Maria Lara-Tejero's research works | Yale-New Haven Hospital and other places

This page lists works of an author who doesn't have a ResearchGate profile or hasn't added the works to their profile yet. It is automatically generated from public (personal) data to further our legitimate goal of comprehensive and accurate scientific recordkeeping. If you are this author and want this page removed, please let us know.

Tetrameric PilZ protein stabilizes stator ring in complex flagellar motor and is required for motility in Campylobacter jejuni

Article

December 2024

11 Reads

Proceedings of the National Academy of Sciences

[...]

Rotation of the bacterial flagellum, the first identified biological rotary machine, is driven by its stator units. Knowledge gained about the function of stator units has increasingly led to studies of rotary complexes in different cellular pathways. Here, we report that a tetrameric PilZ family protein, FlgX, is a structural component underneath the stator units in the flagellar motor of Campylobacter jejuni . FlgX forms a stable tetramer that does not bind cyclic di-GMP (c-di-GMP), unlike other canonical PilZ domain–containing proteins. Cryoelectron tomography and subtomogram averaging of flagellar motors in situ provide evidence that FlgX interacts with each stator unit and plays a critical role in stator ring assembly and stability. Furthermore, FlgX is conserved and was most likely present in the common ancestor of the phylum Campylobacterota . Overall, FlgX represents a divergence in function for PilZ superfamily proteins as well as a player in the key stator–rotor interaction of complex flagellar motors.

Cryo-EM structure of the bacterial effector protein SipA bound to F-actin reveals a unique mechanism for filament stabilization

December 2023

16 Reads

The bacterial pathogen Salmonella spp. modulates cellular processes by delivering effector proteins through its type III secretion systems. Among these effectors, SipA facilitates bacterial invasion and promotes intestinal inflammation. The mechanisms by which this effector carries out these functions are incompletely understood although SipA's ability to modulate actin dynamics is central to some of these activities. Here we report the cryo-EM structure of SipA bound to filamentous actin. We show that this effector stabilizes actin filaments through unique interactions of its carboxy terminal domain with four actin subunits. Furthermore, our structure-function studies revealed that SipA's actin-binding activity is independent from its ability to stimulate intestinal inflammation. Overall, these studies illuminate critical aspects of Salmonella pathogenesis, and provide unique insight into the mechanisms by which a bacterial effector modulates actin dynamics.

Download

LRRK2 is a component of the RAB32-dependent host defence pathway against Salmonella
a, Western blot analysis of cell lysates of parental and CRISPR–Cas9-generated Lrrk2⁻/⁻ Raw264.7 or DC2.4 cells. IFNγ: interferon gamma. b,c, Raw264.7 or DC2.4 parental (control) and Lrrk2⁻/⁻ cells were infected with either wild-type S. Typhi (b) (MOI 6) or S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI 3) (c) encoding a luciferase-based itaconate biosensor, and the levels of luciferase in the cell lysates were measured 20 h after infection. Each circle or square represents a single luciferase measurement. The mean ± s.d. and P values of the indicated comparisons (unpaired two-tailed Student’s t-test) are shown (n = 6 for each category). Nluc: nanoluciferase. d, Alternatively, Raw264.7 parental (control) and Lrrk2⁻/⁻ cells were infected with S. Typhi strains encoding an eGFP-based itaconate biosensor (MOI 6) and the percentage of bacterial cells expressing eGFP were determined 5 h after infection. Each square and circle represent the mean of an individual experiment in which at least 200 infected cells were examined. The P value (unpaired two-tailed Student’s t-test) of the indicated comparison is shown. Infected cells were fixed, stained with 4,6-diamidino-2-phenylindole (DAPI) (blue) to visualize nuclei, and stained with an anti-Salmonella LPS antibody along with Alexa 594-conjugated anti-rabbit antibody (red) to visualize all bacteria. Representative fields of infected cells are shown (scale bar, 5 µm). e, Itaconate levels in BMDMs obtained from the indicated mice before and after LPS treatment to induce the expression of IRG1. Values represent the mean ± s.d. of three independent measurements (n = 3 for each category). f–k, Raw264.7 or DC2.4 parental (control) and Lrrk2⁻/⁻ cells, as well as BMDMs from C57BL/6 or Lrrk2⁻/⁻ mice, were infected with either wild-type S. Typhi (MOI 6), wild-type S. Typhimurium (MOI 3) or an S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI 3) (as indicated) and the number of CFUs was determined 20 h after infection. Each square or circle represents the CFU in an independent measurement. The mean ± s.d. and P values (unpaired two-tailed Student’s t-test) of the indicated comparisons are shown (n = 6 for each category). l,m, C57BL/6 or Lrrk2⁻/⁻ mice were intraperitoneally infected with wild-type S. Typhimurium (l) or the ∆gtgE ∆sopD2 isogenic mutant derivative (m) (10² CFU), and 4 days after infection, bacterial loads in the spleen of the infected animals were determined. Each circle or square represents the CFU of the spleen of an individual animal. The mean ± s.d. and P values (unpaired two-tailed Student’s t-test) of the indicated comparisons are shown (n = 6 for each category).
Source data

The kinase activity of LRRK2 is required for its contribution to the RAB32-dependent pathogen restriction pathway
a,b, Raw264.7 (a) or HT29 (b) cells were treated with LPS or infected with the indicated bacterial strains for the indicated times. The activation of LRRK2, assessed by its phosphorylation at S935, was then analysed by immunoblotting with the indicated antibodies. c,d, Raw264.7 or DC2.4 cells were pre-treated with the LRRK2 inhibitor GSK2578215A for 90 min, infected with either wild-type S. Typhi (MOI 6) (c) or the S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI 3) (d), both encoding a luciferase-based itaconate biosensor, and the levels of luciferase in the cell lysates were measured 20 h after infection. Each circle or square represents a single luciferase measurement. The mean ± s.d. and P values (unpaired two-tailed Student’s t-test) of the indicated comparisons are shown (n = 6 for each category). e,f, Raw264.7 or DC2.4 cells were pre-treated with the LRRK2 inhibitor GSK2578215A for 90 min, infected with wild-type S. Typhi (MOI 6) (e) or the S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI 3) (f), and the number of CFUs was determined 20 h after infection. Each circle or square represents a single measurement. The mean ± s.d. and the P values (unpaired two-tailed Student’s t-test) of the indicated comparisons are shown (n = 6 for each category).
Source data

LRRK2 scaffolds the formation of the RAB32–IRG1 complex
a,b, LRRK2 interacts with RAB32 (a). HEK-293T cells were transiently co-transfected with a plasmid expressing GFP–LRRK2 and a plasmid expressing FLAG–RAB32. Twenty hours after transfection, cells were infected with the S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI = 3), and 4 h after infection, cell lysates were analysed by immunoprecipitation (IP) and immunoblotting with antibodies against the FLAG epitope and GFP, respectively. The quantification of the intensity of the GFP–LRRK2 band is shown in b. Each circle, square or triangle represents a measurement in an independent experiment. The mean ± s.d. and P value (two-way ANOVA) of the indicated comparisons are shown (n = 3 for each category). c,d, LRRK2 interacts with IRG1 (c). HEK-293T cells were transiently co-transfected with a plasmid expressing GFP–LRRK2 and a plasmid expressing FLAG–IRG1. Twenty hours after transfection, cells were infected with the S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI = 3), and 4 h after infection, cell lysates were then analysed by immunoprecipitation and immunoblotting with antibodies against the FLAG epitope and GFP, respectively. The quantification of the intensity of the GFP–LRRK2 band is shown in d. Each circle, square or triangle represents a measurement in an independent experiment. The mean ± s.d. and P value (two-way ANOVA) of the indicated comparisons are shown (n = 3 for each category). e,f, LRRK2 promotes the formation of the RAB32–IRG1 complex (e). Raw264.7 parental (control) or Lrrk2−/− cells stably expressing FLAG–RAB32 were left uninfected or infected with the S. Typhimurium ∆gtgE ∆sopD2 mutant strain (MOI 3) for 18 h. Cell lysates were then analysed by immunoprecipitation and immunoblotting with antibodies against the FLAG epitope, endogenous IRG1 or LRRK2, and β-actin (as a loading control). The quantification of the intensity of the IRG1 band relative to the intensity of the RAB32 band is shown in f. Each circle, square or triangle represents a measurement in an independent experiment. The mean ± s.d. and P values (two-way ANOVA) of the indicated comparisons are shown (n = 3 for each category).
Source data

Intimate association of the SCV with the mitochondria observed by cryo-ET
a, Cryo-fLM of cultured HeLa cells stably expressing IRG1–GFP (green) and infected with S. Typhi encoding an mCherry itaconate biosensor (red). Specimens were vitrified in liquid ethane 3 h post infection. The white dashed line marks the cell boundary. The yellow dotted square region was targeted for further imaging analysis. b, SEM image of the S. Typhi-infected HeLa cell shown in a before cryo-FIB milling. The two rectangular boxes show areas targeted for ablation during the cryo-FIB milling. c, SEM image of the cryo-lamella (<200 nm thick) containing the target bacteria. Cryo-fLM signals (green: IRG1–GFP; red: mCherry itaconate biosensor) are overlayed on the SEM image. d, Cryo-ET image of the highlighted area in c showing close association of the SCV and mitochondria. e,f, Inter-membrane tethers bridge the SCV–mitochondria interface. Zoomed-in image of the tomographic slice at the SCV–mitochondria interface highlighted in d (e). Yellow and green transparent lines overlay the vacuolar and mitochondria membranes, respectively. White arrows denote the inter-membrane tethers. A 3D rendering of the SCV–mitochondria interface (z = 110 slices) (f). Bacterial, vacuolar and mitochondrial membranes are shown in magenta, yellow and green, respectively; bacterial ribosomes are shown in grey and inter-membrane tethers are shown in white. Following the subtomogram averaging of the inter-membrane tethers, segmented volume of the tether was mapped back into the original tomogram using the recalculated coordinates and Euler parameters. g–k, Additional examples of the SCV–mitochondria association in HeLa cells. Cryo-ET image showing close association of the SCV and mitochondria (g). The indicated zoomed-in regions of the tomographic slice at the SCV–mitochondria interfaces are shown in h and i, and the corresponding 3D renderings are shown in j and k (z = 197 slices). The colour scheme indicating bacterial, vacuolar and mitochondrial membranes, bacterial ribosomes and inter-membrane tethers is as indicated in f. l–p, Inter-membrane tethers are also observed at the SCV–mitochondrial interface in BMDMs infected with S. Typhi. BMDMs isolated from C57BL/6 mice were cultured on cryo-EM grids and infected with S. Typhi encoding the mCherry itaconate biosensor for 1 h, and mCherry expressing S. Typhi cells were targeted for cryo-FIB milling and cryo-ET imaging. Cryo-ET image showing close association of the SCV and mitochondria (l). The indicated zoomed-in regions of the tomographic slice at the SCV–mitochondria interfaces are shown in m and n, and the corresponding 3D renderings are shown in o and p (z = 88 slices). q, Measurement of the length of the vacuolar membrane (VM) and mitochondrial outer membrane (MiOM) interface. The X–Y plane density profiling function in tomographic software IMOD was used to measure the maximum length of the intimate contact between the VM and MiOM in both HeLa (blue) and BMDM (red) cells. Measurements were taken in ten interfaces (N = 10) from three independent experiments for both HeLa and BMDM cells. Dots in this scatter dot plot represent raw data. Solid lines within the box represent mean values (HeLa: 163.5 ± 95.6 nm, BMDM: 247.2 ± 162.7 nm). Two-tailed Welch’s test resulted in a P value of 0.1291, which suggests no significant difference between the distances that VM and MiOM make in HeLa and BMDM cells. r, Measurement of the inter-membrane distance between VM and MiOM. The X–Y plane density profiling function in tomographic software IMOD was used to measure distance between VM and MiOM in both HeLa (blue) and BMDM (red) cells. Measurements were taken at 30 interfaces (N = 30) from three independent experiments in both HeLa and BMDM cells. Dots in this scatter dot plot represent raw data. Solid lines within the box represent mean values (HeLa: 15.82 ± 3.04 nm; BMDM: 16.06 ± 4.35). Two-tailed Welch’s test resulted in a P value of 0.8098, which suggests no significant difference between the inter-membrane spacings measured in HeLa and BMDM cells. s, Quantification of inter-membrane tethers in HeLa and BMDM cells. Positions of inter-membrane tethers are identified as a part of particle picking procedure for the subsequent subtomogram averaging. Numbers of inter-membrane tethers in both HeLa and BMDM cells are divided by the total number of mitochondria that show visible inter-membrane tethers. t, A 2- and 3D cross-section of the subtomogram average map. The vertical length of the tether perpendicular to the membranes is ~15 nm. Mitochondrial inner membrane (MiIM) and MiOM are shown in green and the SCV membrane (VM) is shown in yellow. M: mitochondria; S: S. Typhi.
Source data

LRRK2 is required for establishing a close association between the SCV and the mitochondria
a–n, Cryo-ET images showing the SCV and mitochondria in BMDMs obtained from C57BL/6 (a–d), BLOC3−/− (e–h) and Lrrk2−/− (i–n) mice, infected for 3 h with a wild-type S. Typhi strain constitutively expressing mScarlet. Tomograms are shown in a,e,i and l, and their respective 3D renderings in b–d(z = 61 slices), f–h (z = 51 slices), j and k, (z = 50 slices) and m and n (z = 71 slices), for C57BL/6 (wild type (WT)), BLOC3−/− and Lrrk2−/− BMDMs, as indicated. The SCV membrane is shown in yellow, mitochondria are shown in green and inter-membrane tethers in white. Note the altered S. Typhi bacterial cell envelope architecture (denoted in pink) in BMDMs from C57BL/6 mice, and its normal appearance (denoted in blue) in BLOC3−/− or Lrrk2−/− BMDMs. Also, while inter-membrane tethers are readily visualized linking the mitochondria and the SCV in BMDMs from C57BL/6 and BLOC3−/− mice (highlighted in the zoomed-in areas of c,d,g and h), no tethers were visualized in BMDMs from Lrrk2−/− mice (i–n), even in the rare occasions when mitochondria and SCVs were seen in close proximity (l–n). o, Quantification of the percentage of SCVs making intimate contact (a distance of ≤25 nm) with mitochondria as observed by cryo-ET. A total of 32 cells were analysed from two independent experiments in WT, BLOC3−/− and Lrrk2−/− BMDM cells. An unpaired t-test was used to determine the statistical significance. n.s., difference not statistically significant (P = 0.8721). p,q, Itaconate delivery to the SCV in BMDMs obtained from C57BL/6, BLOC3−/− or Lrrk2−/− mice (as indicated). BMDMs were infected with wild-type S. Typhi (MOI = 6) encoding a luciferase-based itaconate biosensor and the levels of luciferase in the cell lysates were measured 3 h after infection. Each circle or square represents a single luciferase measurement. The mean ± s.d. and P values of the indicated comparisons (unpaired two-tailed Student’s t-test) are shown. r, Quantification of the percentage of bacteria contained within SCVs in close contact with mitochondria that show envelope alterations. A total of 21 bacteria were examined in two independent experiments with WT and BLOC3−/− BMDM cells. An unpaired t-test was used to determine the statistical significance (P = 0.0045).
Source data

Parkinson’s disease kinase LRRK2 coordinates a cell-intrinsic itaconate-dependent defence pathway against intracellular Salmonella

August 2023

156 Reads

15 Citations

Nature Microbiology

[...]

Cell-intrinsic defences constitute the first line of defence against intracellular pathogens. The guanosine triphosphatase RAB32 orchestrates one such defence response against the bacterial pathogen Salmonella, through delivery of antimicrobial itaconate. Here we show that the Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) orchestrates this defence response by scaffolding a complex between RAB32 and aconitate decarboxylase 1, which synthesizes itaconate from mitochondrial precursors. Itaconate delivery to Salmonella-containing vacuoles was impaired and Salmonella replication increased in LRRK2-deficient cells. Loss of LRRK2 also restored virulence of a Salmonella mutant defective in neutralizing this RAB32-dependent host defence pathway in mice. Cryo-electron tomography revealed tether formation between Salmonella-containing vacuoles and host mitochondria upon Salmonella infection, which was significantly impaired in LRRK2-deficient cells. This positions LRRK2 centrally within a host defence mechanism, which may have favoured selection of a common familial Parkinson's disease mutant allele in the human population.

View access options

The sorting platform in the type III secretion pathway: From assembly to function

Literature Review

June 2023

43 Reads

2 Citations

BioEssays

Jose EDUARDO Soto

María Lara-Tejero

The type III secretion system (T3SS) is a specialized nanomachine that enables bacteria to secrete proteins in a specific order and directly deliver a specific set of them, collectively known as effectors, into eukaryotic organisms. The core structure of the T3SS is a syringe-like apparatus composed of multiple building blocks, including both membrane-associated and soluble proteins. The cytosolic components organize together in a chamber-like structure known as the sorting platform (SP), responsible for recruiting, sorting, and initiating the substrates destined to engage this secretion pathway. In this article, we provide an overview of recent findings on the SP's structure and function, with a particular focus on its assembly pathway. Furthermore, we discuss the molecular mechanisms behind the recruitment and hierarchical sorting of substrates by this cytosolic complex. Overall, the T3SS is a highly specialized and complex system that requires precise coordination to function properly. A deeper understanding of how the SP orchestrates T3S could enhance our comprehension of this complex nanomachine, which is central to the host-pathogen interface, and could aid in the development of novel strategies to fight bacterial infections.

Fig. 1. Coupling AlphaFold 2 (AF2) modeling and in vivo photo-cross-linking to map the needle complex interface with the sorting platform protein OrgA. (A) Schematic representation and cryo-ET structure (17) of the Salmonella T3SS. (B) Structure of the 2:1 PrgH-OrgA complex generated using AlphaFold multimer (PrgH in tan and OrgA in green). Insets show the cross-linkable residues in the PrgH-OrgA interfaces mapped onto the structures. (C and D) Whole cell lysates of S. Typhimurium strains expressing OrgA 3FLAG and the indicated pBpa-containing PrgH mutants after exposure to UV light or left untreated. Samples were analyzed by western blot with antibodies to the NC base (red channel, anti-rabbit) or the FLAG epitope (green channel, anti-mouse) in OrgA. (E) Whole cell lysates of S. Typhimurium strains expressing 3FLAG PrgH and the indicated pBpa-containing OrgA variant that have been exposed to UV light or left untreated. Samples were analyzed by western blot with antibodies to the FLAG epitope. (F) Bottom view of the proposed rearrangement of the PrgH N ring upon OrgA docking. Western blots shown in all panels are representative of three biological replicates.

Fig. 2. Defining the SpaO-OrgA interface. (A) AlphaFold multimer model of the OrgA-SpaO complex (OrgA in green and SpaO in blue). Inset shows the crosslinkable residues between OrgA and SpaO mapped onto the structures. (B and C) Whole cell lysates of S. Typhimurium strains chromosomally expressing OrgA 3FLAG (B) or 2HA SpaO (C) and the indicated pBpa-containing 2HA SpaO (B) or OrgA 3FLAG (C) variants that have been exposed to UV light or left untreated. Samples were analyzed by western blot with antibodies directed to the HA (green channel, anti-mouse) or FLAG (red channel, anti-rabbit) epitopes. Western blots shown in all panels are representative of three biological replicates.

Fig. 4. Systematic OrgB photo-cross-linking analyses identify the sorting platform cradle interfaces. (A and B) Identification of cross-linkable amino acids in the OrgB-SpaO interface. Crystal structure of SpaO (red) in complex with the N-terminal region of OrgB 1-30 (gray) (PDB ID code 4YX7) showing (in green) the OrgB residues (K20 and R21) selected for pBpa incorporation (A). Whole cell lysates of S. Typhimurium strains expressing 3FLAG SpaO and the indicated OrgB M45 pBpa variants after exposure to UV light (or left untreated) were analyzed by western blot with antibodies directed against the FLAG (red channel, anti-rabbit) or M45 (green channel, anti-mouse) epitopes (B). (C and D) Structural modeling and site-directed photo-cross-linking define OrgB-InvC interface. Enlarged view of the modeled InvC (red)-OrgB (gray) interface (C). The OrgB residues chosen for pBpa incorporation are shown in green (C). Whole cell lysates of S. Typhimurium strains expressing InvC 3FLAG and the indicated OrgB M45 pBpa variant, exposed to UV light or left untreated, were analyzed by immunoblot using antibodies directed to the FLAG (red channel, anti-rabbit) or M45 (green channel, anti-mouse) epitopes. The cross-link between InvC 3FLAG and OrgB M45 is indicated. Western blots shown in all panels are representative of three biological replicates.

Fig. 7. Proposed pathway for the assembly of the Salmonella sorting platform. (1-3) OrgA docks onto PrgH N concomitantly to PrgH ring assembly. OrgB engages the C-terminal region of SpaO triggering conformational change that makes it permissive for binding to PrgH-bound OrgA and completing the assembly of the pods. (4 and 5) Assembly of the OrgB cradle provides a structural scaffold to recruit InvC, facilitating its oligomerization. (6) Incorporation of InvI onto the InvC hexamer results in a functional type III secretion sorting platform.

Assembly and architecture of the type III secretion sorting platform

December 2022

90 Reads

6 Citations

Proceedings of the National Academy of Sciences

Jose EDUARDO Soto

Jorge E. Galán

María Lara-Tejero

Type III secretion systems are bacterial nanomachines specialized in protein delivery into target eukaryotic cells. The structural and functional complexity of these machines demands highly coordinated mechanisms for their assembly and operation. The sorting platform is a critical component of type III secretion machines that ensures the timely engagement and secretion of proteins destined to travel this export pathway. However, the mechanisms that lead to the assembly of this multicomponent structure have not been elucidated. Herein, employing an extensive in vivo cross-linking strategy aided by structure modeling, we provide a detailed intersubunit contact survey of the entire sorting platform complex. Using the identified cross-links as signatures for pairwise intersubunit interactions in combination with systematic genetic deletions, we mapped the assembly process of this unique bacterial structure. Insights generated by this study could serve as the bases for the rational development of antivirulence strategies to combat several medically important bacterial pathogens.

Download

Assembly and architecture of the type III secretion sorting platform

September 2022

58 Reads

Jose EDUARDO Soto

Jorge E. Galán

María Lara-Tejero

Type III secretion systems are bacterial nanomachines specialized in protein delivery into target eukaryotic cells. The structural and functional complexity of these machines demand highly coordinated mechanisms for their assembly and operation. The sorting platform is a critical component of type III secretion machines that ensures the timely engagement and secretion of proteins destined to travel this export pathway. However, the mechanisms that lead to the assembly of this multi-component structure have not been elucidated. Herein, employing structure modeling and an extensive in vivo crosslinking strategy, we provide a detailed inter-subunit-contact survey of the entire sorting platform complex. Using the identified crosslinks as signatures for pairwise inter-subunit interactions in combination with systematic genetic deletions, we mapped the assembly process of this unique bacterial structure. Insights generated by this study could serve as the bases for the development of anti-virulence strategies to combat several medically important bacterial pathogens.

Download

Figure 1. Identification of CI-M6PR as a typhoid toxin-interacting protein. (a and b) Purified FLAG-tagged wild-type typhoid toxin or its PltB S35A mutant unable to bind glycosylated receptor proteins (a) were used in affinity purification experiments (outlined in a) to identify typhoid toxin-interacting proteins, which led to the identification of CI-M6PR (see Figure 1-source data 2). (b) The interaction between typhoid toxin and CI-M6PR was verified in Salmonella Typhi-infected cells. Henle-407 cells were infected with S. Typhi expressing FLAG-tagged CdtB for 24 hr and the interaction between typhoid toxin and endogenous CI-M6PR was probed by affinity purification with a FLAG antibody (directed to the CdtB subunit of typhoid toxin) and western blot (with antibodies to both FLAG and anti-CI-M6PR). (c and d) Co-localization of the S. Typhi-containing vacuole and CI-M6PR. Henle-407 cells were infected with S. Typhi for the indicated times and examined by immunofluorescence with differentially labeled antibodies to S. Typhi and CI-M6PR. The quantification of the co-localization is shown in (d). Values (Mander's overlap coefficient) represent the degree of co-localization between CI-M6PR and S. Typhi and are the mean ± SEM. ****: p<0.0001. Scale bar = 5 µm. SCV: Salmonella-containing vacuole; CI-M6PR: cation-independent mannose-6-phosphate receptor. WCL: whole-cell lysates; IP: immunoprecipitation.

Typhoid toxin sorting and exocytic transport from Salmonella Typhi infected cells

May 2022

58 Reads

11 Citations

eLife

[...]

Typhoid toxin is an essential virulence factor for Salmonella Typhi, the cause of typhoid fever in humans. This toxin has an unusual biology in that it is produced by Salmonella Typhi only when located within host cells. Once synthesized, the toxin is secreted to the lumen of the Salmonella -containing vacuole from where it is transported to the extracellular space by vesicle carrier intermediates. Here we report the identification of the typhoid toxin sorting receptor and components of the cellular machinery that packages the toxin into vesicle carriers, and exports it to the extracellular space. We found that the cation-independent mannose-6-phosphate receptor serves as typhoid toxin sorting receptor and that the coat protein COPII and the GTPase Sar1 mediate its packaging into vesicle carriers. Formation of the typhoid toxin carriers requires the specific environment of the Salmonella Typhi-containing vacuole, which is determined by the activities of specific effectors of its type III protein secretion systems. We also found that Rab11B and its interacting protein Rip11 control the intracellular transport of the typhoid toxin carriers, and the SNARE proteins VAMP7, SNAP23, and Syntaxin 4 their fusion to the plasma membrane. Typhoid toxin's cooption of specific cellular machinery for its transport to the extracellular space illustrates the remarkable adaptation of an exotoxin to exert its function in the context of an intracellular pathogen.

Download

Typhoid toxin sorting and exocytic transport from Salmonella Typhi infected cells

August 2021

69 Reads

Typhoid toxin is an essential virulence factor for Salmonella Typhi, the cause of typhoid fever in humans. This toxin has an unusual biology in that it is produced by Salmonella Typhi only when located within host cells. Once synthesized, the toxin is secreted to the lumen of the Salmonella-containing vacuole from where it is transported to the extracellular space by vesicle carrier intermediates. Here we report the identification of the typhoid toxin sorting receptor and the cellular machinery that packages the toxin into vesicle carriers, and exports it to the extracellular space. We found that the cation-independent mannose-6-phosphate receptor serves as typhoid toxin sorting receptor and that the coat protein COPII and the GTPase Sar1 mediate its packaging into vesicle carriers. Formation of the typhoid toxin carriers requires the specific environment of the Salmonella Typhi-containing vacuole, which is determined by the activities of specific effectors of its type III protein secretion systems. We also found that Rab11B and its interacting protein Rip11 control the intracellular transport of the typhoid toxin carriers, and the SNARE proteins VAMP7, SNAP23, and Syntaxin 4 their fusion to the plasma membrane. Typhoid toxin's cooption of specific cellular machinery for its transport to the extracellular space illustrates the remarkable adaptation of an exotoxin to exert its function in the context of an intracellular pathogen.

Download

Itaconate is an effector of a Rab GTPase cell-autonomous host defense pathway against Salmonella

Article

July 2020

178 Reads

103 Citations

Science

[...]

Rab32 puts itaconate where it's needed Myeloid cells can restrict the replication of intracellular bacterial pathogens such as Salmonella using a Rab family guanosine triphosphatase called Rab32. However, the underlying mechanisms remain unclear. Chen et al. report that Rab32 and its exchange factor, BLOC3, interact with aconitate decarboxylase 1 (IRG1). This complex enables the direct delivery of IRG1's antimicrobial product, itaconate, from the mitochondria to Salmonella -containing vacuoles. Itaconate concentrations in vacuoles correlated with bacterial survival, highlighting the biological relevance of this metabolite during infections. Similar findings in Escherichia coli –infected cells suggest that this is a more general phenomenon in which mitochondria and the Rab32 pathway play a critical role in antibacterial host defense. Science , this issue p. 450

Mechanisms of substrate recognition by a typhoid toxin secretion-associated muramidase

January 2020

105 Reads

18 Citations

eLife

Typhoid toxin is a virulence factor for the bacterial pathogen Salmonella Typhi, which causes typhoid fever in humans. After its synthesis by intracellular bacteria, typhoid toxin is secreted into the lumen of the Salmonella-containing vacuole by a secretion mechanism strictly dependent on TtsA, a specific muramidase that facilitates toxin transport through the peptidoglycan layer. Here we show that substrate recognition by TtsA depends on a discrete domain within its carboxy terminus, which targets the enzyme to the bacterial poles to recognize YcbB-edited peptidoglycan. Comparison of the atomic structures of TtsA bound to its substrate and that of a close homolog with different specificity identified specific determinants involved in substrate recognition. Combined with structure-guided mutagenesis and in vitro and in vivo crosslinking experiments, this study provides an unprecedented view of the mechanisms by which a muramidase recognizes its peptidoglycan substrate to facilitate protein secretion.

Download

... Rab GTPases also act upstream of LRRK2 to promote its membrane recruitment and activation (Dhekne et al., 2023;Eguchi et al., 2018;Gomez et al., 2019;Lian et al., 2023;Pfeffer, 2022;Purlyte et al., 2018;Unapanta et al., 2023;Vides et al., 2022;Wang et al., 2023a). However, although Rab GTPases promote LRRK2 kinase activity in specific contexts, recent knockout (KO) mouse studies revealed the persistence of partial or even full LRRK2 kinase activity in multiple tissues even after depletion of key Rab proteins implicated in LRRK2 activation (Dhekne et al., 2023;Kalogeropulou et al., 2020). ...
Reference:
A STING–CASM–GABARAP pathway activates LRRK2 at lysosomes

Parkinson’s disease kinase LRRK2 coordinates a cell-intrinsic itaconate-dependent defence pathway against intracellular Salmonella

Citing Article
Publisher preview available
August 2023

Nature Microbiology

[...]

... Pathogenic Gram-negative bacteria infect animals and plants by secreting type-3 (T3) proteins known as type-3 effectors (T3Es), type-3 translocators (T3Ts), and T3 chaperones (B€ uttner, 2012). T3Es and T3Ts are secreted via a nanoscale syringe-shaped apparatus, a pilus in phytopathogenic bacteria, and a needle in zoopathogenic bacteria, with the aid of T3 chaperones (Soto & Lara-Tejero, 2023). During infection, T3 chaperones help the secreted proteins to remain unfolded to facilitate transport toward the bacterial-eukaryotic interface (Domingues et al., 2014). ...
Reference:
Plant PI4P is required for bacteria to translocate type‐3 effectors

The sorting platform in the type III secretion pathway: From assembly to function

Citing Article
June 2023

BioEssays

Jose EDUARDO Soto

María Lara-Tejero

... The macromolecular assembly spans the inner (IM) and outer membranes (OM). Two distinct parts can be differentiated in the system: the needle complex and the sorting platform (Lara-Tejero and Galań, 2019;Soto et al., 2022). At the base of the needle complex there is a multi-ring structure that spans the inner membrane, formed by PrgH and PrgK subunits (SctD and SctJ subunits in the unified nomenclature). ...
Reference:
Primary architecture and energy requirements of Type III and Type IV secretion systems

Assembly and architecture of the type III secretion sorting platform

Citing Article
Full-text available
December 2022

Proceedings of the National Academy of Sciences

Jose EDUARDO Soto

Jorge E. Galán

María Lara-Tejero

... Typhoid toxin has a unique biology, as it is exclusively produced by intracellular Salmonella and delivered to the extracellular space via a receptor-mediated exocytic transport pathway [10][11][12] . Through autocrine and paracrine routes, typhoid toxin enters various cell types via receptor-mediated retrograde transport to its subcellular destination 13 . ...
Reference:
Mitochondrial injury induced by a Salmonella genotoxin triggers the proinflammatory senescence-associated secretory phenotype

Typhoid toxin sorting and exocytic transport from Salmonella Typhi infected cells

Citing Article
Full-text available
May 2022

eLife

[...]

... This approach has led to the identification of a single metabolite that enhances the predictive accuracy for mortality risk. Initially, itaconic acid underwent extensive investigation in the field of basic medical sciences, with a growing emphasis on its potent antibactnate (4-OI) [28][29][30][31][32], which has demonstrated the ability to inhibit the replication of SARS-CoV-2. Through activation of Nrf2, an antioxidant and anti-inflammatory transcription factor, 4-OI induces an antiviral response capable of suppressing the replication of SARS-CoV-2 and other pathogenic viruses, such as herpes simplex virus 1 (HSV-1) and vaccinia virus [33]. ...
Reference:
Integration of metabolomics methodologies for the development of predictive models for mortality risk in elderly patients with severe COVID-19

Itaconate is an effector of a Rab GTPase cell-autonomous host defense pathway against Salmonella

Citing Article
July 2020

Science

[...]

... Once synthesized by intracellular S. Typhi, typhoid toxin is secreted from the bacteria into the lumen of the Salmonella-containing vacuole (SCV) by a novel protein secretion system named Type 10 Secretion System (T10SS) [19][20][21] . The pivotal component of this secretion system is TtsA (typhoid toxin secretion protein A), a specialized peptidoglycan hydrolase responsible for actively releasing the toxin through the bacterial cell wall without causing bacterial cell lysis 20,22 . ...
Reference:
Cytolysin A is an intracellularly induced and secreted cytotoxin of typhoidal Salmonella

Mechanisms of substrate recognition by a typhoid toxin secretion-associated muramidase

Citing Article
Full-text available
January 2020

eLife

... Despite the conservation of these genes, our sequencing efforts, spanning approximately 800 nucleotides, have revealed variation around a conserved 150-nucleotide sequence block. The functional implications of these sequence discrepancies within the genes of the export apparatus remain unclear; however, it is conceivable that they are related to the specific effector arsenals of individual bacterial strains, as the proteins of the export apparatus interact with the T3Es and mediate their passage through the bacterial cell wall (Butan et al. 2019). Furthermore, the observed sequence heterogeneity raises the possibility that the primers are inefficient in detecting T3SS in our isolate spectrum. ...
Reference:
Effects of T3SS-positive Pseudomonas isolates on sugar beet growth stimulation and pathogen resistance

High-resolution view of the type III secretion export apparatus in situ reveals membrane remodeling and a secretion pathway

Citing Article
November 2019

Proceedings of the National Academy of Sciences

[...]

... Salmonella Typhi and related toxigenic Salmonella have evolved a chimeric typhoid toxin implicated in typhoid fever, immune evasion and chronic Salmonella carriage [9,14,34]. ...
Reference:
Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

Alternate subunit assembly diversifies the function of a bacterial toxin

Citing Article
Full-text available
August 2019

Nature Communications

[...]

... Our findings may indicate that toxin-induced DDRs elicit antimicrobial responses, which suppress Salmonella bacteraemia during typhoid fever. : bioRxiv preprint toxin or a toxin-negative (TN) strain lacking the genes pltB pltA and cdtB [16]. ...
Reference:
Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

Investigation of the role of typhoid toxin in acute typhoid fever in a human challenge model

Citing Article
Full-text available
July 2019

Nature Medicine

[...]

... The assembly of the sorting platform marks the onset of type III protein secretion. Escorted by dedicated chaperones and aided by regulatory proteins, substrates are sequentially loaded onto the sorting platform in a hierarchical fashion: first early substrates comprising the subunits that form the inner rod and the extracellular needle, followed by the translocases that facilitate passage of the effector proteins through the target host cell membrane, and last the effector proteins that modulate cellular functions (15,19). ...
Reference:
Assembly and architecture of the type III secretion sorting platform

The Type III Secretion System Sorting Platform

Citing Chapter
June 2019

Current Topics in Microbiology and Immunology

María Lara-Tejero

Maria Lara-Tejero’s research while affiliated with Yale-New Haven Hospital and other places

What is this page?

Publications (110)

Citations (32)