Viollier, P. | Search bioRxiv

bioRxiv2023

β-helical protein PerA orchestrates antibiotic uptake across the Caulobacter outer membrane via converging stress signaling systems

While large membrane-impermeable antibiotics cannot traverse a lipid barrier, spacious importers such as TonB-dependent receptors (TBDRs) can mistakenly ferry antibiotics across the bacterial outer membrane (OM). We discovered that loss of PerA, an enigmatic {beta}-helix protein in the OM of the oligotrophic -proteobacterium Caulobacter crescentus, reprograms the OM TBDR proteome from ChvT that imports the glycopeptide antibiotic vancomycin to an uncharacterized TBDR (BugA) that confers sensitivity to the polypeptide antibiotic bacitracin. Both antibiotics are large zinc-binding molecules that target the peptidoglycan, echoing the zinc stress response induces destabilization of PerA. Inactivation of PerA launches two conserved and interwoven envelope stress programs that remodel the OM with TBDRs, a tripartite multidrug efflux pump and periplasmic proteases. Thus, unanticipated entry routes for antibiotics emerge in stressed diderm bacteria that may be treatable with membrane impermeable antimicrobials owing to an underlying transcriptional stress response pathways coordinated by a novel type of OM regulator.

microbiology↗

bioRxiv2019

Rationally designed oral vaccines can set an evolutionary trap for Salmonella Typhimurium

Introductory paragraphThe ability of gut bacterial pathogens to escape immunity by antigenic variation, particularly via changes to surface-exposed antigens, is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2, 3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here we demonstrated this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: A long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase-variation4, 5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen specific IgA and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of oral vaccines, we rationally induced IgA responses blocking all of these trajectories, which selected for Salmonella mutants carrying deletions of the O-antigen polymerase wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents, complement), predation (bacteriophages), and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

immunology↗

bioRxiv2016

A Novel DNA-binding Protein Coordinates Asymmetric Chromosome Replication and Chromosome Partitioning

Bacterial chromosome replication is regulated from a single replication origin (ori) that receives cell cycle signals. Following replication, bacteria often use the parABS partition system with a centromere-like parS locus to place the chromosomes into the daughter cells. Our knowledge of cell cycle regulation is incomplete and we searched for novel regulators of chromosome replication. Here we show that in the cell cycle model Caulobacter crescentus a novel DNA-binding protein promotes both the initiation of chromosome replication and the earliest step of chromosome partitioning. We used biochemical fractionation to identify a protein (OpaA) that preferentially binds to mutated ori DNA that also increases ori-plasmid replication in vivo. OpaA represents a previously unknown class of DNA-binding proteins. opaA gene expression is essential and sufficient OpaA levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified the genomic binding sites for OpaA, with the strongest associations at the parABS locus near ori. Using molecular-genetic and fluorescence microscopy experiments, we showed that OpaA also promotes the first step of chromosome partitioning, the initial separation of the duplicated parS loci following ori replication. This separation occurs before the parABS mechanism and it coincides with the regulatory step that splits the symmetry of the chromosomes so that they are placed at distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that OpaA coordinates replication with the poorly understood mechanism of early chromosome separation. opaA lethal suppressor and antibiotic experiments argue that future studies be focused on the mechanistic roles for transcription and translation at this critical step of the cell cycle.\n\nAuthor SummaryLike all organisms, bacteria must replicate their chromosomes and move them into the newly dividing cells. Eukaryotes use non-overlapping phases, first for chromosome replication (S-phase) followed by mitosis (M-phase) when the completely duplicated chromosomes are separated. However, bacteria combine both phases so chromosome replication and chromosome separation (termed chromosome \"partitioning\") overlap. In many bacteria, including Caulobacter crescentus, chromosome replication initiates from a single replication origin (ori) and the first duplicated regions of the chromosome immediately begin \"partitioning\" towards the cell poles long before the whole chromosome has finished replication. This partitioning movement uses the centromere-like DNA called \"parS\" that is located near the ori. Here we identify a completely novel type of DNA-binding protein called OpaA and we show that it acts at both ori and parS. The timing and coordination of overlapping chromosome replication and partitioning phases is a special regulatory problem for bacteria. We further demonstrate that OpaA is selectively required for the initiation of chromosome replication at ori and likewise that OpaA is selectively required for the initial partitioning of parS. Therefore, we propose that OpaA is a novel regulator that coordinates chromosome replication with the poorly understood mechanism of early chromosome separation.

microbiology↗