Coxiella burnetii type IVB secretion system region I genes are expressed early during the infection of host cells - PubMed (original) (raw)

Coxiella burnetii type IVB secretion system region I genes are expressed early during the infection of host cells

John K Morgan et al. FEMS Microbiol Lett. 2010 Oct.

Abstract

Analysis of the Coxiella burnetii RSA 493 (Nine Mile phase I strain) genome revealed ORFs with significant homology to the type IVB secretion system (T4BSS) of Legionella pneumophila. The T4BSS genes exist primarily at two loci, designated regions I (RI) and II. In C. burnetii, little is known about the T4BSS regions and the role they play in establishing and/or maintaining infection. Coxiella burnetii T4BSS RI contains genes arranged in three linkage groups: (1) icmW→CBU1651→icmX, (2) icmV→dotA→CBU1647, and (3) icmT→icmS→dotD→dotC→dotB→CBU1646. We used reverse transcriptase (RT)-PCR to demonstrate transcriptional linkage within the groups, and that icmX, icmV, and icmT are transcribed de novo by 8 h post infection (hpi). We then examined the transcript levels for icmX, icmW, icmV, dotA, dotB, and icmT during the first 24 h of an infection using quantitative RT-PCR. The expression initially increased for each gene, followed by a decrease at 24 hpi. Subsequently, we analyzed IcmT protein levels during infection and determined that the expression increases significantly from 8 to 24 hpi and then remains relatively constant. These data demonstrate temporal changes in the RNA of several C. burnetii T4SS RI homologs and the IcmT protein. These changes correspond to early stages of the C. burnetii infectious cycle.

© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1. Coxiella burnetii T4BSS Region I gene linkage

(a) Physical map of C. burnetii T4BSS RI. Solid arrows represent T4BSS homologs. Open arrows represent non-T4BSS ORFs. Gene designations are indicated above each ORF. Diamond-ended lines indicate the position of primers and DNA products that would result from RT-PCR amplification. (b) Agarose gel image(s) showing RT-PCR products corresponding to co-transcribed genes from Linkage Group i, ii, and iii (correlating to the diamond tipped lines in (a). L, 100 bp DNA ladder (size designated on left). +RT, with reverse transcriptase. −RT, without reverse transcriptase. C, DNA control.

Fig. 2

Fig. 2. RT-PCR detection of C. burnetii T4BSS transcripts, icmT, icmV, and icmW

RNA template was isolated at 8 hpi from rifampicin-treated (+Rif) and mock-treated (−Rif) cells. L, 100 bp DNA ladder (size designated on left). +RT/−Rif, with reverse transcriptase and mock-treated. −RT/−Rif, without reverse transcriptase and mock-treated. +RT/+Rif, with reverse transcriptase and rifampin-treated. −RT/+Rif, without reverse transcriptase and rifampin-treated. C, DNA control.

Fig. 3

Fig. 3. C. burnetii icmX, icmW, icmV, dotA, dotB, and icmT transcript levels during the early stages of infection

(a–f) Fold changes in mRNA levels relative to 0 hpi. An equal amount of total RNA from each sample was analyzed by RT-qPCR. The time (in hpi) when total RNA was harvested is indicated below the X-axis. Results represent the mean of three biological samples with no fewer than three technical replicates of each sample. Standard error bars represent the combined standard error of the mean (S.E.M.) per time point.

Fig. 4

Fig. 4. Relative IcmT expression over a time course of infection

(a) Representative dual channel IFA micrograph (400× magnification) images of C. burnetii infected Vero cells 24 hpi. Left panel - Alexa® 488 indicates RαIcmT antibody binding. Right panel - Alexa® 555 indicates Guinea pig anti-whole C. burnetii antibody. Inset images are 8 bit (gray scale) conversions of the color images. Representative Regions of Interest (ROI) are shown (arrows). (b) Immunoblot using RαIcmT to probe total protein from purified C. burnetii (lane 2), Vero cells (lane 3), blank lane (lane 4), and purified recombinant IcmT (rIcmT, lane 5). Lane 1 contains the protein size ladder. (c) Pixel density ratios of IcmT to whole C. burnetii (488:555) from digital images captured at 0, 8, 16, 24, 48, 96, and 168 hpi were converted to quotient values relative to 0 hpi. Error bars represent the S.E.M. for each respective quotient value.

Similar articles

Cited by

References

    1. Akporiaye ET, Rowatt JD, Aragon AA, Baca OG. Lysosomal response of a murine macrophage-like cell line persistently infected with Coxiella burnetii. Infect Immun. 1983;40:1155–1162. - PMC - PubMed
    1. Andreas Untergasser HN, Rao Xiangyu, Bisseling Ton, Geurts René, Leunissen Jack AM. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Research 2007. 2007;35:W71–W74. doi:10.1093/nar/gkm306. - PMC - PubMed
    1. Beare PA, Unsworth N, Andoh M, et al. Comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus Coxiella. Infect Immun. 2009;77:642–656. - PMC - PubMed
    1. Berger KH, Isberg RR. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol. 1993;7:7–19. - PubMed
    1. Beron W, Gutierrez MG, Rabinovitch M, Colombo MI. Coxiella burnetii localizes in a Rab7-labeled compartment with autophagic characteristics. Infect Immun. 2002;70:5816–5821. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources