Active invasion of bacteria into living fungal cells

  1. Nadine Moebius
  2. Zerrin Üzüm
  3. Jan Dijksterhuis
  4. Gerald Lackner
  5. Christian Hertweck  Is a corresponding author
  1. Leibniz Institute for Natural Product Research and Infection Biology, Germany
  2. CBS-KNAW Fungal Biodiversity Centre, Netherlands
  3. Friedrich Schiller University, Germany
8 figures and 4 tables


Microscopic image of Burkholderia rhizoxinica (green) residing in the cytosol of Rhizopus microsporus.

The GFP encoding B. rhizoxinica cells can re-colonize the sterile R. microsporus, then induce fungal sporulation. The endobacterium is transmitted via fungal vegetative spores (upper right corner).
Schematic view of the organization of the type 2 secretion system (T2SS) gene clusters from various bacterial species.

The T2SS gene loci of the two R. microsporus endosymbiotic bacterial strains B. rhizoxinica and Burkholderia endofungorum, as well as the squid endosymbiont Vibrio fischeri, Escherichia coli, and the human pathogens Burkholderia pseudomallei and Pseudomonas aeruginosa are displayed here. The spaces between the arrows represent non-adjacent genes (single genes are located further away on the genome), while lines indicate closely linked genes.
Figure 3 with 3 supplements
Photographs and microscopy images of R. microsporus hyphae several days after inoculation with B. rhizoxinica wt or mutant strains in 6-well plates.

The pictures present the infection of R. microsporus with wt or mutant B. rhizoxinica in the following order: (A) B. rhizoxinica wt, (B) B. rhizoxinica Δchi::Kanr, (C) B. rhizoxinica ΔgspC::Kanr, (D)—B. rhizoxinica ΔgspD::Kanr, (E)—control (no bacteria added). Spore formation is visible in (A), while spore formation was not detected in (BE) even after 5 days of co-incubation.
Figure 3—figure supplement 1
Knock out strategy of site directed mutagenesis to B. rhizoxinica.

(A)—Illustration of PCR strategy and primers used to confirm mutant genotypes. (B)—Confirmation of B. rhizoxinica ΔgspD::Kanr by gel electrophoresis of PCR products amplified from genomic DNA of B. rhizoxinica ΔgspD::Kanr or wt, respectively. Primers were combined in individual PCR reactions as indicated in above. (C) Confirmation of B. rhizoxinica ΔgspC::Kanr by PCR. (D) Confirmation of B. rhizoxinica Δchi::Kanr by PCR. (E) Confirmation of B. rhizoxinica Δcbp::Kanr by PCR. (F) Confirmation of B. rhizoxinica Δchts::Kanr by PCR. (G) Confirmation of B. rhizoxinica Δchi::Kanr by PCR.
Figure 3—figure supplement 2
Fluorescence microscopy carried out with B. rhizoxinica wt and mutant strains B. rhizoxinica ΔgspD, B. rhizoxinica ΔgspC and B. rhizoxinica Δchi in a 3 day co-culture with sterile R. microsporus.

Whereas wt bacteria clearly localize within the fungal hyphae, no endobacteria carrying mutations in either the chitinase gene or the components of the T2SS are detectable within the living hyphae (upper part). Scale bar represents 10 µm.
Figure 3—figure supplement 3
Lytic potential of B. rhizoxinica secretome.

(A)—Proteolytic assay using skim milk agar plates. Aliquots of the supernatants of B. rhizoxinica wt, B. rhizoxinica ΔgspD::Kanr, B. rhizoxinica ΔgspC::Kanr, respectively are incubated on filter disks on the plate. (B)—Chitinolytic plate assay using B. rhizoxinica culture supernatant on agar plate supplemented with 0.05% colloidal chitin and stained with Calcofluor.
2D gel analysis of the secretomes of wild type and mutants.

(A) B. rhizoxinica wt, (B) B. rhizoxinica ΔgspC::Kanr, (C) B. rhizoxinica ΔgspD::Kanr and (D) B. rhizoxinica wt in co-culture with R. microsporus. Chitin-binding protein (I-5, III-14, III-15, IV-6, II-5), chitinase (I-6, III-9) and chitosanase (I-3, I-4, III-10, III-11, IV-3, IV-4, IV-5, II-3) were identified. List of identified proteins is shown in Table 2. In general, each gel was loaded with 100 μg of TCA-precipitated proteins from the culture supernatant.
Figure 5 with 1 supplement
Functional analyses of chitinolytic enzymes.

(A) Chitinase activity in cell-free culture supernatants of an E. coli harboring recombinant chitinase from B. rhizoxinica after incubation for 30, 60, and 90 min, in comparison to the activity of a recombinant S. lividans enzyme. Error bars indicate the standard deviation of three individual experiments. (B) Calcofluor staining of R. microsporus hyphae. (C) Phylogenetic analysis of chitinases from the GH family 18; protein sequences were retrieved from NCBI and comprise subfamily A and B sequences. (D) Chitin-binding assay performed using acid released crab shell chitin and the supernatant of B. rhizoxinica wt. SDS page of the non-bound fraction (S), the bound protein fraction (F1) and the pelleted chitin with the rest of the bound protein (F2). The three indicated proteins were identified using MALDI-TOF. (E) Gene expression assay for T2SS and chitinolytic proteins. The expression of T2SS genes gspC and gspD as well as chi, cbp and chts in B. rhizoxinica were monitored using RT qPCR in pure culture, in co-cultivation with a cured host (R. microsporus) and after re-infection of the cured host. The gene rpoB was used as an internal standard for the calculation of expression levels and normalization. The expression of all five genes is substantially increased in the wt during co-cultivation, while expression levels after re-infection decreased to nearly the level in pure wt culture. Error bars indicate standard deviation.
Figure 5—figure supplement 1
Multiple alignment of the chitinase protein sequences was performed based on the three-dimensional structure of the ChiB sequence from S. marcescens (pdb1e15) and Chi from P. furiosus (2dsk).

Red and blue characters indicate α-helices and β-strands, respectively. The DXDXE motif is highlighted in yellow. Alignment was created using PROMALS3D. The CID motif of subfamily A chitinases in S. marcescens in underlined in orange, it is absent in B. rhizoxinica and P. furiosus sequences.
Course of infection of B. rhizoxinica (Br) to R. microsporus (Rm) observed by scanning cryo-electron microscopy.

(AD) Attachment/adherence of bacteria to fungal hyphae after 1 hr and (FH) 20 hr of co-cultivation; (CD, FH) bacterial and fungal cell walls start to merge; (D) fibrillar structures connecting a bacterial cell to the hyphal surface; (EH) fusion of cell walls and the intrusion of bacterial cells into the fungal hyphae. White arrows mark areas of particular interest. (IJ) Scanning electron microscopy of a co-culture of sterile R. microsporus with B. rhizoxinica ΔgspD (I) and B. rhizoxinica Δchi (J). Attachment of both mutant strains to the hyphal surface is visible, however, no intimate contact or fusion events could be observed. Scale bars represent 5 µm.
Evidence for the lack of active engulfment of B. rhizoxinica by R. microsporus.

(A) Bacteria (green) attaches to fungal hyphae within 1 hr. (B) The endocytotic activity of the fungus can be observed by the red vesicles that are present all around the hyphae and highly accumulated at the apical tip. After the infection, no fungal membrane is visible around the bacterium.
Model of processes involved in bacterial invasion.

Chitinase as well as other effector proteins are secreted via a bacterial T2SS and induce a local dissolution of the fungal cell wall. This enables bacteria to enter and colonize the fungal cell and induce sporulation.


Table 1

Annotation of the B. rhizoxinica T2SS gene cluster
GeneProposed function of encoded proteinHomolog in Klebsiella oxytocaPercent identity (BlastP)Homolog in Burkholderia pseudomalleiPercent identity (BlastP)
gspCConnecting inner and outer membrane complexpulCgspC61
gspDSecretin, outer membrane pore formationpulD39gspD64
gspECytoplasmitc ATPase, energy for translocation of pseudopilinspulE54gspE89
gspFAnchoring protein, inner membrane platform for pseudopilinspulF46gspF83
gspGMajor prepilin-like protein, pilus-like structure formationpulG56gspG87
gspHPseudopilin subunit, pilus-like structure formationpulH70gspH66
gspIPseudopilin subunit, pilus-like structure formationpulI44gspI66
gspJPseudopilin subunit, pilus-like structure formationpulJ43gspJ55
gspKPseudopilin subunit, pilus-like structure formationpulK26gspK62
gspLAnchoring protein, inner membrane platform for pseudopilinspulL27gspL57
gspMAnchoring protein, inner membrane platform for pseudopilinspulM24gspM56
gspNConnecting inner and outer membrane complexpulN29gspN63
gspOPrepilin, inner membrane peptidasepulO43gspO60
Table 2

Secretome proteins identified by MALDI-TOF
Spot no.Accesion no. (NCBI)Protein indetificationTotal Mass0 (kDA)Total pIMascot scoreMatching peptidesSequence coverage (%)SignalP prediction (y/n)
I-1gi│312169059Glutamate/aspartate-binding protein32.49.664.21340.1y
I-2gi│312169059Glutamate/aspartate-binding protein32.49.6114.01849.5y
I-3gi│312167773Chitosanase (EC
I-4gi│312167773Chitosanase (EC
I-5gi│312168534Chitin-binding protein27.07.785.2849.2y
I-6gi│312168091Chitinase (EC
II-1gi│312169059Glutamate/aspartate-binding protein32.49.6107.02051.5y
II-2gi│312169059Glutamate/aspartate-binding protein32.49.6114.01846.8y
II-3gi│312167773Chitosanase (EC
II-4gi│312168534Chitin-binding protein27.07.780.4729.1y
III-1gi│312169059Glutamate/aspartate-binding protein32.49.6133.02051.9y
III-2gi│312169059Glutamate/aspartate-binding protein32.49.696.21951.2y
III-3gi│312167620Toluene transport system Ttg2d protein23.79.569.0530.5y
III-4gi│312168022Ribosome recycling factor (RRF)
III-5gi│312169310Adenosylhomocysteinase (EC
III-6gi│312166966Chaperone protein DnaK69.74.9222.03048.2n
III-7gi│31216888860 kDa chaperonin GroEL57.45.197.71641.0n
III-8gi│312169323S-adenosylmethionine synthetase (EC
III-9gi│312167529Protein translation elongation factor Tu (EF-Tu)43.15.2148.02266.9n
III-10gi│312167773Chitosanase (EC
III-11gi│312167773Chitosanase (EC
III-12gi│312168091Chitinase (EC
III-13gi│312168185Peptidyl-prolyl cis-trans isomerase (EC
III-14gi│312168534Chitin-binding protein27.07.7101.01259.8y
III-15gi│312168534Chitin-binding protein27.07.760.6534.8y
III-16gi│312167051Superoxide dismutase (EC
III-17gi│312168185Peptidyl-prolyl cis-trans isomerase (EC
III-18gi│312167149Inorganic pyrophosphatase (EC
III-19gi│31216779534 kDa membrane antigen precursor21.86.885.51360.7y
IV-1gi│312169059Glutamate/aspartate-binding protein32.49.6151.02362.6y
IV-2gi│312169059Glutamate/aspartate-binding protein32.49.698.61545.5y
IV-3gi│312167773Chitosanase (EC
IV-4gi│312168534Chitin-binding protein27.07.795.3737.3y
Table 3

Bacterial and fungal strains
Burkholderia rhizoxinica HKI-0454Wild type, isolated from Rhizopus microsporus ATCC62417*
Rhizopus microsporus ATCC62417Fungal host harboring bacterial endosymbionts, isolated from rice seedlings
B. rhizoxinica ΔgspD::KanrT2SS mutant B. rhizoxinica with deletion of gspDThis study
B. rhizoxinica ΔgspC::KanrT2SS mutant B. rhizoxinica with deletion of gspCThis study
B. rhizoxinica Δchit::KanrB. rhizoxinica with deletion of chitinase geneThis study
B. rhizoxinica Δcbp::KanrB. rhizoxinica with deletion of chitin-binding protein geneThis study
B. rhizoxinica Δchts::KanrB. rhizoxinica with deletion of chitosanase geneThis study
B. rhizoxinica/pHKT4B. rhizoxinica wt harboring a RFP expression vectorThis study
B. rhizoxinica ΔgspD::Kanr/pHKT2B. rhizoxinica gspD mutant harboring a GFP expression vectorThis study
B. rhizoxinica ΔgspC::Kanr/pHKT2B. rhizoxinica gspC mutant harboring a GFP expression vectorThis study
B. rhizoxinica Δchit::Kanr/pHKT2B. rhizoxinica chit mutant harboring a GFP expression vectorThis study
B. rhizoxinica Δcbp::Kanr/pHKT2B. rhizoxinica cbp mutant harboring a GFP expression vectorThis study
B. rhizoxinica Δchts::Kanr/pHKT2B. rhizoxinica chts mutant harboring a GFP expression vectorThis study
R. microsporus + B. rhizoxinica ΔgspD::Kanr/pHKT2Reinfected cured fungal host with gspD mutant harboring a GFP expressing vectorThis study
R. microsporus + B. rhizoxinica ΔgspC::Kanr/pHKT2Reinfected cured fungal host with gspC mutant harboring a GFP expressing vectorThis study
R. microsporus + B. rhizoxinica Δchit::Kanr/pHKT2Reinfected cured fungal host with chit mutant harboring a GFP expressing vectorThis study
R. microsporus + B. rhizoxinica Δcbp::Kanr/pHKT2Reinfected cured fungal host with cbp mutant harboring a GFP expressing vectorThis study
R. microsporus + B. rhizoxinica Δchts::Kanr/pHKT2Reinfected cured fungal host with chts mutant harboring a GFP expressing vectorThis study
Escherichia coli BL21(DE3)/pET28a-ChiE. coli with expression vector harboring chitinase geneThis study
  1. *

    Partida-Martinez and Hertweck (2005) Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 437:884–888.

  2. Ibaragi (1973) Studies on rice seedling blight. I. Growth injury caused by Rhizopus sp. under high temperature. Ann. Phytopathol. Soc. Jpn 39:141–144.

Table 4

Primers used in this study
NameOligo sequence
Primers used for generating KO mutantsTII_D_fl1_fw5′-GCTACGGATCCCTGCCAGGTATTGCCGTATT-3′
Primers used for RT-qPCR studiesTIISS_D_RT_F5′-GAGCAGCGATACCAACATCC-3′
Primers used for control of generated mutantsArm_A_rv (Kan)5′-AGTGACAACGTCGAGCACAG-3′

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  1. Nadine Moebius
  2. Zerrin Üzüm
  3. Jan Dijksterhuis
  4. Gerald Lackner
  5. Christian Hertweck
Active invasion of bacteria into living fungal cells
eLife 3:e03007.