Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon.

The virulence of enteropathogenic Escherichia coli (EPEC) depends on a type III secretion system (T3SS), a membrane-spanning apparatus that injects effector proteins into the cytoplasm of target enterocytes. The T3SS, or injectisome, is a self-assembled nanomachine whose biogenesis and function rely on the ordered secretion of three distinct categories of proteins: early, middle, and late type III substrates. In EPEC, this hierarchical secretion is assisted by several cytosolic protein complexes at the base of the injectisome. Among these, the sorting platform is involved in the recognition and sequential loading of the different classes of T3-substrates. In addition, a heterotrimeric gatekeeper complex, also known as a molecular switch, operates in concert with components of the T3SS export apparatus to guarantee the delivery of middle substrates prior to late substrate secretion. In this study, we showed that the sorting platform is differentially required for the secretion of distinct categories of substrates. Moreover, we demonstrated a cooperative interplay and protein–protein interactions between the sorting platform and the gatekeeper complex for proper middle and late substrate docking and secretion. Overall, our results provide new insights into the intricate molecular mechanisms that regulate protein secretion hierarchy during T3SS assembly. IMPORTANCE: Enteropathogenic Escherichia coli employs a type III secretion system to deliver virulence proteins directly into host cells, disrupting multiple cellular processes to promote infection. This multiprotein system assembles in a precise stepwise manner, with specific proteins being recruited and secreted at distinct stages. The sorting platform and the gatekeeper complex play critical roles in regulating this process, but their cooperative mechanism has not been fully elucidated. Here, we reveal a novel functional interaction between these two components, which is critical for hierarchical substrate recognition and secretion. These findings advance our understanding of the molecular mechanisms underlying bacterial virulence and suggest new potential targets for antimicrobial strategies aimed at disrupting T3SS function.

Salmonella enterica serovar Typhimurium ST213 is an emergent multidrug-resistant sequence type associated with the food chain, and gastrointestinal and invasive infections in North America. Here, we applied genomic and phenotypic analyses to illustrate the diversity and evolution of sequence type ST213. The population structure and evolutionary history of ST213 strains, particularly the North American isolates (NA-ST213) distinguish them from other S. Typhimurium sequence types, including European ST213 strains. NA-ST213 isolates were distributed in four co-circulating lineages with distinct multidrug resistance profiles and unique phage and CRISPR spacers patterns that could have shaped their local microevolution. Compared to the SL1344 reference strain, NA-ST213 demonstrated reduced adherence and internalization in cultured eukaryotic cell lines but exhibited more efficient replication and intracellular survival. This study underscores the relevance of studying an emergent S. Typhimurium sequence type and the events leading to its diversification beyond the well-characterized reference strains and worldwide predominant sequence types. However, it must also serve as a cautionary tale of the potential health risk the NA-ST213 may represent; particularly when there is a close relationship with pandemic sequence types such as the monophasic ST34.

Introduction: Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form “attaching and effacing” (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood.Methods: To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis.Results and discussion: We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR’s alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

Introduction. Enteropathogenic Escherichia coli (EPEC) strains pose a significant threat as a leading cause of severe childhood diarrhoea in developing nations. EPEC pathogenicity relies on the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE), facilitating the secretion and translocation of bacterial effector proteins. Gap Statement. While the regulatory roles of PerC (plasmid-encoded regulator) and GrlA (global regulator of LEE-activator) in ler expression and LEE gene activation are well-documented in the EPEC prototype strain E2348/69, understanding the variability in LEE gene expression control mechanisms among clinical EPEC isolates remains an area requiring further investigation. Aim. This study aims to explore the diversity in LEE gene expression control mechanisms among clinical EPEC isolates through a comparative analysis of secretion profiles under defined growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Methodology. We compared T3SS-dependent secretion patterns and promoter expression in both typical EPEC (tEPEC) and atypical EPEC (aEPEC) clinical isolates under growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Additionally, we conducted promoter reporter activity assays, quantitative real-time PCR and Western blot experiments to assess gene expression activity. Results. Significant differences in T3SS-dependent secretion were observed among tEPEC and aEPEC strains, independent of LEE sequence variations or T3SS gene functionality. Notably, a clinical tEPEC isolate exhibited increased secretion levels under repressive growth conditions and in the absence of both PerC and GrlA, implicating an alternative mechanism in the activation of Ler (LEE-encoded regulator) expression. Conclusion. Our findings indicate that uncharacterized LEE regulatory mechanisms contribute to phenotypic diversity among clinical EPEC isolates, though their impact on clinical outcomes remains unknown. This challenges the conventional understanding based on reference strains and highlights the need to investigate beyond established models to comprehensively elucidate EPEC pathogenesis.

The Escherichia coli common pilus (ECP) is a chaperone–usher pilus widely distributed in E. coli and other enterobacteria, where it mediates adhesion, biofilm formation, and colonization. Citrobacter rodentium , a surrogate model for the human attaching-and-effacing pathogens enteropathogenic and enterohemorrhagic E. coli , contains a conserved ecp operon ( Cr - ecp ) whose regulation has substantially diverged from E. coli . Cr-ecp expression is induced under static DMEM growth at temperatures below 37 °C, driven by a σ⁷⁰-dependent promoter embedded in a regulatory region of approximately 410 bp. We identified an essential distal regulatory element (DRE) between positions − 378 and − 359 relative to the transcription start site, with a 10-bp core required for promoter activity. In contrast to the EcpR-dependent model in E. coli , the ecpR ortholog is dispensable under the conditions tested. The nucleoid-associated proteins IHF and H-NS bind the regulatory region; IHF is strictly required for activation even when H-NS is absent. These findings define a regulatory architecture distinct from E. coli ecp .