The Shigella ProU system is required for osmotic tolerance and virulence

Abstract

To cope with hyperosmotic stress encountered in the environments and in the host, the pathogenic as well as non-pathogenic microbes use diverse transport systems to obtain osmoprotectants. To study the role of Shigella sonnei ProU system in response to hyperosmotic stress and virulence, we constructed deletion and complementation strains of proV and used an RNAi approach to silence the whole ProU operon. We compared the response between wild type and the mutants to the hyperosmotic pressure in vitro, and assessed virulence properties of the mutants using gentamicin protection assay as well as Galleria mellonella moth larvae model. In response to osmotic stress by either NaCl or KCl, S. sonnei highly up-regulates transcription of proVWX genes. Supplementation of betaine greatly elevates the growth of the wild type S. sonnei but not the proV mutants in M9 medium containing 0.2 M NaCl or 0.2 M KCl. The proV mutants are also defective in intracellular growth compared with the wild type. The moth larvae model of G. mellonella shows that either deletion of proV gene or knockdown of proVWX transcripts by RNAi significantly attenuates virulence. ProU system in S. sonnei is required to cope with osmotic stress for survival and multiplication in vitro, and for infection.

Role of Proline in Pathogen and Host Interactions

“…Deletion of proP and proU in the E. coli strain HU734 resulted in slower growth in high-osmolality human urine but did not impact urinary tract colonization in mice (55). Studies of other bacteria showed that the ProU transporter is important for infection of moth larvae by Shigella sonnei (143). Also, ProP was concluded to be critical for survival of S. enterica serovar Typhimurium in desiccated environments such as stainless steel surfaces (75).…”

A Novel sRNA in Shigella flexneri That Regulates Tolerance and Virulence Under Hyperosmotic Pressure

“…In pathogenic bacteria, response to osmotic stress is a critical mechanism for survival during environmental osmotic stress as well as for establishing infection. For instance, the ProU system functions to allow S. sonnei to tolerate hyperosmotic stress in vitro , while also facilitating the survival and proliferation of the bacteria within stressful intracellular niches (Mahmoud et al, 2017 ). Herein, we observed that when exposed to hyperosmotic stress, Ssr54 was downregulated and survival of the Ssr54 deletion strain increased.…”

“…Meanwhile, the two efflux pumps, AcrAB and MdtJI, of Shigella are reportedly associated with resistance to bile salts and the extrusion of toxic compounds (Leuzzi et al, 2015 ; Nickerson et al, 2017 ). Hence, developing strategies for tolerating changes in environmental osmolarity, is critical for pathogenic bacteria to not only permit survival during environmental osmotic stress, but also to establish infection (Mahmoud et al, 2017 ). Specifically, maintaining a stable osmotic balance between the cell cytoplasm and outer environment is an important challenge for all cell types, particularly bacteria.…”

How to manage stress: Lessons from an intracellular pathogen

“…In addition, both intra-and extracellular bacterial pathogens use a variety of means to adapt to metabolic and environmental challenges that they encounter during an infection. In this regard, recent work from Mahmoud et al 1 suggest that one such environmental condition that intracellular pathogenic bacteria encounter and respond to upon entry into infected cells is cytosolic hyperosmotic stress.…”

“…8 In recent work, Mahmoud et al, report another advance, which points to the importance of osmoregulation during intracellular infection. 1 Following up on an earlier observation with Shigella flexneri, 9 an intracellular gastrointestinal pathogen, the investigators demonstrate that the closely related Shigella sonnei ProU transport system is transcriptionally up-regulated in vitro under hyperosmotic conditions. The ProU transport system, encoded in the proVWX operon, is comprised of 3 components: ProV, an ATPase, ProW, an inner membrane protein, and ProX, a periplasmic substrate binding component, that work together to likely transport betaine and proline betaine into the Shigella cytoplasm.…”