Biology subjects

Rojas, C. M.

Publications and source records attributed to Rojas, C. M..

6 recordsLinked to original sources

bioRxiv2026

The Pseudomonas protegens PBL3 secretome exerts antimicrobial activity against Burkholderia glumae by disrupting cell envelope homeostasis and impairing bacterial replication

The effectiveness of antimicrobials used to manage bacterial infections is increasingly threatened by the emergence of antimicrobial resistance. Many conventional antimicrobials act on a single defined target that pathogens can circumvent through target modification or pathway adaptation. In contrast, antimicrobials that disrupt multiple cellular processes may impose greater fitness costs and reduce the likelihood of resistance development, making their discovery a priority for pharmaceutical and agricultural applications. We previously demonstrated that the environmental bacterium Pseudomonas protegens PBL3 secretes a complex mixture of molecules (secretome) with antimicrobial activity against several plants and human pathogens. However, the mechanism underlying this activity remained unknown. Here, we used the plant pathogen Burkholderia glumae to investigate the mode of action of the P. protegens PBL3 secretome. We showed that the P. protegens PLB3 secretome causes extensive cell envelope damage that results in cell death and interferes with bacterial multiplication. Transcriptomic analysis further revealed broad disruption of processes associated with cell-envelope biogenesis and homeostasis and bacterial replication. Collectively, these findings demonstrate that the P. protegens PBL3 secretome acts as a multi-target antimicrobial that could be harnessed in the development of therapeutics to control bacterial infections of agricultural and medical importance.

microbiology↗

bioRxiv2026

AtNHR2A and AtNHR2B participate in unconventional protein secretion in response to environmental stress

The Arabidopsis thaliana nonhost resistance proteins 2A (AtNHR2A) and 2B (AtNHR2B) play crucial roles in plant immunity as the single mutants Atnhr2a and Atnhr2b and the double mutant Atnhr2bAtnhr2a are susceptible to the non-adapted pathogen Pseudomonas syringae pv. tabaci that is unable to infect wild-type Col-0 plants. The localization of fluorescent versions of AtNHR2A and AtNHR2B to compartments of the endomembrane system together with their interaction with secreted proteins suggested a function in endomembrane-mediated secretory processes participating in plant immunity. Comparative apoplastic proteomics analysis between wild type Col-0 and the double mutant Atnhr2bAtnhr2a after treatment P. syringae pv. tabaci, revealed that AtNHR2A and AtNHR2B are indeed required for the secretion of proteins containing N-terminal signal peptides that occurs through the conventional protein secretion pathway. In this work, we leveraged these apoplastic proteomics datasets to identify proteins lacking N-terminal signal peptide and expected to be secreted through unconventional secretion pathway(s). We discovered that AtNHR2A and AtNHR2B are also required for the secretion of proteins through an unconventional secretion pathway that, intriguingly, included proteins previously associated with abiotic stress. These findings led us to define the subcellular dynamics of AtNHR2A and AtNHR2B, and through co-localization analyses and the use of vesicle trafficking inhibitors, we uncovered their trafficking pathways transitioning through Golgi-dependent and Golgi-independent pathways to ultimately reach the central vacuole. Our findings suggest that AtNHR2A and AtNHR2B participate in a multivesicular bodies-vacuole-mediated unconventional secretion pathway that results in the release of proteins involved in plant responses to environmental stresses.

plant biology↗

bioRxiv2026

Comparative genomics analysis reveals that antimicrobial activity in Pseudomonas protegens PBL3 is associated with gene clusters participating in multiple cellular functions

The environmental bacterium Pseudomonas protegens PBL3 has antagonistic activity against the bacterial pathogen Burkholderia glumae, the causal agent of the rice disease Bacterial Panicle Blight (BPB). The antimicrobial activity of the P. protegens PBL3 was found in the bacteria-free secreted fraction (secretome), but the specific molecules, as well as the genetic basis conferring activity, have not been identified. To elucidate the genes and biosynthetic pathways governing the antimicrobial activity of the P. protegens PBL3 secretome, we used comparative genomics, by leveraging six Pseudomonas spp. strains, with available genomic sequences and exhibiting contrasting antimicrobial activities against B. glumae. We hypothesized that Pseudomonas spp. strains with antimicrobial activity against B. glumae have conserved genes with P. protegens PBL3 and those genes are absent in the strains lacking activity. To test this hypothesis, we performed comparative genomics analysis across the six strains using two complementary approaches, anvio and progressiveMauve, and using P. protegens PBL3 as the reference genome. This analysis revealed 188 genes uniquely present in antimicrobial-producing strains. Seven of those genes were associated with biosynthetic gene clusters predicted to encode secondary metabolites; additional genes were grouped into twenty-five contiguous clusters with functions associated with secretion, signal transduction, regulation, transport/efflux, carbohydrate metabolism and one with additional uncharacterized function. Altogether, this study provided a complex and multi-functional network of candidate genes in antimicrobial-producing strains, suggesting that P. protegens PBL3 employs not only classical biosynthetic pathways but also integrated regulatory, metabolic, and export modules to synthesize and deploy antimicrobials. ImportanceBacterial plant diseases can be controlled through biological control, a strategy wherein beneficial microorganisms known as biological control agents (BCAs) interfere with the biological activities of pathogens through several mechanisms. One of these mechanisms is antibiosis, by which antimicrobial molecules produced by the BCA prevent pathogen multiplication or actively kill the pathogen. While this mechanism of antibiosis has been widely recognized, the specific molecules associated with the antimicrobial activity are not always identified, given their diverse and complex chemical structures as well as the unique and intricate biosynthetic pathways. This study unraveled different pathways underlying the antimicrobial activity in the environmental bacterium Pseudomonas protegens PBL3 to advance the discovery of effective antimicrobials to control bacterial plant diseases.

microbiology↗

bioRxiv2025

PERINATAL ORGANOPHOSPHATE FLAME RETARDANT EXPOSURE ALTERS ADULT HPA AXIS FUNCTION AND AVOIDANCE BEHAVIOR IN A SEX-SPECIFIC MANNER IN MICE

Organophosphate flame retardants (OPFRs) are ubiquitous flame-retardant additives with endocrine-disrupting properties. Despite increasing evidence that OPFRs impact neurodevelopment, their effects on the neuroendocrine stress response remain poorly understood. To examine their long-term impact on stress regulation, we treated pregnant C57Bl/6J dams to a mixture of tris(1,3-dichloro-2-propyl) phosphate (TDCPP), triphenyl phosphate (TPP), and tricresyl phosphate (TCP; 1 mg/kg each) from gestational day (GD) 7 through postnatal day (PND) 14. Adult offspring (8-9 weeks of age) were then challenged with acute stressors, including 1 h restraint or a 6-day acute variable stress (AVS) paradigm. Perinatal OPFR exposure produced persistent, sex-specific alterations in the hypothalamic-pituitary-adrenal (HPA) axis and stress-related neurocircuitry. Following 1 h restraint, OPFR-treated females showed heightened serum corticosterone. In addition, gene expression analysis revealed sex-dependent disruptions in key stress-regulatory pathways after OPFR treatment and 1 h restraint in the hypothalamus (Crhr1, Crhr2, Ptpn5) and pituitary (Crhr1, Pomc, Nr3c1). Females demonstrated more differences in adrenal gene expression related to steroidogenesis (Mc2r, Cyp11b2) and catecholamine biosynthesis (Dbh, Pnmt), with OPFR-treated groups having blunted responses. OPFR AVS females displayed reduced corticosterone and Crh mRNA in the hypothalamus, and downregulated Pacap/Pac1r expression in the bed nucleus of the stria terminalis (BNST), accompanied by increased behavioral avoidance and immobility. In males, OPFR exposure led to increased BNST Pacap and Pac1r, expression, along with hyperactivity and avoidance behaviors. Together, these findings demonstrate that early-life OPFR exposure induces lasting, sex-specific dysregulation of the HPA axis and associated stress circuits, highlighting OPFRs as developmental neuroendocrine disruptors with implications for mood and stress-related disorders.

neuroscience↗

bioRxiv2022

Alterations to the broad-spectrum formin inhibitor SMIFH2 improve potency

SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.

biochemistry↗

bioRxiv2021

Pseudomonas syringae pv. tomato DC3000 Effector HopG1 is a multi-faceted protein that Triggers Necrotic Cell Death that is attenuated by the Nonhost Resistance 2B (AtNHR2B) Protein

The plant pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) has become a paradigm in plant-bacteria interactions due to its ability to cause disease in the model plant Arabidopsis thaliana. Pst DC3000 uses the type III secretion system to deliver type III secreted effectors (T3SEs) directly into the plant cytoplasm. Pst DC3000 T3SEs contribute to pathogenicity by suppressing plant defense responses and targeting plants physiological processes. Although the complete repertoire of effectors encoded in the Pst DC3000 genome have been identified, the specific function for most of them remains to be elucidated. The mitochondrial-localized T3E HopG1, suppresses plant defense responses and promotes the development of disease symptoms. Here, we show that HopG1 triggers necrotic cell death that enables the growth of non-adapted pathogens. We further showed that HopG1 interacts with the plant immunity-related protein AtNHR2B and that AtNHR2B attenuates HopG1-virulence functions.

plant biology↗