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. 2007 Sep;65(5):1193-217.
doi: 10.1111/j.1365-2958.2007.05860.x. Epub 2007 Jul 23.

Analysis of the RpoS regulon in Borrelia burgdorferi in response to mammalian host signals provides insight into RpoS function during the enzootic cycle

Melissa J Caimano  1 , Radha Iyer, Christian H Eggers, Cynthia Gonzalez, Elizabeth A Morton, Michael A Gilbert, Ira Schwartz, Justin D Radolf
Affiliations

Analysis of the RpoS regulon in Borrelia burgdorferi in response to mammalian host signals provides insight into RpoS function during the enzootic cycle

Melissa J Caimano et al. Mol Microbiol. 2007 Sep.

Abstract

Borrelia burgdorferi (Bb) adapts to its arthropod and mammalian hosts by altering its transcriptional and antigenic profiles in response to environmental signals associated with each of these milieus. In studies presented here, we provide evidence to suggest that mammalian host signals are important for modulating and maintaining both the positive and negative aspects of mammalian host adaptation mediated by the alternative sigma factor RpoS in Bb. Although considerable overlap was observed between genes induced by RpoS during growth within the mammalian host and following temperature-shift, comparative microarray analyses demonstrated unequivocally that RpoS-mediated repression requires mammalian host-specific signals. A substantial portion of the in vivo RpoS regulon was uniquely upregulated within dialysis membrane chambers, further underscoring the importance of host-derived environmental stimuli for differential gene expression in Bb. Expression profiling of genes within the RpoS regulon by quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed a level of complexity to RpoS-dependent gene regulation beyond that observed by microarray, including a broad range of expression levels and the presence of genes whose expression is only partially dependent on RpoS. Analysis of Bb-infected ticks by qRT-PCR established that expression of rpoS is induced during the nymphal blood meal but not within unfed nymphs or engorged larvae. Together, these data have led us to postulate that RpoS acts as a gatekeeper for the reciprocal regulation of genes involved in the establishment of infection within the mammalian host and the maintenance of spirochetes within the arthropod vector.

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Figures

Fig. 1
Fig. 1
A. Borrelia burgdorferi wild-type (WT) c162 was cultivated at 23°C, following a temperature-shift to 37°C at pH 7.5 and 6.8, and within DMCs. RpoSi was cultivated in vitro following temperature-shift to 37°C in the absence (−) or presence (+) of 5 mM IPTG. Whole-cell lysates were separated by SDS-PAGE and then either silver-stained or immunoblotted using polyclonal sera directed against FlaB, DbpA and Lp6.6. B. Wild-type strain 297 Bb, cultivated initially at 23°C, were serially passaged either in vitro in BSK-H medium at 37°C or within DMCs for a total of five passages (P1–P5). Whole-cell lysates were separated by SDS-PAGE and then silver-stained or immunoblotted using polyclonal sera directed against OspE. Molecular mass markers (kDa) are indicated.
Fig. 2
Fig. 2
Summary of genes upregulated or downregulated by RpoS within DMCs by replicon. Numbers represent the total number of genes identified for each replicon for each category. Plasmids lp36, lp38 and lp56 are either divergent or missing from strain 297; pending the availability of complete plasmid sequences for Bb 297, B31 plasmid locations are used for the corresponding strain 297 orthologues. No genes on lp25 were induced by RpoS. No genes on cp26, cp32s, lp21, lp25, lp28-3 or lp56 were found to be downregulated by RpoS during DMC cultivation.
Fig. 3
Fig. 3
Genes encoded on lp54 found to be differentially regulated by RpoS. The locations of differentially regulated lp54-borne genes are indicated by their position on the replicon. For upregulated genes, transcriptional orientation is indicated by open (+ strand) or shaded (− strand) bars. Arrows are used to indicate transcriptional orientation of downregulated genes. The wild type/rpoS mutant relative expression levels are indicated on the left using a logarithmic scale.
Fig. 4
Fig. 4
qRT-PCR analysis of representative core genes upregulated by RpoS both in vitro and within DMCs. Values represent the average transcript copy number for each gene normalized per 100 copies of flaB. Bars indicate the standard error of the mean. Normalized transcript copy numbers between wild-type and rpoS mutant Bb under each condition were compared using an unpaired t-test and are significantly different (P ≤ 0.05).
Fig. 5
Fig. 5
A. qRT-PCR analysis of genes up-regulated by RpoS exclusively within DMCs. bb0729 (gltP), though not included in the DMC dataset, was examined here based on semi-quantitative RT-PCR data demonstrating its co-transcription with bb0728 (see text). Values represent the average transcript copy number of each gene normalized per 100 copies of flaB. Bars indicate the standard error of the mean. Normalized transcript copy numbers between wild-type and rpoS mutant Bb under each condition were compared using an unpaired t-test and are significantly different (P ≤ 0.05) except where indicated; ns, not significant. B. qRT-PCR analysis of genes up-regulated by RpoS exclusively in vitro. Values represent the average transcript copy number of each gene normalized per 100 copies of flaB. Bars indicate the standard error of the mean. Normalized transcript copy numbers between wild-type and rpoS mutant Bb under each condition were compared using an unpaired t-test and are significantly different (P ≤ 0.05).
Fig. 6
Fig. 6
qRT-PCR analysis of genes downregulated by RpoS. Values represent the average copy number of each gene normalized per 100 copies of flaB. Bars indicate the standard error of the mean. Normalized transcript copy numbers between wild-type and rpoS mutant Bb under each condition were compared using an unpaired t-test and are significantly different (P ≤ 0.05) except where indicated. ns, not significant.
Fig. 7
Fig. 7
qRT-PCR analysis of rpoS in Bb strain 297-infected I. scapularis ticks. Bb-infected ticks were assayed for expression of rpoS as nymphs (Flat Nymph), during nymphal engorgement (Fed Nymphs) and immediately following larval acquisition (Fed Larvae), then normalized per 100 copies of flaB. Bars indicate the standard error of the mean.
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