Treponema denticola TroR is a manganese- and iron-dependent transcriptional repressor

Treponema denticola harbours a genetic locus with significant homology to most of the previously characterized Treponema pallidum tro operon. Within this locus are five genes ( troABCDR ) encoding for the components of an ATP-binding cassette cation-transport system ( troABCD ) and a DtxR-like transcriptional regulator ( troR ). In addition, a σ 70 -like promoter and an 18 bp region of dyad symmetry were identified upstream of the troA start codon. This putative operator sequence demonstrated similarity to the T. pallidum TroR (TroR Tp ) binding sequence; however, the position of this motif with respect to the predicted tro promoters differed. Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroR Td ) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members. In the present study, we show that TroR Td is a manganese- and iron-dependent transcriptional repressor using Escherichia coli reporter constructs and in T. denticola . In addition, we demonstrate that although TroR Td possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroR Td . Based upon these findings, we propose that TroR Td represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72265/1/j.1365-2958.2008.06418.x.pd

Burkholderia pseudomallei and Burkholderia mallei vaccines: Are we close to clinical trials?

B. pseudomallei is the cause of melioidosis, a serious an often fatal disease of humans and animals. The closely related bacterium B. mallei, which cases glanders, is considered to be a clonal derivative of B. pseudomallei. Both B. pseudomallei and B. mallei were evaluated by the United States and the former USSR as potential bioweapons. Much of the effort to devise biodefence vaccines in the past decade has been directed towards the identification and formulation of sub-unit vaccines which could protect against both melioidosis and glanders. A wide range of proteins and polysaccharides have been identified which protective immunity in mice. In this review we highlight the significant progress that has been made in developing glycoconjugates as sub-unit vaccines. We also consider some of the important the criteria for licensing, including the suitability of the "animal rule" for assessing vaccine efficacy, the protection required from a vaccine and the how correlates of protection will be identified. Vaccines developed for biodefence purposes could also be used in regions of the world where naturally occurring disease is endemic

Identification of Circulating Bacterial Antigens by In Vivo Microbial Antigen Discovery

Detection of microbial antigens in clinical samples can lead to rapid diagnosis of an infection and administration of appropriate therapeutics. A major barrier in diagnostics development is determining which of the potentially hundreds or thousands of antigens produced by a microbe are actually present in patient samples in detectable amounts against a background of innumerable host proteins. In this report, we describe a strategy, termed in vivo microbial antigen discovery (InMAD), that we used to identify circulating bacterial antigens. This technique starts with “InMAD serum,” which is filtered serum that has been harvested from BALB/c mice infected with a bacterial pathogen. The InMAD serum, which is free of whole bacterial cells, is used to immunize syngeneic BALB/c mice. The resulting “InMAD immune serum” contains antibodies specific for the soluble microbial antigens present in sera from the infected mice. The InMAD immune serum is then used to probe blots of bacterial lysates or bacterial proteome arrays. Bacterial antigens that are reactive with the InMAD immune serum are precisely the antigens to target in an antigen immunoassay. By employing InMAD, we identified multiple circulating antigens that are secreted or shed during infection using Burkholderia pseudomallei and Francisella tularensis as model organisms. Potential diagnostic targets identified by the InMAD approach included bacterial proteins, capsular polysaccharide, and lipopolysaccharide. The InMAD technique makes no assumptions other than immunogenicity and has the potential to be a broad discovery platform to identify diagnostic targets from microbial pathogens

Hcp1 is not produced by <i>B. thailandensis</i> during growth in minimal media.

<p>Whole cell lysates were prepared from overnight cultures of <i>B. thailandensis</i> E264 grown in LB4G, M9G, M9CG or M9TG, and then assayed for Hcp1 production by Western immunoblotting using anti-BtHcp1 polyclonal rabbit serum. A whole cell lysate prepared from <i>B. thailandensis</i> DW503 (pBtvirAG) grown overnight in LB was used as a positive control (+ve) for Hcp1 expression. The protein band corresponding to Hcp1 is indicated by the black arrowhead.</p

Minimal media supplements influence <i>B. mallei</i> Hcp1 production.

<p>Whole cell lysates were prepared from overnight cultures of <i>B. mallei</i> SR1A, ATCC 23344, NCTC 3708 and NCTC 3709 grown in LB4G, M9G, M9CG or M9TG, and then assayed for Hcp1 production by Western immunoblotting using anti-BmHcp1 polyclonal rat serum. The protein band corresponding to Hcp1 is indicated by the black arrowhead.</p

Iron and zinc inhibit <i>B. mallei</i> T6SS-1 expression.

<p>Whole cell lysates were prepared from overnight cultures of <i>B. mallei</i> SR1A grown in: (A) M9CG, M9TG or M9TG-C; (B) M9CG alone (none), M9CG supplemented with copper, iron, magnesium, manganese, nickel and zinc (pooled; 10 µM each), M9CG individually supplemented with copper, iron, magnesium, manganese, nickel or zinc (Cu, Fe, Mg, Mn, Ni or Zn; 10 µM each) or; (C) M9CG alone (none), M9CG supplemented with iron and zinc (Fe/Zn, 10 µM each), 2x iron (2x Fe, 20 µM) or 2x zinc (2x Zn, 20 µM); and then assayed for Hcp1 production by Western immunoblotting using anti-BmHcp1 polyclonal rat serum. The protein band corresponding to Hcp1 is indicated by the black arrowheads. (D) Transcript levels of <i>bimA</i>, <i>tssA</i>, <i>hcp1</i>, <i>virG, tssM</i> and <i>dnaK</i> were determined by qRT-PCR using gene specific primers and dual-labeled Taqman probes. RNA was harvested from <i>B. mallei</i> strains grown for 8 h in M9CG (none) or M9CG plus iron and zinc (Fe/Zn; 10 µM each). Relative mRNA levels were calculated using the ΔΔC_T method and represent fold changes in comparison to M9CG. All values have been normalized to the internal control, <i>rpoA</i>. Results represent the means and standard deviations of three independent experiments performed in triplicate.</p