension was boiled at 95ºC for 5 min and treated with 25 µL StrataClean resin (Agilent, Cat. 400714) for 1 min and centrifuge at 10,000 xg to isolate genomic DNA in the supernatant. PCR was performed on this sample to amplify the insert for Sanger sequencing.

Bacterial strains and culture conditions

All the Mpn strains generated in this work derive from the wild-type strain M129-B7 (ATTC 29342, subtype 1) and are described in Supp. Table 6. All strains were grown in Hayflick modified medium (ISSN:0036-8075) with 20% horse serum at 37 °C and 5% CO2 in tissue culture flasks (Corning). Hayflick broth was supplemented with chloramphenicol (20 µg mL−1) for transformants selection. For cloning purposes, the E. coli NEB 5-alpha High Efficiency strain (New England Biolabs) was grown at 37 °C in LB broth or on LB agar plates supplemented with ampicillin (100 µg mL−1).

NanoLuc assay

Luciferase sequence within some of the constructs was used as a reporter system. Luminescence was detected using Nano-Glo® Luciferase Assay (Promega, Cat. N1110). Briefly, different strains were grown in 25 cm2 flasks for 72 h (Mycoplasma pneumoniae late exponential phase [57]) in Hayflick media with selection antibiotics. Supernatant was collected and passed through 0.1 μm filter for bacteria elimination, and cells were washed three times with PBS and harvested by scraping off in a 500 µL volume. 50 µL of supernatant and cell suspension of each strain was plated and 50 µL of the working reagent was added for immediate luminescence detection using Tecan Infinite M200 device, set up for 500 ms integration time, 50 ms settle time. Luminescence was normalized by total protein concentration of the cell culture using BCA Protein Assay (Thermo Scientific, Cat. 23225).

HaloTag assay

The HaloTag® reporter protein is an engineered, catalytically inactive hydrolase derivative, which rapidly forms an irreversible covalent bond with HaloTag® ligands (PROMEGA) under physiological conditions. Its specificity lays on the lack of endogenous expression by biological systems. Briefly, HaloTag sequence is introduced in a vector to be integrated into the genome and expressed by Mpn strains. After reaching the exponential phase, cells from culture flasks were washed 3 times with warmed PBS and scraped in 300 µL of PBS. Cells were centrifuged at 10,000 xg for 10 min and resuspended in a PBS diluted solution of 1 µM fluorescent ligand (cell-impermeant Alexa Fluor 488 ligand Cat. G1001) and incubated for 2 h at 37 °C. Cells were washed 3 times and fluorescence intensity was measured in a fluorimeter (Tecan Infinite M200).

SpyCatcher/SpyTag

The SpyCatcher/SpyTag system is based on Streptococcus pyogenes extracellular proteins that are stabilized by spontaneous intramolecular iso-peptide bonds. The CnaB2 domain of fibronectin binding protein generates a bond between Lys1 and Asp117 catalyzed by Glu77. It has been stably splited into a larger component that is the incomplete immunoglobulin-like domain (termed SpyCatcher) of 138 residues that contains the reactive Lys and catalytic Glu, and a shorter peptide (SpyTag) of 13 residues that includes the reactive Asp (PDB ID: 4MLI) [58, 59]. Briefly, strains expressing liposignals and fused to SpyCatcher protein were cultured in Hayflick media 72 h at 37 °C, 5% CO2. Quasar670 labeled SpyTag (sample ID: Q670-P201118, Peptide Synthesis Facility Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain) of sequence Q670-RGVPHIVMVDAYKRYK-amide was added to each culture flask in a final concentration of 1 µM and incubated at 37 °C overnight. Supernatant and pellet were collected for western blot assay.

Western blot

Mpn strains were cultured for 72 h at 37 °C, 5% CO2 in a 25 cm2 flask with antibiotic- supplemented Hayflick. 500 µL of supernatant was collected and filtered through 0.1 μm filter. In parallel, cells were washed 3 times with PBS and scraped in PSB- SDS 1% buffer with protease inhibitor cocktail (Promega, cat. G6521) and sonicated for complete lysis. Both supernatant and lysate were prepared in 4x fluorescent compatible sample buffer (Invitrogen, cat. LC2570) and heated at 95 °C for 5 min. Samples were run in NuPAGE™ 4–12% Bis-Tris Protein Gels (Invitrogen, cat. WG1402) with MES SDS running buffer - novex (Fisher scientific, cat. NP0002) for 40 min at 200 V. iBlot gel transfer stacks nitrocellulose (Invitrogen, cat. IB301001) were used for transference and fluorescence was detected using iBright Imaging System.

Growth curves

For bacterial growth, pre-cultures of 5 mL Hayflick media with 1:1000 inoculates were prepared for each of the tested constructs. After reaching exponential phase, cells were scraped into Hayflick media and total protein was measured by BCA Protein Assay. Volumes were adjusted to the same protein concentration and 200 uL were plated in 96 multi-well plate and cultured in a Tecan Infinite 200 Pro plate reader. Growth was observed by measuring the ratios of optical density (OD) at 430/560 nm and 600 nm each hour until stationary phase.

Data analysis

Statistical analysis and plots were performed in R (R Core Team (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/) employing Rstudio environment (RStudio Team (2020). RStudio: Integrated Development for R. RStudio, PBC, Boston, MA URL http://www.rstudio.com/.).

Data availability

The datasets analysed during the current study as well as all strains and plasmids generated in this work are available from the corresponding author on reasonable request.

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Acknowledgements

We acknowledge the support of the Spanish Ministry of Science, Innovation and Universities through the Centro de Excelencia Severo Ochoa (CEX2020-001049-S, MCIN/AEI /10.13039/501100011033), the Generalitat de Catalunya through the CERCA programme and to the EMBL partnership. We are grateful to the CRG Core Technologies Programme for their support and assistance in this work.

Funding

This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreements No 670216 and 101020135). We also acknowledge the support of the Spanish Ministry of Science, Innovation and Universities through the Plan Nacional PID2021-122341NB-I00 funded by MICIU/AEI/10.13039/501100011033/FEDER, UE.

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Author notes

  1. Yamile Ana and Daniel Gerngross author contributed equally to the work.

Authors and Affiliations

  1. Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona, 08003, Spain

    Yamile Ana, Daniel Gerngross & Luis Serrano

  2. Lab Automation Facility, Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

    Daniel Gerngross

  3. Universitat Pompeu Fabra (UPF), Barcelona, Spain

    Luis Serrano

  4. ICREA, Pg. Lluís Companys 23, Barcelona, 08010, Spain

    Luis Serrano

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Contributions

YA, DG and LS conceptualized the study; YA and DG performed the experiments; YA, DG and LS did the data analysis; YA wrote the original draft of the manuscript; DG and LS reviewed and edited the manuscript; LS supervised the study; LS acquired the funding for the study. All authors read and approved the final manuscript.

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12934_2024_2574_MOESM1_ESM.png

Supplementary Material 1: Supplementary Figure 1. A A phylogenetic tree of complete Mpn lipoproteins sequences was performed to determine the presence of homologs for further analysis. B Multiple linear alignment of liposignals belonging to secreted lipoproteins of Mpn. C Multiple linear alignment of liposignals from poorly or non-secreted Mpn lipoproteins. Homologues were excluded for multiple linear alignment

12934_2024_2574_MOESM2_ESM.png

Supplementary Material 2: Supplementary Figure 2. A Scheme of SpyCatcher/SpyTag strategy for supernatant and cell surface detection of SpyCatcher fused to different liposignals. B Correlation of the retention/secretion ratio from WT liposignals obtained in NanoLuc-HaloTag (fluorescence/luminescence) and SpyCatcher/SpyTag (pellet/supernatant) assays. C Correlation of the retention/secretion ratio from MPN058 mutants and MPN643 elongated liposignals obtained in NanoLuc-HaloTag a (fluorescence/luminescence) and SpyCatcher/SpyTag (pellet/supernatant). Pearson’s correlation was calculated, resulting in a coefficient > 0.6 in both cases

12934_2024_2574_MOESM3_ESM.png

Supplementary Material 3: Supplementary Figure 3. A SpyCatcher/SpyTag assay was performed, and fluorescence was assessed by western blot of 10 µg per cell lysate and supernatant samples (determined by BCA assay). MPN647 liposignal fused to SpyCatcher was added as a high secretion liposignal control according to MS data of secreted lipoproteins (Supp. Table 3). B Growth curves of MPN643 variants

Supplementary Material 4

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Supplementary Material 9

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Ana, Y., Gerngross, D. & Serrano, L. Heterologous protein exposure and secretion optimization in Mycoplasma pneumoniae. Microb Cell Fact 23, 306 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-024-02574-z

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