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. 2024 Sep 4;20(9):e1012533.
doi: 10.1371/journal.ppat.1012533. eCollection 2024 Sep.

Stenotrophomonas maltophilia uses a c-di-GMP module to sense the mammalian body temperature during infection

Yan Wang  1   2 , Kai-Ming Wang  1   3 , Xin Zhang  4 , Wenzhao Wang  5 , Wei Qian  1   6 , Fang-Fang Wang  1   6
Affiliations

Stenotrophomonas maltophilia uses a c-di-GMP module to sense the mammalian body temperature during infection

Yan Wang et al. PLoS Pathog. .

Abstract

The body temperature of Warm-blooded hosts impedes and informs responses of bacteria accustomed to cooler environments. The second messenger c-di-GMP modulates bacterial behavior in response to diverse, yet largely undiscovered, stimuli. A long-standing debate persists regarding whether a local or a global c-di-GMP pool plays a critical role. Our research on a Stenotrophomonas maltophilia strain thriving at around 28°C, showcases BtsD as a thermosensor, diguanylate cyclase, and effector. It detects 37°C and diminishes c-di-GMP synthesis, resulting in a responsive sequence: the periplasmic c-di-GMP level is decreased, the N-terminal region of BtsD disengages from c-di-GMP, activates the two-component signal transduction system BtsKR, and amplifies sod1-3 transcription, thereby strengthening the bacterium's pathogenicity and adaptation during infections in 37°C warm Galleria mellonella larvae. This revelation of a single-protein c-di-GMP module introduces unrecognized dimensions to the functional and structural paradigms of c-di-GMP modules and reshapes our understanding of bacterial adaptation and pathogenicity in hosts with a body temperature around 37°C. Furthermore, the discovery of a periplasmic c-di-GMP pool governing BtsD-BtsK interactions supports the critical role of a local c-di-GMP pool.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. BtsD enhances pathogenicity and in vivo adaptability of S. maltophilia at 37°C.
(A) G. mellonella larvae killing assay, demonstrating larval mortality following injection of specified bacterial cultures at 28°C and 37°C, respectively. The data averages are presented, with the original data dots and errors shown in S1A Fig. (B) Bacterial count assay, displaying the numbers of viable bacteria after injection and collection from G. mellonella larvae. The column and line charts depicted are representative of three independent experiments with comparable outcomes. Data in the bacterial count assay are presented as mean ± SD of three independent replicates. Statistical significance was evaluated using the two-tailed unpaired Student’s t-test. The p values of each of the specified two groups are presented upside. WT-EV: the wide-type strain bearing empty pBBR1MCS2 vectors; ΔbtsD-EV: the btsD deletion mutant bearing empty pBBR1MCS2 vectors; CbtsD: the complementary strain constitutively expressing btsD in btsD deletion background; CbtsDΔGGDEF: the complementary strain constitutively expressing the recombinant btsDΔGGDEF, with the GGDEF domain deleted, in btsD deletion background.
Fig 2
Fig 2. BtsD is a thermosensor regulating periplasmic levels of c-di-GMP.
(A) The secondary structures of BtsD and its recombinant proteins. SP represents the signal peptide identified by SignalIP 4.0, FN3 represents the FN3 domain under the SMART accession number SM000060, and GGDEF represents the GGDEF domain under the accession number SM000267. The recombinant variants are as follows: BtsDΔSP, with the SP deleted; BtsDΔGGDEF, with the GGDEF domain deleted; BtsDΔ(SP-GGDEF), with both the SP and GGDEF domain deleted; BtsDΔ(SP-FN3), with both the SP and FN3 domain deleted; and BtsDGGDEF containing the only GGDEF domain. (B) Western blot analyses revealed the presence of BtsD in the periplasm of the strain expressing His-tagged wild-type BtsD, as opposed to the strain expressing recombinant BtsDΔSP with the SP deleted. To exclude cross-contamination, the α subunit of RNA polymerase, GL004030, and β-lactamase were used as controls for cytosolic, membrane, and periplasmic proteins, respectively. BtsD: the strain expressing the His-tagged wild-type BtsD; BtsDΔSP: the strain expressing the His-tagged BtsDΔSP. (C) Periplasmic c-di-GMP levels quantified by LC-MS/MS. The isolated periplasmic fractions, verified to be free of contamination by Western blot analysis, were subjected to LC-MS/MS for c-di-GMP concentration measurement. The data are presented as mean ± SD of three independent replicates. Statistical significance was evaluated using the two-tailed unpaired Student’s t-test. The p values for each of the specified two groups are presented upside. Details of the strains are described above. (D) Reduced levels of c-di-GMP generated by purified BtsDΔSP at 37°C compared to 28°C. BtsDΔ(SP-GGDEF) served as the negative control, and bands of DncV products were used to indicate the location of c-di-GMP separated by thin layer chromatography (TLC). DncV is a c-di-GMP synthetase from Escherichia coli. All presented data are representatives of three independent replicates, yielding consistent outcomes.
Fig 3
Fig 3. BtsD triggers the BtsKR to enhance the pathogenicity and in vivo adaptability at 37°C.
(A) Locations of btsD, btsK, and btsR in the genome of S. maltophilia CGMCC 1.1788 and primers used to investigate the operon structure. (B) Secondary structures of both BtsK and BtsR predicted by SMART with the vertical bar indicating the transmembrane region. HAMP, HisKA, HAPase_c, REC, and Trans_reg_C domains were indicated. SensorBtsK denotes the recombinant BtsK containing only the sensor domain, while BtsKΔsensor refers to the recombinant BtsD with its sensor domain deleted. (C) Validation of the operon btsR-btsK-btsD by RT-PCR. RT represents PCR using cDNA reversely transcribed from total RNAs as templates. While -RT served as the negative control using total RNAs without reverse transcription, and DNA as the positive control with genomic DNA as templates. (D) and (E) G. mellonella larvae killing assay, demonstrating larval mortality following injection of specified bacterial cultures at 28°C and 37°C, respectively. For (D) and (E), the data averages are presented, with the original data dots and errors shown in S1B and S1C Fig, respectively. (F) Bacterial count assay, displaying the numbers of viable bacteria after injection and collection from G. mellonella larvae. Data in (F) are presented as mean ± SD of three independent replicates. Statistical significance was evaluated using the two-tailed unpaired Student’s t-test, with p < 0.05 indicating a statistically significant difference. all the shown data are representative of three independent experiments with comparable outcomes. WT-EV is described above. ΔbtsK-EV and ΔbtsR-EV: the btsK and btsR deletion mutant carrying empty pBBR1MCS2 vectors; CbtsK and CbtsR: the complementary strains constitutively expressing btsK and btsR in btsK and btsR deletion background, respectively; CbtsKH264A and CbtsRD58A: the complementary strains constitutively expressing the recombinant btsK with its His264-encoding sequences replaced by an Ala-encoding sequences and the recombinant btsR with its Asp58-encoding sequences replaced by an Ala-encoding sequences; Δ(btsD-btsR)-EV: the mutant with both btsD and btsR deleted, carrying empty pBBR1MCS2 vectors; Δ(btsD-btsR)-CbtsD, Δ(btsD-btsR)-CbtsR, and Δ(btsD-btsR)-CbtsRD58A: the complementary strains constitutively expressing btsD, btsR, and btsRD58A in a background where both btsD and btsR are deleted, respectively.
Fig 4
Fig 4. The BtsD-BtsK-BtsR pathway upregulates sod1-3 transcription, enhancing pathogenicity and in vivo adaptability at 37°C.
(A) G. mellonella larvae killing assay, demonstrating larval mortality following injection of specified bacterial cultures at 28°C and 37°C, respectively. The data averages are presented, with the original data dots and errors shown in S1D Fig. (B) Bacterial count assay, displaying the numbers of viable bacteria after injection and collection from G. mellonella larvae. (C) The ChIP-qPCR assay quantifying binding of His-tagged BtsR or BtsRD58A with the sod1-3 promoter in the presence of BtsD or its recombinant form BtsDΔGGDEF, or in its absence with the non-tagged BtsR as the negative control. The presence or recombination of both BtsD and BtsR in the used strains is denoted below the panel. The recombination of BtsR includes BtsR-his6, the His-tagged BtsR, BtsRD58A-his6, the His-tagged BtsRD58A, and BtsR, the wild type BtsR. BtsD and BtsDΔGGDEF, as described above, are denoted by a + sign to indicate their presence. (D) The transcript levels of sod1-3 quantified by qRT-PCR in the presence or absence of BtsR at 28°C and 37°C. -28 and -37 represent the temperatures used to culture the specified strains. The shown data are representative of three independent experiments with comparable outcomes. Data are presented as mean ± SD of three independent replicates. Statistical significance was evaluated using the two-tailed unpaired Student’s t-test. The p values for each of the specified two groups are presented upside. ΔbtsD-Csod1-3 and Δ(btsD-btsR)-Csod1-3: the strains constitutively expressing the sod1-3 cluster in the backgrounds where btsD or both btsD and btsR are deleted. The other strains are described above.
Fig 5
Fig 5. BtsD reduces BtsKR phosphorylation by enabling its N-terminal region to bind with BtsK′s sensor domain.
(A) BtsK and BtsR constitutes a TCS. The upper panel shows phosphorylation bands of BtsK and BtsR. BtsK was incubated with excessive ATP to evaluate its autokinase activity, while BtsR was incubated with phosphorylated BtsK to access the phosphotransfer from BtsK to BtsR. BtsKH264A and BtsRD58A served as negative controls. The lower panel presents a Coomassie brilliant blue-stained gel indicating the protein amounts used in the assay. (B) BtsDΔSP inhibited the autokinase activity of BtsK by interacting with its sensor domain, which was reversed by preincubating BtsDΔSP with c-di-GMP. Preincubation of BtsK with BtsDΔSP or the preincubated BtsDΔSP and c-di-GMP mixture was conducted before the autophosphorylation assay. All reactions were conducted at 37°C, except for one reaction specified to be 28°C. (C) The N-terminal region of BtsD (BtsDΔ(SP-GGDEF)) bound with both c-di-GMP and the sensor domain of BtsK (SensorBtsK), and preincubation of BtsDΔ(SP-GGDEF) with c-di-GMP prevented the binding of BtsDΔ(SP-GGDEF) with SensorBtsK. MST assays were used the check binding affinities between indicated molecules. BtsDΔ(SP-GGDEF) & c-di-GMP represents a preincubated mixture of BtsDΔ(SP-GGDEF) and c-di-GMP. Data are presented as mean ± SD of three independent replicates. (D) The N-terminal region of BtsD reduced phosphorylation levels of both BtsK and BtsR, which was reversed by preincubating it with c-di-GMP. Preincubation of BtsDΔ(SP-GGDEF) or the preincubated mixture of BtsDΔ(SP-GGDEF) and c-di-GMP with BtsK or both BtsK and BtsR was done before initiating phosphorylation assays. (E) Unphosphorylated BtsR showed higher binding affinity with the sod1-3 promoter than phosphorylated BtsR. BtsR was incubated with labeled sod1-3 promoter probes with or without the competition of unlabeled probes (cold probes) to access its binding with the sod1-3 promoter. Relative folds of the cold probes to the labeled probes were indicated. Preincubating BtsR with BtsK was done to obtain phosphorylated BtsR, and BtsKH264A served as a negative control for preincubation with BtsR. All the shown data are representative of three independent experiments with comparable outcomes. All the proteins are described above. -P denotes the phosphorylation bands of the specified proteins.
Fig 6
Fig 6. The BtsD-BtsK-BtsR system detects 37°C to regulate S. maltophilia′s pathogenicity and in vivo adaptability.
BtsD acts as a thermosensor, detecting 37°C to decrease its c-di-GMP synthetase activity, reducing the periplasmic c-di-GMP level and thereby releasing its N-terminal region from binding with c-di-GMP. The release allows the N-terminal region to interact with the sensor domain of BtsK, causing the inhibition of BtsK′s autokinase activity, leading to decreased phosphorylation levels of both BtsK and its paired BtsR. Consequently, BtsR binds to the sod1-3 promoter, enhancing sod1-3 transcription and thereby boosting the pathogenicity and in vivo adaptability of S. maltophilia during its invasion of G. mellonella larvae at 37°C.
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F-FW is supported by Science & Technology Fundamental Resources Investigation Program grant 2022FY101100, National Science Foundation of China grant 32070076, and Youth Innovation Promotion Association CAS grant 2020091. The funders had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript.

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