How to manage stress: Lessons from an intracellular pathogen

Many studies aimed at advancing our understanding of the mechanisms that promote survival of bacterial pathogens focus on deciphering the means by which these well-adapted microbes manipulate host cellular processes to their own advantage. For example, pathogens often utilize specialized protein delivery systems to directly inject effector proteins into the mammalian cell cytosol that inhibit host immune responses. 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.

Bacteria are highly sensitive to changes in environmental osmolarity, in part, due to their high surface area to volume ratio. Under hyperosmotic conditions, water fluxes across the membrane lead to structural and functional changes in the cell that initially lead to an influx of potassium ions (K⁺) into the bacterial cytosol. This immediate response is followed by the uptake and/or synthesis of glycine betaine, and other organic osmolytes, which minimally perturb cellular functions even when present at high intracellular concentrations. The latter is mediated via a variety of osmosensors/transporters whose abundance are controlled largely at the transcriptional level.^2,3 Transporters involved in organic osmolyte uptake serve as paradigms for the study of osmoadaptive mechanisms in bacteria and include ProP (a proton symporter belonging to the major facilitator family), BetT and BetU (sodium symporters in the betaine-carnitine-choline transporter family), and ProU (an ATP-hydrolyzing system belonging to the ATP-binding cassette (ABC transport family) (for review, see refs ^2-4).

Most of our current knowledge regarding bacterial osmosensing systems is from studies focused on bacteria grown in defined media. There is also evidence that osmoregulation is required by bacterial pathogens to establish and replicate in specific environmental niches encountered during an infection. For example, through the import of carnitine, the OpcU osmoregulatory system of Listeria monocytogenes, a gastrointestinal pathogen, promotes intestinal persistence⁵ as well as resistance to bile, a biologic detergent, found in high concentrations in the small intestines.⁶ Similarly, the uropathogenic E. coli ProP osmoregulatory system is observed to promote growth of this pathogen within human urine and the murine urinary tract,⁷ while the expression of the Mycobacterium tuberculosis proXVWZ operon, which encodes a betaine glycine ABC transporter, mediates osmolyte uptake and promotes bacterial replication within infected macrophages.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. As compared to wild type S. sonnei strains, those that lack or no longer express proV demonstrate viability defects when grown under hyperosmotic conditions. The growth defects are not rescued in the presence of the osmoprotectant betaine, suggesting that a functional ProU system is required for betaine transport in S. sonnei. Furthermore, strains impaired in proV expression, exhibit slower doubling times post-invasion of this intracytoplasmic pathogen into HEK293 cells. The slower growth is not due to a loss of invasion capacity or the loss of the expression of proteins encoded on its major virulence plasmid, thus suggesting that the Shigella ProU osmosensing system plays a key role in promoting the growth and replication of Shigella within the cytosol of infected cells.

The earlier observation by Lucchini et al. that the transcriptional activity of the proVWX operon is markedly elevated upon infection of both epithelial cells and macrophages⁹ coupled with the demonstration by Mahmoud et al. that this operon plays a role in promoting Shigella survival within infected cells is intriguing. In vitro studies suggest that the ProU system senses the ionic strength of the environment.^2,3 This suggests that the bacteria encounter a hyperosmotic condition, during an early stage of their intracellular lifestyle, likely upon entry into the host cell cytosol. However, given that the cytosol of bacteria, at least when grown in standard laboratory media, is ∼300 mM,¹⁰ similar to that of the cytosol of human cells, which likely reflect that of serum osmolarity (∼290 mM), it seems unlikely for osmolarity to be the stress signal. Perhaps, as suggested by Lucchini et al.,⁹ alterations in concentrations of specific ions or other signatures unique to the mammalian cell cytosol, serve as indicators of osmotic stress, which affect transcriptional regulation of the proU operon via changes in DNA supercoiling.¹¹ Of note, expression of proVWX is observed with intracellular Shigella but not Salmonella,^9,12 suggesting that some component within the cytosol, as compared to remodeled phagosomes, the niche of intracellular Salmonella, may serve as an osmotic stress signal.

Alternatively, there is evidence form numerous studies that the Shigella outer membrane and cell wall are remodeled upon entry into host cells, perhaps resulting in differences in permeability, upon entry into host cells. For example, Paciello et al. observe that upon entry into epithelial cells, intracellular Shigella remodels its lipopolysaccharide (LPS) to evade recognition by the host innate immune system.¹³ Similarly, using shotgun proteomics to globally characterize the protein content of Shigella grown in broth versus within the epithelial cell cytosol,¹⁴ Pieper et al. observe marked differences, both increases and decreases, in the levels of multiple outer membrane proteins. While these studies did not observe significant increases in levels of detectable ProV or ProW and only a 2-fold increase in ProX, they did observe a significant increase in the level of OsmC, a protein whose expression is induced by hyperosmolarity.¹⁴ Thus, perhaps remodeling of the cell wall in response to the host cell environment results in at least transient changes in osmotic sensitivity that induces proVWX expression. Many questions still remain, but clearly future work focused on studying the adaptation responses of Shigella and other pathogens in their niche environments will likely advance our understanding of the unique stressors both intra- and extracellular pathogens encounter during the course of an infection. Such studies have the potential to lead to the identification of novel target pathways for the development of anti-bacterial agents, perhaps even host-based, a pressing need in this era of escalating antibiotic resistance.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work is supported by grants from the National Science Foundation NSF IOS 1615858 (to AJ) and the National Institute of Allergy and Infectious Diseases NIAID RO1AI064285 (to CFL).