Biology subjects

Kamanova, J.

Publications and source records attributed to Kamanova, J..

7 recordsLinked to original sources

bioRxiv2025

CluSeek: Bioinformatics Tool to Identify and Analyze Gene Clusters

Gene clusters are key structural and functional units that encode diverse phenotypes in genomes, from metabolism to pathogenesis. As genome sequencing expands, tools for systematic exploration of this growing data are increasingly needed. We present CluSeek, an open-source, Python-based platform for discovering, visualizing, and analyzing gene clusters across all GenBank data. Unlike existing tools, CluSeek does not rely on predefined cluster types or reference libraries but enables mining of any gene neighborhoods containing colocalized homologs of user-specified genes. It features an intuitive graphical interface suitable for non-bioinformaticians and is freely available at https://cluseek.com. We demonstrate the versatility of Cluseek in two distinct case studies: (i) mining of specialized metabolites, where CluSeek uncovered over 16 new classes containing the bioactivity-enhancing 4-alkyl-L-proline moiety, previously known in only three Golden Era antibiotic classes; and (ii) analysis of type III secretion systems present in Bordetella species, revealing previously unrecognized taxonomic distribution, and genetic variants, including gene multiplications and novel components with potential functional significance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/676505v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1767e2eorg.highwire.dtl.DTLVardef@5603e3org.highwire.dtl.DTLVardef@1193475org.highwire.dtl.DTLVardef@1c32e83_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Cytotoxicity and effector cooperation in the type III secretion system of Aeromonas schubertii

The type III secretion system (T3SS) is an important virulence factor of Gram-negative bacteria, including the genus Aeromonas, a group of aquatic bacteria capable of both mutualistic and pathogenic interactions. Aeromonas species are increasingly recognized as opportunistic human pathogens. The type strain A. schubertii ATCC 43700 encodes two distinct T3SSs located in the Aeromonas pathogenicity islands 1 and 2, hereby designated as API1 and API2, respectively. Presented work investigates the role of API1 and API2 in A. schubertii-induced cytotoxicity and identifies novel type III secretion effectors. HeLa cell infections showed that API1, but not API2, is essential for cellular cytotoxicity resulting in both apoptotic and necrotic cell death. The {Delta}API1 mutant failed to induce cytotoxicity, whereas the wild-type (WT) and {Delta}API2 strains induced comparable cytotoxic effects. Proteomic analysis identified 7 candidate effectors secreted by the API1 injectisome under low-calcium conditions. These included two previously characterized effectors, AopH and AopO of A. salmonicida, and five novel effectors hereby named AopI, AopJ, AopL, AopT, and AopU, whose injection into host cells via API1 was validated using a split luciferase reporter system. Functional analysis revealed distinct roles for these effectors. AopL, homologous to the VopQ effector of Vibrio parahaemolyticus, accelerated caspase 3-independent necrosis, while AopI, homologous to ExoY of Pseudomonas aeruginosa, suppressed caspase activation and necrosis, indicating a pro-survival function. These results show the role of API1 injectisome in the cytotoxicity of A. schubertii and expand our understanding of T3SS-mediated host- pathogen interactions in Aeromonas species. ImportanceThis work demonstrates that the API1 injectisome is an important cytotoxicity determinant in A. schubertii and identifies novel effectors and their distinct contributions to host cell cytotoxicity, including the pro-survival effect of AopI and the cytotoxic effect of AopL. This interplay highlights a fine-tuned balance between pro-survival and cytotoxic mechanisms which is orchestrated by A. schubertii effectors.

microbiology↗

bioRxiv2025

Limited response of primary nasal epithelial cells to Bordetella pertussis infection and the effector protein BteA

Bordetella pertussis is a Gram-negative coccobacillus that causes whooping cough or pertussis, a respiratory disease that has recently experienced a resurgence. Upon entering the respiratory tract, B. pertussis colonizes the airway epithelium and attaches to ciliated cells. Here, we used primary human nasal epithelial cells (hNECs) cultured at the air-liquid interface, and investigated their interaction with the B. pertussis B1917, focusing on the role of the type III secretion system effector protein BteA. In this model, which resembles the epithelial cells of nasal epithelium in vivo, B. pertussis B1917 initially replicated in the overlying mucus and scarcely colonized the cell cilia. The colonization led to a gradual decline in epithelial barrier function, as shown by measurements of trans-epithelial electrical resistance (TEER) and staining of the tight junction protein zonula occludens 1 (ZO-1). The decrease in TEER occurred independently of the cytotoxic effector protein BteA. Transcriptomic and proteomic analyses of hNECs showed only moderate changes following infection, primarily characterized by increased mucus production, including upregulation of mucin MUC5AC. No profound response to BteA was detected. Furthermore, the infection did not induce production of inflammatory cytokines, suggesting that B. pertussis B1917 evades recognition by hNECs in this model system. These findings suggest that the bacterium may utilize the mucus layer in the airways as a protective niche to minimize epithelial recognition and damage. ImportanceThe nasal epithelium is the initial site where B. pertussis comes into contact with the host during respiratory infection. This work established human nasal epithelial cells (hNECs) cultured at the air-liquid interface (ALI) as an in vitro model to investigate B. pertussis infection. Using this system, we were able to show that in the early stages of colonization the clinical isolate B. pertussis B1917 replicates in the mucus without disrupting epithelial barrier function. Infection results in moderate transcriptomic and proteomic changes and is characterized by increased mucus production and minimal inflammatory signaling.

microbiology↗

bioRxiv2024

The Bordetella effector protein BteA induces host cell death by disruption of calcium homeostasis

Bordetella pertussis is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca2+ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca2+ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death. ImportanceThe respiratory pathogens, Bordetella pertussis and Bordetella bronchiseptica, exhibit cytotoxicity towards a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA effector. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

microbiology↗

bioRxiv2024

Bsp22 protein polymerization drives dynamic adaptation of the Bordetella type III secretion system injectisome

Many gram-negative bacteria are equipped with a type III secretion system (T3SS) injectisome, enabling the direct translocation of effector proteins from the bacterial cytoplasm into host cells. In the case of Bordetella bronchiseptica, a respiratory pathogen of diverse mammals, the T3SS injectisome exhibits a unique needle tip filament formed by the Bsp22 protein, essential for bacterial persistence in mice. Here, we used B. bronchiseptica and B. pertussis with in-frame insertions of short peptide tags into the Bsp22 to elucidate its formation and characteristics via super-resolution imaging with fluorophore-labeled nanobodies and biarsenic probes. We report that on abiotic surfaces, Bsp22 forms flexible filaments that protrude up to several {micro}m from bacterial cells. In these conditions, Bsp22 filaments grow continuously without any apparent growth control, with Bsp22 subunits being added at the distal end. Remarkably, during host cell infection, the growth of Bsp22 filaments is constrained, accompanied by downregulated Bsp22 protein production. When infecting HeLa cells, some Bsp22 filaments form a short physical bridge between the bacterium and the host cell surface. In nasal epithelium, where B. bronchiseptica colonization is specifically restricted to ciliated cells, Bsp22 filaments align parallel to the cilia, pointing towards the cell apical surface. These results highlight the context-specific and environment-influenced dynamic modulation of the length and orientation of Bordetella needle tip filament, uncovering the adaptability of the Bordetella T3SS injectisome in response to conditions encountered during host cell infection.

microbiology↗

bioRxiv2020

Lipid binding by N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA

The classical Bordetella species, B. pertussis and B. bronchiseptica, employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector into the host cells. Upon injection, BteA localizes to the cytosolic leaflet of lipid rafts via its N-terminal lipid raft targeting (LRT) domain and induces cell death. The plasma membrane targeting and cytotoxicity mechanisms of BteA are poorly understood. Using protein-lipid overlay assay and surface plasmon resonance, we showed here that the recombinant LRT domain, which adopts a four-helix bundle topology of membrane localization domains, specifically binds negatively charged membrane phospholipids. The binding affinity for phosphatidylinositol 4,5-bisphosphate (PIP2)-containing liposomes with Kd ~450 nM was higher than for those enriched in phosphatidylserine (Kd ~1.2 M) while both phospholipids were required for plasma membrane targeting in yeast cells. The membrane association of LRT further depended on its electrostatic and hydrophobic interactions and involved a loop L1-located leucine residue. Importantly, charge-reversal substitutions within the L1 region disrupted plasma membrane localization of BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the plasma membrane of host cells is, hence, dispensable for the effector cytotoxicity. Author summaryThe respiratory pathogens of humans and other animals, Bordetella pertussis and Bordetella bronchiseptica, produce a type III secretion system effector protein BteA. This effector consists of two functional domains, an N-terminal lipid raft targeting (LRT) domain, and a cytotoxic C-terminal domain, which induces non-apoptotic and caspase-1-independent host cell death. We found here that the LRT domain of BteA associates with plasma membrane by binding to negatively charged phospholipids. We further discovered that the mechanism of membrane association by LRT is reminiscent of the one used by three other diverse families of toxins: clostridial glucosyltransferase toxins, multifunctional-autoprocessing RTX toxins (MARTX), and Pasteurella multocida-like toxins. Intriguingly, we also report that plasma membrane targeting by the LRT domain does not contribute to cytotoxic activity of BteA during B. bronchiseptica infection. Overall, our work elucidated the mechanism of plasma membrane association by LRT, and further provided the basis for future research on cellular activities of BteA and the mechanism of BteA-induced cell death.

microbiology↗

bioRxiv2020

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. Deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteA{Delta}A503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, production of Bp BteA{Delta}A503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response. Author summaryPertussis remains the least-controlled vaccine-preventable infectious disease and the mechanisms by which Bordetella pertussis subverts defense mechanisms of human airway mucosa remain poorly understood. We found that B. pertussis had the cytotoxic activity of its type III secretion system-delivered effector BteA strongly attenuated by insertion of an alanine residue at position 503 as compared to the BteA homologue of the animal pathogen B. bronchiseptica. This functional adaptation reduced the capacity of B. pertussis to suppress host inflammatory response and may contribute to an acute course of the pulmonary form of human infant pertussis.

microbiology↗