NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli

Michael J Barnett; Jully Pinheiro; Jeremy R Keown; Jacob Biboy; Joe Gray; Ioana-Wilhelmina Lucinescu; Waldemar Vollmer; Robert P Hirt; Augusto Simoes-Barbosa; David C Goldstone

doi:10.1371/journal.ppat.1011563

. 2023 Aug 16;19(8):e1011563. doi: 10.1371/journal.ppat.1011563

NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli

Michael J Barnett ¹, Jully Pinheiro ¹, Jeremy R Keown ¹, Jacob Biboy ², Joe Gray ³, Ioana-Wilhelmina Lucinescu ³, Waldemar Vollmer ², Robert P Hirt ³, Augusto Simoes-Barbosa ^1,^*,^#, David C Goldstone ^1,^4,^*,^#

Editor: Patricia J Johnson⁵

PMCID: PMC10461829 PMID: 37585473

Abstract

Trichomonas vaginalis is a human protozoan parasite that causes trichomoniasis, a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a shift to a dysbiotic vaginal microbiome that is depleted of lactobacilli. Studies on co-cultures have shown that vaginal bacteria in eubiosis (e.g. Lactobacillus gasseri) have antagonistic effects on T. vaginalis pathogenesis, suggesting that the parasite might benefit from shaping the microbiome to dysbiosis (e.g. Gardnerella vaginalis among other anaerobes). We have recently shown that T. vaginalis has acquired NlpC/P60 genes from bacteria, expanding them to a repertoire of nine TvNlpC genes in two distinct clans, and that TvNlpCs of clan A are active against bacterial peptidoglycan. Here, we expand this characterization to TvNlpCs of clan B. In this study, we show that the clan organisation of NlpC/P60 genes is a feature of other species of Trichomonas, and that Histomonas meleagridis has sequences related to one clan. We characterized the 3D structure of TvNlpC_B3 alone and with the inhibitor E64 bound, probing the active site of these enzymes for the first time. Lastly, we demonstrated that TvNlpC_B3 and TvNlpC_B5 have complementary activities with the previously described TvNlpCs of clan A and that exogenous expression of these enzymes empower this mucosal parasite to take over populations of vaginal lactobacilli in mixed cultures. TvNlpC_B3 helps control populations of L. gasseri, but not of G. vaginalis, which action is partially inhibited by E64. This study is one of the first to show how enzymes produced by a mucosal protozoan parasite may contribute to a shift on the status of a microbiome, helping explain the link between trichomoniasis and vaginal dysbiosis. Further understanding of this process might have significant implications for treatments in the future.

Author summary

Trichomoniasis is a frequent infection of the human urogenital tract. The causative agent is Trichomonas vaginalis, a protozoan parasite that lives on the mucosal surface of the vagina and has to interact with bacteria naturally residing on this site (i.e. microbiota). It has been shown that the composition of bacterial species of the vaginal microbiota differs between infected and non-infected women, but what drives this microbial shift during infection to an unbalanced microbiota (i.e. dysbiosis) remains unknown. A previous study from our group revealed that T. vaginalis acquired NlpC/P60 genes from bacteria (or TvNpCs), known for producing enzymes that break down peptidoglycan which is the main component of bacterial cell walls. Here, we expand this characterization to show that different TvNpCs have complementary activities helping the protozoan to control populations of vaginal lactobacilli in mixed cultures in laboratory. This study advances our knowledge on the interaction of mucosal pathogens with the microbiota, a topic of increasingly importance for improving treatments including against trichomoniasis and morbidities associated with a dysbiotic microbiota.

Introduction

Trichomonas vaginalis is an extracellular protozoan parasite that infects the urogenital tract of humans causing trichomoniasis. With about a quarter of a billion cases each year, trichomoniasis ranks as the most common nonviral sexually transmitted infection in the world affecting more people than Syphilis, Neisseria gonorrhoeae and Chlamydia trachomatis combined [1]. Trichomoniasis causes vaginitis, prostatitis and urethritis with women carrying a disproportional morbidity burden including increased chances of developing pelvic inflammatory disease [2], cervical cancer [3, 4], increased risk of acquiring and transmitting HIV [5, 6] and, concerningly, significantly adverse pregnancy outcomes [2–4, 7, 8].

The vaginal microbiome of women of reproductive age has been classified into five community state types (CSTs), where the dominance of a single species of Lactobacillus has been allocated to four of these communities and representing ~75% of asymptomatic women [9]. Trichomoniasis has been correlated with the species-diversified CST where, conversely, host-protective lactobacilli are excluded or less dominant [10, 11]. This CST is formed by mostly anaerobic bacteria with the dominance of Prevotella, Gardnerella, Megasphera, Snethia and Atopobium vaginae coincidently found in women experiencing a dysbiotic condition known as bacterial vaginosis [9–11]. These observations do not necessarily imply on causation, but at least suggest that T. vaginalis may shape this microbiome [12]. Indeed, vaginal lactobacilli exert protective effects against T. vaginalis [13, 14] while vaginal dysbiotic bacteria engage synergistically with this protozoan [15–17]. Whether a causation relationship may exist between T. vaginalis and the status of the vaginal microbiome, the underlying molecular mechanisms of this microbial interaction remain poorly understood.

The adaptation of T. vaginalis to the urogenital tract of humans is thought to be recent, relative to evolution of trichomonads as parasites of various hosts and mucosal sites, and was followed by genome expansion including the lateral acquisition of genes from bacteria [18–20]. We have recently identified that T. vaginalis acquired bacterial NlpC/P60 enzymes, likely from two events of lateral gene transfer and followed by gene duplication resulting in nine genes that fall into clans A and B [21]. NlpC/P60 proteins were identified early on as cysteine proteases (clan CA, family C40) [18], and broadly classified under the cysteine/histidine dependant aminohydrolase/peptidase super family (CHAP) [22]. NlpC/P60-containing enzymes typically hydrolyse the stem peptides of bacterial peptidoglycan (PG). The expansion of this gene family in T. vaginalis, a eukaryotic organism that lacks PG, suggests these enzymes may play important role in mediating interactions between the parasite and the bacterial members of the human urogenital tract microbiota.

The lateral acquisition of bacterial genes targeting PG has been observed in other eukaryotes [23], and considered as an adaptive trait to target specific bacteria [24]. Therefore, targeting PG might represent one of the mechanisms by which T. vaginalis shapes the vaginal microbiota contributing to the parasite’s capacity to thrive at this mucosal site. It is also possible this trait may have even evolved earlier in this lineage, and thus shared across other species of trichomonads that are known for parasitizing other mucosal sites and hosts. Although this has yet to be demonstrated, infection by Trichomonas gallinae that parasitizes the mucosa of the upper digestive tract of pigeons was found to cause significant alterations on the gut microbiota with depletion of lactobacilli [25].

We have previously shown that two members of T. vaginalis NlpC/P60 enzymes, both belonging to clan A, are functional DL-endopeptidases degrading stem peptides of PG [21]. These enzymes, TvNlpC_A1 and TvNlpC_A2, are secreted by the protozoan and may confer an adaptive advantage for controlling and/or exploiting bacterial populations. Here, we extend the characterization of TvNlpCs to two other members of the clan B (TvNlpC_B3 and B5). We show the presence of the two gene family clans in other trichomonads, characterize the structure of one the T. vaginalis clan B enzyme with and without a ligand, and we demonstrate that TvNlpCs of clans A and B have complementary cellular and enzymatic capabilities that support T. vaginalis on controlling populations of vaginal lactobacilli in mixed cultures.

Results

The diversity and phylogeny of NlpC/P60 genes in T. vaginalis and other trichomonads

A total of nine genes encoding NlpC/P60 homologues were initially annotated in the draft genome of Trichomonas vaginalis [18] and detailed sequence comparisons identified two distinct subfamilies, called clans A and B, with respective members named TvNlpC_A1-4 (four members) and TvNlpC_B1-5 (five members) (S1A Table), derived from likely two distinct lateral gene transfers from distinct Gram-positive bacteria donors [21, 26]. Published transcriptomics data [27, 28] indicated that at least one member of each family is expressed in different strains, with a total of seven genes with evidence of transcription (S1B Table). In addition, evidence for protein expression was provided from both total proteomics analyses of trophozoites and pseudocysts (TvNlpC_A2 and TvNlpC_B4) [29] and from phagolysosome-enriched fractions (TvNlpC_A2 and TvNlpC_A3) [30] (S1C Table). TvNlpC_A2 was also detected in the secretome of T. vaginalis [31].

In an attempt to further resolve the origins of the TvNlpC genes, we investigated the existence of homologues in the draft genomes of Trichomonas gallinae [32] and Trichomonas tenax [33] and searched for additional similar bacterial homologues to the genes from the Trichomonas lineage. A total of eight and six NlpC/P60 homologues were identified in the draft genome of T. gallinae and T. tenax, respectively, and four NlpC/P60 homologues were also identified in the genome annotation of the trichomonad Histomonas meleagridis [34] (S1C Table). Phylogenetic analyses focusing on the Trichomonas spp and Histomonas NlpC/P60 protein sequences and a selection of their most similar bacterial homologues established that all three Trichomonas species have members of the subfamilies NlpC_A and NlpC_B and that these were acquired by a shared common ancestor through two likely distinct events of lateral gene transfer. Despite the generally poorly resolved topology, characterized by some branches with low bootstrap values that are unstable upon variation in the evolutionary models and taxa sampling used for phylogenetic inferences (as per two examples in S1 Fig), the phylogeny with a broader sampling of Trichomonads sequences (Fig 1) is consistent with and support our initial conclusions based on the T. vaginalis NlpC/P60 sequences only [21]. The lateral acquisition of NlpC/P60 genes from bacteria is a feature of T. vaginalis that is shared with other mucosal-dwelling Trichomonads.

T. vaginalis NlpC/P60 genes participate on protozoa-lactobacilli interactions

The expansion of this gene family in T. vaginalis, and now revealed in other Trichomonads (Fig 1), suggests that these peptidoglycan hydrolases might be important for the interaction of these mucosal-dwelling protozoans with the local microbiota of the host. Here, we wanted to understand whether TvNlpC genes of clans A and B might share complementary activities on the interaction of T. vaginalis with host-protective vaginal lactobacilli. To test this, we incubated T. vaginalis alone or with vaginal Lactobacillus gasseri ATCC 9853 in a serum-free defined media (SFM). During the incubation period, samples were taken for microscopy, colony forming units (CFU) of lactobacilli and differential expression analysis of the nine TvNlpC genes (Fig 2).

Fig 2 — **(A)** Spotting single and mixed cultures of Tv and Lg on MRS-agar plates (selective for lactobacilli) shows that the population of Lg is reduced in numbers when in mixed culture with the protozoan. **(B)** The interaction of Tv and Lg results in co-aggregation. Parasites clump together in the presence of the bacteria (top left), but not in the absence of them (bottom left), after 8 hours of incubation. The diameter of twenty random cell clumps show that their sizes vary and tend to increase with time of incubation (right, *p-value < 0.01 from Games-Howell pairwise test). **(C)** RT-qPCR assays show the fold-change on the expression levels of TvNlpC/P60 genes across time, when comparing Tv in the presence versus absence of Lg. All TvNlpC/P60 genes of clan A and B were included in this analysis, except for TvNlpC_B1 which did not produce a significant C_T value. Results show that all genes with measurable expression are upregulated in T. *vaginalis* when in mixed culture with lactobacilli at 8 hours.

We noticed a reduction in the population of lactobacilli upon coincubation with T. vaginalis. This reduction in bacterial numbers was visibly different from the control (i.e. lactobacilli alone), particularly at 8 hours (Fig 2A). Notably, this reduction was accompanied by the formation of clumps of live parasites of varied sizes which increased with the time of incubation (Fig 2B). When comparing the expression of TvNlpC genes, despite some fluctuations, we found that all TvNlpC genes with detectable expression were upregulated when the protozoan was co-incubated with lactobacilli at the latest time point (Fig 2C). The maximum response was for TvNlpC_A3 with ~15-fold upregulation at 8 hours. The expression of TvNlpC_B1 was not detectable with a significant C_T value and thus excluded. Together, these findings demonstrate that vaginal L. gasseri induced aggregation of parasites which concomitantly upregulate the expression of TvNlpC genes leading to a reduction in bacterial population.

We have previously shown a gain of function phenotype when overexpressing an unregulated copy of TvNlpC_A1 and A2 in T. vaginalis, with parasites gaining the ability of controlling populations of the model bacterium Escherichia coli in mixed culture [21]. Here, we expanded this analysis to TvNlpC_B3 and B5, two members belonging to the other gene family clan (S1 Table and Fig 1). To do this, all four transfected T. vaginalis overexpressing one of the four HA-tagged TvNlpC genes (A1, A2, B3 or B5) were co-incubated with lactobacilli (Fig 3). T. vaginalis transfected with an empty vector (i.e. not expressing any protein of interest) were used as control.

Fig 3 — **(A)** Spotting mixed cultures of T. *vaginalis* and lactobacilli on MRS-agar plates reveals a greater reduction on bacterial numbers when the protozoan is expressing an unregulated, HA-tagged copy of TvNlpC_A1, TvNlpC_A2, TvNlpC_B3 or TvNlpC_B5 as compared to the empty vector. **(B)** Immunofluorescence microscopy and Western blot show that TvNlpC_B3 and _B5 are located in organelles. (Top panel) Immunofluorescence of T. *vaginalis* cells transfected with HA-tagged TvNlpC_B3 and _B5 and stained with FITC-conjugated antibody and DAPI. (Bottom panel) Cell fractionation of T. *vaginalis* transfected with HA-tagged TvNlpC_B3 and _B5 followed by Western blots, probed with antibodies against the HA-tagged protein (Anti-HA) or the organellar marker ferredoxin (Anti-Fd).

We observed that the unregulated expression of an additional copy of any of the TvNlpC genes in T. vaginalis enhanced the ability of the parasite to control the population of vaginal strain of L. gasseri in mixed cultures, with noticeable differences at 4 and 8 hours of incubation, as compared to the control (Fig 3A). We then undertook immunofluorescence, cell fractionation and Western blotting to localise the HA-tagged TvNlpC_B3 and _B5 in T. vaginalis cells (Fig 3B). TvNlpC_A1 and A2 were found on the surface and in the secretion of T. vaginalis [21, 31]. Here, TvNlpC_B3 and _B5 were detected as punctate spots in T. vaginalis cells by immunofluorescence. While staining for TvNlpC_B3 was uniformly dispersed in the cell, TvNlpC_B5 was found within larger areas by the nucleus (Fig 3B, top). This observation was consistent with the detection of both proteins mostly within the organellar fraction on Western blots (Fig 3B, bottom). Despite different cellular localizations, gain of function experiments indicate that TvNlpC genes of both clans A and B contribute to aiding the parasite to control populations of vaginal L. gasseri in mixed cultures.

T. vaginalis NlpC_B3 and NlpC_B5 share substrate specificity

To investigate the substrate specificity of TvNlpC_B3 and TvNlpC_B5, purified recombinant proteins, and their predicted catalytically inactive versions TvNlpC_B3 (C53S) and TvNlpC_B5 (C52S), were incubated with PG isolated from two different bacterial strains. The E. coli MC1061Δ6LDT strain lacks all known LD-transpeptidases resulting on a PG that is rich in tetrapeptides [35]. The E. coli CS703-1 strain lacks multiple Penicillin-binding proteins resulting on a pentapeptide-rich PG [36]. A control sample contained no protein. The samples were then incubated with the muramidase cellosyl and the resulting muropeptides were analysed by HPLC, as described previously [37].

As expected, neither of the catalytically inactive versions were able to digest PG (Fig 4). Both TvNlpC_B3 and TvNlpC_B5 with native catalytic residue were active against both types of PG, showing the substantial reduction of the main substrate peaks. Both enzymes digested the cross-linked TetraTetra dimer but not the Tetra monomer in the tetrapeptide-rich PG (Fig 4A), producing the disaccharide dipeptide (Di, peak 2 in Fig 4) and the disaccharide tetrapeptide linked to D-Ala-m-Dap from a second peptide (Tetra- D-Ala-m-Dap, peak 1 in Fig 4). Both enzymes were also active on pentapeptide-rich PG, cleaving the TetraPenta dimer to produce Di and the disaccharide pentapeptide linked to D-Ala-m-Dap from a second peptide (peak 3 in Fig 4). In addition, both enzymes also cleaved in, albeit with less efficiency, monomeric or dimeric pentapeptide stems (light grey arrows in Fig 4B). When cleaving in the Penta monomer, the Penta-D-Ala-m-Dap product was detected (peak 3, Fig 4B). All products of cleavage were confirmed by mass spectrometry analysis. The in vitro endopeptidase activities of the recombinant TvNlpC_B3 and TvNlpC_B5 were also verified against purified PG of L. gasseri ATCC 9853 (S2 Fig).

These results confirm that both enzymes cleave between D-iGlu (position 2) and m-Dap (position 3) of pentapeptides or the donor part of cross-linked peptides, i.e. the same cleavage site as for Tse1, an ’attacking’ PG hydrolase secreted by the type 6 secretion system of Pseudomonas aeruginosa [38, 39]. We have previously shown that TvNlpC_A1 and A2 were not capable of digesting pentapeptides in PG [21]. In conclusion, despite the redundant activities of TvNlpC_B3 and B5, they complement the activities of TvNlpC_A1 and A2.

E64 binds to TvNlpC_B3 catalytic site in vitro and limits its activity in mixed cultures

We have previously shown that the NlpC/P60 domains of TvNlpC_A1 and _A2 adopt the expected papain-like fold [40] and exhibit open and highly accessible active sites [21]. In addition, the N-termini form two bacterial SH3 domains with a shared arrangement relative to the NlpC/P60 domains [21]. The SH3 domain is a feature lacking among TvNlpC proteins of the clan B [21], thus determining the structure of a TvNlpC of this clan could give us additional insights in the function of this gene family. Moreover, no structures of NlpC/P60 domain proteins have been determined with their ligand to date. Thus, the mechanisms of how these proteins achieve their specificity is unknown. To help elucidate key residues in the active site environment, we undertook crystallisation experiments with and without the cysteine protease inhibitor E64, obtaining crystals that were then used for structural characterization of the TvNlpC_B3 protein.

The final model of TvNlpC_B3, refined to an R/Rfree of 0.158/0.170 (full data collection and refinement statistics in S2 Table), and contains the contiguous segment from Asn23 to Tyr137 (Fig 5). As expected, TvNlpC_B3 exhibits an α+β, papain-like fold, consisting of 4 α-helices and 5 β-sheets arranged in antiparallel fashion. This fold is highly conserved across all NlpC/P60 and CHAP domains [41]. The active site residues of TvNlpC_B3 form a catalytic triad, identified as Cys53, His99 and His111 using homology with family members [22] [41], (Fig 1). Helix2 contains the active site cysteine at the N-terminus of the helix, positioning it in close proximity with His99 and His111 (Fig 5). His111 is also responsible for the single unusual rotamer of our model and is supported by well-defined electron density. Similarly to other Cys-His-His peptidases, the positioning of His111 appears to orientate His99 for facilitating enzymatic activity (via hydrogen bonding between ND1 atom of His111 and the NE2 atom of His99) [42].

The active site groove runs from the N-terminus of Helix α2, between β-strands 2 and 3 and across the β-sheet ending at strand 4 (Fig 5). This groove occupies a full face of the domain, making it a dominant feature of the otherwise globular protein (S3 Fig). The active site cysteine is located centrally within the grove, with the imidazole groups of histidine residues on one side, and the hydroxyl of Ser54 on the other, altogether forming the groove’s floor. The serine side of the groove terminates on the R group of the highly conserved Arg71 (where the first SH3b domain of NlpC_A1 would interact with the NlpC/P60 domain) (Fig 5).

The structure of TvNlpC_B3 bound to the inhibitor E64 was determined by molecular replacement using the Apo enzyme structure, and refined to an R/Rfree of 0.183/0.198 (full data collection and refinement statistics in S2 Table). Examination of the electron density in the active site revealed a small extra fragment of density connected to the active site cysteine (Cys53) sulfur atom (S4 Fig). While this fragment is considerably smaller that the entire E64 molecule, as explained ahead, we believe it is a fragment of the E64 molecule. Mass spectrometry of the sample used for crystallisation revealed masses for two major species which correlate to TvNlpC_B3 bound to E64 (14,046 Da), and TvNlpC_B3 bound to a 131 kDa fragment (13,821 Da), with the latter being the major species (S4 Fig). The fragment of E64 present in our structure is well supported by density, and represents this 131 kDa fragment, suggesting that the breakdown of the E64 molecule and selective crystallization of this complex.

We modelled the E64 fragment covalently bound to the Cys53 via the C2 atom. The carboxyl group on C1 forms polar interactions with the carboxyl group of Tyr41, the ND1 atom of His99, and the main chain nitrogen of Cys53. The Tyr41 residue is conserved, and is suggested to have a role in regulating the nucleophilicity of Cys53, or in stabilising the substrate and/or tetrahedral intermediate during catalysis [43]. The carboxyl group from C3 of the E64 fragment forms no interactions with the protein and is exposed to solvent. The carboxyl from C4 forms a network of hydrogen bonds between sidechain of Ser54 and the mainchain nitrogen of Ala73. N1 is the last visible atom of the E64 fragment and co-ordinates a water molecule with the mainchain nitrogen of Lys74, this water would likely be displaced when considering the intact E64 molecule. No major differences in the protein were observed between the E64 bound, and apo TvNlpC_B3 structures RMSD of all atoms = 0.103) indicating a lack of conformational change on inhibitor binding.

The structure determination of TvNlpC_B3 with E64 (Fig 5) led us to evaluate whether this cell-permeable cysteine protease inhibitor might have a measurable effect on the ability of T. vaginalis to control populations of lactobacilli in mixed cultures. In addition, we wanted to see if the bactericidal activity of T. vaginalis observed in mixed cultures (Fig 2A) may apply to an important vaginal dysbiotic bacterium (i.e. G. vaginalis). To evaluate these hypotheses, co-incubation experiments of TvNlpC_B3-transfected T. vaginalis with vaginal L. gasseri or G. vaginalis were carried out in the absence or presence of E64 (Fig 6).

We confirmed that the unregulated expression of TvNlpC_B3 enhances the control of lactobacilli, but not of G. vaginalis (Fig 6A), by T. vaginalis in mixed cultures. E64 was seen to partially prevent the action of T. vaginalis on controllling lactobacilli populations. When quantifying these reductions on bacterial numbers by CFU (Fig 6B), we observed that T. vaginalis was up to ~8 times more effective on controlling lactobacilli when transfected with TvNlpC_B3 than with the empty vector. Although E64 did not completely block this action by the parasite, it inhibited the enhanced activity of TvNlpC_B3-transfected T. vaginalis against lactobacilli by up to ~9-fold at the latest time-point (Fig 6B). Therefore, in agreement with the ligand-protein structure, this cysteine protease inhibitor significantly reduces the ability of the protozoan on controlling populations of L. gasseri in mixed cultures.

Discussion

In this study, we reported that the two-clan configuration of laterally acquired NlpC/P60-containing peptidases (i.e. NlpC_A and NlpC_B subfamilies) is also present in trichomonads other than T. vaginalis. The conservation of both subfamilies, with the preservation of the catalytic triad Cys-His-His, in the phylogeny of trichomonads with genomes available highlights two events of lateral gene transfer in a common ancestor that likely took place in a pigeon-infecting species [20, 44]. This was followed by gene duplications and amelioration, leading to optimized enzymes that may target various bacteria in the different context of hosts (i.e. pigeons or mammals) and mucosal surfaces (i.e. oral cavity and upper digestive tract or the urogenital tract). The structural configurations identified for the TvNlpC were also found among the sequences from T. gallinae and T. tenax with or without SH3b domains. In addition, the H. meleagridis was found to encode four homologues most similar to the Clan B enzymes, despite weak support values. H. meleagridis is an important bird pathogen that cannot be grown axenically, but requires Gram-negative bacteria to grow in vitro [34]. While this study showed that TvNlpCs might help T. vaginalis to compete out lactobacilli, in the case of H. meleagridis, these enzymes may be even more critical by aiding the parasite to feed on bacteria for some essential metabolites. Studies will be needed to assess the function and the importance of NlpC/P60-containing peptidases among these trichomonads.

For T. vaginalis, we have previously shown that two NlpC/P60 enzymes of clan A (TvNplC_A1 and _A2) are functional DL-endopeptidases that cleave bacterial peptidoglycan (PG) with similar structure and redundant activity [21]. On the other hand, TvNlpCs of clan B lack SH3 domains. We depicted here the papain-like structure of the TvNlpC_B3 with a fragmented E64 molecule. To our knowledge, this is the first NlpC/P60-containing peptidase to be co-crystalized with a ligand shedding light in the structure-function of these enzymes. TvNlpC_B3 and _B5 were shown to digest tetra and pentapeptides in PG. TvNplC_A1 and _A2 were limited to digest tetrapeptides only, and thus possibly optimized to digest the mature form of PG on bacterial cell walls [21]. Hence, this study shows that the PG hydrolytic activities between TvNlpCs of clans A and B complement each other. More studies will be necessary for further understanding the specificity and complementary activities of all nine TvNlpC enzymes.

We now know as well that at least eight out of the nine TvNlpC genes are indeed expressed in T. vaginalis cells, complementing expression evidence that was initially based on transcriptomics and proteomics data generated from T. vaginalis in the absence of any bacteria [27–31]. Importantly, the expression of these genes respond positively to the presence of lactobacilli (with up to 15-fold upregulation) helping explain why previous omics studies may have missed on detecting the expression of some TvNlpC genes. For instance, TvNlpC_B3 and _B5 were never detected in previous proteomics studies [29–31]. The positive response of TvNlpC gene expression to lactobacilli indicates that T. vaginalis should recognize environmental cues, unknown at this stage, that might be related to this vaginal bacterium.

TvNlpC_B3 and _B5 were found to be located in organelles, whereas both TvNplC_A1 and _A2, were shown on the surface and to be secreted by T. vaginalis [21]. TvNlpC_B3 and _B5 were not found in the proteome of lysosome-enriched fractions [30], but lack of their detection in these organelles may be explained by not having bacteria triggering upregulation of TvNplC gene expression. For example, despite remarking similarity between the secreted TvNplC_A1 and _A2 [21], only the latter was detected in the T. vaginalis secretome [31]. Further studies will be needed for determining the final organellar destination of TvNlpC_B3 and _B5, as well as the cellular localization of other TvNlpCs.

T. vaginalis is known for being both secretory and phagocytic [45]. Phagocytosis by T. vaginalis has been reported elsewhere [46–49], involving the transitioning to the amoeboid morphology of this protozoan and other dramatic changes at ultrastructural cellular level [48, 49]. T. vaginalis has been shown to phagocyte host cells, yeast and bacteria to a different extent [46–49] and this broad phagocytic capacity was found to the correlate with the virulence of the strains [48, 49]. Limited evidence suggests that phagocytosis by T. vaginalis may contribute to pathogenesis, the vaginal biome changes and nutrition acquisition by the parasite. We envisage that TvNlpCs may be employed by this protozoan parasite to attack vaginal bacteria extracellularly or intracellularly following phagocytosis. However, more investigation is needed to understand how these extra- and intracellular TvNlpCs exactly reach the PG in the bacterial cell wall and the possible involvement of phagocytosis in this process.

We found that the endogenous upregulation of TvNlpC gene expression by lactobacilli is accompanied by a reduction of these bacteria in mixed cultures and that the overexpression of these enzymes in transfected cells enhances this phenotype. On the other hand, the unregulated expression of TvNlpC_B3 in T. vaginalis did not lead to reducing G. vaginalis population in mixed cultures. In alignment to the co-crystal structure of TvNlpC_B3 with E64, we showed that this cysteine-protease inhibitor supresses the action of T. vaginalis on lactobacilli significantly. This observation is in agreement with E64 reducing the cytotoxicity of T. vaginalis in response to the secretion of cysteine-proteases [45]. This inhibitor, however, does not completely block the bacteriolytic action of T. vaginalis suggesting that additional factors other than TvNlpCs (e.g. amidases and antimicrobial peptides) might also contribute to the efficacy of this parasite controlling bacterial populations. Further studies are needed to understand the complementary activities of all nine NlpC/P60 enzymes of T. vaginalis and their preference for targeting vaginal bacteria with specificity.

Because PG is the major structural component of bacterial cell walls and essential for maintaining the integrity of bacterial cells, it is not surprising to find that employing PG-degrading enzymes (i.e. amidases and peptidases) for controlling bacterial population may be a widely spread strategy that has co-evolved with hosts across the tree of life and in concert with their autochthonous microorganisms. This strategy is often employed by bacteriophages [50], has been reported in predatory bacteria [51] and between non-predatory bacteria, with the latter delivering these enzymes via type IV and VI secretion systems [38, 52]. More recently, Photobacterium damselae subsp. piscicida (a pathogenic bacterium of marine fish) was shown to secrete PnpA, a NlpC/P60 with notable specificity [53]. PnpA was shown to cleave PG of competing bacteria, giving a competitive advantage to the pathogen. Differently to our work, instead of using purified PG, the authors employed insoluble sacculi which might help preserve the structural features of the cell wall required for targeting bacteria with specificity. PnpA is secreted and, similarly to TvNlpC_A1 and TvNlpC_A2 [21], it remains to be described how these enzymes reach the PG of their bacterial targets.

PG-degrading enzymes have been claimed as potential antibacterial genes that were laterally acquired by eukaryotes from bacteria [54]. These acquisitions have enhanced innate immune function, as described in the deer tick Ixodes scapularis that employs an amidase to limit the proliferation of the bacterial agent of the Lyme disease Borrelia burgdorferi [24]. In protozoan parasites, TvNplCs are the only examples of functional PG hydrolases described to date, and our study is showing that this might be a feature on the evolution of other Trichomonads infecting different mucosal sites and host species. Such investigations will help understand the complexity, dynamics and evolution of host-parasite-microbiome interactions. In perspective, further studies will help clarify how trichomoniasis is accompanied by vaginal dysbiosis which knowledge might contribute to the development of antimicrobial therapies that concomitantly reduce co-morbidities associated with this infection and the dysbiotic microbiome.

Materials and methods

Identification of NlpC/P60 genes in the draft genomes of Trichomonas spp and sequence comparisons

Local tblastn searches against the draft genome sequences of T. tenax (Strain NIH4 ATCC 30207, GenBank assembly accession: GCA_900231805.1) and T. gallinae (isolate XT1081-07, GenBank assembly accession: GCA_008369845.1) were performed with T. vaginalis NlpC/P60 proteins as query, identifying 6 and 8 NlpC/P60 ORFs for the respective species. ORFinder (https://www.ncbi.nlm.nih.gov/orffinder/) was used to identify full-length coding sequences of each gene candidate. BlastP searches at the NCBI were performed to identify close homologues among bacteria. These searches also identified hits derived from proteins annotated in the recently published genomes of Histomonas meleagridis [34]. The four proteins sequence from only one of two strains of Histomonas meleagridis were considered. Proteins aligned to homologues from T. vaginalis and selected proteins from bacteria using SEAVIEW [55] further supported the identification of full ORFs in T. gallinae and T. tenax. Slightly editing the original alignment (104 residues) [21] led to 103 well-aligned residues, i.e. matching hypothesis of site homology across the selected bacteria and Trichomonads’ sequences (S1 Data). Maximum likelihood phylogenetic inferences were performed with IQ-TREE and considered several evolutionary models, both homogenous models and protein mixture models [56, 57]. Protein domain organisation was analysed using InterProScan (https://www.ebi.ac.uk/interpro/search/sequence/) [58].

Single and mixed cultures of protozoan and vaginal bacteria

Trichomonas vaginalis genome strain G3 was cultured in TYM medium [59] supplemented with 10% heat-inactivated horse serum, 10 U/ml penicillin, and 10 μg/ml streptomycin (Invitrogen). Lactobacillus gasseri ATCC 9857, a strain of human vaginal origin, was grown in MRS (deMan, Rogosa, and Sharpe) medium without serum. Gardnerella vaginalis ATCC 14018 was grown in 1685 NYC III medium supplemented with 5% of glucose and 10% heat-inactivated horse serum. All three microorganisms were grown at 37°C without agitation in microaerophilic condition, i.e. in standard 15 ml Falcon tubes (12 cm high) filled up with 15.5 ml of media to reduce oxygen exchange. For the coincubation assays, the following procedures were taken. The number and viability of T. vaginalis cells, grown from frozen stocks in the absence of antibiotics for at least three days and with daily passages, were assessed under a hemocytometer. The number and viability of bacterial cells were assessed by flow cytometry as previously described [13]. Microbial media was removed by centrifugation and cells (>95% viability) were washed and resuspended in antibiotic- and serum-free keratinocyte media (K-SFM; Invitrogen). T. vaginalis (2–5 × 10⁵ cells/ml) was mixed with bacteria in a 1:3 ratio in 1 ml volume and in a 24-well tissue culture plate and incubated at 37 °C under anaerobiosis (Oxoid AnaeroGen Compact, Thermo Scientific). As controls, bacteria and T. vaginalis were treated and incubated under the same conditions but in the absence of each other. Cultures were mixed by pipetting, before collecting samples for plating on agar. For the time points indicated, images of the microbes settled on the plastic surface of the wells were taken under the inverted microscope (EC3 Leica camera) at 200X magnification. Twenty random clumps of T. vaginalis were measured from their longest diameter distance. Additionally, for every time point, 5 μl of undiluted cultures were spotted and 50 μl of cultures diluted in sterile water were spread on MRS-agar or NYC-agar plates (which procedure is selective for lactobacilli or G. vaginalis respectively) and grown at 37°C under atmospheric air for L. gasseri or under anaerobiosis for G. vaginalis (Oxoid AnaeroGen Compact, Thermo Scientific) for 24-48h. The spot plates were photographed, and CFU counts were obtained from the spread plates indicated, 0.1 mM E-64 was added to the mixed cultures at time zero from a 10 mM stock prepared in sterile water. Control wells without E-64 received the same volume of sterile water, instead. The introduction of an episomal copy of a HA-tagged TvNlpC_B3 and _B5 (TVAG_042760 and TVAG_411960, respectively) or an empty plasmid in T. vaginalis was achieved by transfection, as previously described [21]. Transfected T. vaginalis were used in the experiments above, where indicated. These co-incubation experiments were run three times independently, with two spread plating replicas for each experiment. Data was quantified across all experiments and results were expressed as a percentage of CFU reduction in comparison to the controls (i.e., in the absence of T. vaginalis).

Gene expression analysis

For gene expression analysis, mixed cultures of protozoa:lactobacilli and single cultures of T. vaginalis were scaled up to 15 ml of K-SFM in one tissue culture dish for each time point. Total RNA was obtained from the mixed and single cultures at each time point by TRIzol (Invitrogen) and subsequently purified by removal of DNA (DNase I, Ambion) and clean-up with RNeasy MinElute (Qiagen). We utilized the same gene nomenclature as previously published [21], i.e. TvNlpC_A1 (TVAG_119910), TvNlpC_A2 (TVAG_457240), TvNlpC_A3 (TVAG_324990) TvNlpC_A4 (TVAG_252970) for TvNlpC/P60 genes of clan A, and TvNlpC_B1 (TVAG_393610), TvNlpC_B2 (TVAG_051010), TvNlpC_B3 (TVAG_042760), TvNlpC_B4 (TVAG_209010) and TvNlpC_B5 (TVAG_411960) for TvNlpC/P60 genes of clan B. Reverse transcription and quantitative real-time PCR (RT-qPCR) were conducted using SuperScript IV Reverse Transcriptase and PowerUP SYBR green Master Mix (Thermo Fisher) in a 7900 HT-Realtime thermocycler (Applied Biosystems). We followed the PCR protocols as exactly detailed in our previous publication [21], with TvNlpC-specific primers listed in here (S3 Table), employing the standard threshold cycle (C_T) method for relative expression analysis after data normalization and using recommended software (SDS 2.3 and RQ Manager 1.2 applications, Applied Biosystems). The expression levels of the genes above were compared between mixed cultures versus single cultures from triplicate experiments.

Subcellular localization of TvNlpC_B3 and _B5

Transfected T. vaginalis cells expressing the HA-tagged TvNlpC_B3 and _B5 were used to assess the subcellular localization of these proteins by two complementary assays. We employed indirect immunofluorescence assay (IFA) with the mouse anti-HA antibody and the FITC-conjugated secondary antibody and cell fractionation followed by Western blot, as described previously [21]. For the Western blots, protein from cell fractions (organelle, cytosol and membrane) and total cell were brought up to equivalent volumes. To help reinforce the lack of detection in any given fraction, loading volumes varied. When probing for the HA-tagged TvNlpC_B3 and _B5 with the anti-HA, we loaded 5X more volume of protein from cell fractions than total cell. When probing for ferredoxin (anti-Fd), a known organellar marker for T. vaginalis, this ratio was inverted. Other than this modification, protocols for IFA, cell fractionation and Western blot were followed as exactly detailed in our previous publication [21].

Expression and purification of TvNlpC_B3 and _B5

Coding sequences for TvNlpC_B3 and _B5 were truncated at the N-terminus by 15 and 16 amino acids respectively, based on the identification of predicted signal sequences using software packages (Phobius and PrediSi) [60, 61]. Inactive enzyme mutants were generated for TvNlpC_B3 and TvNlpC_B5 by targeting the active site cysteine (C53S and C52S, respectively) identified via sequence homology with other family members. Sequences were cloned into pET47b expression plasmid with an N-terminal hexa His-tag and 3C-protease cleavage site (detailed in S2 Data), with proteins expressed in Escherichia coli strain BL21(DE3). Bacterial cells were grown in LB to an OD₆₀₀ of 0.6–0.8, and expression induced at 18°C by the addition of 1 mM IPTG. Cells were harvested by centrifugation and resuspended in 20 mM TRIS/HCl pH 7.8, 300 mM NaCl, 0.5 mM TCEP, 10% v/v glycerol. Proteins were purified by immobilised metal affinity chromatography and eluted in 10 mM TRIS/HCl, pH 8.0, 300 mM NaCl, 300 mM Imidazole. To remove the N-terminal tag, pooled protein was incubated with 3C protease and dialysed into 10 mM TRIS/HCl pH 8.0, 150 mM NaCl, 0.1 mM TCEP overnight. Final purification was carried out by size exclusion chromatography using a Superdex 75 16/60 column. Fractions containing pure protein were pooled and concentrated using 3,000 molecular weight cut-off spin-concentrator and stored at -20°C until required.

Enzymatic assay for TvNlpC_B3 and _B5

PG from Escherichia coli strains MC1061Δ6LDT (tetrapeptide-rich PG) or CS703-1 (pentapeptide-rich PG) was incubated with different TvNlpC proteins (10 μM) in 20 mM Tris/HCl pH 7.5, 150 mM NaCl for 4 h at 37 ⁰C in a thermomixer set at 800 rpm. A control sample contained no protein. The reaction was stopped by heating the samples at 100 ⁰C for 10 min. To release the muropeptides from the PG, the pH was adjusted to 4.8 and muramidase cellosyl (Hoechst, Frankfurt am Main, Germany) was added to a final concentration of 50 μg/ml prior to incubation overnight at 37⁰C in a thermomixer set at 800 rpm. The reaction was stopped by heating the samples at 100⁰C for 10 min on a dry heat block and the sample was centrifuged at 13,000 ×g for 10 min. The supernatant was supplemented with an equal volume of 0.5 M sodium borate pH 9.0 and ~1 mg sodium borohydride powder, followed by an incubation of 30 min at ambient temperature to reduce the muropeptides. The samples were then acidified to pH 4.0–4.5 using 20% phosphoric acid. The resulting reduced muropeptides were separated by HPLC using a 250 × 4.6 mm 3 μm Prontosil 120-3-C18 AQ reversed-phase column (Bischoff, Leonberg, Germany) as described [37]. The eluted muropeptides were detected by their absorbance at 205 nm. Products of TvNlpC versions were collected and analysed by mass spectrometry as described [62, 63]. The measured neutral masses were: Di, 698.053 and 698.067 (theoretical: 698.286); Tetra-D-Ala-m-Dap, 1184.309 (theoretical: 1184.530); Penta-D-Ala-m-Dap, 1255.353 (theoretical: 1255.567). The PG of L. gasseri ATCC 9847 was purified as previously described for other Gram-positive bacteria [64], and subjected to the same treatments described above.

Crystallisation and structure determination of TvNlpC_B3

Crystallisation of the purified recombinant TvNlpC_B3 was undertaken by sitting-drop vapour diffusion using the JSCG+, PACT Premier, and MORPHEUS screens (Molecular Dimensions) in Intelliplate HT 96 well plates with a 50 μL reservoir. Crystallisation drops consisting of a 1:1 and 2:1 protein:reservoir ratio in a total volume of 300 nL were dispensed using an Oryx automated robotic crystallisation system (Douglas Instruments). All crystallisation experiments took place at 18°C. TvNlpC_B3 crystals were grown in JSCG+ condition E4; 1.26 M Ammonium sulfate, 0.1 M TRIS, pH 8.5. The cysteine protease inhibitors E64 was added to the purified TvNlpC_B3 in at least 5:1 molar ratio, and left for an hour to incubate at room temperature. The resulting mixture was then screened on the JCSG+ 96HT tray for crystal growth. Crystals grew in the same conditions as the apo crystals and were cryo-protected with the addition of 20% w/v glycerol, before flash-frozen and stored under liquid nitrogen to be shipped to the Australian Synchrotron (Melbourne, Victoria, Australia) for data collection using the MX1 and MX2 beamlines. Data were indexed and integrated using either MosFlm software suite [65] or XDS software package. Integrated reflections were used as input to the AIMLESS pipeline of the CCP4 software suite [66, 67]. A 5% Free-R reflection set was maintained for TvNlpC_B3 models with and without E64.

Crystallographic phases for TvNlpC_B3 were estimated by molecular replacement with PHASER [68]. A truncated NlpC/P60 domain of TvNlpC_A1 (PDBid: 6BIO) [21] was used as a search model. Molecular replacements were chosen by a TFZ value over 8.0, and RFZ over 4.0. Successful molecular replacement was visualised in COOT software [69], and the electron density maps scrutinised for interpretable electron density with logical differences between the residues of the search model, and the expected residues of the crystallised protein. Structural models were refined with iterative cycles of refinement in REFMAC5 [70] from the CCP4 software suite. After REFMAC refinement, the model and associated density, as well as Fo-Fc density maps were visualised for real-space refinement and manipulation in COOT software [69]. Refinement cycles were monitored with crystallographic R/Rfree factors, and RMS bond angle and bond length. Due to the ratio of unique observations to atom parameters in the final model (observ/param = 2.47) and high resolution of the dataset, full anisotropic B factors were included in the final refinement. The final model was validated using the Molprobity web server (http://molprobity.biochem.duke.edu/), and the PDB submission validation web server (https://validate-rcsb-1.wwpdb.org/).

Supporting information

S1 Fig. A comparison of phylogenetic relationship between bacterial and Trichomonads NlpC/P60 protein sequences from two distinct evolutionary models.

Different models and taxa sampling were used to investigate the phylogenetic relationships between the identified NlpC/P60 protein sequences from the three species of Trichomonas: T. vaginalis, T. tenax and T. gallinae. Sequences from the fourth Trichomonad Histomonas meleagridis were also included in addition to close bacterial homologues to the Trichomonads homologues. Maximum likelihood based phylogenetic inferences used: (i) the best fitting identified homogenous model for the protein alignment using the automatic model selection function as implemented in iq-tree (see Material and Methods sections for mode details) (model LG+G+I, shown on the right and that also corresponds to the phylogeny shown in Fig 1) or (ii) protein mixture models, one such phylogeny is shown on the left (LG4X+F) that allow different amino acid composition and evolutionary rates across the alignment. The protein mixture models were used, as they are considered to be more reliable in extracting phylogenetic signal from divergent sequences. The branches with the lower support values (from 1000 ultrafast bootstraps) are also those corresponding to sections of the phylogenies that are sensitive to the model used. These differences between these two phylogenies highlight the lack of phylogenetic signal in that short alignment of only 103 residues.

(TIF)

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S2 Fig. Endopeptidase activity of TvNlpC_B3 and TvNlpC_B5 against purified peptidoglycan of L. gasseri.

The catalytic-active and -inactive versions of TvNlpC_B3 (A) and TvNlpC_B5 (B), as indicated, were incubated with PG from L. gasseri. Muropeptides were released by cellosyl, reduced with sodium borohydride and separated by HPLC. Cleavage products (red arrows), that are absent from the -inactive enzyme controls, indicate activity of these enzymes against the PG of L. gasseri.

(TIF)

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S3 Fig. A comparison of the active site groove between TvNlpC_A1 and TvNlpC_B3.

This comparison shows that TvNlpC_B3 exhibits a more open and accessible groove shape than TvNlpC_A1, including an absence of the nearby extended loop structure between strands S1 and S2 (highlighted blue). A nearby SH3 domain (highlighted pink) modulates the groove in TvNlpC_A1.

(TIF)

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S4 Fig. Electron density of the TvNlpC_B3 active site before and after refinement with fragment of E64.

For reference, 2Fo-Fc is shown at 1.0 sigma blue, positive and negative Fo-Fc at 3.0 sigma are shown in green and red respectively. (A) Apo TvNlpC_B3, (B) TvNlpC_B3 with E64 prior to and after building of E64 fragment, and (C) final model of TvNlpC_B3 with E64 fragment modelled. (D) Mass spectroscopy analysis of TvNlpC_B3 incubated with E64. Three major peaks were observed corresponding to TvNlpC_B3 (peak 1), TvNlpC_B3 and the E64 fragment (peak 2) and TvNlpC_B3 and E64 (peak 3). (E) The fragment identified in both the crystallography and mass spectroscopy analysis corresponds to a 131Da fragment of E64.

(TIF)

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S1 Table. Features of NlpC/P60 genes and proteins of Trichomonads with draft genomes available.

(A) Features of the nine TvNlpC genes and proteins of clans A (A1 to A4 in blue) and B (B1 to B5 in red); (B) Evidence for the expression of TvNlpCs at transcript and protein level as previously reported; (C) NlpC/P60 genes identified in Trichomonads: Trichomonas gallinae, Trichomonas tenax and Histomonas meleagridis.

(XLSX)

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S2 Table. Data collection and refinement statistics for the crystal structure of TvNlpC_B3 with and without the inhibitor E64.

(DOCX)

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S3 Table. List of forward and reverse primers for the nine TvNlpC genes, which were used in quantitative real-time PCR.

(XLSX)

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S1 Data. Data supporting site homology hypothesis across the selected bacteria and Trichomonads’ sequences used for phylogeny.

(FST)

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S2 Data. TvNlpC_B3 and TvNlpS_B5 amino acid sequences detailing the N-terminal signal truncations used for E. coli cloning, expression and purification of these recombinant proteins.

(DOCX)

Click here for additional data file.^{(14KB, docx)}

Data Availability

All relevant data are within the manuscript and Supporting Information files.

Funding Statement

This work was supported by the Maurice & Phyllis Paykel Trust and the Faculty Research Development Fund from the University of Auckland (AS-B). WV was supported by the BBSRC (BB/W013630/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1011563.r001

Decision Letter 0

Patricia J Johnson, Dominique Soldati-Favre

14 Feb 2023

Dear Dr. Simoes-Barbosa,

Thank you very much for submitting your manuscript "NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The additional data should be included in the revision: (1) include data showing that the NlpC/P60 hydrolases produced by T. vaginalis break down the peptidoglycan of Lactobacillus, (2) compare the killing effect of T. vaginalis on several bacteria, not just lactobacilli, to rule out a generalized bactericidal activity of the parasite, (3) data addressing whether phagocytosis is responsible for lactobacillus killing and (4) report T. vaginalis viability at the endpoint of experiments where the parasite is incubated at high concentrations for long periods.

All minor issues raised by the reviewers should also be addressed.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Patricia J. Johnson, PhD

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

All minor issues raised by the reviewers should also be addressed.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors describe the role of dipeptidoglycan/hydrolases produced by the protozoan Trichomonas vaginalis, and hypothesize the role of these enzymes in vaginal dysbiosis, specifically describing the effect of these enzymes on lactobacilli. In addition, they describe the active site of the enzymes.

The paper is clear, well written, all experimental procedures are clearly described, and the conclusions are very interesting.

I have only to report some minor points, which might improve the understanding of the paper for a reader unfamiliar with the mechanisms of pathogenicity of T.vaginalis.

Reviewer #2: Previous work has demonstrated that the vaginal microbiome is altered and dysbiotic in patients infected with the protozoan T. vaginalis, but those studies have not yet shown the mechanism underlying this connection. Here, the authors suggest a potentially causal relationship between T. vaginalis infection and the change in vaginal microbiome composition. Building upon their own previous studies, the authors show that T. vaginalis has the capability to directly antagonize bacteria through production of bacterial peptidoglycan-targeting NlpC/P60 hydrolases. Furthermore, they show that this organism, as well as several evolutionarily related organisms, produce two separate classes of NlpC/P60 hydrolases that break down peptidoglycan in different ways. The authors show a) that expression of T. vaginalis NlpC/P60 hydrolases is upregulated in the presence of lactobacilli and b) that survival of lactobacilli is decreased by co-culture with T. vaginalis (and this bacterial inhibition is enhanced when the T. vaginalis overexpresses NlpC/P60 hydrolases). Together, these results suggest that T. vaginalis may exert influence on the vaginal microbiome by producing enzymes that create an unfavorable environment for lactobacilli. I am pleased recommend publication after the authors address some key issues listed below.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: No Major Issues

Reviewer #2: The authors do not provide, or speculate about, a mechanism through which T. vaginalis inhibits lactobacilli specifically. One might guess that the NlpC/P60 hydrolases produced by T. vaginalis directly break down the peptidoglycan of Lactobacillus species; however, the authors do not show this. Instead, the authors show that the T. vaginalis NlpC/P60 hydrolases can break down the peptidoglycan of two genetically engineered strains of E. coli. The work could be greatly improved by performing the experiments shown in Figure 4 with peptidoglycan isolated from Lactobacillus (and ideally other known bacterial members of the vaginal microbiome) rather than just that of E. coli.

The authors show that lactobacilli are killed in the presence of T. vaginalis, but do not show the effects of T. vaginalis on any other bacteria. Without these key comparisons, the results in Figure 2A could be due to a general bactericidal activity of T. vaginalis.

While gain of function phenotype was demonstrated by overexpressing one of the four TvNlpC_A1, A2, B3 and B5, the loss of function was not demonstrated. Would be good if the authors could generate and evaluate TvNlpC deletion strains, if possible.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Results:

The experiments described demonstrate a reduction of lactobacilli in the supernatant. Is it possible that the number of bacteria is reduced as a result of phagocytosis by protozoa? This possibility needs to be discussed, along with the possibility of phagocytosis activation under the experimental conditions. Do the authors have data that can describe whether phagocytosis-related genes are upregulated under the experimental conditions described?

Lane 206-277. This section is very long. Is it possible to reduce the number of words?

Discussion:

The role of phagocytosis of lactobacilli in reducing the number of these bacteria needs to be better discussed

Material and Methods:

Lane 383. The authors state that the culture conditions are microaerophilic. They should report at least the height of the tubes, to demonstrate the distance of the cultured cells from the interface of air and oxygen

Lane 389: The number of cells is very high: the concentration of 2-5 x 106, especially for long incubation periods, is not physiological for T.vaginalis. The authors should report cell viability under these conditions

396. Is the sample collected after shaking the cultures? If the cultures are not shaken, the bacteria tend to sediment.

Reviewer #2: Figure 2B. Quantification of cell clumps size does not provide much of additional value as there is no statistical comparison and there is a variability in sizes.

Figure 3B. Scale bars are missing.

Line 102: Authors claim that crystal structure was obtained with bound ‘biological ligand’. E64 inhibitor is broad spectrum cysteine peptidase inhibitor (papain, cathepsin B, cathepsin L, calpain and staphopain) isolated from Aspergillus japonicus. E64 is not a natural ligand for TvNlpCs.

Figure 3. WB. Anti-Fd. More details are required to describe what is Anti-Fd (from material and method section it is ferredoxin).

Line 182. TvNlpC_B3 and B5 could act on E.coli PG. Why not Lactobacillus PG? If TvNlpC_B3 and B5 can cleave Lactobacillus PG, it will provide insights on mechanism of controlling Lactobacillus population.

Figure 4. Typos for labels: B3 and B3 (C53S) mislabeled as B5 and B(C52S).

Line 233. Typo: ‘S2 Fig’ instead of ’Fig. S2’

Line 249. Though LC-MS data of TvNlpC_B3 with bound E64 inhibitor was mentioned, no data was demonstrated to confirm covalent biding of E64 to TvNlpC_B3. Moreover, a good negative control would be LC-MS data of TvNlpC_B3(C53S) + E64.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Pier Luigi Fiori

Reviewer #2: No

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PLoS Pathog. 2023 Aug 16;19(8):e1011563. doi: 10.1371/journal.ppat.1011563.r002

Author response to Decision Letter 0

29 Jun 2023

Attachment

Submitted filename: Response to editor and reviewers.docx

Click here for additional data file.^{(24.8KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1011563.r003

Decision Letter 1

Patricia J Johnson, Dominique Soldati-Favre

18 Jul 2023

Dear Dr. Simoes-Barbosa,

We are pleased to inform you that your manuscript 'NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Patricia J. Johnson, PhD

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The new version of the manuscript is definitely improved. The authors have responded to criticism

Reviewer #2: (No Response)

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: The manuscript may be published in the present form

Reviewer #2: While the authors have not addressed all the Reviewers' critiques experimentally, I appreciate the new data, revisions and discussion that are not included. I am pleased to recommend acceptance of the manuscript.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The manuscript may be published in the present form

Reviewer #2: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Pier Luigi Fiori

Reviewer #2: No

PLoS Pathog. doi: 10.1371/journal.ppat.1011563.r004

Acceptance letter

Patricia J Johnson, Dominique Soldati-Favre

8 Aug 2023

Dear Dr. Simoes-Barbosa,

We are delighted to inform you that your manuscript, "NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. A comparison of phylogenetic relationship between bacterial and Trichomonads NlpC/P60 protein sequences from two distinct evolutionary models.

(TIF)

Click here for additional data file.^{(2.7MB, tif)}

S2 Fig. Endopeptidase activity of TvNlpC_B3 and TvNlpC_B5 against purified peptidoglycan of L. gasseri.

(TIF)

Click here for additional data file.^{(389.6KB, tif)}

S3 Fig. A comparison of the active site groove between TvNlpC_A1 and TvNlpC_B3.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S4 Fig. Electron density of the TvNlpC_B3 active site before and after refinement with fragment of E64.

(TIF)

Click here for additional data file.^{(2.3MB, tif)}

S1 Table. Features of NlpC/P60 genes and proteins of Trichomonads with draft genomes available.

(XLSX)

Click here for additional data file.^{(21.9KB, xlsx)}

S2 Table. Data collection and refinement statistics for the crystal structure of TvNlpC_B3 with and without the inhibitor E64.

(DOCX)

Click here for additional data file.^{(17.3KB, docx)}

S3 Table. List of forward and reverse primers for the nine TvNlpC genes, which were used in quantitative real-time PCR.

(XLSX)

Click here for additional data file.^{(12.6KB, xlsx)}

S1 Data. Data supporting site homology hypothesis across the selected bacteria and Trichomonads’ sequences used for phylogeny.

(FST)

Click here for additional data file.^{(6.3KB, fst)}

S2 Data. TvNlpC_B3 and TvNlpS_B5 amino acid sequences detailing the N-terminal signal truncations used for E. coli cloning, expression and purification of these recombinant proteins.

(DOCX)

Click here for additional data file.^{(14KB, docx)}

Attachment

Submitted filename: Response to editor and reviewers.docx

Click here for additional data file.^{(24.8KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and Supporting Information files.

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PERMALINK

NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli

Michael J Barnett

Jully Pinheiro

Jeremy R Keown

Jacob Biboy

Joe Gray

Ioana-Wilhelmina Lucinescu

Waldemar Vollmer

Robert P Hirt

Augusto Simoes-Barbosa

David C Goldstone

Roles

Abstract

Author summary

Introduction

Results

The diversity and phylogeny of NlpC/P60 genes in T. vaginalis and other trichomonads

Fig 1. Phylogenetic relationship of NlpC/P60 proteins from Trichomonas, Histomonas and a selection of their close bacterial homologues.

T. vaginalis NlpC/P60 genes participate on protozoa-lactobacilli interactions

Fig 2. The interaction of Trichomonas vaginalis (Tv) and Lactobacillus gasseri (Lg) results on the reduction of bacterial population, parasite aggregation and upregulation on the expression of TvNlpC/P60 genes.

Fig 3. Gain of function phenotype and cytolocalization of TvNlpC genes in T. vaginalis.

T. vaginalis NlpC_B3 and NlpC_B5 share substrate specificity

Fig 4. Enzymatic activity of TvNlpC_B3 and TvNlpC_B5.

E64 binds to TvNlpC_B3 catalytic site in vitro and limits its activity in mixed cultures

Fig 5. The structure of TvNlpC_B3.

Fig 6. The activity of TvNlpC_B3-transfected T. vaginalis against vaginal bacteria from mixed cultures.

Discussion

Materials and methods

Identification of NlpC/P60 genes in the draft genomes of Trichomonas spp and sequence comparisons

Single and mixed cultures of protozoan and vaginal bacteria

Gene expression analysis

Subcellular localization of TvNlpC_B3 and _B5

Expression and purification of TvNlpC_B3 and _B5

Enzymatic assay for TvNlpC_B3 and _B5

Crystallisation and structure determination of TvNlpC_B3

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Patricia J Johnson

Dominique Soldati-Favre

Roles

Author response to Decision Letter 0

Decision Letter 1

Patricia J Johnson

Dominique Soldati-Favre

Roles

Acceptance letter

Patricia J Johnson

Dominique Soldati-Favre

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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