Incorporation of fluorophore-cholesterol conjugates into liposomal and mycobacterial membranes.

Fluorescently-labeled steroids that emit intense blue light in nonpolar solvent (λem (CH2Cl2)≈440nm, ΦF=0.70) were prepared by treating cholesteryl chloroformate with 4-amino-1,8-naphthalimides. The lipid portion of the conjugates embeds into liposomal membrane bilayers in minutes, leaving the fluorophore exposed to the external aqueous environment. This causes a 40-nm red-shift in λem and significant quenching. DFT optimizations predict the conjugates to be about 30Å long when fully extended, but rotation about the linker group can bring the compounds into an 'L'-shape. Such a conformation would allow the cholesteryl anchor to remain parallel to the acyl chains of a membrane while the fluorescent group resides in the interfacial region, instead of extending beyond it. When incubated with Mycobacterium smegmatis mc2 155, a bacterial species known to use natural cholesterol, the labeled steroids support growth and can be found localized in the membrane fraction of the cells using HPLC. These findings demonstrate stable integration of fluorescent cholesterols into bacterial membranes in vivo, indicating that these compounds may be useful for evaluating cholesterol uptake in prokaryotic organisms.

The ferrous iron transporter FtrABCD is required for the virulence of Brucella abortus 2308 in mice.

Iron transport has been linked to the virulence of Brucella strains in both natural and experimental hosts. The genes designated BAB2_0837-0840 in the Brucella abortus 2308 genome sequence are predicted to encode a CupII-type ferrous iron transporter homologous to the FtrABCD transporter recently described in Bordetella. To study the role of the Brucella FtrABCD in iron transport, an isogenic ftrA mutant was constructed from B. abortus 2308. Compared with the parental strain, the B. abortus ftrA mutant displays a decreased capacity to use non-haem iron sources in vitro, a growth defect in a low iron medium that is enhanced at pH 6, and studies employing radiolabelled FeCl3 confirmed that FtrABCD transports ferrous iron. Transcription of the ftrA gene is induced in B. abortus 2308 in response to iron deprivation and exposure to acid pH, and similar to other Brucella iron acquisition genes that have been examined the iron-responsiveness of ftrA is dependent upon the iron response regulator Irr. The B. abortus ftrA mutant exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host.

SodA is a major metabolic antioxidant in Brucella abortus 2308 that plays a significant, but limited, role in the virulence of this strain in the mouse model.

The gene designated BAB1_0591 in the Brucella abortus 2308 genome sequence encodes the manganese-cofactored superoxide dismutase SodA. An isogenic sodA mutant derived from B. abortus 2308, designated JB12, displays a small colony phenotype, increased sensitivity in vitro to endogenous superoxide generators, hydrogen peroxide and exposure to acidic pH, and a lag in growth when cultured in rich and minimal media that can be rescued by the addition of all 20 amino acids to the growth medium. B. abortus JB12 exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, but this attenuation is limited to the early stages of infection. Addition of the NADPH oxidase inhibitor apocynin to infected macrophages does not alleviate the attenuation exhibited by JB12, suggesting that the basis for the attenuation of the B. abortus sodA mutant is not an increased sensitivity to exogenous superoxide generated through the oxidative burst of host phagocytes. It is possible, however, that the increased sensitivity of the B. abortus sodA mutant to acid makes it less resistant than the parental strain to killing by the low pH encountered during the early stages of the development of the brucella-containing vacuoles in macrophages. These experimental findings support the proposed role for SodA as a major cytoplasmic antioxidant in brucella. Although this enzyme provides a clear benefit to B. abortus 2308 during the early stages of infection in macrophages and mice, SodA appears to be dispensable once the brucellae have established an infection.

Mur regulates the gene encoding the manganese transporter MntH in Brucella abortus 2308.

MntH is the only high-affinity manganese transporter identified in Brucella. A previous study showed that MntH is required for the wild-type virulence of Brucella abortus 2308 in mice (Anderson ES, et al., Infect. Immun. 77:3466-3474, 2009) and indicated that the mntH gene is regulated in a manganese-responsive manner in this strain by a Mur homolog. In the study presented here, the transcriptional start site for mntH in B. abortus 2308 was determined by primer extension analysis. Specific interactions between Mur and the mntH promoter region were demonstrated in an electrophoretic mobility shift assay (EMSA), and a Mur binding site was identified in the -55 to -24 region of the mntH promoter by DNase I footprint analysis. The specificity of the interaction of Mur with the putative Mur box was further evaluated by EMSA employing oligonucleotides in which the consensus nucleotides in this region were substituted. These studies not only confirm a direct role for Mur in the Mn-responsive regulation of mntH expression in Brucella abortus 2308 but also identify the cis-acting elements upstream of mntH that are responsible for this regulation.

The iron-responsive regulator irr is required for wild-type expression of the gene encoding the heme transporter BhuA in Brucella abortus 2308.

Irr and RirA, rather than Fur, serve as the major iron-responsive regulators in the alphaproteobacteria. With only a few exceptions, however, the relative contributions of these transcriptional regulators to the differential expression of specific iron metabolism genes in Brucella strains are unclear. The gene encoding the outer membrane heme transporter BhuA exhibits maximum expression in Brucella abortus 2308 during growth under iron-deprived conditions, and mutational studies indicate that this pattern of bhuA expression is mediated by the iron-responsive regulator Irr. Specifically, a bhuA-lacZ transcriptional fusion does not produce elevated levels of ß-galactosidase in response to iron deprivation in the isogenic irr mutant BEA5, and, unlike the parental strain, B. abortus BEA5 cannot utilize heme as an iron source in vitro and is attenuated in mice. A derivative of the bhuA-lacZ transcriptional fusion lacking the predicted Irr binding site upstream of the bhuA promoter does not produce elevated levels of ß-galactosidase in response to iron deprivation in the parental B. abortus 2308 strain, and a direct and specific interaction between a recombinant version of the Brucella Irr and the bhuA promoter region was observed in an electrophoretic mobility shift assay. Despite the fact that it lacks the heme regulatory element linked to the iron-responsive degradation of its counterpart in Bradyrhizobium japonicum, readily detectable levels of Irr were found only in B. abortus 2308 cells by Western blot analysis following growth under iron-deprived conditions.

Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host.

Brucella strains produce abortion and infertility in their natural hosts and a zoonotic disease in humans known as undulant fever. These bacteria do not produce classical virulence factors, and their capacity to successfully survive and replicate within a variety of host cells underlies their pathogenicity. Extensive replication of the brucellae in placental trophoblasts is associated with reproductive tract pathology in natural hosts, and prolonged persistence in macrophages leads to the chronic infections that are a hallmark of brucellosis in both natural hosts and humans. This review describes how Brucella strains have efficiently adapted to their intracellular lifestyle in the host.

The manganese transporter MntH is a critical virulence determinant for Brucella abortus 2308 in experimentally infected mice.

The gene designated BAB1_1460 in the Brucella abortus 2308 genome sequence is predicted to encode the manganese transporter MntH. Phenotypic analysis of an isogenic mntH mutant indicates that MntH is the sole high-affinity manganese transporter in this bacterium but that MntH does not play a detectable role in the transport of Fe(2+), Zn(2+), Co(2+), or Ni(2+). Consistent with the apparent selectivity of the corresponding gene product, the expression of the mntH gene in B. abortus 2308 is repressed by Mn(2+), but not Fe(2+), and this Mn-responsive expression is mediated by a Mur-like repressor. The B. abortus mntH mutant MWV15 exhibits increased susceptibility to oxidative killing in vitro compared to strain 2308, and a comparative analysis of the superoxide dismutase activities present in these two strains indicates that the parental strain requires MntH in order to make wild-type levels of its manganese superoxide dismutase SodA. The B. abortus mntH mutant also exhibits extreme attenuation in both cultured murine macrophages and experimentally infected C57BL/6 mice. These experimental findings indicate that Mn(2+) transport mediated by MntH plays an important role in the physiology of B. abortus 2308, particularly during its intracellular survival and replication in the host.

The AraC-like transcriptional regulator DhbR is required for maximum expression of the 2,3-dihydroxybenzoic acid biosynthesis genes in Brucella abortus 2308 in response to iron deprivation.

Phenotypic evaluation of isogenic mutants derived from Brucella abortus 2308 indicates that the AlcR homolog DhbR (2,3-dihydroxybenzoic acid [2,3-DHBA] biosynthesis regulator) modulates the expression of the genes involved in 2,3-DHBA production, employing 2,3-DHBA or brucebactin as a coinducer.

Brucella abortus requires the heme transporter BhuA for maintenance of chronic infection in BALB/c mice.

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA (Brucella heme utilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.

Truncation of herpes simplex virus type 2 glycoprotein B increases its cell surface expression and activity in cell-cell fusion, but these properties are unrelated.

Formation of small polykaryons by cell-cell fusion is characteristic of herpes simplex virus (HSV) lesions, but the great majority of viruses isolated from such lesions produce only limited cell fusion in tissue culture. Because of this, HSV laboratory strains that produce extensive cell fusion (syncytium formation) in culture are regarded as variants or mutants. Furthermore, the rarity of clinical isolates able to produce syncytia in culture suggests that extensive cell fusion is deleterious in vivo. Mutations that confer a syncytial phenotype can then be regarded as bypassing a mechanism that normally limits cell fusion. Determination of how these mutations, some of which are in the cytoplasmic tail of glycoprotein B (gB), lead to syncytium formation will likely reveal how fusion is controlled. Here we show the following. (i) Truncation of the cytoplasmic tail of HSV type 2 gB (gB-2) by a minimum of 25 residues or a maximum of 49 residues produces a syncytial phenotype. (ii) Truncation by 20 to 49 residues increases cell fusion when gB-2 is coexpressed with only gD-2, gH-2, and gL-2. (iii) Truncation by 25 or more residues removes a potential endocytosis motif and increases gB-2 cell surface expression. (iv) Mutation of this motif increases gB-2 cell surface expression but does not increase fusogenic activity, whereas mutation of another potential endocytosis motif does not increase surface expression but does increase fusogenic activity. Therefore, syncytial mutations in the cytoplasmic tail of gB-2 do not act by increasing cell surface levels of the protein.