Biology subjects

Bernal-Bayard, J.

Publications and source records attributed to Bernal-Bayard, J..

6 recordsLinked to original sources

bioRxiv2026

Genome-scale TraDIS reveals dynamic and conserved fitness requirements of Salmonella Typhimurium across sequential host niches during porcine infection

Non-typhoidal salmonellosis remains a major global cause of foodborne gastrointestinal disease, with pigs representing an important reservoir of Salmonella enterica serovar Typhimurium. The emergence of host-adapted, multidrug-resistant lineages has further reinforced the need to understand the genetic basis of bacterial persistence and pathogenicity within physiologically relevant hosts. Here, we applied transposon-directed insertion sequencing (TraDIS) to systematically define the genetic requirements of a highly host-adapted, multidrug-resistant S. Typhimurium DT104 isolate across sequential host niches during porcine infection. A high-density transposon mutant library ([~]1.2 million mutants) was subjected to in vivo selection in the ileal mucosa and mesenteric lymph nodes, as well as ex vivo infection of primary porcine neutrophils. Across all conditions, we identified 1,813 conditionally essential genes, revealing strong niche-specific fitness signatures and a progressive increase in selective stringency along the infection route. Ileal colonization was primarily driven by determinants of invasion, motility and lipopolysaccharide biosynthesis. In contrast, survival within neutrophils depended on resistance to antimicrobial stresses and extensive metabolic rewiring, whereas persistence in lymph nodes required a broader functional repertoire integrating virulence, motility, envelope remodelling and metabolic adaptation, including bacterial microcompartment-associated pathways. Despite this marked heterogeneity, we identified a conserved porcine host-conditioned essential genome, comprising core invasion functions, RNA metabolism and genome maintenance pathways, and central metabolism. Notably, the outer membrane lipid asymmetry system Mla and the twin-arginine translocation (Tat) pathway emerged as conserved bottlenecks for in vivo fitness across all host-associated environments. Together, these findings establish a hierarchical model of S. Typhimurium adaptation during porcine infection and provide a systems-level view of tissue-specific and conserved genetic requirements underpinning persistence in a major zoonotic reservoir.

microbiology↗

bioRxiv2025

Ubiquitylome Rewiring by Bacterial E3 Ligases Reveals Multifaceted Host Subversion

Salmonella enterica has evolved an arsenal of effector proteins secreted via type III secretion systems (T3SS) to manipulate host cell functions. Among these, the NEL family E3 ubiquitin ligases (SlrP, SspH1, and SspH2) are known to modulate immune signaling, but the breadth of their impact on the host ubiquitylome remains unexplored. In this study, we have performed a global proteomic analysis to identify host proteins ubiquitylated in response to expression of these three effectors in human cells. Using enrichment strategies combined with mass spectrometry under conditions where the proteasome is active or not, we identified 214 putative substrates of ubiquitylation. Gene ontology and KEGG pathway analysis revealed enrichment in pathways related to RNA processing, ribosome biogenesis, cytoskeleton organization, chromatin remodeling, and vesicular trafficking. In vitro ubiquitylation assays validated five novel substrates and revealed differential substrate specificity and patterns of ubiquitin chain topology among the effectors. Notably, expression of SspH1 in Saccharomyces cerevisiae disrupted polysome profiles in a ligase activity dependent manner, indicating a direct impact of the bacterial effector on translation of eukaryotic cells. Comparison with previously published global interactomes and ubiquitylomes supports a model in which Salmonella NEL effectors subvert a broader range of host pathways through targeted ubiquitylation. Our findings uncover new roles for NEL ubiquitin ligases in host manipulation and provide a holistic analysis of their effects on the ubiquitylome of the host cell, constituting a valuable resource for the study of bacterial pathogenesis and infection biology.

microbiology↗

bioRxiv2025

Provision of Preferred Nutrients to Macrophages Enables Salmonella to Replicate Intracellularly Without Relying on Type III Secretion Systems

Intracellular survival and replication within macrophages are key virulence determinants of Salmonella enterica serovar Typhimurium. This phenomenon is traditionally attributed to the activity of its two Type III Secretion Systems (T3SS) and their associated effectors. A critical challenge for these bacteria is acquiring nutrients from inside the host cell. Thus, they modulate the metabolism of host cells to replicate. Given the metabolic plasticity of macrophages, a key unresolved question is how their metabolic heterogeneity shapes intracellular Salmonella replication. By using human primary macrophages and live-cell imaging to monitor bacterial dynamics at the single-cell level, we revealed that Salmonella does not replicate in all infected cells. However, supplementation with specific carbon sources used by Salmonella during infection accelerated bacterial replication and increased the proportion of macrophages showing replicative bacteria. Remarkably, this occurred even in the absence of functional T3SSs, as a {Delta}prgH/{Delta}ssaV double mutant was able to replicate in a subset of infected cells under favorable nutrient conditions. These phenotypes are further amplified in macrophages with higher glycolytic activity, such as the murine RAW 264.7 cell line. Further analyses demonstrated that enhanced Salmonella replication is not strictly dependent on host glycolytic activity but is instead driven by the ability of the host cell to take up the nutrients Salmonella prefers for its replication early during infection. In summary, our findings suggest that the dependence of Salmonella on its T3SSs for intracellular replication can be bypassed when host cells provide optimal access to key nutrients and highlight the impact of metabolic heterogeneity in shaping intracellular bacterial replication during infection of macrophages.

microbiology↗

bioRxiv2025

Lack of RNase HI affects virulence in Salmonella enterica

Bacterial virulence and antibiotic resistance are interconnected global threats that can synergize to multiply their adverse effects on health and economy. Two proposed strategies to address this dual challenge are non-biocidal inhibition of virulence traits (anti-virulence strategy) and manipulation of eco-evolutionary dynamics in bacterial populations to hinder dissemination of antibiotic resistance (anti-resistance strategy). Both strategies require identifying factors involved in bacterial gene expression, fitness and evolution. R-loops strongly influence these traits, as well as fitness of bacteria carrying antibiotic resistance mutations. This makes the enzyme responsible for R-loop degradation, the RNase HI, a promising target for anti-resistance approaches. Interestingly, the involvement of R-loops in gene expression could also make RNase HI a potential target for anti-virulence strategies. In this study, we explored this possibility by investigating the effects of RNase HI deficiency on the pathogenicity of Salmonella enterica. We found that the absence of RNase HI alters the expression of genes associated with virulence both at the population and single-cell levels, and both ex vivo and during infection of mammalian cells. Furthermore, we observed that RNase HI depletion causes defects in phenotypes associated with virulence, such as motility and biofilm formation. Lack of RNase HI also reduces the ability of Salmonella to survive within macrophages. However, lack of RNase HI does not significantly affect the invasion of mammalian epithelial cells or their immune response. Overall, our results demonstrate a pleiotropic influence of R-loops on bacterial virulence, suggesting that RNase HI could be a target for anti-virulence strategies. These findings provide a theoretical framework for the development of dual anti-resistance and anti-virulence interventions based on RNase HI targeting.

microbiology↗

bioRxiv2022

Escherichia coli membrane microdomain SPFH protein HflC interacts with YajC and contributes to aminoglycoside and oxidative stress tolerance

Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs) also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. By contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner-membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner-membrane FMMs and the regulation of cellular processes. Here we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCEEukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane micro-domains. These domains are also called lipid rafts and contain proteins of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but were mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacteria E. coli is altered in absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli.

microbiology↗

bioRxiv2022

Chromosomal position of ribosomal protein genes impacts long term evolution of Vibrio cholerae.

It is unclear how gene order within the chromosome influences bacterial evolution. The genomic location of genes encoding the flow of genetic information is biased towards the replication origin (oriC) in fast-growing bacteria. To study the role of chromosomal location on cell physiology we relocated the S10-spec- locus (S10), harboring half of ribosomal protein genes, to different chromosomal positions in the fast-growing pathogen V. cholerae. We found that growth rate, fitness and infectivity inversely correlated the distance between S10 and oriC. To gain insight into the evolutionary effect of ribosomal protein genomic position, we evolved strains bearing S10 at its current oriC-proximal location or derivatives where the locus far from it, at the chromosomal termini. All populations increased their growth rate along the experiment regardless S10 genomic location. However, the growth rate advantage of an oriC-proximal location persisted along experimental evolution indicating that suppressor mutations cannot compensate S10 genomic position. An increment in biofilm forming capacity was another common trait observed along the experiment. Deep sequencing of populations showed on average 1 mutation fixed each 100 generations, mainly at genes linked to flagellum biosynthesis regulation, lipopolysaccharide synthesis, chemotaxis, biofilm and quorum sensing. We selected fast-growing clones displaying a [~]10% growth rate increment. We found that they harbored inactivating mutations at, among other sites, the flagellum master regulators flrAB. The introduction of these mutations into naive V. cholerae strains resulted in a [~]10% increase of growth rate. Our study therefore demonstrates that the location of ribosomal protein genes conditions the evolutionary trajectory of growth rate in the long term. While genomic content is highly plastic in prokaryotes, gene order is an underestimated factor that conditions cellular physiology and lineage evolution. The lack of suppression enables artificial gene relocation for genetic circuit reprogramming.

microbiology↗