The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence

Mycobacterium tuberculosis and its close relative Mycobacterium marinum infect macrophages and induce the formation of granulomas, organized macrophage-rich immune aggregates. These mycobacterial pathogens can accelerate and co-opt granuloma formation for their benefit, using the specialized secretion system ESX-1, a key virulence determinant. ESX-1-mediated virulence is attributed to the damage it causes to the membranes of macrophage phagosomal compartments, within which the bacteria reside. This phagosomal damage, in turn, has been attributed to the membranolytic activity of ESAT-6, the major secreted substrate of ESX-1. However, mutations that perturb ESAT-6 membranolytic activity often result in global impairment of ESX-1 secretion. This has precluded an understanding of the causal and mechanistic relationships between ESAT-6 membranolysis and ESX-1-mediated virulence. Here, we identify two conserved residues in the unstructured C-terminal tail of ESAT-6 required for phagosomal damage, granuloma formation and virulence. Importantly, these ESAT-6 mutants have near-normal levels of secretion, far higher than the minimal threshold we establish is needed for ESX-1-mediated virulence early in infection. Unexpectedly, these loss-of-function ESAT-6 mutants retain the ability to lyse acidified liposomes. Thus, ESAT-6 virulence functions in vivo can be uncoupled from this in vitro surrogate assay. These uncoupling mutants highlight an enigmatic functional domain of ESAT-6 and provide key tools to investigate the mechanism of phagosomal damage and virulence.

NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia—a locus for craniofacial sensory nerves—were used to examine how NGF affects the Ca2+-dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca2+-dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca2+-independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181–197 and 198–206) to support CAP-evoked Ca2+-dependent exocytosis at different intensities of stimulation.

Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice

A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5 end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5 resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330- 338) is important for two events that drive NHEJ, stimulating ligation and inhibiting resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly through inhibiting Dna2-mediated resection.

The C-terminal domain of SEC-10 is fundamental for exocyst function, Spitzenkorper organization and cell morphogenesis in Neurospora crassa.

The exocyst is a conserved multimeric complex that participates in the final steps of the secretion of vesicles. In the filamentous fungus Neurospora crassa, the exocyst is crucial for polar growth, morphology, and the organization of the Spitzenkorper (Spk), the apical body where secretory vesicles accumulate before being delivered to the plasma membrane. In the highly polarized cells of N. crassa, the exocyst subunits SEC-3, SEC-5, SEC-6, SEC-8, and SEC-15 were previously found localized at the plasma membrane of the apices of the cells, while EXO-70 and EXO-84 occupied the frontal outer layer of the Spk, occupied by vesicles. The localization of SEC-10 had remained so far elusive. In this work, SEC-10 was tagged with the green fluorescent protein (GFP) either at its N- or C-terminus and found localized at the plasma membrane of growing hyphal tips, similar to what was previously observed for some exocyst subunits. While expression of an N-terminally tagged version of SEC-10 at its native locus was fully viable, expression of a C-terminally tagged version at its native locus resulted in severe hyphal growth and polarity defects. Additionally, a sec-10 knockout mutant in a heterokaryotic state (with genetically different nuclei) was viable but showed a strongly aberrant phenotype, confirming that this subunit is essential to maintain hyphal morphogenesis. Transmission electron microscopy analysis revealed the lack of a Spk in the SEC-10-GFP strain, suggesting a critical role of the exocyst in the vesicular organization at the Spk. Mass spectrometry analysis revealed fewer peptides of exocyst subunits interacting with SEC-10-GFP than with GFP-SEC-10, suggesting an essential role of the C-terminus of SEC-10 in exocyst assembly and/or stability. Altogether, our data suggest that an unobstructed C-terminus of SEC-10 is indispensable for the exocyst complex function and that a GFP tag could be blocking important subunit-subunit interactions.

Foot-and-mouth disease virus 3A hijacks Sar1 and Sec12 for ER remodeling in a COPII-independent manner

Positive-stranded RNA viruses modify host organelles to form replication organelles (ROs) for their replication. Enteroviral 3A protein has been demonstrated to be highly associated with the COPI pathway, in which factors work on the ER-to-Golgi intermediate and the Golgi. However, Sar1, a COPII factor exerting coordinated action at endoplasmic reticulum (ER) exit sites, rather than COPI factors, is required for foot-and-mouth disease virus (FMDV) replication. Therefore, we thought that deep understanding of FMDV 3A was the key to explaining the differences and to unlocking the secret of FMDV RO formation. In this study, FMDV 3A was confirmed as a peripheral membrane protein capable of modifying the ER into vesicle-like structures, which were neither COPII vesicles nor autophagosomes. When the C-terminus of 3A was truncated, it would be located at the ER without vesicular modification. This change was revealed by mGFP and APEX2 fusion constructs observed by fluorescence microscopy and electron tomography, respectively. Referring to other 3A truncation, the minimal region for modification was aa 42–92. Furthermore, we found that the remodeling was related to two COPII factors, Sar1 and Sec12. Both interacted with 3A, but their binding domains on 3A were different. Finally, we hypothesized that the N-terminus of 3A would interact with Sar1 as its C-terminus simultaneously interacted with Sec12, which possibly would enhance Sar1 activation. On the ER membrane, two active Sar1 were connected by 3A with regions of aa 42–59 and aa 76–92, causing curvature of the membrane. This mechanism is distinct from the traditional COPII pathway and should be crucial for FMDV RO formation.

Potentiation of drug toxicity through virus latency reversal promotes preferential elimination of HIV infected cells

Eliminating latently infected cells is a highly challenging, indispensable step towards the overall cure for HIV/AIDS. We recognized that the unique HIV protease cut site (Phe-Pro) can be reconstructed using a potent toxin, monomethyl auristatin F (MMAF), which features Phe at its C-terminus. We hypothesized that this presents opportunities to design prodrugs that are specifically activated by the HIV protease. To investigate this, a series of MMAF derivatives was synthesized and evaluated in cell culture using latently HIV-infected cells. Cytotoxicity of compounds was enhanced upon latency reversal by up to 11-fold. In a mixed cell population, nanomolar concentrations of the lead compound depleted predominantly the HIV-infected cells and in doing so markedly enriched the pool with the uninfected cells. Despite expectation, mechanism of action of the synthesized toxins was not as HIV protease-specific prodrugs, but likely through the synergy of toxicities between the toxin and the reactivated virus.

WNK kinases sense molecular crowding and rescue cell volume via phase separation

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Ratiometric Electrochemical Biosensor Based on Internally Controlled Duplex PCR for Detection of Mycobacterium Tuberculosis

The pathogenic bacteria Mycobacterium tuberculosis (MTB) is responsible for tuberculosis, which is well known as the globally leading cause of death. The likelihood of false negative interpretation as well as potential influence from intrinsic and extrinsic factors are considerably minimized by the incorporation of internal control (IC) detection in the developed assay platform. Ratiometric electrochemical (REC) biosensor for detection of MTB was developed based on the IC integration via duplex PCR (dPCR) and a dual-signal electrochemical readout. The MTB- or IC-specific PNA probe was labeled with methylene blue (MB) or ferrocene (FC), respectively at the C terminus, producing a strong square wave voltammetry signal. Interaction of the ICdPCR product could induce changes in the dynamics of these two redox-labeled PNA probes (MTB-MB and IC-FC) that were attached to the screen-printed gold electrode via formation of a self-assembled monolayer. Using this MB as a reporter and FC as an IC, the REC ICdPCR biosensor achieved a broad detection range from 10 fM to 10 nM and a detection limit of 1.26 fM, corresponding to approximately 2.5 bacteria cells. The REC ICdPCR biosensor was applied to MTB measurement in practical samples, exhibiting high accuracy and more importantly high practicability.

Pericentriolar matrix integrity relies on cenexin and Polo-Like Kinase (PLK)1

Polo-Like-Kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosome's pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa) and zebrafish embryos in parallel to phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation and that a conserved C-terminal PLK1 binding site between humans, zebrafish, and out to cnidaria is required for PCM maintenance through PLK1-dependent substrate phosphorylation events. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexin's C-terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to anchor PLK1 moderating its potential to phosphorylate PCM substrates required for PCM maintenance and function.

A novel plasmid entry exclusion system in pKPC_UVA01, a promiscuous conjugative plasmid carrying the bla KPC carbapenemase gene

Conjugative plasmids are the principal mediator in the emergence and spread of antibiotic resistance genes in Enterobacterales. Plasmid entry-exclusion (EEX) systems can restrict their transfer into the recipient bacteria carrying closely related plasmids. In this study, we have identified and characterized a novel plasmid entry exclusion system in a carbapenem resistance plasmid pKPC_UVA01, responsible for widespread dissemination of the bla KPC carbapenemase gene among Enterobacterales in the United States. The identified eex gene in the recipient strain of different Enterobacterales species inhibits the conjugation transfer of pKPC_UVA01 plasmids at a range of 200-400 fold, and this inhibition was found to be a dose-dependent function of the EEX protein in recipient cells. The C-terminus truncated version of eex or eex with an early termination codon at the C-terminus region alleviates inhibition of conjugative transfer. Unlike the strict specificity of plasmid exclusion by the known EEX protein, the newly identified EEX in the recipient strain can inhibit the transfer of IncP and IncN plasmids. The eex gene from the plasmid pKPC_UVA01 is not required for conjugative transfer but is essential in the donor bacteria for entry exclusion of this plasmid. This is a novel function of a single protein that is essential in both donor and recipient bacteria for entry exclusion of a plasmid. This eex gene is found to be distributed in multi-drug resistance plasmids similar to pKPC_UVA01 in different Enterobacterales species and may contribute to the stability of this plasmid type by controlling its transfer.