Fig 1.
B. burgdorferi tolerates a narrow range of osmolarity (250 to 650 mOsm).
(A) A drawing of an I. scapularis tick showing the measurements used to determine scutal index: A = idiosoma length, B = scutal width [35]. (B) Development of the scutal index during feeding of nymph and adult ticks. Each point represents the average of the measurements of 15 ticks. (C) Osmolarity of the blood meal isolated from the midgut of nymph or adult ticks during the feeding as a function of the scutal index. Each red circle denotes the osmolarity measured in the blood meal of an infected, feeding tick. Each blue circle denotes the osmolarity measured in the blood meal of an uninfected, feeding tick. Osmolarity was measured with Wescor vapor pressure osmometer as described in the Methods section.
Fig 2.
The effects of osmolarity on the physiology, cell morphology and motility of B. burgdorferi.
(A) Rate of growth of the wild-type strain B31-A3 from 50 to 1,250 mOsm under anaerobic (90% N2, 5% CO2, 5% H2), microaerobic (90% N2, 5% CO2, 5%O2) and aerobic (78% N2, 21% O2, 0.05% CO2) conditions at 34°C and E. coli in aerobic conditions at 34°C. (B) Spirochetes, grown at different osmolarities, were examined by dark-field microscopy. Under each photograph of cells is the average length of 200 cells from 5 independent cultures (C) The motility of cells was evaluated by assessing 200 cells per culture condition (e.g., 250 mOsm) by dark-field microscopy.
Fig 3.
The effects of osmolarity on specific proteins regulated by the RpoN-RpoS regulatory cascade.
B. burgdorferi strains B31-A3, B31-A3ΔrpoN and B31-A3ΔrpoS were grown in 250, 450 and 650 mOsm BSK-II to mid-log phase and cell lysates (40 μg of protein/lane) were analyzed by immunoblotting. (A) B31-A3 lysates (40 μg of protein/lane) were probed with OspA, OspC, DbpA, BBA66, RpoN, RpoS, BosR, Rrp1 and Rrp2 antigen-specific antisera. (B) B31-A3ΔrpoN, and (C) B31-A3ΔrpoS cell lysates probed with the same antigen-specific antisera. (D) B31-A3 (40 μg protein/lane) cell lysates probed with serum from mice infected by B. burgdorferi via tick bite.
Fig 4.
Gene expression of genes encoding crucial regulatory proteins and virulence factors at 250, 450 and 650 mOsm.
The expression of genes encoding regulatory proteins (rrp1, bosR, rrp2, rpoS, rpoN), virulence factors (ospC, dbpA, bba66, bb0844, ospA) and rpoD analyzed by qRT-PCR in B. burgdorferi B31-A3. RNA isolated from cells grown at 250, 450 and 650 mOsm. The gene expression was normalized to enoS.
Fig 5.
Rrp1 is required for the survival of B. burgdorferi at higher osmolarity.
(A) Rate of growth of B31-5A4, B31-5A4Δhk1 and B31-5A4Δrrp1 mutants at osmolarities ranging from 150 to 1050 mOsm under microaerobic growth conditions. (B) Growth of strain B31-5A4 and 5A4Δrrp1 mutant in BSK-II at various osmolarities. Cells were quantified by colonies on BSK-II plating media. (C) hk1 expression was analyzed by relative qRT-PCR in B. burgdorferi B31-A3 normalized to enoS. (D) Quantitation of c-di-GMP in B31-5A4, B31-5A4Δhk1 and B31-5A4Δrrp1 mutants.
Fig 6.
The ProU system and its role in osmotolerance.
(A) In vitro expression of the three genes of the proU locus: proV, proW and proX at 250, 450 and 650 mOsm. Gene expression was normalized to enoS. (B) proV expression during nymph feeding. (C) Rate of growth of strains B31-A3, B31-A3proX and B31-A3proX pSABG1 at osmolarities ranging from 150 to 1050 mOsm under microaerobic conditions. The arrow denotes growth at 300 mOsm. (D) Immunoblot of cell lysates (40μg of protein/lane) of B. burgdorferi B31-A3 and B31-A3proX cells grown at 300, 450 and 650 mOsm to mid-log phase and probed with OspC-specific antisera.
Fig 7.
gltP and its potential role in osmotolerance.
(A) In vitro expression of the putative glutamate transporters (bb0401 and gltP). Gene expression was normalized to enoS. (B) gltP expression during nymph feeding (C). Rate of growth from 150 to 1050 mOsm in microaerobic condition. (D) Immunoblots of B. burgdorferi B31-5A18 and B31-5A18gltP grown at 300, 450 and 650 mOsm to mid-log phase and cell lysates (40μg of protein/lane) probed with OspC-specific antisera.
Fig 8.
Expression of genes encoding putative ion transporters.
(A) Expression analyses of different ion transporter genes of B. burgdorferi grown in BSK-II at 250, 450 and 650 mOsm. (B) Same as A except measured in unfed, partially fed and replete I. scapularis infected with B. burgdorferi. Gene expression was normalized to enoS.
Fig 9.
Model for generation of osmotic stress and its effect on regulatory pathway during the enzootic cycle.
During acquisition, spirochetes encounter an increase of the osmolarity, which requires gene regulation by Hk1-Rrp1. The activation of Hk1-Rrp1 system increases the level of c-di-GMP which triggers metabolic adaptation to the new environment and also affects cell motility [7, 37, 62]. After feeding, the depletion of nutrients activates RelBbu-dependent gene expression which promotes long-term survival in the midgut [3]. During the second feeding, RelBbu regulatory effects decrease, restoring normal growth in a nutrient replenished environment. Other relevant physiological changes (decreasing osmolarity, increased temperature, etc.) stimulate Rrp2-RpoN-RpoS-dependent virulence factors required for the transmission and successful colonization of a new host.