Pyridoxal phosphate synthases PdxS/PdxT are required for Actinobacillus pleuropneumoniae viability, stress tolerance and virulence

Fang Xie; Gang Li; Yalei Wang; Yanhe Zhang; Long Zhou; Chengcheng Wang; Shuanghong Liu; Siguo Liu; Chunlai Wang

doi:10.1371/journal.pone.0176374

Abstract

Pyridoxal 5’-phosphate (PLP) is an essential cofactor for numerous enzymes involved in a diversity of cellular processes in living organisms. Previous analysis of the Actinobacillus pleuropneumoniae S-8 genome sequence revealed the presence of pdxS and pdxT genes, which are implicated in deoxyxylulose 5-phosphate (DXP)-independent pathway of PLP biosynthesis; however, little is known about their roles in A. pleuropneumoniae pathogenicity. Our data demonstrated that A. pleuropneumoniae could synthesize PLP by PdxS and PdxT enzymes. Disruption of the pdxS and pdxT genes rendered the pathogen auxotrophic for PLP, and the defective growth as a result of these mutants was chemically compensated by the addition of PLP, suggesting the importance of PLP production for A. pleuropneumoniae growth and viability. Additionally, the pdxS and pdxT deletion mutants displayed morphological defects as indicated by irregular and aberrant shapes in the absence of PLP. The reduced growth of the pdxS and pdxT deletion mutants under osmotic and oxidative stress conditions suggests that the PLP synthases PdxS/PdxT are associated with the stress tolerance of A. pleuropneumoniae. Furthermore, disruption of the PLP biosynthesis pathway led to reduced colonization and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model. The data presented in this study reveal the critical role of PLP synthases PdxS/PdxT in viability, stress tolerance, and virulence of A. pleuropneumoniae.

Citation: Xie F, Li G, Wang Y, Zhang Y, Zhou L, Wang C, et al. (2017) Pyridoxal phosphate synthases PdxS/PdxT are required for Actinobacillus pleuropneumoniae viability, stress tolerance and virulence. PLoS ONE 12(4): e0176374. https://doi.org/10.1371/journal.pone.0176374

Editor: Digby F. Warner, University of Cape Town, SOUTH AFRICA

Received: August 3, 2016; Accepted: April 10, 2017; Published: April 27, 2017

Copyright: © 2017 Xie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This research was supported by grants from Fund for Agro-scientific Research in the Public Interest (201303034, http://www.moa.gov.cn/), SGL, National Natural Science Foundation of China (No. 31100103, http://www.nsfc.gov.cn), FX, Natural Science Foundation of Heilongjiang Province of China (No. C2016067, http://www.hljkjt.gov.cn/), CLW, Natural Science Foundation of Heilongjiang Province of China (No. QC2016044, http://www.hljkjt.gov.cn/), FX, the project of Harbin Science and Technology innovative talents (No. 2015RQQYJ073, http://www.hrbinfo.gov.cn/), CLW, the State's Key Project of Research and Development Plan (No. 2016YFD0500700, http://www.most.gov.cn/index.htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Vitamin B6 is an essential cofactor in a multitude of cellular enzymatic reactions and is required in the metabolism of carbohydrates, amino acids, and fatty acids [1]. Pyridoxal 5’-phosphate (PLP) is the biochemically active form of vitamin B6 [2]. To date, studies have identified more than 160 enzymes that are functionally dependent on PLP [3, 4]. Vitamin B6 plays an important role in living organisms, and humans and animals must obtain it from food. By contrast, bacteria, fungi, and plants are able to synthesize PLP via de novo pathways.

Bacteria possess two distinct de novo PLP biosynthesis pathways, referred to as deoxyxylulose 5-phosphate (DXP)-dependent pathway and DXP-independent pathway [5, 6]. Escherichia coli and other members of the γ-subdivision of proteobacteria adopt the DXP-dependent pathway, which involves two enzymes PdxA and PdxJ. In contrast, the DXP-independent pathway is found in most eubacteria, fungi, protozoa, archaea, plants and metazoan, and requires PdxS and PdxT enzymes [5, 7]. As shown in Fig 1A, by converting glutamine into glutamate, PdxT generates ammonia, which is used by PdxS to synthesize PLP from a 5 and a 3 carbon sugar, such as ribose 5-phosphate and glyceraldehyde 3-phosphate [8, 9]. Although the de novo PLP biosynthesis pathways have been found in non-pathogenic bacteria, such as Escherichia coli and Bacillus subtilis [5, 10], the relation between PLP biosynthesis pathways and bacterial pathogenicity remains poorly understood. However, a previous study on Mycobacterium tuberculosis suggested that PLP biosynthesis was also essential for bacterial survival and virulence [8].

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Fig 1. Enzymatic activity of PdxS and PdxT.

(A) Substrates and product of a typical PLP synthase. (B) Effect of glutamine (0–15 mM) on glutaminase activity of PdxS and PdxT. (C) Effect of ribose 5-phosphate (0–1.5 mM) on PLP synthesis of PdxS and PdxT. (D) Effect of glyceraldehyde 3-phosphate (0–1.0 mM) on PLP synthesis of PdxS and PdxT. The kinetic constants were estimated by fitting to the Michaelis-Menten equation.

https://doi.org/10.1371/journal.pone.0176374.g001

Actinobacillus pleuropneumoniae is a Gram-negative bacterial pathogen responsible for porcine pleuropneumonia, which is a severely contagious respiratory disease that causes major economic losses for the swine industry worldwide [11]. Effective survival and persistence in pigs is a critical hindrance for A. pleuropneumoniae eradication [11, 12]. Recent analysis of the A. pleuropneumoniae S-8 genome sequence revealed the presence of the pdxS and pdxT genes [13]. Additionally, a previous study revealed that both pdxS and pdxT genes were downregulated after inactivation of the clpP gene which is required for stress tolerance in A. pleuropneumoniae [14].

To date, the vitamin B6 biosynthesis pathway has not been characterized in A. pleuropneumoniae. Furthermore, whether A. pleuropneumoniae adopts the DXP-independent pathway or whether the PLP synthases PdxS/PdxT are required for viability, stress tolerance, and virulence of A. pleuropneumoniae remain unclear. In the present study, we identified and characterized the function of enzymes PdxS and PdxT in the vitamin B6 biosynthesis pathway in A. pleuropneumoniae. We constructed knockouts in the pdxS and pdxT genes, respectively and investigated the role of PdxS and PdxT in viability, stress tolerance, and virulence of A. pleuropneumoniae.

Materials and methods

Ethics statement

Animal experiments were approved by Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (CAAS) and carried out in strict accordance with animal ethics guidelines and approved protocols (Heilongjiang-SYXK-2011–022). All efforts were made to minimize animal suffering.

Bacterial strains and growth conditions

The bacterial strains, plasmids, and primers used in this study are described in S1 and S2 Tables. The A. pleuropneumoniae strains were cultured in a brain heart infusion (BHI) medium supplemented with 10 μg/mL nicotinamide dinucleotide (NAD) (Sigma-Aldrich, USA). For the culture of A. pleuropneumoniae transconjugants (single crossovers), BHI medium was supplemented with 10 μg/mL of NAD and 7 μg/mL of chloramphenicol. E. coli β2155 was grown in Luria-Bertani (LB) medium supplemented with 1 mM diaminopimelic acid (DAP) (Sigma-Aldrich, USA). The chemically defined medium (CDM) was prepared as previously described [15], without the addition of NH₄Cl. All strains were routinely grown at 37°C.

Protein expression and purification

The coding sequences of A. pleuropneumoniae pdxS and pdxT genes were PCR-amplified from S-8 genomic DNA using specific primers SF/SR and TF/TR (S1 Table). The digested PCR products were ligated with NdeI/XhoI-digested pET22b(+) (Novagen). The recombinant plasmids were confirmed by sequencing and used to transform into E. coli BL21 (DE3). The expression of the each target protein was induced for 18 h at 16°C with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) in LB broth containing 50 μg/ml ampicillin. The His6-tag fusion proteins were loaded onto a Ni Sepharose 6 Fast Flow column (GE Healthcare, United States) and purified as previously described [16]. The recombinant protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China).

Glutaminase activity assay

Glutaminase activity was measured as described previously [17]. Samples of 8 μM PdxS, 8 μM PdxT, or 8 μM mixture of both proteins was incubated with 10 mM glutamine, 6 units of bovine glutamate dehydrogenase, and 0.5 mM 3-acetylpyridine adenine dinucleotide (APAD; Sigma) in a total reaction volume of 300 μl of 50 mM Tris-Cl (pH 8.0). The reaction mixture was incubated at 37°C. The absorbance of each sample was read at a wavelength of 363 nm (OD₃₆₃).

PLP formation assay

PLP formation assay was performed using a procedure as previously described [17]. Samples of 8 μM PdxS, 8 μM PdxT, or 8 μM mixture of both proteins was incubated with 0.5 mM ribose 5-phosphate, 1mM DL-glyceraldehyde 3-phosphate, 10 mM glutamine or 10 mM ammonium sulfate was added to a final volume of 300μl of 50 mM Tris-HCl (pH 8.0). The reaction mixture was incubated at 37°C. The absorbance of each sample was read at a wavelength of 414 nm (OD₄₁₄).

Construction of gene deletion mutants and complemented strains

The primers used for the construction of the deletion mutants S-8ΔpdxS and S-8ΔpdxT are listed in S1 Table. Primers SUF/SUR and SDF/SDR were used to amplify the two segments flanking with the pdxS gene. Using single-overlap extension PCR (SOE PCR), the fragment with a 487 bp internal deletion in the pdxS gene (from nt 54 to 539) was generated, and cloned into the conjugative vector pEMOC2 [18] to construct plasmid pEM△pdxS. Using E. coli β2155 and a single-step transconjugation system [19, 20], plasmid pEM△pdxS was applied to introduce the pdxS mutation into the S-8 strain. After two homologous recombination steps, the A. pleuropneumoniae S-8ΔpdxS mutant was verified by sequencing and PCR analyses using primers SCF/SCR and RTSF/RTSR (Fig 2A).

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Fig 2. Chromosomal inactivation of the pdxS and pdxT genes.

(A) Schematic representation of the A. pleuropneumoniae pdxS locus. The figure shows the binding locations for the oligonucleotide primers used to amplify the two flanking regions (1265 bp and 1294 bp, respectively) used in the construction of the pEM△pdxS plasmid and the diagnostic PCR analysis of the S-8ΔpdxS mutant (1106 bp) and WT S-8 strains (1573 bp). The S-8ΔpdxS mutant contains a 487 bp deletion (shadowed domain) in the pdxS gene. (B) (A) Schematic representation of the A. pleuropneumoniae pdxT locus. The figure shows the binding locations for the oligonucleotide primers used to amplify the two flanking regions (1377 bp and 1387 bp, respectively) used in the construction of the pEM△pdxT plasmid and the diagnostic PCR analysis of the S-8ΔpdxT mutant (660 bp) and WT S-8 strains (1151 bp). The S-8ΔpdxT mutant contains a 491 bp deletion (shadowed domain) in the pdxT gene. (C) PCR identification of the S-8ΔpdxS mutant using the primers SCF/SCR. For lane 1, the WT S-8 strain (1573 bp); for lane 2, the S-8ΔpdxS mutant (1106 bp); for lane 3, the complemented S-8ΔpdxScomp strain (1573 and 1106 bp). (D) PCR identification of the S-8ΔpdxT mutant using the primers SCF/TCR. For lane 1, the WT S-8 strain (2151bp); for lane 2, the S-8ΔpdxT mutant (1660 bp); for lane 3, the complemented S-8ΔpdxTcomp strain (1660 and 1264 bp). (E) PCR identification of the S-8ΔpdxS mutant using the primers RTSF/RTSR. For lane 1, the WT S-8 strain (214 bp); for lane 2, the S-8ΔpdxS mutant; for lane 3, the complemented S-8ΔpdxScomp strain (214 bp). (F) PCR identification of the S-8ΔpdxT mutant using the primers RTTF/RTTR. For lane 1, the WT S-8 strain (267 bp); for lane 2, the S-8ΔpdxT mutant; for lane 3, the complemented S-8ΔpdxTcomp strain (267 bp). For lane M, DL2000 DNA marker (from top to bottom: 2000, 1000, 750, 500, 250, and 100 bp).

https://doi.org/10.1371/journal.pone.0176374.g002

The deletion of pdxT gene in A. pleuropneumoniae S-8 was performed using the same procedure as described above. The primers TUF/TUR, and TDF/TDR were used to generate a 491 bp internal deletion in the pdxT gene (from nt 7 to 497), and the S-8ΔpdxT mutant was confirmed by PCR analyses with the primers SCF/TCR and RTTF/RTTR (Fig 2B).

The promoter of PdxS and PdxT was predicted using the BPROM program (http://www.softberry.com/). The pLpdxS plasmid was constructed by cloning the 1573-bp PCR product amplified with the primers SCF/SCR (S1 Table), which contained the entire pdxS open reading frame (ORF) and the upstream region to include the native promoter, into plasmid pLS88 [21]. Using SOE PCR, the 1264-bp fragment containing the upstream promoter and entire pdxT ORF was generated, and ligated into the plasmid pLS88 to yield plasmid pLpdxT plasmid (Fig 2A and 2B). The plasmids pLpdxS and pLpdxT were electroporated into S-8ΔpdxS and S-8ΔpdxT, respectively for trans complementation. The complemented strains were selected on BHI agar containing 20 μg/mL of kanamycin, and were verified by PCR using the primers SCF/SCR or TCF/TCR.

RNA isolation and qRT-PCR

For RNA isolation, A. pleuropneumoniae strains were grown to mid-logarithmic phase in 3 ml of BHI medium. The cultures were harvested by centrifugation at 10,000 g at 4°C. Total RNA was extracted using RNeasy kit (Qiagen) and complementary DNA (cDNA) was synthesized using the PrimeScript RT reagent kit (TaKaRa, Japan) according to the manufacturer's instructions. The primers used for analysis of gene expression are listed in S2 Table. The cDNA samples were amplified using SYBR Green I (TakaRa). Real-time polymerase chain reactions were performed in a MicroAmp Optical 96-well reaction plate using qTOWER 2.2 system (Analytikjena, Germany). The quantitative qRT-PCR experiments were performed in triplicate, with recF as an internal control [22].

In vitro growth assays

In vitro growth assays were conducted as previously described [23]. The A. pleuropneumoniae wild-type (WT) S-8, S-8ΔpdxS mutant, S-8ΔpdxT mutant and complemented strains S-8ΔpdxScomp and S-8ΔpdxTcomp were grown in 3 ml of BHI medium supplemented with 10 μM PLP for 18 h, then washed three times with CDM medium to remove residual PLP and diluted to optical densities at 600 nm (OD₆₀₀) of 0.1. The fresh cultures in 10 ml of CDM medium were respectively supplemented with 10 μM PLP (Sigma), 1 mM ammonium sulfate or not supplemented any chemicals. The cultures were then incubated at 37°C. The OD₆₀₀ values were recorded at an interval of 2 h using the Eppendorf BioPhotometer (Eppendorf, Germany).

In vitro stress assays

In vitro stress assays were performed as described previously [14]. The A. pleuropneumoniae WT S-8, S-8ΔpdxS mutant, S-8ΔpdxT mutant and complemented strains S-8ΔpdxScomp and S-8ΔpdxTcomp were grown to OD₆₀₀ 0.8 in BHI medium supplemented with 10 μM PLP. We inoculated bacteria at different time points according to the growth curves to try to get all strains to achieve the OD600 of 0.8 at the same time. In addition, we inoculated bacteria at an interval of 0.5 h to make sure that. Cells (10⁸ CFU/mL) of each strain from the broth cultures were washed three times with un-supplemented BHI medium, and harvested by centrifugation at 4000 g for 10 min. To test the tolerance of the cells to oxidative stress, cells of each strain were resuspended in 1 ml of BHI medium containing 5 mM H₂O₂ and incubated in the presence or absence of PLP for 45 min at 37°C. To test the tolerance of the cells to osmotic stress, cells of each strain were resuspended in 1 ml of BHI medium containing 0.4 M NaCl and incubated in the presence or absence of PLP for 45 min at 37°C. Cells of each strain were resuspended in BHI medium in the presence or absence of PLP as controls and incubated for 45 min at 37°C. All of the cultures of each stress assay were washed three times with BHI medium to remove residual H₂O₂ or NaCl, and serially diluted and cultured on BHI agar plates supplemented with 10 μM PLP. The cell count was determined after 24 h of incubation. Values of stress resistance were calculated as [(stressed sample CFU/ ml)/ (control sample CFU/ ml)]×100.

Transmission electron microscopy

The A. pleuropneumoniae WT S-8, S-8ΔpdxS mutant, S-8ΔpdxT mutant and complemented strains S-8ΔpdxScomp and S-8ΔpdxTcomp were cultivated in BHI medium supplemented with 10 μM PLP at 37°C for 16 h, then washed three times with un-supplemented BHI medium to remove residual PLP and diluted to optical densities at 600 nm (OD₆₀₀) of 0.05. The fresh cultures were grown to mid-exponential growth phase in 10 ml of BHI medium with or without 10 μM PLP. The cells were washed three times with PBS and fixed for 2 h in 2% osmic acid. Dehydration was performed in upgraded ethanol, and then the samples were embedded in SPI-Pon 812 resin (emicron) for two days. Resin-soaked sample blocks were polymerized at 70°C, and samples were counterstained with uranyl acetate and lead citrate. Electron micrographs were recorded with a transmission electron microscope (JEM-1200EX, JEOL, Japan).

Mouse in vivo experiments

BALB/c mouse has been acknowledged as an appropriate model for A. pleuropneumoniae infection [24, 25]. Specific-pathogen-free, six-week-old female BALB/c mice (Beijing Vital River Laboratory Animal Co., Ltd.) were used for virulence evaluation of A. pleuropneumoniae WT S-8, S-8ΔpdxS mutant, and S-8ΔpdxT mutant strains. Briefly, all A. pleuropneumoniae strains were cultured in BHI medium supplemented with 10 μM PLP at 37°C, and harvested during the mid-exponential phase and washed three times with sterile PBS. A total of 160 mice were randomly divided into 16 groups (n = 10/group). For each strain, five experimental groups were inoculated intraperitoneally with 100 μL of PBS containing varying concentrations of bacterial suspension (10⁵–10⁹ CFU). Non-infected mice in the control group were inoculated with 100 μL of sterile PBS (pH 7.4). Animal suffering was reduced with buprenorphine (0.05 mg/kg), given subcutaneously for analgesia every 12 hours during the first 72 hours after infection. The health status and the weight of the mice were monitored twice daily for a 14-day period and humane endpoints used to determine if the mice met criteria to be euthanized [26]. These criteria included weight loss >10–15%, lethargy, inability to stand, anorexia or flocked together for more than 6 hours. Mice meeting criteria were euthanized by cervical dislocation under isoflurane anesthesia. The 50% lethal dose (LD₅₀) of A. pleuropneumoniae WT strain S-8, S-8ΔpdxS mutant, and S-8ΔpdxT mutant strains were calculated as previously described by Reed-Muench [27].

Enumeration of bacterial load in organs

A total of 15 specific-pathogen-free, six week-old female BALB/c mice were randomly divided into 3 groups (n = 5), and each group was intraperitoneally administered with 1.0×10⁷ CFU of WT S-8 strain, S-8ΔpdxS mutant, and S-8ΔpdxT mutant strains respectively. Three (3) days post-infection, mice from each group were humanely euthanized and the organs of lung, liver, and kidney were removed aseptically. Samples were weighed, and homogenized using a tissue homogenizer (100 mg weight/ml of PBS). Viable counts in serial dilutions of homogenates were determined following culture on BHI agar plates for 24 h at 37°C.

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 5.01 (GraphPad Software Inc., USA). The data are expressed as the means +/- standard deviation. P-values less than 0.05 were considered statistically significant.

Results

PdxS and PdxT are involved in PLP synthesis in A. pleuropneumoniae

The ORFs of pdxS and pdxT genes are 888-bp and 576-bp in length, respectively, and encode the putative pyridoxal 5'-phosphate synthase subunits PdxS and PdxT. Searching the respective conserved domains in NCBI database revealed that PdxS has SOR_SNZ domain which represents a family of pyridoxal 5'-phosphate synthase and PdxT has SNO domain which represents glutamine amidotransferase family. Both PdxS and PdxT shared a high identity in the amino acid sequences with their orthologous sequences in Bacillus subtilis (84 and 55%, respectively) and Streptococcus pneumoniae (64 and 41%, respectively). In this study, to determine the activities of PdxS and PdxT in vitamin B6 formation in vitro, A. pleuropneumoniae pdxS and pdxT genes were overexpressed in E. coli BL21 (DE3) and the recombinant His6-tag fusion proteins were purified using Ni²⁺-affinity chromatography. SDS—PAGE analysis showed that the recombinant proteins, with the molecular weight of approximately 35.3 kD and 23.4 kD (S1 Fig), respectively, were obtained when the cells were induced with 0.5 mM IPTG at 16°C for 18 h.

Glutaminase activity of PdxT was only detected in the presence of its partner protein PdxS. As shown in Fig 1B, varying the concentration of glutamine in the assay produced typical Michaelis-Menten kinetics, the catalytic constants K_M and k_cat were 1.72±0.14 mM and 6.12±0.14 min^-1 respectively. Furthermore, the ribose 5-phosphate and glyceraldehyde 3-phosphate were served as substrates of PLP synthase PdxS and PdxT, and tested for their ability to form PLP. The kinetic constants were determined as K_M = 159.8±13.5 μM, k_cat = 0.020±0.0005 min^-1 for ribose 5-phosphate (Fig 1C), and K_M = 175.7±23.0 μM, k_cat = 0.023±0.0011 min^-1 for glyceraldehyde 3-phosphate, respectively (Fig 1D).

A. pleuropneumoniae S-8ΔpdxS and S-8ΔpdxT mutants are auxotrophic for PLP

To demonstrate the function of PLP biosynthesis in A. pleuropneumoniae, pdxS and pdxT-knockout mutants, S-8ΔpdxS and S-8ΔpdxT, respectively, were generated using the double-crossover homologous recombination approach and confirmed by PCR and sequencing (Fig 2). The results of qRT-PCR showed that the transcription levels of the downstream genes were unaffected, confirming that the mutations in S-8ΔpdxS and S-8ΔpdxT strains were nonpolar (S2 Fig). Complementation of the S-8ΔpdxS and S-8ΔpdxT were achieved using the plasmid pLpdxS and pLpdxT, respectively (Fig 2). The complemented mutants were designated S-8ΔpdxScomp and S-8ΔpdxTcomp. The results of qRT-PCR showed that the transcriptions of pdxS in S-8ΔpdxS and pdxT in S-8ΔpdxT were virtually undetectable and partially restored in the complemented strains.

The growth properties of the A. pleuropneumoniae WT S-8, S-8ΔpdxS, S-8ΔpdxT, S-8ΔpdxScomp, and S-8ΔpdxTcomp strains were investigated in vitro. Upon cultivation in the absence of PLP, both S-8ΔpdxS and S-8ΔpdxT mutants exhibited severe growth defects (Fig 3A). It is suggested that PLP is essential for A. pleuropneumoniae growth in vitro. We therefore analyzed the growth of S-8ΔpdxS and S-8ΔpdxT mutants in the presence PLP supplementation, the defective growth of these mutants could be recovered, fundamentally similar to growth curves of the WT strain S-8 (Fig 3B). The growth curves of the S-8, S-8ΔpdxScomp, and S-8ΔpdxTcomp strains were similar in the absence and presence of PLP. These data suggest critical roles for PdxS and PdxT proteins in A. pleuropneumoniae PLP biosynthesis.

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Fig 3. Growth characteristics of the A. pleuropneumoniae strains.

The growth curves of the WT S-8, S-8ΔpdxS, S-8ΔpdxT, S-8ΔpdxScomp strain and S-8ΔpdxTcomp strains in the absence (A) or presence (B) of PLP. Overnight culture of each strain was inoculated into fresh CDM medium with or without 10μM PLP and grown at 37°C for 10 h. Growth was monitored by OD₆₀₀ at an interval of 2 h.

https://doi.org/10.1371/journal.pone.0176374.g003

The PLP synthesis activity of PdxS is not dependent on PdxT in the presence of ammonium

The growth properties of the A. pleuropneumoniae WT S-8, S-8ΔpdxS mutant, S-8ΔpdxT mutant and complemented strains S-8ΔpdxScomp and S-8ΔpdxTcomp were also analyzed in the presence of ammonium supplementation in vitro. With ammonium sulfate supplementation, the defective growth rate of S-8ΔpdxT mutant was partially restored, but the defective growth of the S-8ΔpdxS mutant remained unchanged (Fig 4A). We then examined PLP synthesis activity of PdxS using ammonium substitute for glutamine in the reaction. Unlike using glutamine as substrate, the individual PdxS could produce PLP in the addition of ammonium (Fig 4B and 4C). The PLP synthase had a specific activity of 996 ± 54 pmol min^-1 mg^-1 using glutamine as substrate, and of 508 ± 38 pmol min^-1 mg^-1 using ammonium sulphate as substrate.

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Fig 4. PLP synthase activity of PdxS in the presence of ammonium.

(A) The growth curves of the WT S-8, S-8ΔpdxS, S-8ΔpdxT mutant, S-8ΔpdxScomp strain and S-8ΔpdxTcomp strains in the presence of ammonium supplementation. Overnight culture of each strain was diluted into fresh CDM medium supplemented with 1mM ammonium sulfate. Bacteria were grown at 37°C for 10 h and growth was monitored by OD₆₀₀ at an interval of 2h. (B) PLP synthase activity of PdxS, PdxT, and the mixture of the two proteins using glutamine as substrate in the reaction. (C) PLP synthase activity of PdxS, PdxT, and the mixture of the two proteins using ammonium substitute for glutamine in the reaction. Points indicate the mean values of three independent assays, and error bars indicate standard deviations.

https://doi.org/10.1371/journal.pone.0176374.g004

A. pleuropneumoniae S-8ΔpdxS and S-8ΔpdxT mutants are sensitive to NaCl and H₂O₂

The viability of the WT S-8, S-8ΔpdxS mutant, S-8ΔpdxT mutant and complemented strains S-8ΔpdxScomp and S-8ΔpdxTcomp were investigated when exposed to oxidative stress and osmotic stress conditions. As shown in S3A Fig, in the absence of PLP and when the cells were exposed to H₂O₂-induced oxidative stress, the viable counts of S-8ΔpdxS and S-8ΔpdxT were much lower than that of the S8 cells. The survival rates of the S-8ΔpdxS mutant and S-8ΔpdxT mutant were 18.19% and 34.73%, respectively, which were significantly lower (P < 0.01) than that of WT S-8 (75.68%) (Fig 5A). To exclude the influence of growth defects of S-8ΔpdxS and S-8ΔpdxT in the absence of PLP, the cells of each strain were also exposed to oxidative stress in the presence of PLP supplementation. The viable counts of S-8ΔpdxS and S-8ΔpdxT were still lower than that of the S8 cells, although increased compared to the viable counts of S-8ΔpdxS and S-8ΔpdxT exposed to oxidative stress in the absence of PLP (S3B Fig). As shown in Fig 5B, in the presence of PLP supplementation, 74.86% of WT S-8 survived. However, S-8ΔpdxS and S-8ΔpdxT exhibited 28.90% (P < 0.01) and 47.16% (P < 0.01) survival rates, respectively. Similar results were obtained when the cells were exposed to osmotic stress. The viable counts of S-8ΔpdxS and S-8ΔpdxT were lower than that of the S8 cells (S3C and S3D Fig). In the absence of PLP, WT S-8 cells under osmotic stress exhibited the 85.89% survival rate, whereas the S-8ΔpdxS mutant and the S-8ΔpdxT mutant respectively exhibited 32.08% (P < 0.01) and 52.13% (P < 0.05) survival rate (Fig 5C). In the presence of PLP supplementation, the survival rate of the S-8ΔpdxS mutant was 38.58%, which was significantly lower (P < 0.01) than that of WT S-8 (86.09%); and the survival rate of S-8ΔpdxT mutant was 59.16%, which was also significantly lower (P < 0.05) than that of WT S-8 (Fig 5D). These results suggest that PLP synthases PdxS/PdxT are linked to resistance to osmotic stress and oxidative stress.

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Fig 5. Response of A. pleuropneumoniae mutants to oxidative and osmotic stresses.

Overnight culture of WT S-8, S-8ΔpdxS, S-8ΔpdxT, S-8ΔpdxScomp strain and S-8ΔpdxTcomp strains were diluted into fresh BHI broth and grown to OD₆₀₀ 0.8. Bacteria were then treated with 5 mM H₂O₂ in the absence (A) and presence (B) of PLP for 45 min, 0.4 M NaCl in the absence (C) and presence (D) of PLP for 45 min. Values of stress resistance were calculated as [(stressed sample CFU/ ml)/ (control sample CFU/ ml)]×100. The data shown are the means of three independent assays, and error bars indicate standard deviations. **, p < 0.01; *, p < 0.05.

https://doi.org/10.1371/journal.pone.0176374.g005

Loss of pdxS and pdxT leads to morphological defects of A. pleuropneumoniae

The effects of PLP on the morphologies of WT S-8, S-8ΔpdxS mutant, S-8ΔpdxT mutant and complemented strains S-8ΔpdxScomp and S-8ΔpdxTcomp were observed using transmission electron microscopy. These data showed that the morphologies of the five strains were similar in the presence of PLP supplementation, and were consistent with the normal morphology of coccobacilli (S4 Fig). However, in the absence of PLP, the S-8ΔpdxS mutant and S-8ΔpdxT mutant exhibited irregular and aberrant shapes, showing holes and deep craters on their surface. S-8ΔpdxS also appeared partially lysed or swollen (Fig 6). Approximately 49% of S-8ΔpdxS and 33.67% of S-8ΔpdxT cells were abnormal cells, whereas S-8, S-8ΔpdxScomp and S-8ΔpdxTcomp strains exhibited only 2.3%, 7.7% and 6% of abnormal cells, respectively (Fig 6D). These data indicate that PLP production is required for maintaining normal cellular morphology of A. pleuropneumoniae.

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Fig 6. Transmission electron microscopy of A. pleuropneumoniae mutants in the absence of PLP.

TEM of WT S-8, S-8ΔpdxS, S-8ΔpdxT, S-8ΔpdxScomp strain and S-8ΔpdxTcomp strains in the mid-log phase in the absence of PLP were carried out. The cell morphology of the S-8ΔpdxS mutant and S-8ΔpdxT mutant exhibited irregular and aberrant shapes, showing holes and deep crater on their surface. S-8ΔpdxS also appeared partially lysed or swollen. Scale bar = 5μm.

https://doi.org/10.1371/journal.pone.0176374.g006

Loss of pdxS attenuates the virulence of A. pleuropneumoniae in the BALB/c mouse model

To address whether pdxS or pdxT deletion affected the virulence of A. pleuropneumoniae, BALB/c mice were inoculated intraperitoneally with WT S-8, S-8ΔpdxS mutant, and S-8ΔpdxT mutant strains at various doses, and LD50 values were determined. The LD₅₀ value of the S-8ΔpdxS mutant was 2.51×10⁸ CFU, higher than 5.01×10⁶ CFU for WT S-8. The virulence of the S-8ΔpdxS mutant was 50.1-fold attenuated compared to that of WT S-8. Conversely, the LD₅₀ value of the S-8ΔpdxT mutant (7.76×10⁷ CFU) was found to be only 15.5-fold lower than WT S-8. These results suggested that the loss of both pdx enzymes attenuates the pathogenicity of A. pleuropneumoniae, with a greater impact observed with the deletion of pdxS.

The capacity of WT S-8, S-8ΔpdxS mutant, and S-8ΔpdxT mutant strains to colonize mice was then tested. The A. pleuropneumoniae load in tissues of systemically infected mice was determined by culturing the lungs, livers, and kidneys homogenates 3 days post-infection. As shown in Fig 7, the viable counts of the S-8ΔpdxS mutant in the lungs, livers and kidneys were significantly lower compared with those of WT S-8 (P<0.01). Similarly, significant differences (P < 0.05) in bacterial loads were also found between the WT S-8-inoculated mice and the S-8ΔpdxT-inoculated mice in lungs. In addition, the bacterial loads in the livers and kidneys of the S-8ΔpdxT-inoculated mice were decreased compared to the WT S-8-inoculated mice, although these differences were not statistically significant (Fig 7). Taken together, the results showed that the S-8ΔpdxS mutant of A. pleuropneumoniae displayed an impaired colonization ability in BALB/c mice.

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Fig 7. Bacterial loads in organs from BALB/c mice infected with A. pleuropneumoniae mutants.

Mice were infected with WT S-8, S-8ΔpdxS, and S-8ΔpdxT mutant strains, and bacterial loads in (A) lung, (B) liver, (C) kidney examined 3 days post infection. The data shown are the means of bacterial colonies from five mice, and error bars indicate standard deviations. **, p < 0.01; *, p < 0.05.

https://doi.org/10.1371/journal.pone.0176374.g007

Discussion

In the A. pleuropneumoniae genome, only the adjacent pdxS and pdxT genes are annotated as belonging to the pdx family. Our results in the present study demonstrated that A. pleuropneumoniae synthesizes PLP via the DXP-independent pathway through the utilization of the two enzymes: the synthase subunit PdxS and the glutaminase subunit PdxT. In this study, we observed that respective disruption of the pdxS and pdxT genes rendered A. pleuropneumoniae auxotrophic for PLP (Fig 3), suggesting the importance of PLP biosynthesis in the growth of A. pleuropneumoniae. In addition, the results of the PLP synthase activity showed that both PdxS and PdxT proteins were required for PLP production in vitro in the presence of glutamine (Fig 4B and 4C). These findings are consistent with the previous observations in other bacteria.

In this study, the supplementation of exogenous ammonium in the growth broth complemented the defective growth rate of the S-8ΔpdxT mutant, but did not influence the defective growth rate of the S-8ΔpdxS mutant (Fig 4A). Similar results have also been reported in the pdxT deletion mutant in S. pneumoniae and B. subtilis whose defective growth rate could also be compensated in the presence of ammonium [9, 28], suggesting that the preliminary function of PdxT protein in PLP synthesis is producing ammonium from glutamine. Therefore, PdxS can produce PLP in the absence of PdxT when the glutamine is replaced by ammonium in the reaction (Fig 4C). These observations in this assay are in agreement with the proposed two-step model for PLP formation in previous studies [29, 30]. In this model, PdxT depends on PdxS to hydrolyze glutamine into ammonium, and PdxS utilizes ammonium for generating PLP by incorporation with other chemicals.

Intriguingly, we found that the PLP synthases PdxS/PdxT were implicated in the stress responses of A. pleuropneumoniae. To the best of our knowledge, this is the first study to show the relation between the PLP synthases PdxS/PdxT and stress response in bacteria. From the data presented here, it appeared that S-8ΔpdxS and S-8ΔpdxT mutants showed different responses in vitro to stress conditions compared with WT S-8. We found that pdxS and pdxT gene deletion caused increased susceptibility of A. pleuropneumoniae to H₂O₂-induced oxidative stress and NaCl-induced osmotic stress (Fig 5). Though these findings were not described in bacteria, PLP formation has been linked to stress tolerance in plants [31–33]. In Arabidopsis thaliana, disruption of PLP synthases lead to reduced plant size, slowed root growth and hypersensitivity to NaCl [31, 32]. In addition, PLP production in A. thaliana has also been associated with defense against cellular oxidative stress since A. thaliana mutants deficient in PLP synthase were shown to be highly sensitive to oxidative stresses [33]. Combining our findings presented here and previous work by others, it is highly likely that PLP synthases PdxS/PdxT play a vital role in osmotic stress and oxidative stress responses in A. pleuropneumoniae. However, the supplementation of exogenous PLP did not reverse the stress responses of the S-8ΔpdxS and S-8ΔpdxT mutants. We speculate that PdxS/PdxT may participate in other critical cellular processes involved in the biosynthesis of important enzymes associated with stress responses; however further investigations are required for confirmation.

Another objective of this study was to clarify whether PLP synthases PdxS/PdxT are essential for A. pleuropneumoniae pathogenicity in a mouse model. The results of this study indicated that the virulence and colonization of the S-8ΔpdxS and S-8ΔpdxT mutants were attenuated (Fig 7), although there were differences regarding the magnitude of attenuation, suggesting that the loss of the PLP biosynthesis pathway influences the pathogenicity of A. pleuropneumoniae in BALB/c mice. These findings are correlated with observations presented by other previous studies made in M. tuberculosis, H. pylori and S. pneumoniae [8, 9, 23]. We subsequently analyzed the probable reasons for attenuated virulence. It is likely that the PLP concentration in mice is insufficient for optimal growth of these A. pleuropneumoniae mutants in vivo. The PLP concentration in plasma of mice has been shown to fluctuate between 100 and 200 nM [34]. However, the addition of 100 nM of PLP to the broth did not significantly compensate the defective growth of the S-8ΔpdxS and S-8ΔpdxT mutants in vitro in our pilot experiments (data not shown), suggesting that the levels of PLP in mice may be too low to support the optimal growth of these mutants, which further affected the colonization and virulence of the S-8ΔpdxS and S-8ΔpdxT mutants. Another probability is the involvement of PLP in the biosynthesis of surface-exposed structures of A. pleuropneumoniae. Indeed, it has been shown that PLP biosynthesis is essential for the synthesis of surface-exposed structures involved in adhesion and colonization in some pathogenic bacteria, such as lipopolysaccharide (LPS) and flagella [23, 35, 36]. In this study, the S-8ΔpdxS and S-8ΔpdxT mutants exhibited aberrant shapes in the absence of PLP (Fig 6). However, whether this change directly affected the surface-exposed structures of A. pleuropneumoniae or then consequently affected the adhesion and colonization of A. pleuropneumoniae, needs further investigation.

Additionally, it is worth noting that the PLP biosynthesis pathway is present in various pathogenic bacteria, but is absent in humans and mammals [37, 38]. Therefore, the de novo pathway of PLP biosynthesis is of particular interest as novel potential drug targets for the therapy of bacterial infections. The data presented in this study demonstrated that disruption of PdxS/PdxT pathway of PLP biosynthesis inhibited the normal growth of A. pleuropneumoniae. This finding offers the new possibility of prevention and treatment of A. pleuropneumoniae infection which ravages the swine industry. In conclusion, the present study revealed the critical role of PLP synthases PdxS/PdxT in viability, stress tolerance, and virulence related to A. pleuropneumoniae. In addition, bacteria are also able to synthesize PLP by a salvage pathway [39], in which PLP is synthesized from other B6 vitamers. Future studies should include attempts to determine whether this pathway is present in A. pleuropneumoniae and its function in A. pleuropneumoniae pathogenicity.

Supporting information

S1 Fig. SDS—PAGE analysis of the recombinant PdxS and PdxT proteins purified with affinity chromatography.

Recombinant PdxS (rPdxS) and PdxT (rPdxT) were separated by 12% SDS-PAGE. Lane M, molecular mass markers.

https://doi.org/10.1371/journal.pone.0176374.s001

(TIF)

S2 Fig. The relative transcription levels of selected genes.

(A) Transcriptional levels of pdxS gene in WT S-8, S-8ΔpdxS, and S-8ΔpdxScomp strains. (B) Transcriptional levels of pdxT gene in WT S-8, S-8ΔpdxT, and S-8ΔpdxTcomp strains. (C) Transcriptional levels of downstream genes of pdxS in WT S-8 and S-8ΔpdxS strains. (D) Transcriptional levels of downstream genes of pdxT in WT S-8 and S-8ΔpdxT strains.

https://doi.org/10.1371/journal.pone.0176374.s002

(TIF)

S3 Fig. Viable counts of A. pleuropneumoniae mutants exposed to oxidative and osmotic stresses.

Overnight culture of WT S-8, S-8ΔpdxS, S-8ΔpdxT, S-8ΔpdxScomp strain and S-8ΔpdxTcomp strains were diluted into fresh BHI broth and grown to OD₆₀₀ 0.8. Bacteria were then treated with 5 mM H₂O₂ in the absence (A) and presence (B) of PLP for 45 min, 0.4 M NaCl in the absence (C) and presence (D) of PLP for 45 min. Viable CFUs of A. pleuropneumoniae were counted. The data shown are the means of three independent assays.

https://doi.org/10.1371/journal.pone.0176374.s003

(TIF)

S4 Fig. Transmission electron microscopy of A. pleuropneumoniae mutants in the presence of PLP supplementation.

TEM of WT S-8, S-8ΔpdxS, S-8ΔpdxT, S-8ΔpdxScomp strain and S-8ΔpdxTcomp strains in the mid-log phase in the presence of PLP supplementation were carried out. The morphologies of the five strains were similar and were consistent with the normal morphology of coccobacilli.

https://doi.org/10.1371/journal.pone.0176374.s004

(TIF)

S1 Table. Characteristics of bacterial strains, plasmids, and primers used in this study.

https://doi.org/10.1371/journal.pone.0176374.s005

(DOCX)

S2 Table. Primers used in qRT-PCR study.

https://doi.org/10.1371/journal.pone.0176374.s006

(DOCX)

Acknowledgments

This research was supported by grants from Special Fund for Agro-scientific Research in the Public Interest (201303034), National Natural Science Foundation of China (31100103), Natural Science Foundation of Heilongjiang Province of China (C2016067 and QC2016044), and the project of Harbin Science and Technology innovative talents (2015RQQYJ073), and the State's Key Project of Research and Development Plan (2016YFD0500700). We thank Paul R. Langford, Janine T. Bossé, and Yanwen Li (Section of Paediatrics, Department of Medicine, Imperial College London, St. Mary’s Campus, United Kingdom) for language polishing and the valuable suggestions on the manuscript.

Author Contributions

Conceptualization: FX CLW.
Data curation: LZ.
Formal analysis: YHZ.
Funding acquisition: SGL FX CLW.
Investigation: SHL LZ YLW.
Methodology: FX CLW.
Project administration: CLW.
Resources: SHL.
Software: GL.
Supervision: CLW.
Validation: CCW YLW.
Visualization: LZ.
Writing – original draft: FX.
Writing – review & editing: CLW SGL.

References

1. Mooney S, Leuendorf JE, Hendrickson C, Hellmann H. Vitamin B6: a long known compound of surprising complexity. Molecules. 2009;14(1):329–51. pmid:19145213
- View Article
- PubMed/NCBI
- Google Scholar
2. Eliot AC, Kirsch JF. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annual review of biochemistry. 2004;73:383–415. pmid:15189147
- View Article
- PubMed/NCBI
- Google Scholar
3. Percudani R, Peracchi A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO reports. 2003;4(9):850–4. pmid:12949584
- View Article
- PubMed/NCBI
- Google Scholar
4. Percudani R, Peracchi A. The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families. BMC bioinformatics. 2009;10:273. pmid:19723314
- View Article
- PubMed/NCBI
- Google Scholar
5. Fitzpatrick TB, Amrhein N, Kappes B, Macheroux P, Tews I, Raschle T. Two independent routes of de novo vitamin B6 biosynthesis: not that different after all. The Biochemical journal. 2007;407(1):1–13. pmid:17822383
- View Article
- PubMed/NCBI
- Google Scholar
6. Mukherjee T, Hanes J, Tews I, Ealick SE, Begley TP. Pyridoxal phosphate: biosynthesis and catabolism. Biochimica et biophysica acta. 2011;1814(11):1585–96. pmid:21767669
- View Article
- PubMed/NCBI
- Google Scholar
7. Tambasco-Studart M, Titiz O, Raschle T, Forster G, Amrhein N, Fitzpatrick TB. Vitamin B6 biosynthesis in higher plants. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(38):13687–92. pmid:16157873
- View Article
- PubMed/NCBI
- Google Scholar
8. Dick T, Manjunatha U, Kappes B, Gengenbacher M. Vitamin B6 biosynthesis is essential for survival and virulence of Mycobacterium tuberculosis. Molecular microbiology. 2010;78(4):980–8. pmid:20815826
- View Article
- PubMed/NCBI
- Google Scholar
9. El Qaidi S, Yang J, Zhang JR, Metzger DW, Bai G. The vitamin B(6) biosynthesis pathway in Streptococcus pneumoniae is controlled by pyridoxal 5'-phosphate and the transcription factor PdxR and has an impact on ear infection. Journal of bacteriology. 2013;195(10):2187–96. pmid:23475965
- View Article
- PubMed/NCBI
- Google Scholar
10. Raschle T, Amrhein N, Fitzpatrick TB. On the two components of pyridoxal 5'-phosphate synthase from Bacillus subtilis. The Journal of biological chemistry. 2005;280(37):32291–300. pmid:16030023
- View Article
- PubMed/NCBI
- Google Scholar
11. Chiers K, De Waele T, Pasmans F, Ducatelle R, Haesebrouck F. Virulence factors of Actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host. Veterinary research. 2010;41(5):65. pmid:20546697
- View Article
- PubMed/NCBI
- Google Scholar
12. Bosse JT, Janson H, Sheehan BJ, Beddek AJ, Rycroft AN, Kroll JS, et al. Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes and infection / Institut Pasteur. 2002;4(2):225–35.
- View Article
- Google Scholar
13. Li G, Xie F, Zhang Y, Wang C. Draft genome sequence of Actinobacillus pleuropneumoniae serotype 7 strain S-8. Journal of bacteriology. 2012;194(23):6606–7. pmid:23144372
- View Article
- PubMed/NCBI
- Google Scholar
14. Xie F, Zhang Y, Li G, Zhou L, Liu S, Wang C. The ClpP protease is required for the stress tolerance and biofilm formation in Actinobacillus pleuropneumoniae. PloS one. 2013;8(1):e53600. pmid:23326465
- View Article
- PubMed/NCBI
- Google Scholar
15. Wagner TK, Mulks MH. A subset of Actinobacillus pleuropneumoniae in vivo induced promoters respond to branched-chain aminoacid limitation. FEMS Immunology and Medical Microbiology. 2006; 48(2):192–204. pmid:16995880
- View Article
- PubMed/NCBI
- Google Scholar
16. Chen L, Dang G, Deng X, Cao J, Yu S, Wu D, et al. Characterization of a novel exported esterase Rv3036c from Mycobacterium tuberculosis. Protein expression and purification. 2014;104:50–6. pmid:25224799
- View Article
- PubMed/NCBI
- Google Scholar
17. Strohmeier M, Raschle T, Mazurkiewicz J, Rippe K, Sinning I, Fitzpatrick TB, et al. Structure of a bacterial pyridoxal 5'-phosphate synthase complex. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(51):19284–9. pmid:17159152
- View Article
- PubMed/NCBI
- Google Scholar
18. Baltes N, Tonpitak W, Hennig-Pauka I, Gruber AD, Gerlach GF. Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence. FEMS microbiology letters. 2003;220(1):41–8. pmid:12644226
- View Article
- PubMed/NCBI
- Google Scholar
19. Dehio C, Meyer M. Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli. Journal of bacteriology. 1997;179(2):538–40. pmid:8990308
- View Article
- PubMed/NCBI
- Google Scholar
20. Oswald W, Tonpitak W, Ohrt G, Gerlach G. A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker. FEMS microbiology letters. 1999;179(1):153–60. pmid:10481100
- View Article
- PubMed/NCBI
- Google Scholar
21. Willson PJ, Albritton WL, Slaney L, Setlow JK. Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi. Antimicrobial Agents and Chemotherapy. 1989; 33(9):1627–30. pmid:2684012
- View Article
- PubMed/NCBI
- Google Scholar
22. Nielsen KK, Boye M. Real-time quantitative reverse transcription-PCR analysis of expression stability of Actinobacillus pleuropneumoniae housekeeping genes during in vitro growth under iron-depleted conditions. Applied and environmental microbiology. 2005;71(6):2949–54. pmid:15932989
- View Article
- PubMed/NCBI
- Google Scholar
23. Grubman A, Phillips A, Thibonnier M, Kaparakis-Liaskos M, Johnson C, Thiberge JM, et al. Vitamin B6 is required for full motility and virulence in Helicobacter pylori. mBio. 2010;1(3).
- View Article
- Google Scholar
24. Chiang CH, Huang WF, Huang LP, Lin SF, Yang WJ. Immunogenicity and protective efficacy of ApxIA and ApxIIA DNA vaccine against Actinobacillus pleuropneumoniae lethal challenge in murine model. Vaccine. 2009;27(34):4565–70. pmid:19520199
- View Article
- PubMed/NCBI
- Google Scholar
25. Seo KW, Kim SH, Park J, Son Y, Yoo HS, Lee KY, et al. Nasal immunization with major epitope-containing ApxIIA toxin fragment induces protective immunity against challenge infection with Actinobacillus pleuropneumoniae in a murine model. Veterinary immunology and immunopathology. 2013;151(1–2):102–12. pmid:23200821
- View Article
- PubMed/NCBI
- Google Scholar
26. Nemzek JA, Xiao HY, Minard AE, Bolgos GL, Remick DG. Humane endpoints in shock research. Shock. 2004;21(1):17–25. Epub 2003/12/17. pmid:14676679
- View Article
- PubMed/NCBI
- Google Scholar
27. Reed L, Muench H. A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol.; 1938. p. 493–7.
- View Article
- Google Scholar
28. Belitsky BR. Physical and enzymological interaction of Bacillus subtilis proteins required for de novo pyridoxal 5'-phosphate biosynthesis. Journal of bacteriology. 2004;186(4):1191–6. pmid:14762015
- View Article
- PubMed/NCBI
- Google Scholar
29. Neuwirth M, Flicker K, Strohmeier M, Tews I, Macheroux P. Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase. Biochemistry. 2007;46(17):5131–9. pmid:17408246
- View Article
- PubMed/NCBI
- Google Scholar
30. Wallner S, Neuwirth M, Flicker K, Tews I, Macheroux P. Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis. Biochemistry. 2009;48(9):1928–35. pmid:19152323
- View Article
- PubMed/NCBI
- Google Scholar
31. Gonzalez E, Danehower D, Daub ME. Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway. Plant physiology. 2007;145(3):985–96. pmid:17873088
- View Article
- PubMed/NCBI
- Google Scholar
32. Denslow SA, Rueschhoff EE, Daub ME. Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress. Plant physiology and biochemistry: PPB / Societe francaise de physiologie vegetale. 2007;45(2):152–61.
- View Article
- Google Scholar
33. Chen H, Xiong L. Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses. The Plant journal: for cell and molecular biology. 2005;44(3):396–408.
- View Article
- Google Scholar
34. Vandekamp JL, Westrick JA, Smolen A. B-6 Vitamer Concentrations in Mouse Plasma, Erythrocytes and Tissues. Nutr Res. 1995;15(3):415–22.
- View Article
- Google Scholar
35. Sheng H, Lim JY, Watkins MK, Minnich SA, Hovde CJ. Characterization of an Escherichia coli O157:H7 O-antigen deletion mutant and effect of the deletion on bacterial persistence in the mouse intestine and colonization at the bovine terminal rectal mucosa. Applied and environmental microbiology. 2008;74(16):5015–22. pmid:18552194
- View Article
- PubMed/NCBI
- Google Scholar
36. Asakura H, Hashii N, Uema M, Kawasaki N, Sugita-Konishi Y, Igimi S, et al. Campylobacter jejuni pdxA affects flagellum-mediated motility to alter host colonization. PloS one. 2013;8(8):e70418. pmid:23936426
- View Article
- PubMed/NCBI
- Google Scholar
37. Knockel J, Muller IB, Butzloff S, Bergmann B, Walter RD, Wrenger C. The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference. The Biochemical journal. 2012;443(2):397–405. pmid:22242896
- View Article
- PubMed/NCBI
- Google Scholar
38. Ma W, Cao W, Zhang B, Chen K, Liu Q, Li Y, et al. Engineering a pyridoxal 5'-phosphate supply for cadaverine production by using Escherichia coli whole-cell biocatalysis. Scientific reports. 2015;5:15630. pmid:26490441
- View Article
- PubMed/NCBI
- Google Scholar
39. Yang Y, Tsui HC, Man TK, Winkler ME. Identification and function of the pdxY gene, which encodes a novel pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate biosynthesis in Escherichia coli K-12. Journal of bacteriology. 1998;180(7):1814–21. pmid:9537380
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Mooney S, Leuendorf JE, Hendrickson C, Hellmann H. Vitamin B6: a long known compound of surprising complexity. Molecules. 2009;14(1):329–51. pmid:19145213
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Eliot AC, Kirsch JF. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annual review of biochemistry. 2004;73:383–415. pmid:15189147
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Percudani R, Peracchi A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO reports. 2003;4(9):850–4. pmid:12949584
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Percudani R, Peracchi A. The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families. BMC bioinformatics. 2009;10:273. pmid:19723314
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Fitzpatrick TB, Amrhein N, Kappes B, Macheroux P, Tews I, Raschle T. Two independent routes of de novo vitamin B6 biosynthesis: not that different after all. The Biochemical journal. 2007;407(1):1–13. pmid:17822383
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Mukherjee T, Hanes J, Tews I, Ealick SE, Begley TP. Pyridoxal phosphate: biosynthesis and catabolism. Biochimica et biophysica acta. 2011;1814(11):1585–96. pmid:21767669
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Tambasco-Studart M, Titiz O, Raschle T, Forster G, Amrhein N, Fitzpatrick TB. Vitamin B6 biosynthesis in higher plants. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(38):13687–92. pmid:16157873
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Dick T, Manjunatha U, Kappes B, Gengenbacher M. Vitamin B6 biosynthesis is essential for survival and virulence of Mycobacterium tuberculosis. Molecular microbiology. 2010;78(4):980–8. pmid:20815826
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. El Qaidi S, Yang J, Zhang JR, Metzger DW, Bai G. The vitamin B(6) biosynthesis pathway in Streptococcus pneumoniae is controlled by pyridoxal 5'-phosphate and the transcription factor PdxR and has an impact on ear infection. Journal of bacteriology. 2013;195(10):2187–96. pmid:23475965
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Raschle T, Amrhein N, Fitzpatrick TB. On the two components of pyridoxal 5'-phosphate synthase from Bacillus subtilis. The Journal of biological chemistry. 2005;280(37):32291–300. pmid:16030023
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Chiers K, De Waele T, Pasmans F, Ducatelle R, Haesebrouck F. Virulence factors of Actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host. Veterinary research. 2010;41(5):65. pmid:20546697
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Bosse JT, Janson H, Sheehan BJ, Beddek AJ, Rycroft AN, Kroll JS, et al. Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes and infection / Institut Pasteur. 2002;4(2):225–35.
View Article
Google Scholar

[46] View Article

[47] Google Scholar

[ref13] 13. Li G, Xie F, Zhang Y, Wang C. Draft genome sequence of Actinobacillus pleuropneumoniae serotype 7 strain S-8. Journal of bacteriology. 2012;194(23):6606–7. pmid:23144372
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref14] 14. Xie F, Zhang Y, Li G, Zhou L, Liu S, Wang C. The ClpP protease is required for the stress tolerance and biofilm formation in Actinobacillus pleuropneumoniae. PloS one. 2013;8(1):e53600. pmid:23326465
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref15] 15. Wagner TK, Mulks MH. A subset of Actinobacillus pleuropneumoniae in vivo induced promoters respond to branched-chain aminoacid limitation. FEMS Immunology and Medical Microbiology. 2006; 48(2):192–204. pmid:16995880
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref16] 16. Chen L, Dang G, Deng X, Cao J, Yu S, Wu D, et al. Characterization of a novel exported esterase Rv3036c from Mycobacterium tuberculosis. Protein expression and purification. 2014;104:50–6. pmid:25224799
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref17] 17. Strohmeier M, Raschle T, Mazurkiewicz J, Rippe K, Sinning I, Fitzpatrick TB, et al. Structure of a bacterial pyridoxal 5'-phosphate synthase complex. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(51):19284–9. pmid:17159152
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref18] 18. Baltes N, Tonpitak W, Hennig-Pauka I, Gruber AD, Gerlach GF. Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence. FEMS microbiology letters. 2003;220(1):41–8. pmid:12644226
View Article
PubMed/NCBI
Google Scholar

[69] View Article

[70] PubMed/NCBI

[71] Google Scholar

[ref19] 19. Dehio C, Meyer M. Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli. Journal of bacteriology. 1997;179(2):538–40. pmid:8990308
View Article
PubMed/NCBI
Google Scholar

[73] View Article

[74] PubMed/NCBI

[75] Google Scholar

[ref20] 20. Oswald W, Tonpitak W, Ohrt G, Gerlach G. A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker. FEMS microbiology letters. 1999;179(1):153–60. pmid:10481100
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref21] 21. Willson PJ, Albritton WL, Slaney L, Setlow JK. Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi. Antimicrobial Agents and Chemotherapy. 1989; 33(9):1627–30. pmid:2684012
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref22] 22. Nielsen KK, Boye M. Real-time quantitative reverse transcription-PCR analysis of expression stability of Actinobacillus pleuropneumoniae housekeeping genes during in vitro growth under iron-depleted conditions. Applied and environmental microbiology. 2005;71(6):2949–54. pmid:15932989
View Article
PubMed/NCBI
Google Scholar

[85] View Article

[86] PubMed/NCBI

[87] Google Scholar

[ref23] 23. Grubman A, Phillips A, Thibonnier M, Kaparakis-Liaskos M, Johnson C, Thiberge JM, et al. Vitamin B6 is required for full motility and virulence in Helicobacter pylori. mBio. 2010;1(3).
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref24] 24. Chiang CH, Huang WF, Huang LP, Lin SF, Yang WJ. Immunogenicity and protective efficacy of ApxIA and ApxIIA DNA vaccine against Actinobacillus pleuropneumoniae lethal challenge in murine model. Vaccine. 2009;27(34):4565–70. pmid:19520199
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

[ref25] 25. Seo KW, Kim SH, Park J, Son Y, Yoo HS, Lee KY, et al. Nasal immunization with major epitope-containing ApxIIA toxin fragment induces protective immunity against challenge infection with Actinobacillus pleuropneumoniae in a murine model. Veterinary immunology and immunopathology. 2013;151(1–2):102–12. pmid:23200821
View Article
PubMed/NCBI
Google Scholar

[96] View Article

[97] PubMed/NCBI

[98] Google Scholar

[ref26] 26. Nemzek JA, Xiao HY, Minard AE, Bolgos GL, Remick DG. Humane endpoints in shock research. Shock. 2004;21(1):17–25. Epub 2003/12/17. pmid:14676679
View Article
PubMed/NCBI
Google Scholar

[100] View Article

[101] PubMed/NCBI

[102] Google Scholar

[ref27] 27. Reed L, Muench H. A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol.; 1938. p. 493–7.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref28] 28. Belitsky BR. Physical and enzymological interaction of Bacillus subtilis proteins required for de novo pyridoxal 5'-phosphate biosynthesis. Journal of bacteriology. 2004;186(4):1191–6. pmid:14762015
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref29] 29. Neuwirth M, Flicker K, Strohmeier M, Tews I, Macheroux P. Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase. Biochemistry. 2007;46(17):5131–9. pmid:17408246
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref30] 30. Wallner S, Neuwirth M, Flicker K, Tews I, Macheroux P. Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis. Biochemistry. 2009;48(9):1928–35. pmid:19152323
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref31] 31. Gonzalez E, Danehower D, Daub ME. Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway. Plant physiology. 2007;145(3):985–96. pmid:17873088
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref32] 32. Denslow SA, Rueschhoff EE, Daub ME. Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress. Plant physiology and biochemistry: PPB / Societe francaise de physiologie vegetale. 2007;45(2):152–61.
View Article
Google Scholar

[123] View Article

[124] Google Scholar

[ref33] 33. Chen H, Xiong L. Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses. The Plant journal: for cell and molecular biology. 2005;44(3):396–408.
View Article
Google Scholar

[126] View Article

[127] Google Scholar

[ref34] 34. Vandekamp JL, Westrick JA, Smolen A. B-6 Vitamer Concentrations in Mouse Plasma, Erythrocytes and Tissues. Nutr Res. 1995;15(3):415–22.
View Article
Google Scholar

[129] View Article

[130] Google Scholar

[ref35] 35. Sheng H, Lim JY, Watkins MK, Minnich SA, Hovde CJ. Characterization of an Escherichia coli O157:H7 O-antigen deletion mutant and effect of the deletion on bacterial persistence in the mouse intestine and colonization at the bovine terminal rectal mucosa. Applied and environmental microbiology. 2008;74(16):5015–22. pmid:18552194
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref36] 36. Asakura H, Hashii N, Uema M, Kawasaki N, Sugita-Konishi Y, Igimi S, et al. Campylobacter jejuni pdxA affects flagellum-mediated motility to alter host colonization. PloS one. 2013;8(8):e70418. pmid:23936426
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref37] 37. Knockel J, Muller IB, Butzloff S, Bergmann B, Walter RD, Wrenger C. The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference. The Biochemical journal. 2012;443(2):397–405. pmid:22242896
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref38] 38. Ma W, Cao W, Zhang B, Chen K, Liu Q, Li Y, et al. Engineering a pyridoxal 5'-phosphate supply for cadaverine production by using Escherichia coli whole-cell biocatalysis. Scientific reports. 2015;5:15630. pmid:26490441
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref39] 39. Yang Y, Tsui HC, Man TK, Winkler ME. Identification and function of the pdxY gene, which encodes a novel pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate biosynthesis in Escherichia coli K-12. Journal of bacteriology. 1998;180(7):1814–21. pmid:9537380
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Ethics statement

Bacterial strains and growth conditions

Protein expression and purification

Glutaminase activity assay

PLP formation assay

Construction of gene deletion mutants and complemented strains

RNA isolation and qRT-PCR

In vitro growth assays

In vitro stress assays

Transmission electron microscopy

Mouse in vivo experiments

Enumeration of bacterial load in organs

Statistical analysis

Results

PdxS and PdxT are involved in PLP synthesis in A. pleuropneumoniae

A. pleuropneumoniae S-8ΔpdxS and S-8ΔpdxT mutants are auxotrophic for PLP

The PLP synthesis activity of PdxS is not dependent on PdxT in the presence of ammonium

A. pleuropneumoniae S-8ΔpdxS and S-8ΔpdxT mutants are sensitive to NaCl and H2O2

Loss of pdxS and pdxT leads to morphological defects of A. pleuropneumoniae

Loss of pdxS attenuates the virulence of A. pleuropneumoniae in the BALB/c mouse model

Discussion

Supporting information

S1 Fig. SDS—PAGE analysis of the recombinant PdxS and PdxT proteins purified with affinity chromatography.

S2 Fig. The relative transcription levels of selected genes.

S3 Fig. Viable counts of A. pleuropneumoniae mutants exposed to oxidative and osmotic stresses.

S4 Fig. Transmission electron microscopy of A. pleuropneumoniae mutants in the presence of PLP supplementation.

S1 Table. Characteristics of bacterial strains, plasmids, and primers used in this study.

S2 Table. Primers used in qRT-PCR study.

Acknowledgments

Author Contributions

References

A. pleuropneumoniae S-8ΔpdxS and S-8ΔpdxT mutants are sensitive to NaCl and H₂O₂