Biology subjects

Velazquez, C.

Publications and source records attributed to Velazquez, C..

6 recordsLinked to original sources

bioRxiv2025

The cryo-EM structure of an adaptor-effector complex reveals the mechanism of a widespread pore-forming toxin family

Pseudomonas putida KT2440 is a plant-beneficial rhizobacterium that encodes multiple Type VI secretion systems (T6SS) to outcompete phytopathogens in the rhizosphere. Among its antibacterial effectors, Tke5 has been identified as a potent pore-forming toxin that disrupts ion homeostasis without causing considerable membrane damage. Tke5 belongs to the BTH_I2691 protein family and harbours an N-terminal marker for the type six secretion system effectors (MIX) motif, previously shown to be required for T6SS-dependent secretion in other systems. Many MIX-containing effectors require T6SS adaptor proteins (Tap) for secretion, but until now, the molecular mechanism for adaptor-effector binding has remained elusive. Here, we report the 2.8 [A] cryo-EM structure of the Tap3-Tke5 complex, providing structural and functional insight into how this effector is recruited by its cognate adaptor protein Tap3. Functional dissection shows that the -helical region of Tke5 is sufficient to kill intoxicated bacteria, while its {beta}-rich region likely contributes to target membrane specificity. These findings suggest a general mechanism of MIX-containing BTH_I2691 proteins for Tap recruitment and toxin activity, contributing to our fundamental understanding of a widespread yet understudied toxin family.

microbiology↗

bioRxiv2025

The Pseudomonas aeruginosa Tse4 toxin assembles ion-selective and voltage-sensitive ion channels to couple membrane depolarisation with K+ efflux

Pseudomonas aeruginosa employs the Type VI secretion system (T6SS) to outcompete other bacteria in its environment. Among the effectors secreted by the T6SS of P. aeruginosa PAO1, Tse4 is known for its potent antibacterial activity. This study elucidates the molecular function of Tse4, which promotes cell depolarisation in competing bacteria. Our results show that Tse4 spontaneously incorporates into lipid monolayers and forms multiionic channels in planar bilayers, with either ohmic conduction or diode-like rectifying currents and a preference for cations over anions. These observations allow us to propose a model of action whereby Tse4 channels couple cell depolarisation with K+ efflux. These insights into Tse4s pore-forming activity enhance our understanding of bacterial competition and exemplify a finely tuned antibacterial strategy, coupling its ability to cause membrane depolarisation with potassium efflux that synergises with other T6SS effectors. These results highlight the sophistication of Pseudomonas aeruginosas competitive arsenal.

microbiology↗

bioRxiv2025

Tke5 is a novel Pseudomonas putida toxin that depolarises membranes killing plant pathogens

The soil bacterium Pseudomonas putida injects toxic proteins into neighbouring competitors, including resilient phytopathogens, using the Type VI secretion system (T6SS). The secretion of toxins endows P. putida with a significant fitness advantage, allowing this biocontrol agent to thrive in plant-related polymicrobial environments and prevent phytopathogen infections. Despite its agricultural significance, the toxin repertoire of P. putida, particularly those secreted via the K2- and K3-T6SSs, remains poorly understood. We present the first comprehensive molecular study of Tke5, a potent toxin encoded within the K3-T6SS, which represents the first comprehensive functional analysis of the BTH_I2691 protein family. Our biophysical data demonstrate that Tke5 is a pore-forming toxin that disrupts bacterial membranes through selective ion transport, inducing membrane depolarisation and cell death. Unlike conventional detergent-like pore-forming toxins, Tke5 preserves overall membrane integrity, avoiding large, non-specific disruptions. This unique mechanism offers a powerful approach to targeting resilient phytopathogens. This study reveals a previously undescribed mode of action within a widespread yet understudied toxin family. Our findings highlight the potential of P. putida as a biocontrol agent, offering alternatives to chemical pesticides by exploiting novel toxin mechanisms. Understanding these bacterial toxins is crucial for developing effective strategies to combat plant pathogens.

microbiology↗

bioRxiv2023

Replication poison treated BRCA1-deficient breast cancers are prone to MRE11 over-resection resulting in single strand DNA accumulation and mitotic catastrophe

BRCA1, BRCA2 and RAD51, key players of homologous recombination (HR) repair, are also involved in stalled DNA replication fork protection and repair. BRCA1-deficiency is encountered in 25% of Triple Negative Breast Cancer (TNBC). Here we investigated the sensitivity of BRCA1-deficient TNBC cell models to gemcitabine a frequently used replication poison that does not alter DNA structure. We show that BRCA1-deficient models, in contrast to their isogenic BRCA1-proficient counterparts, are superiorly sensitive to gemcitabine, accumulate massive levels of single strand DNA (ssDNA), in absence of RPA and RAD51 signals and elevated double strand break (DSB) numbers leading to cell death. Remarkably, ssDNA accumulation in gemcitabine-treated BRCA1-deficient cells was strongly diminished by the MRE11 inhibitor mirin, while it did not affect ssDNA levels resulting from PARP inhibitor olaparib treatment. The central role of MRE11 DNA resection strongly suggested that replication fork reversal may be important in response to replication poisoning by gemcitabine in BRCA1-deficient models. Furthermore, we demonstrate that gemcitabine-treated BRCA1-deficient cells showing massive ssDNA accumulation slipped into mitosis and produced mitotic bridges and micronuclei (MN) showing strong BrdU and {gamma}H2AX staining. Noticeably these BrdU-positive MN and DNA bridges triggered cGAS sensing. Our data, thus, strongly suggest that gemcitabine treatment could be beneficial in BRCA1-deficient TNBC both in terms of cancer cell death, but possibly as well in terms of antitumor immune response.

cancer biology↗

bioRxiv2022

Neurobehavioral precursors of compulsive cocaine-seeking in dual fronto-striatal circuits

Only some individuals using drugs recreationally eventually become addicted, and persist in drug seeking and taking despite adverse consequences. The neurobehavioral determinants of this individual vulnerability have not been fully elucidated. We report that in drug naive rats the future tendency to develop compulsive cocaine seeking is characterised by behavioral stickiness-related functional hypoconnectivity between the prefrontal cortex and posterior dorsomedial striatum in combination with impulsivity-related structural alterations in the infralimbic cortex, anterior insula and nucleus accumbens. These findings show that the vulnerability to develop compulsive cocaine seeking behavior stems from pre-existing structural or functional changes in two distinct cortico-striatal systems that underlie deficits in impulse control and goal-directed behavior.

neuroscience↗

bioRxiv2019

Galectin-3 as a new negative checkpoint of the immune response is the key target for effective immunotherapy against prostate cancer

Prostate cancer (PCa) is a major health problem worldwide. Taxol derivatives-based chemotherapies or immunotherapies are usually proposed depending on the symptomatic status. In the case of immunotherapy, tumors develop robust immune escape mechanisms that abolish any protective response. However, Docetaxel has been shown to enhance the effectiveness of immunotherapy in a variety of cancers, but to date, the mechanism is still unknown. Herein, we showed first that Galectin-3 (Gal-3) expressed by prostate tumor cells is the principal immunological checkpoint responsible of the failure of immunotherapy; and that Docetaxel leads to the inhibition of Gal-3 expression in PCa cells as well as in clinical samples of mCRPC patients promoting a Th1 response. We thus optimized a prostate cancer animal model that undergoes surgical resection of the tumor like prostatectomy to mimic what is usually performed in patients. More importantly, using low and nontoxic doses of taxane prior to immunotherapy, we were able to directly impact the activation and proliferation of CD8+ cytotoxic T cells through reducing the number of CD8+CD122+CD28-T cells and highly control tumor recurrence. Thus, Gal-3 expression by PCa cells is a key inhibitor for the success of immunotherapy, and low doses of Docetaxel with noncytotoxic effect on leukocyte survival should be used prior to vaccination for all PCa patients. This combined treatment sequence right after surgery would promote the preconditioning of the tumor microenvironment, allowing for effective anti-tumor immunotherapy and can be transferred rapidly to clinical therapeutic protocols.

immunology↗