Instituto de Biología Molecular y Celular de Rosario

En este estudio se ha evaluado el impacto de un choque térmico agu­do sobre el desarrollo embrionario del pez cebra (Danio rerio). El estresor (1 h; 38 °C) fue aplicado al inicio de la gastrulación en la especie (6 horas post fecun­dación (hpf)). Embriones de la línea transgénica Tg(hsp70l:dn-fgfr1a-EGFP), con inhibición inducible de la vía del factor de crecimiento de fibroblastos (Fgf), fueron utilizados para evaluar los efectos del estresor. El marco temporal de mo­nitorización englobó la embriogénesis en la especie (hasta las 72 hpf). Se eva­luaron los siguientes parámetros: supervivencia (0 hpf, 24 hpf, 48 hpf y 72 hpf), eclosión (48 hpf y 72 hpf), malformaciones (72 hpf) y comportamiento (24 hpf). Nuestros resultados indican que el choque térmico no resulta letal en el marco de evaluación seleccionado, pero provoca una reducción de la tasa de eclosión y un elevado número de malformaciones en la línea Tg(hsp70l:dn- fgfr1a-EGFP). Además, nuestros datos muestran alteraciones en el comportamiento a 24 hpf. Estos hallazgos evidencian que estresores agudos de muy corta duración pueden provocar serias alteraciones fisiológicas en etapas del desarrollo temprano, cuan­do el embrión muestra una mayor vulnerabilidad.

Heme is an essential molecule for most organisms, yet some parasites, like Trypanosoma cruzi, the causative agent of Chagas disease, cannot synthesize it. These parasites must acquire heme from their hosts, making this process critical for their survival. In the midgut of the insect vector, T. cruzi epimastigotes are exposed to both hemoglobin (Hb) and free heme resulting from its degradation. Despite the importance of this nutrient, how different heme sources influence parasite gene expression remains poorly understood.Here, we showed that heme restitution either as hemin or Hb to heme-starved parasites induces an early and distinct transcriptional response in T. cruzi epimastigotes. Using RNA sequencing at 4- and 24-hours post-supplementation, we identified gene subsets commonly or uniquely regulated by each heme source, including genes putatively linked to heme acquisition and metabolism. The study includes the first focused characterization of CRAL/TRIO domain-containing protein (TcCRAL/TRIO), a novel heme-responsive hemoprotein. Our results provide a more detailed picture of T. cruzi biology and highlights heme acquisition as a promising point of vulnerability to control parasite proliferation.

Abstract Background Carbapenem-resistant Klebsiella pneumoniae CRKP) represents a critical public health threat, with limited treatment options due to its resistance to last-line antibiotics. Cefiderocol (FDC), a novel siderophore cephalosporin, has shown efficacy against CRKP; however, resistance has emerged. This study characterizes a spontaneous FDC-resistant subpopulation (IHC216) derived from a KPC producing K. pneumoniae strain (KPNMA216). To understand this phenotype, we focused on genomic, transcriptional, and phenotypic adaptations.Figure 1.(A) Expression of genes coding for siderophores (irp1, iucA and entB) and siderophores transporters (fepA, cirA, iroN, fiU and fecA) and β-lactamase blaKPC163 in the KPNMA216 and IHC216 strains. The data shown of qRT-PCR are mean ± SD. Fold changes were calculated using ΔΔCt analysis. At least three independent biological samples were tested using four technical replicates. Statistical significance (P < 0.05) was determined by two-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism (GraphPad software, San Diego, CA, USA). Significance was indicated by: *P < 0.05, **P < 0.01, ***P < 0.001, and **** P < 0.0001. (B) Biofilm formation in tubes quantified by crystal violet and capsule density in KPC216 and IHC216 strains. three independent biological samples were tested using four technical replicates. A representative image is shown. Statistical analysis was determined by t test (p < 0.05), using GraphPad Prism (GraphPad software, San Diego, CA, USA). Methods Whole-genome sequencing (WGS) was performed to identify genetic mutations associated with FDC resistance. Quantitative real-time PCR (qRT-PCR) was used to assess gene expression changes related to iron acquisition, antibiotic resistance, oxidative stress response, and cell wall synthesis. Antimicrobial susceptibility testing was conducted using minimum inhibitory concentration (MIC) assays. Capsule and biofilm formation were evaluated using standard biochemical assays. Results IHC216 exhibited an increase in FDC MIC compared to the wild-type strain (from 8 to 32 ug/ml). While FDC resistance developed, meropenem MIC decreased from 32 mg/L to 0.5 mg/L, and imipenem MIC from 48 mg/L to 3 mg/L, demonstrating collateral susceptibility. WGS identified mutations in genes linked to transcriptional regulation and membrane permeability. qRT-PCR analysis revealed significant downregulation of key iron acquisition genes and upregulation of alternative iron uptake pathways (Fig.1a). blaKPC-163 expression was markedly reduced, correlating with restored susceptibility to carbapenems (Fig. 1a). Additionally, increased capsule production and biofilm formation were observed (Fig.1b). Conclusion This study highlights the intricate genetic and transcriptional adaptations underlying FDC resistance in KPC-producing K. pneumoniae. The observed collateral susceptibility to carbapenems offers potential treatment strategies that exploit this vulnerability. Understanding these resistance mechanisms is critical for optimizing therapeutic approaches against CRKP infections. Disclosures Robert A. Bonomo, MD, Merck: Grant/Research Support|Shinogi: Grant/Research Support|VenatoRx: Grant/Research Support

The pathogenicity of Gram-negative bacteria is mediated by multiple virulence factors that likely include secreted Outer Membrane Vesicles (OMVs) that can act as a cargo for delivery of enzymes or toxins to target tissues. Here, we have studied the effects on the host of OMVs prepared from one of the most potent pathogens of Drosophila melanogaster, Serratia marcescens. OMV injection leads to the apparent demise of flies within few hours. We identify a number of host defenses that somewhat protect it from the action of OMVs, namely the systemic humoral immunity pathway Immune deficiency, Prophenol Oxidases 1&2, and the redox active enzymes Dual oxidase, NADPH-oxidase, and Nitric Oxygen Synthase. In contrast, unidentified hemocyte function(s) and the circulating protease Hayan promote the pathogenicity of OMVs. Mechanistically, we find that OMVs promote the activation of the JNK pathway and the transient expression of the pro-apoptotic genes head-involution defective and reaper in at least neurons. Our data suggest that mitochondrially-derived reactive oxygen species promote neuronal cell death that leads to the paralysis of OMV-injected flies. We identify the metalloprotease PrtA as a major virulence factor of OMVs and show that the injection of purified PrtA mimics most of the effects of OMVs. Finally, our data further indicate that PrtA contributes to the pathogenicity of injected Serratia marcescens. This study underscores the potential for OMVs to act as virulence factors that efficiently target the nervous system in vivo despite the blood brain barrier.

Carbapenem resistance in the gram-negative opportunistic pathogen Acinetobacter baumannii primarily stems from the overexpression of acquired class D serine β-lactamases, known as OXA carbapenemases. These enzymes exhibit weak carbapenemase activity and possess lipoprotein signal peptides. While the kinetic and structural aspects of OXA enzymes have been characterized, their biogenesis pathway has received little attention, despite potentially offering novel therapeutic targets. Here, we investigated the biosynthetic process of the OXA-58 carbapenemase in the model A. baumannii strain ATCC17978. [3H]palmitate labeling confirmed that the OXA-58 precursor is lipidated in vivo. Replacing the OXA-58 lipobox cysteine with alanine through site-directed mutagenesis demonstrated that, while the lipoprotein pathway is not essential for productive OXA-58 synthesis, it is crucial for achieving the high cellular OXA-58 levels A. baumannii needs to efficiently overcome carbapenem challenge. Lipidation significantly increased OXA-58 hydrophobicity, directing the carbapenemase to a membrane location, likely the outer membrane (OM), after periplasmic translocation. This specific localization is a critical step for accumulating the high periplasmic OXA-58 concentration necessary for carbapenem resistance. Furthermore, lipidation enabled the selective recruitment of OXA-58 into outer membrane vesicles (OMVs), revealing a novel disposal mechanism for surplus OXA-58 production. In conclusion, the A. baumannii lipoprotein biosynthetic pathway facilitates both the high periplasmic OXA-58 concentration essential for a more efficient carbapenem resistance and the accompanying selective removal of surplus OXA-58 production via OMV. These features were likely powerful drivers in the selection of the lipoprotein pathway for the overproduction of OXA carbapenemases among contemporary A. baumannii strains subjected to carbapenem challenge.

Abstract Pair correlation functon(pCF) is a powerful approach that has gained increasing popularity in recent years for studying protein dynamics in living cells; however, its application remains limited by methodological constraints and the absence of standardized protocols. In this work, we optimize key aspects of pCF and dual color-pCF analysis. We demonstrate that spectral bleed-through as low as 1% produces spurious cross-correlations, underscoring the need for rigorous correction. To address this, we introduce a robust signal demixing strategy that removes false positive correlations while preserving genuine molecular interactions. This advance expands the reliability of correlation analyses in complex live-cell environments. Applying this approach, we investigated the nucleocytoplasmic shuttling of the splicing factor RBM10 in living cells expressing dengue virus (DENV) NS5 polymerase under poly (I:C)-triggered innate immune response. Our findings reveal that RBM10 and NS5 not only interact but also co-shuttle between the nuclear and cytoplasmic compartments. Furthermore, RBM10 transport is differentially modulated by both NS5 and innate immune induction, reinforcing the role of DENV in altering host nuclear transport. Overall, this work establishes signal demixing as a key advance for live-cell correlation techniques and demonstrates its potential for uncovering complex host-virus interactions.

Background: Chronic Chagas disease can affect multiple organs, most notably the heart and gastrointestinal tract, and in some cases, the nervous system. However, the underlying pathophysiological mechanisms of this parasitic infection remain incompletely understood. Summary: Evidence from studies in both mice with acute Trypanosoma cruzi (T. cruzi) infection and in patients with Chagas disease has revealed a range of immune-neuroendocrine alterations and metabolic disruptions. In this review, we highlight key findings in human Chagas disease related to these abnormalities and discuss their potential contributions to disease pathogenesis. Key Messages: In the context of chronic Chagas disease, the neuroendocrine-immune axis operates as a dynamic interface, integrating systemic immune-endocrine processes with localized responses in the central nervous system (CNS), with each component influencing disease advancement and organ-specific pathology through distinct yet interconnected mechanisms.

Sperm capacitation involves proteolytic remodeling of membrane proteins, including components of the CatSper calcium channel, which is essential for hyperactivation and male fertility. Here, we identify the seminal protease inhibitor SPINK3, a known decapacitation factor that suppresses premature capacitation in the female tract, as the first physiological inhibitor of CATSPER1 processing. In mouse sperm, SPINK3 blocks capacitation-induced CATSPER1 cleavage, preserving a subpopulation with intact CatSper channels and lacking pTyr development in the flagellum. SPINK3 localizes to the outer surface of the sperm principal piece membrane in a CatSper-dependent but non-quadrilateral pattern, stabilizes membrane organization, and delays cholesterol efflux. These results reveal SPINK3 as a multifunctional regulator of capacitation, shaping sperm subpopulations in the female reproductive tract.

Seminal plasma proteins play key roles in maintaining the survival and functional integrity of spermatozoa after ejaculation. Semenogelin-1 (SEMG1) is the predominant protein in the seminal plasma and a major structural component of the semen coagulum in humans and mice. SEMG1 binds EPPIN (Epididymal protease inhibitor) on the sperm surface, transiently inhibiting the acquisition of sperm motility and hyperactivation after ejaculation. Nevertheless, the molecular mechanisms underlying SEMG1-mediated inhibition of sperm hyperactivation remain unclear. We hypothesize that the SEMG1 effects are associated with the inhibition of CatSper, a sperm-specific calcium channel crucial for triggering hyperactivation. We observed that recombinant mouse SEMG1 (mSEMG1) inhibited both progressive motility and hyperactivation in a concentration-dependent manner. Notably, hyperactivation was not recovered upon NH4Cl-induced alkalinization, suggesting that the effects of mSEMG1 are downstream of capacitation-associated intracellular alkalinization. Electrophysiological recordings revealed that mSEMG1 impaired CatSper currents at concentrations near its IC50 for inhibiting sperm hyperactivation. Recombinant mSEMG1 truncations mSEMG1Q32-V118 and mSEMG1R98-G375, but not mSEMG1Y221-G375, independently inhibited sperm hyperactivation and CatSper currents similar to full-length mSEMG1. However, only mSEMG1R98-G375 displayed similar EPPIN-binding capacity comparable to full-length mSEMG1, whereas mSEMG1Q32-V118 and mSEMG1Y221-G375 showed only minor binding. Our findings reveal that mouse SEMG1 comprises two distinct functional domains within the Q32-V118 and R98-S220 sequences, which contribute to the inhibition of sperm hyperactivation by targeting the CatSper channel, both independently and dependent on EPPIN binding. These functional domains represent promising prototypes for the design of spermiostatic molecules, offering additional avenues for non-hormonal male contraception targeting spermatozoa.