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Decorin-binding proteins B and A (DbpB and DbpA) are thought to play important roles in Borrelia burgdorferi pathogenesis by serving as adhesins for the extracellular matrix. It has been established that the expression of DbpBA is governed by the Rrp2-RpoN-RpoS regulatory pathway. However, the precise mechanism underlying the control of DbpBA expression has been unclear. In particular, it has been unknown whether RpoS influences DbpBA expression directly or indirectly (through an additional regulatory molecule[s]). Here, employing a wild-type B. burgdorferi strain and a dbpBA-deficient mutant, we analyzed the 5' genetic elements of the dbpBA operon using deletion analysis, coupled with luciferase reporter assays, quantitative reverse transcription PCR, and immunoblot analyses. A minimal promoter, encompassed within 70 bp upstream of the ATG start codon of dbpBA, was identified and found to be necessary and sufficient to initiate dbpBA transcription. The minimal dbpBA promoter was responsive to environmental stimuli such as temperature, pH, and whole blood. Two in silico-identified inverted repeat elements were not involved in the response of dbpBA expression to in vitro stimulation by environmental factors. The expression of dbpBA from the minimal promoter was abolished when rpoS was inactivated. In addition, the targeted mutagenesis of a C at position -14 within the extended -10 region of dbpBA, which has been postulated to be strategic for E[[sigma].sup.S] binding in Escherichia coli, abolished dbpBA expression in B. burgdorferi. These combined data suggest that the Rrp2-RpoN-RpoS pathway controls dbpBA expression by the direct binding of RpoS to an RpoS-dependent promoter. However, given that there remains a distinct difference between the expression of DbpBA and other genes under the direct control of RpoS (e.g., OspC), our findings do not preclude the existence of another layer of gene regulation that may contribute to the modulation of DbpBA expression via an as-yet unknown mechanism. doi: 10.1128/JB.01616-09

Rrp2 is the sole [[sigma].sup.54]-dependent transcriptional activator present in the Borrelia burgdorferi genome. We showed that recombinant Rrp2 binds to DNA in a sequence-nonspecific manner. During infection, Rrp2 activates [[sigma].sup.54]-dependent rpoS expression without an apparent upstream enhancer element commonly associated with other [[sigma].sup.54]-dependent transcriptional activators.

The RpoN--RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, is ostensibly required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN and rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN--RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS.

The first application of the isopropyl-[beta]-D-thiogalactopyranoside (IPTG)-inducible lac repressor/operator system to Borrelia burgdorferi genetics which could be also be used to assess the regulation of other genes potentially involved in virulence expression by Borrelia burgdorferi is demonstrated.

The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in Borrelia burgdorferi. However, evidence to support whether RpoN controls rpoS directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls rpoS in B. burgdorferi.

Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu $4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization ofF. tularensis OMPs.

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi, ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS ([[sigma].sup.s]) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for [[sigma].sup.s]-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for E[[sigma].sup.s] binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin [A.sub.1], further supporting its [[sigma].sup.s] character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of [[sigma].sup.s] to a [[sigma].sup.s]-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.

A 38-kDa lipoprotein of Treponema pallidum (Tp38) was predicted to be a periplasmic sugar-binding protein based on its sequence similarity to the glucose/galactose-binding (MglB) protein of Escherichia coli (P. S. Becker, D. R. Akins, J. D. Radolf, and M. V. Norgard, Infect. Immun. 62:1381-1391, 1994). Inasmuch as glucose is believed to be the principal, if not sole, carbon and energy source for T. pallidum and is readily available to the spirochete during its obligate infection of humans, we hypothesized that Tp38 may serve as the organism's requisite glucose receptor. For the present study, a nonacylated recombinant form of Tp38 was coexpressed with GroES and GroEL in E. coli to facilitate the isolation of soluble, properly folded Tp38. The highly sensitive method of intrinsic fluorescence spectroscopy, predicated on the manner in which tryptophan residues reside and move within protein microenvironments, was then used to assess sugar binding to Tp38. The intrinsic fluorescence of Tp38 was essentially unaltered when it was exposed to D-mannose, D-fucose, D-ribose, L-glucose, or L-galactose, but it changed markedly in the presence of D-glucose, and to a lesser extent, D-galactose, indicating binding. The [K.sub.d] values for D-glucose and D-galactose binding to Tp38 were 152.2 [+ or -] 20.73 nM and 251.2 [+ or -] 55.25 nM, respectively. Site-directed mutagenesis of Trp-145, a residue postulated tD contribute to the sugar-binding pocket in a manner akin to the essential Trp-183 in E. coli MglB, abolished Tp38's conformational change in response to D-glucose. The combined data are consistent with Tp38 serving as a glucose receptor for T. pallidum. These findings potentially have important implications for syphilis pathogenesis, particularly as they may pertain to glucose-mediated chemotactic responses by T. pallidum.

The expression and activation of Toll-like receptors (TLRs) was investigated in leprosy, a spectral disease in which clinical manifestations correlate with the type of immune response mounted toward Mycobacterium leprae. TLR2-TLR1 heterodimers mediated cell activation by killed M. leprae, indicating the presence of triacylated lipoproteins. A genome-wide scan of M. leprae detected 31 putative lipoproteins. Synthetic lipopeptides representing the 19-kD and 33-kD lipoproteins activated both monocytes and dendritic cells. Activation was enhanced by type-1 cytokines and inhibited by type-2 cytokines. In addition, interferon (IFN)-[gamma] and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced TLR1 expression in monocytes and dendritic cells, respectively, whereas IL-4 downregulated TLR2 expression. TLR2 and TLR1 were more strongly expressed in lesions from the localized tuberculoid form (T-lep) as compared with the disseminated lepromatous form (L-lep) of the disease. These data provide evidence that regulated expression and activation of TLRs at the site of disease contribute to the host defense against microbial pathogens., Author(s): Stephan R. Krutzik [1, 2]; Maria Teresa Ochoa [1]; Peter A. Sieling [1]; Satoshi Uematsu [3]; Yolanda W. Ng [1]; Annaliza Legaspi [1]; Philip T. Liu [1, 2]; Stewart [...]

Antibiotic resistance is a challenge for the control of bacterial infections. In an effort to explore unconventional avenues for antibacterial drug development, we focused on the FMN‐transferase activity of the enzyme Ftp from the syphilis spirochete, Treponema pallidum (Ftp_Tp). This enzyme, which is only found in prokaryotes and trypanosomatids, post‐translationally modifies proteins in the periplasm, covalently linking FMN (from FAD) to proteins that typically are important for establishing an essential electrochemical gradient across the cytoplasmic membrane. As such, Ftp inhibitors potentially represent a new class of antimicrobials. Previously, we showed that AMP is both a product of the Ftp_tp‐catalyzed reaction and an inhibitor of the enzyme. As a preliminary step in exploiting this property to develop a novel Ftp_Tp inhibitor, we have used structural and solution studies to examine the inhibitory and enzyme‐binding properties of several adenine‐based nucleosides, with particular focus on the 2‐position of the purine ring. Implications for future drug design are discussed. PDB Code(s): 7MGT; [ABSTRACT FROM AUTHOR]

IgM immunoblotting and PCR analysis of blood and cerebrospinal fluid samples may help identify babies with congenital syphilis who have an infection of the nervous system. This was the conclusion of a study of 148 babies.

Crystal structure reveals that a 4 degree tilt of the C-terminal domain, encompassing residues 190 through 308, in the apo-TroA zinc-free periplasmic metal-binding protein of Treponema pallidum, renders the protein to exhibit a closed conformation than its counterpart zinc-bound TroA.

TroA was initially thought to be a Treponema pallidum outer membrane protein with porin-like characteristics. However, subsequent studies indicated that it actually is a periplasmic substrate-binding protein involved in the transport of metals across the treponemal cytoplasmic membrane. Additional physicochemical studies were performed to resolve the divergent viewpoints with regards to this protein. Findings showed that TroA binds zinc in a 1:1 molar stoichiometric ratio. TroA does not seem to have structural characteristics consistent with those of bacterial porins.

An analysis of circular plasmid-specific DNA sequences from Borrelia burgdorferi has identified a genetic locus, designated 2.9, containing an operon of four genes and open reading frames (ORFs) encoding multiple repeated motifs. Data obtained using molecular genetic techniques suggest that the operon and ORF are differentially expressed in vitro.

Chemiluminescence analysis using an ampicillin-digoxigenin conjugate reveals the penicillin-binding propensity of the 47-kDa lipoprotein immunogen of Treponema palladium (Tpp47). Tpp47 displays carboxy-peptidase activity, which depends on the presence of zinc. Tpp47 expression in Escherichia coli causes cell membrane abnormalities during modified peptidoglycan biosynthesis. The results facilitate the use of E. coli molecular genetics to determine the relationship between syphilis pathogenesis and T. palladium membrane biology.

Borrelia burgdorferi and Treponema pallidum lipoproteins are bound covalently to fatty acids. Palmitate and stearate are the two dominant fatty acids. Fatty acids bound to lipoproteins are different from cellular lipid-bound fatty acids. Fatty acids of acyl transferase lipoprotein control fatty acid utilization during lipoprotein synthesis. Lipoprotein acylation is analyzed for inflammatory and physiological functions of the molecules.

The study of the structure of Treponema pallidum using the freeze-fracture election microscope indicates the presence of intramembrane particles (IMP) in convex and concave OM regions. The OM particles are situated away from cytoplasmic membrane-peptidoglycan and are surface exposed. The motion of the OM proteins in T. pallidum is limited due to periplasmic regions and peptidoglycan cytoplasmic membrane interactions.

Spirochetal Borrelia burgdorferi B31 and N40 strains possess outer surface protein A (OspA) and (OspB) with poor immunogenic reactions as a parasitic stratagem to cause and maintain Lyme borreliosis. Linear bodies oriented perpendicularly to the bacterial axis are present in the outer membrane (OM) of B31 and N40 isolates. Ospa and OspB aggregation with specific antisera does not change the dispersal pattern of OM particles.

* Objectives.--To characterize the neonatal IgG and IgM response to specific Treponema pallidum antigens in the cerebrospinal fluid (CSF) of infants with congenital syphilis. Design.--Cross-sectional survey. Setting.--Newborn nursery and neonatal intensive care unit of a county hospital in Dallas, Tex. Participants.--Twenty-one infants born to mothers with reactive serologic tests for syphilis were enrolled. Group 1 consisted of six infants with clinical and laboratory evidence of congenital syphilis; group 2, six asymptomatic infants born to mothers with untreated syphilis; and group 3, nine asymptomatic infants whose mothers were treated for syphilis before delivery. Selection Procedures.--Random sample. Measurements and Results.--Immunoblotting was used to examine the IgM and IgG reactivities of neonatal serum and CSF against T pallidum antigens. Among serum samples of all group 1 infants, a specific IgM response to T pallidum antigens with apparent molecular masses of 47, 45, and 17 kd was observed. Cerebrospinal fluid IgM reactivity to a 47-kd antigen of T pallidum was seen in four group 1 infants. Serum samples from two group 2 and three group 3 infants demonstrated IgM reactivity against the 47-kd antigen and, in some cases, against the 45-kd antigen of T pallidum. None of 15 group 2 and 3 infants had a positive CSF IgM immunoblot result. The IgG reactivity in CSF was similar in the three groups and was directed against T pallidum antigens with apparent molecular masses of 72, 59, 47, 45, 42, 37, 34, 17, and 15 kd. Conclusions.--A specific IgM response to T pallidum antigens, particularly the 47-kd antigen, was detected in the CSF of some infants with clinical and laboratory evidence of congenital syphilis. The potential usefulness of this test for the diagnosis of congenital neurosyphilis merits further study. (AJDC. 1992;146:1171-1175)

Abstract Background The maintenance of Borrelia burgdorferi in its complex tick-mammalian enzootic life cycle is dependent on the organism's adaptation to its diverse niches. To this end, the RpoN-RpoS regulatory pathway in B. burgdorferi plays a central role in microbial survival and Lyme disease pathogenesis by up- or down-regulating the expression of a number of virulence-associated outer membrane lipoproteins in response to key environmental stimuli. Whereas a number of studies have reported on the expression of RpoS and its target genes, a more comprehensive understanding of when activation of the RpoN-RpoS pathway occurs, and when induction of the pathway is most relevant to specific stage(s) in the life cycle of B. burgdorferi, has been lacking. Results Herein, we examined the expression of rpoS and key lipoprotein genes regulated by RpoS, including ospC, ospA, and dbpA, throughout the entire tick-mammal infectious cycle of B. burgdorferi. Our data revealed that transcription of rpoS, ospC, and dbpA is highly induced in nymphal ticks when taking a blood meal. The RpoN-RpoS pathway remains active during the mammalian infection phase, as indicated by the sustained transcription of rpoS and dbpA in B. burgdorferi within mouse tissues following borrelial dissemination. However, dbpA transcription levels in fed larvae and intermolt larvae suggested that an additional layer of control likely is involved in the expression of the dbpBA operon. Our results also provide further evidence for the downregulation of ospA expression during mammalian infection, and the repression of ospC at later phases of mammalian infection by B. burgdorferi. Conclusion Our study demonstrates that the RpoN-RpoS regulatory pathway is initially activated during the tick transmission of B. burgdorferi to its mammalian host, and is sustained during mammalian infection.