Biology subjects

Berisio, R.

Publications and source records attributed to Berisio, R..

8 recordsLinked to original sources

bioRxiv2025

Structural studies of mycobacterial HptG reveal a silent state and offer insights into TLR4 activation

HtpG of Mycobacterium tuberculosis (HtpGMtb) is an ATP-dependent chaperone that assists the correct folding of nascent and stress-accumulated misfolded proteins, in concert with other chaperones. Beside playing a role in stress response, it is able to elicit an immune response against M. tuberculosis infection by activating Dendritic Cells in a TLR4-mediated manner. However, we lack a full understanding of the molecular determinants of HtpGMtb catalytic activity and TLR4 activation, due to the lack of structural and biophysical data. Here, we report the first crystal structure of HtpGMtb, in complex with the non-hydrolysable form of ATP, AMPPNP. The crystal structure reveals that the HtpGMtb dimer adopts a conformationally silent structure, that precludes the dimerisation of the chaperone catalytic domains needed for ATP hydrolysis. Also, binding studies show that HtpGMtb directly interacts with TLR4 with a nanomolar affinity, and that this interaction allows HtpGMtb dimer to engage two TLR4 molecules. This finding suggests that activation of TLR4 by HtpGMtb is due to its ability to induce intra-cellular receptor dimerisation in an LPS-like mode.

biophysics↗

bioRxiv2025

DepoCatalog: Mapping the Diversity of 105 Recombinant Klebsiella Phage Depolymerases Across Sequence, Structure, and Substrate Specificity

A validated catalog of 105 recombinant depolymerases from Klebsiella phages covers 58 KL-types. 46 novel enzymes from prophages, jumbo phages, and common phages are linked to any known enzymatic activity against 14 classical serotypes and 12 genome-defined KL-types. Using activity-based profiling, structure prediction, and domain dissection, we developed a function-guided classification and a five-class structural catalog. This framework reveals highly specific enzymes active against up to three capsule types. K47 CPS was degraded by three diverse protein groups. Structurally similar depolymerases degrading particular CPS were found in distinct phage taxa, with highly conserved enzymes in Drulisvirus specific to K1-, K2-types. The exclusive depolymerases were found in siphoviruses targeting K2 and K62 serotypes. A case study of five structurally similar enzymes degrading KL22/KL37/KL111 and KL25/KL119 capsules suggested specificity switching via amino acid changes or C-domain modification. Klebsiella phage depolymerases catalog sheds light on their diversity, evolution, and potential application.

microbiology↗

bioRxiv2025

Characterization of Mycobacterium tuberculosis rhodanese-like sulfurtransferase SseA and its newly identified activator Rv3284: A new pathway to exploit as a drug target?

Tuberculosis (TB) remains a critical global health challenge, with Mycobacterium tuberculosis (Mtb) causing 10.8 million cases and 1.25 million deaths in 2023. Key issues include the dormant state of Mtb, resistant to drugs and immune responses, and the emergence of multi-drug-resistant strains. This underscores the need for new therapeutic targets and deeper research into Mtb pathogenesis and immunology. A potential drug target is the enzyme thiosulfate-sulfurtransferase SseA, which plays a role in macrophage infection by Mtb and its resistance to oxidative stress. SseA belongs to the rhodanese-like enzyme family, which catalyzes sulfur transfer reactions essential for Mtb survival. In our research, we identified a new protein (Rv3284), hereinafter referred to as SufEMtb due to its high homology with E. coli SufE, that interacts with SseA and modulates its activity. Sequence analysis and AI molecular modelling revealed detailed insights into their interaction that can contribute to the modulation of SseA activity. This research provides a mechanistic explanation to the need of a partner for SseA activation. Indeed, we propose that SufEMtb enhances SseA enzymatic function by binding to its non-catalytic N-terminal domain and bringing the active sites of the two proteins in close proximity, thus preparing for the activation-enhancing conformational change in a regulatory loop of SseA. This interaction is crucial for the effective enzyme activity and the maintenance of redox homeostasis in Mtb, making the SseA-SufEMtb protein complex a potential target for new TB therapies.

biochemistry↗

bioRxiv2024

Structural studies of KCTD1 and its disease-causing mutant P20S provide insights into the protein function and misfunction

Members of the KCTD protein family play key roles in fundamental physio-pathological processes. A plethora of literature studies have demonstrated their involvement in cancer, neurodevelopmental disorders, and genetic diseases. Despite two decades of intense investigations, the definition of structure-(mis)function relationships for these proteins is still rather limited. Here, we derived atomic-level structural data on KCTD1, by determining the crystal structure of its P20S mutant, which causes the scalp-ear-nipple syndrome, and performing molecular dynamics simulations. In addition to the expected folded domains (BTB and CTD) the crystal structure unravels that also the N-terminal region that precedes the BTB domain (preBTB) adopts a folded polyproline II (PPII) state. The global structure of the KCTD1 pentamer is characterized by an intricate architecture in which the different subunits mutually exchange domains to generate a closed domain swapping motif. In this framework, the BTB domain of each chain makes peculiar contact with the preBTB and the CTD regions of an adjacent chain. Indeed, the BTB-preBTB interaction is made of a PPII-PPII recognition motif whereas the BTB-CTD contacts are mediated by an unusual (+/-) helix discontinuous association. The inspection of the protein structure, along with the data that emerged from the MD data, provides a clear explanation of the pathogenicity of the SENS mutation P20S and unravels the role of the BTB-preBTB interaction in the insurgence of the disease. Finally, the presence of potassium bound to the central cavity of the CTD pentameric assembly provides insights into the role of the protein in metal homeostasis.

biochemistry↗

bioRxiv2020

A Universal Stress Protein upregulated by hypoxia may contribute to chronic lung colonisation and intramacrophage survival in cystic fibrosis.

Universal stress proteins (USPs) are ubiquitously expressed in bacteria, plants and eukaryotes and play a lead role in adaptation to environmental conditions. In Gram negative bacteria they enable adaption of bacterial pathogens to the conditions encountered in the human niche, including hypoxia, oxidative stress, osmotic stress, nutrient deficiency or acid stress, thereby facilitating colonisation. We previously reported that all six USP proteins encoded within a low-oxygen responsive locus in Burkholderia cenocepacia showed increased abundance during chronic colonisation of the CF lung. However, the role of USPs in chronic infection is not known. Using mutants derived from B. cenocepacia strain, K56-2, we show that USP76 is required for growth and survival in many conditions associated with the CF lung including, hypoxia, acidic conditions, oxidative stress. Moreover, it is involved in attachment to host epithelial cells, but not virulence. It also has a role in survival in macrophages isolated from people with CF. In contrast, another USP encoded in the same locus, USP92 had no effect on host cell attachment or oxidative stress, but was responsible for a 3-fold increase in virulence. Overall this shows that these USPs, both upregulated during chronic infection, have distinct roles in Burkholderia pathogenesis and may support the survival of B. cenocepacia in the CF lung. Specifically, USP76 is involved in its survival within CF macrophages, a hallmark of Burkholderia infection.

microbiology↗

bioRxiv2020

Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei

Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the Tier one select agents Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using RT-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the MIC of FA 4-fold in its isogenic mutant. We furthermore showed that the Bucl8 pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8 pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth. Author SummaryBurkholderia pseudomallei and Burkholderia mallei are highly infectious and multidrug resistant bacteria that are classified by the National Institute of Allergy and Infectious Diseases as Tier one select agents partly due to the intrinsic multidrug resistance associated with expression of the efflux pumps. To date, only few efflux pumps predicted in Burkholderia spp. have been studied in detail. In the current study we introduce Bucl8, an outer membrane component of an unreported putative efflux pump with a unique extended extracellular portion that forms a collagen triple helix and binds fibrinogen. We demonstrate Bucl8s role in fusaric acid resistance by defining its operon via bioinformatic and transcriptional analyses, as well as by employing loss-of-function and gain-of-function genetic approaches. Our studies also implicate the Bucl8-associated pump in metabolic and physiologic homeostasis. Understanding how Bucl8 efflux pump contributes to Burkholderia pathology will foster development of pump inhibitors targeting transport mechanism or identifying potential surface-exposed vaccine targets.

microbiology↗

bioRxiv2020

The DNA mimic protein BCAS0292 is involved in the regulation of virulence of Burkholderia cenocepacia

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. B. cenocepacia possesses a large genome encoding multiple virulence systems, stress response proteins and a large locus that responds to low oxygen. We previously identified BCAS0292, an acidic protein encoded on replicon 3. Deletion of the BCAS0292 gene resulted in altered abundance of >1000 proteins; 46 proteins became undetectable while 556 proteins showed >1.5-fold reduced abundance, suggesting BCAS0292 is a global regulator. Moreover, the {triangleup}BCAS0292 mutant showed a range of pleiotropic effects: virulence, host-cell attachment and motility were reduced, antibiotic susceptibility was altered and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. Structural analysis revealed BCAS0292 presents a dimeric {beta}-structure with a negative electrostatic surface. Further, the {Delta}BCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. We propose that BCAS0292 acts as a DNA-mimic, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.

microbiology↗

bioRxiv2020

An engineered stable mini-protein to plug SARS-Cov2 Spikes

AO_SCPLOWBSTRACTC_SCPLOWThe novel betacoronavirus SARS-CoV-2 is the etiological agent of the current pandemic COVID-19. Like other coronaviruses, this novel virus relies on the surface Spike glycoprotein to access the host cells, mainly through the interaction of its Receptor Binding Domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2). Therefore, molecular entities able to interfere with binding of the SARS-CoV-2 Spike protein to ACE2 have a great potential to inhibit viral entry. Starting from the available structural data on the interaction between SARS-CoV-2 Spike protein and the host ACE2 receptor, we here engineered a mini-protein with the aim of creating a soluble and stable Spike interactor. This mini-protein, which was recombinantly produced in high yields, possesses a stable helical conformation and is able to interact with the RBD of glycosylated Spike protein from SARS-CoV-2 with nanomolar affinity, as measured by microscale thermophoresis. By plugging the Spike protein, our mini-protein constitutes a valid tool for the development of treatments against different types of coronavirus.

biophysics↗