Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains.

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.

Microscopy-based phenotypic profiling of infection by Staphylococcus aureus clinical isolates reveals intracellular lifestyle as a prevalent feature.

Staphylococcus aureus is increasingly recognized as a facultative intracellular pathogen, although the significance and pervasiveness of its intracellular lifestyle remain controversial. Here, we applied fluorescence microscopy-based infection assays and automated image analysis to profile the interaction of 191 S. aureus isolates from patients with bone/joint infections, bacteremia, and infective endocarditis, with four host cell types, at five times post-infection. This multiparametric analysis revealed that almost all isolates are internalized and that a large fraction replicate and persist within host cells, presenting distinct infection profiles in non-professional vs. professional phagocytes. Phenotypic clustering highlighted interesting sub-groups, including one comprising isolates exhibiting high intracellular replication and inducing delayed host death in vitro and in vivo. These isolates are deficient for the cysteine protease staphopain A. This study establishes S. aureus intracellular lifestyle as a prevalent feature of infection, with potential implications for the effective treatment of staphylococcal infections.

Functional screenings reveal different requirements for host microRNAs in Salmonella and Shigella infection.

MicroRNAs (miRNAs) are increasingly recognized for their role in infection by bacterial pathogens, although the effect of each individual miRNA remains largely unknown. Here, we used a comparative genome-wide microscopy-based functional screening approach to identify miRNAs controlling infection by two bacterial pathogens-Salmonella enterica serovar Typhimurium and Shigella flexneri. Despite the similarities between these pathogens, we found infections to be controlled by largely non-overlapping subsets of miRNAs, seemingly reflecting different requirements prompted by their distinct intracellular lifestyles. By characterizing a small subset of miRNAs chosen among the strongest inhibitors of Shigella infection, we discovered that miR-3668, miR-4732-5p and miR-6073 exert a selective effect on Shigella infection by impairing bacterial actin-based motility by downregulating N-WASP. Additionally, by identifying let-7i-3p miRNA as a strong inhibitor of Salmonella replication and performing in-depth analysis of its mechanisms of action, we showed that this miRNA specifically inhibits Salmonella infection via modulation of endolysosomal trafficking and the vacuolar environment by targeting the host RGS2 protein. These findings illustrate two paradigms underlying miRNA-mediated regulation of bacterial infection, acting as part of the host response to infection, or as part of bacterial strategies to modulate the host environment and favour pathogenesis.

Shedding light on microRNA function via microscopy-based screening.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally modulate gene expression and orchestrate a wide range of biological and pathological processes. The use of high-throughput screening technologies, in particular microscopy-based screenings (also known as high-content screenings), coupled with genome-wide libraries for modulation of miRNA levels, allow for comprehensive functional analysis of each member of the miRNome in different phenotypic cell-based assays. The wealth of information obtained from such screenings spans across various fields of research, including cancer, cardiovascular, cell reprogramming, and infection biology. Here, we provide an overview of the rationale for performing screenings using synthetic libraries of miRNA mimics and inhibitors, and of the microscopy-based miRNA screenings performed to date. Moreover, a list of resources available for such endeavor is provided. Finally, we describe a detailed procedure for a case study where microscopy-based screening using a library of miRNA mimics was performed to identify miRNAs that control infection of epithelial cells by the bacterial pathogen Salmonella. The methodologies described here can be easily adapted for screenings addressing other biological questions.

Stress-induced host membrane remodeling protects from infection by non-motile bacterial pathogens.

While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re-infection by this and other non-motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens.