It is possible the effects of these anti-microbial peptides require environments that are more acidic than that conferred with BSKII medium at pH6.8, although lower pH as such has a higher growth inhibitory effect on Lyme spirochetes. 7SDMs BpiP only.(TIF) ppat.1009535.s001.tif (157K) GUID:?1F7EBDF0-63EE-411D-B267-4B9828CF1062 S2 Fig: Sensitivity of mutant to murine antimicrobial peptides. All three strains (wt, mt and ct) were propagated at 105/ml in BSKII growth medium at pH 6.8/32C with 100 g/ml of mCRAMP. Cells were enumerated every 24 hours using dark field microscopy. The cultures were produced in triplicate and one out of two impartial experiments is shown. Statistical analysis of the difference in the number of wt and mt or ct and mt spirochetes was done by unpaired test.(TIF) ppat.1009535.s002.tif (83K) GUID:?A1AA239F-EC7B-4F46-A50F-E0AE0BED53B1 S3 Fig: Flow-chart depicting the steps used to determine the interactions of BpiP with borrelial PG. BpiP (wild type and site-specifically altered proteins) were crosslinked to purified PG (devoid of any bound proteins) using the crosslinker DTSSP. The complexes were pulled down by high speed centrifugation and the samples boiled to release the bound proteins and supernatant was separated from PG and analyzed for levels of BpiP by immunoblot analysis using anti-BpiP serum generated against C-terminal region of BpiP.(TIF) ppat.1009535.s003.tif (257K) GUID:?BAB14E5D-5A36-44E0-9131-AE8595F2A966 S4 Fig: Absence of an growth defect in mutant. Wild type (B31/A3), mt and ct strains were diluted from stationary phase (1108 Fam162a bacteria ml?1) cultures, re-seeded at 5105 bacteria ml?1 in BSKII medium and enumerated every 24 hours using dark field microscopy under different growth conditions. The cultures were produced in triplicate, with three impartial trials. Error bars indicate standard error. Levels of significance were decided ex229 (compound 991) using two-way ANOVA with = 95%.(TIF) ppat.1009535.s004.tif (47K) GUID:?F9D03CB9-D3B1-4BAA-99B7-2898196FE3E3 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract The Peptidoglycan (PG) cell wall of the Lyme disease (LD) spirochete, mutant displayed no defect under growth conditions with comparable levels of several virulence-related proteins. However, the burden of mutant in C3H/HeN mice at day 14, 28 and 62 post-infection was significantly lower compared to control strains. No viable mutant was re-isolated from any tissues at day 62 post-infection although mutant was able to ex229 (compound 991) colonize immunodeficient SCID at day 28 post-infection. Acquisition or transmission of mutant by larvae or nymphs respectively, from and to mice, was significantly lower compared to control strains. Further analysis of mutant revealed ex229 (compound 991) increased sensitivity to vancomycin, osmotic stress, lysosomal extracts, human antimicrobial peptide cathelicidin-LL37, complement-dependent killing in the presence of day 14 post-infection mouse serum and increased internalization of CFSC-labeled mutant by macrophages and dendritic cells compared to control strains. These studies demonstrate the importance of accessory protein/s involved in sustaining integrity of PG and cell envelope during different phases of infection. Author summary results in attenuation of contamination in immunocompetent C3H/HeN or BALB/c mice unlike in immunodeficient SCID mice. The mutant is usually more susceptible to effects of vancomycin, osmotic stress, lysosomal extracts, antimicrobial peptides, complement-dependent killing and increased phagocytosis by macrophages and dendritic cells. These observations exhibited that cellular and soluble factors of the host immune response limit the colonization of mutant in immunocompetent hosts. Understanding the role of cell wall components and its accessory factors in the survival of during tick and mammalian phases of infection is usually anticipated to advance strategies to reduce the incidence of Lyme disease. Introduction Lyme disease is the most common tick-borne infectious disease in the US with more than 40,000 confirmed and around 300,000 estimated infections occurring each year according to Centers for Disease Control and Prevention [1]. The causative agent of Lyme disease, tick. has a compact genome with limited metabolic capabilities [2C4]. As an extreme auxotroph, is usually constrained by 1) environmental signals; 2) limited/variable levels of key nutrients; and 3) effects of numerous anti-microbial factors impacting its survival in ticks and vertebrate hosts [2, 5]. To overcome these bottlenecks, undergoes extensive remodeling of its cell envelopecomprising of an inner cytoplasmic membrane, the peptidoglycan (PG) cell wall and the outer membrane with a constellation of primarily lipoproteinsconnecting its metabolism and survival strategies to availability/transport of nutrients to survive and colonize highly divergent hosts. The molecular mechanisms and key determinants that contribute to remodeling and structural integrity of the borrelial cell envelope facilitating host-specific adaptation of are unclear.