The crosstalk between type III secretion system and bacterial cell physiology in Yersinia
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Philipps-Universität Marburg
Abstract
The type III secretion system (T3SS) is an essential virulence factor for a broad range of Gram-negative bacteria. T3SS secretion enables a direct translocation of effector proteins from the bacterial cytoplasm into the target host cell. Despite the high degree of conservation of the T3SS machinery, its regulation and role during host infection is species-specific, spanning from immune cell neutralization during host colonization to the establishment of inflammation at the suited niche. This, combined with the challenge of investigating T3SS secretion in vivo, caused a high discrepancy between the degree of characterization of T3SS machinery's mechanistic properties and T3SS regulation during infection. The knowledge that a complex interplay between secretion and bacterial cell physiology exists is rooted in the discovery of the T3SS machinery itself; secreting cells experience growth retardation. This phenotype is now regarded as secretion-associated growth inhibition (SAGI), but its molecular mechanism is far from being understood.
In the first part of this work, I further explored the impact of T3SS secretion on the cellular physiology of Yersinia enterocolitica. Yersinia is a suited model organism for studying T3SS regulation because of its homogenous induction of secretion under standard laboratory conditions. I localized and quantified the chromosomal and the T3SS-encoding Yersinia virulence plasmid (pYV) DNA to study the effects of T3SS secretion induction on the bacterial cell. I showed that upon T3SS machinery activation, the pYV is promptly upregulated in its copy number and re-localized towards the cell membrane. The plasmid rearrangements were shown to be followed by a broader chromosomal DNA re-organization. Ongoing studies are being conducted to determine if secreting cells undergo chromosomal replication or segregation failure during SAGI. It is hypothesized that bacteria deploy a mechanism called transertion to increase the efficiency of T3SS assembly or T3SS substrates (Yops) secretion. Transertion stands for coupled transcription, translation, and simultaneous protein insertion at the site of action. The presence of the target gene at the protein insertion site is a requirement during transertion. As the pYV plasmid localization showed to respond to induction of T3SS secretion, we tracked T3SS effector transcripts using mRNA fluorescent in situ hybridization coupled with super-resolution microscopy during secretion. The lack of yop mRNA enrichment at the membrane in T3SS-active cells argues against a key involvement of transertion in T3SS assembly or secretion in Yersinia.
To efficiently colonize the host, a group of Gram-negative pathogens, including Salmonella enterica and Pseudomonas aeruginosa, overcome SAGI by expressing the T3SS in a bistable way. This allows relying on a subset of cells being T3SS-active to establish infection while the rest of the population replicates and spreads. In pathogens like Shigella flexneri and Yersinia enterocolitica, division of labor has not been described so far. In the second part of this study, I investigated a previously uncharacterized level of T3SS regulation that Yersinia may employ to counteract SAGI during infection. Yersinia primarily replicates extracellularly within microcolonies, and I showed that replication is enabled by T3SS inhibition triggered by cell density and sensed throughout the colony. The downregulation is specific and reversible and is achieved via modulation of the essential activator of the T3SS, VirF, by the CsrABC system. I propose that this previously unknown, density-driven T3SS repression is crucial for Yersinia replication and dissemination following the initial stages of host colonization, during which the T3SS plays a vital role in evading the innate immune response. This study challenges the long-held assumption that there is no coordinated behavior in the regulation of T3SS.