Clofazimine for Treatment of Cryptosporidiosis in Human Immunodeficiency Virus Infected Adults: An Experimental Medicine, Randomized, Double-blind, Placebo-controlled Phase 2a Trial.

BACKGROUND: We evaluated the efficacy, pharmacokinetics (PK), and safety of clofazimine (CFZ) in patients living with human immunodeficiency virus (HIV) with cryptosporidiosis. METHODS: We performed a randomized, double-blind, placebo-controlled study. Primary outcomes in part A were reduction in Cryptosporidium shedding, safety, and PK. Primary analysis was according to protocol (ATP). Part B of the study compared CFZ PK in matched individuals living with HIV without cryptosporidiosis. RESULTS: Twenty part A and 10 part B participants completed the study ATP. Almost all part A participants had high viral loads and low CD4 counts, consistent with failure of antiretroviral (ARV) therapy. At study entry, the part A CFZ group had higher Cryptosporidium shedding, total stool weight, and more diarrheal episodes compared with the placebo group. Over the inpatient period, compared with those who received placebo, the CFZ group Cryptosporidium shedding increased by 2.17 log2 Cryptosporidium per gram stool (95% upper confidence limit, 3.82), total stool weight decreased by 45.3 g (P = .37), and number of diarrheal episodes increased by 2.32 (P = .87). The most frequent solicited adverse effects were diarrhea, abdominal pain, and malaise. One placebo and 3 CFZ participants died during the study. Plasma levels of CFZ in participants with cryptosporidiosis were 2-fold lower than in part B controls. CONCLUSIONS: Our findings do not support the efficacy of CFZ for the treatment of cryptosporidiosis in a severely immunocompromised HIV population. However, this trial demonstrates a pathway to assess the therapeutic potential of drugs for cryptosporidiosis treatment. Screening persons living with HIV for diarrhea, and especially Cryptosporidium infection, may identify those failing ARV therapy. CLINICAL TRIALS REGISTRATION: NCT03341767.

Invasion of hepatocytes by Plasmodium sporozoites requires cGMP-dependent protein kinase and calcium dependent protein kinase 4.

Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasite's subsequent intracellular differentiation in the liver is the first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second-messengers - cGMP mediated by the parasite's cGMP-dependent protein kinase (PKG), and Ca2+ , mediated by the parasite's calcium-dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.

Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile.

Mycobacterium tuberculosis belongs to a large family of soil bacteria which can degrade a remarkably broad range of organic compounds and utilize them as carbon, nitrogen and energy sources. It has been proposed that a variety of mycobacteria can subsist on alternative carbon sources during latency within an infected human host, with the help of enzymes such as nitrilotriacetate monooxygenase (NTA-Mo). NTA-Mo is a member of a class of enzymes which consist of two components: A and B. While component A has monooxygenase activity and is responsible for the oxidation of the substrate, component B consumes cofactor to generate reduced flavin mononucleotide, which is required for component A activity. NTA-MoB from M. thermoresistibile, a rare but infectious close relative of M. tuberculosis which can thrive at elevated temperatures, has been expressed, purified and crystallized. The 1.6 Å resolution crystal structure of component B of NTA-Mo presented here is one of the first crystal structures determined from the organism M. thermoresistibile. The NTA-MoB crystal structure reveals a homodimer with the characteristic split-barrel motif typical of flavin reductases. Surprisingly, NTA-MoB from M. thermoresistibile contains a C-terminal tail that is highly conserved among mycobacterial orthologs and resides in the active site of the other protomer. Based on the structure, the C-terminal tail may modulate NTA-MoB activity in mycobacteria by blocking the binding of flavins and NADH.

Structure of aldose reductase from Giardia lamblia.

Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP(+)-binding site located within the eight ß-strands of the interior.

The Seattle Structural Genomics Center for Infectious Disease (SSGCID).

The NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium established to apply structural genomics approaches to potential drug targets from NIAID priority organisms for biodefense and emerging and re-emerging diseases. The mission of the SSGCID is to determine approximately 400 protein structures over the next five years. In order to maximize biomedical impact, ligand-based drug-lead discovery campaigns will be pursued for a small number of high-impact targets. Here we review the center's target selection processes, which include pro-active engagement of the infectious disease research and drug therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. This is followed by a brief overview of the SSGCID structure determination pipeline and ligand screening methodology. Finally, specifics of our resources available to the scientific community are presented. Physical materials and data produced by SSGCID will be made available to the scientific community, with the aim that they will provide essential groundwork benefiting future research and drug discovery.

Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design.

In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD(+) interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N(6)-(1-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (6e), N(6)-(substituted-naphthalenemethyl)-2'-deoxy-2'-(substituted-benzamido)adenosine analogues were investigated. N(6)-(1-Naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (6m), N(6)-[1-(3-hydroxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (7m), N(6)-[1-(3-methoxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (9m), N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (11e), and N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC(50)'s of these compounds were in the range 2-12 microM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure-activity relationships of this class of compounds, a library of 240 N(6)-(substituted)-2'-deoxy-2'-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N(6) and C2' substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5'-amido substituents and found that 2',5'-dideoxy-2'-(3,5-dimethoxybenzamido)-5'-(diphenylacetamido)adenosine (49) displays an IC(50) of 60-100 microM against the three parasite GAPDH's.

TcRho1, a farnesylated Rho family homologue from Trypanosoma cruzi: cloning, trans-splicing, and prenylation studies.

Rho GTPases are members of the Ras superfamily and are involved in signal transduction pathways, including maintenance of cell morphology and motility, cell cycle progression, and transcription activation. We report the molecular identification in trypanosomatids (Trypanosoma cruzi) of the first member of the Rho family. The cloned Rho protein, TcRho1, shares approximately 40% homology with other members of the Rho family. Southern blot analysis revealed that TcRHO1 is a single copy gene per haploid genome, and Northern blot assays showed a transcript of 1200 nucleotides in length. Mapping the 5'-untranslated region of TcRHO1 transcripts revealed at least five different transcripts derived from differential trans-splicing. Three of the five transcripts contain the trans-splicing site within the coding region of the TcRHO1 gene. TcRho1 also contains the C-terminal sequence CQLF (CAAX motif), which is predicted to direct post-translation prenylation of the cysteine residue. A synthetic peptide containing this C-terminal motif, when tested against Q-Sepharose chromatography fractions from T. cruzi cytosol, was shown to be efficiently farnesylated, but not geranylgeranylated, despite the fact that the CAAX motif with X = Phe specifies geranylgeranylation by mammalian protein geranylgeranyltransferase I. Furthermore, immunoblot analyses of epimastigote protein with anti-S-farnesylcysteine methyl ester and anti-TcRho1 antisera strongly suggested that TcRho1 is farnesylated in vivo. The farnesylation of proteins such as Rho GTPases could be the basis for the selective cytotoxic action of protein farnesyltransferase inhibitors on trypanosomatids versus mammalian cells.

LYT1 protein is required for efficient in vitro infection by Trypanosoma cruzi.

Trypanosoma cruzi invasion of host cells involves several discrete steps: attachment, parasite internalization mediated by recruitment and fusion of host cell lysosomes, and escape from the parasitophorous vacuole to liberate amastigotes to multiply freely in the cytosol. This report describes the initial characterization of the LYT1 gene and the demonstration that the gene product is involved in cell lysis and infectivity. Mutational analysis demonstrated that deletion of LYT1 resulted in attenuation of infection, which was associated with diminished hemolytic activity. Reintroduction of LYT1 restored infectivity in null mutants, confirming the critical role of LYT1 in infection. Additionally, in vitro stage transition experiments with LYT1-deficient lines showed that these parasites converted to extracellular amastigote-like cells and metacyclic trypomastigotes more rapidly than wild-type parasites, suggesting that the diminished infectivity was not a result of the LYT1 deficiency that affected the parasite's ability to complete the life cycle.

Potent anti-Trypanosoma cruzi activities of oxidosqualene cyclase inhibitors.

Trypanosoma cruzi is the protozoan agent that causes Chagas' disease, a major health problem in Latin America. Better drugs are needed to treat infected individuals. The sterol biosynthesis pathway is a potentially excellent target for drug therapy against T. cruzi. In this study, we investigated the antitrypanosomal activities of a series of compounds designed to inhibit a key enzyme in sterol biosynthesis, oxidosqualene cyclase. This enzyme converts 2,3-oxidosqualene to the tetracyclic product, lanosterol. The lead compound, N-(4E,8E)-5,9, 13-trimethyl-4,8, 12-tetradecatrien-1-ylpyridinium, is an electron-poor aromatic mimic of a monocyclized transition state or high-energy intermediate formed from oxidosqualene. This compound and 27 related compounds were tested against mammalian-stage T. cruzi, and 12 inhibited growth by 50% at concentrations below 25 nM. The lead compound was shown to cause an accumulation of oxidosqualene and decreased production of lanosterol and ergosterol, consistent with specific inhibition of the oxidosqualene cyclase. The data demonstrate potent anti-T. cruzi activity associated with inhibition of oxidosqualene cyclase.

Candida dubliniensis fungemia in a solid organ transplant patient: case report and review of the literature.

We report a case of Candida dubliniensis fungemia in a solid organ transplant patient, which, to our knowledge is the first such case in this patient population. C. dubliniensis is a recently described, emerging fungal pathogen, thus far, found in AIDS and a limited number of other immunosuppressed patients. It is of interest and concern because it can be misidentified as C. albicans and it may be resistant to azole antifungal agents. This case illustrates the need to be aware of emerging pathogens in new host populations and that new techniques used to identify yeast species may provide more accurate identification.

Adenosine analogues as inhibitors of Trypanosoma brucei phosphoglycerate kinase: elucidation of a novel binding mode for a 2-amino-N(6)-substituted adenosine.

As part of a project aimed at structure-based design of adenosine analogues as drugs against African trypanosomiasis, N(6)-, 2-amino-N(6)-, and N(2)-substituted adenosine analogues were synthesized and tested to establish structure-activity relationships for inhibiting Trypanosoma brucei glycosomal phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycerol-3-phosphate dehydrogenase (GPDH). Evaluation of X-ray structures of parasite PGK, GAPDH, and GPDH complexed with their adenosyl-bearing substrates led us to generate a series of adenosine analogues which would target all three enzymes simultaneously. There was a modest preference by PGK for N(6)-substituted analogues bearing the 2-amino group. The best compound in this series, 2-amino-N(6)- [2''(p-hydroxyphenyl)ethyl]adenosine (46b), displayed a 23-fold improvement over adenosine with an IC(50) of 130 microM. 2-[[2''-(p-Hydroxyphenyl)ethyl]amino]adenosine (46c) was a weak inhibitor of T. brucei PGK with an IC(50) of 500 microM. To explore the potential of an additive effect that having the N(6) and N(2) substitutions in one molecule might provide, the best ligands from the two series were incorporated into N(6),N(2)-disubstituted adenosine analogues to yield N(6)-(2''-phenylethyl)-2-[(2'' -phenylethyl)amino]adenosine (69) as a 30 microM inhibitor of T. brucei PGK which is 100-fold more potent than the adenosine template. In contrast, these series gave no compounds that inhibited parasitic GAPDH or GPDH more than 10-20% when tested at 1.0 mM. A 3.0 A X-ray structure of a T. brucei PGK/46b complex revealed a binding mode in which the nucleoside analogue was flipped and the ribosyl moiety adopted a syn conformation as compared with the previously determined binding mode of ADP. Molecular docking experiments using QXP and SAS program suites reproduced this "flipped and rotated" binding mode.

Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92.

By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%-100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.

Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei.

Protein prenylation occurs in the protozoan that causes African sleeping sickness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs. We have cloned the alpha- and beta-subunits of T. brucei protein farnesyltransferase (TB-PFT) using nucleic acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites. TB-PFT is expressed in both bloodstream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli. Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent alpha-helices. Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase. TB-PFT prefers Gln and Met at the X position but not Ser, Thr, or Cys, which are good substrates for mammalian protein farnesyltransferase. A structural homology model of the active site of TB-PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimetic inhibitors.

Multiple alleles of Treponema pallidum repeat gene D in Treponema pallidum isolates.

Two new tprD alleles have been identified in Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in one T. pallidum subsp. pertenue isolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating that tprD2 is expressed during infection.

The tprK gene is heterogeneous among Treponema pallidum strains and has multiple alleles.

We have previously shown that the TprK antigen of T. pallidum, Nichols strain, is predominantly expressed in treponemes obtained 10 days after infection and that the hydrophilic domain of TprK is a target of opsonic antibodies and confers significant protection against homologous challenge. The T. pallidum genome sequence reported the presence of a single copy of the tprK gene in the Nichols strain. In the present study we demonstrate size heterogeneity in the central portions of the TprK hydrophilic domains of 14 treponemal isolates. Sequence analysis of the central domains and the complete open reading frames (ORFs) of the tprK genes confirms this heterogeneity. Further, multiple tprK sequences were found in the Nichols-defined tprK locus in three isolates (Sea 81-4, Bal 7, and Bal 73-1). In contrast, only a single tprK sequence could be identified in this locus in the Nichols strain. Alignment of the DNA and deduced amino acid sequences of the whole tprK ORFs shows the presence of seven discrete variable domains flanked by highly conserved regions. We hypothesize that these heterogeneous regions may be involved in antigenic heterogeneity and, in particular, evasion of the immune response. The presence of different tprK alleles in the tprK locus strongly suggests the existence of genetically different subpopulations within treponemal isolates.

Expression of Trypanosoma cruzi surface antigen FL-160 is controlled by elements in the 3' untranslated, the 3' intergenic, and the coding regions.

The FL-160 surface antigen gene family of T. cruzi consists of hundreds of members of 160 kDa glycoproteins expressed in trypomastigotes, but not in epimastigotes. Steady-state levels of FL-160 mRNA were 80 to 100-fold higher in trypomastigotes than in epimastigotes, yet transcription rates were equivalent between the lifecycle stages. Luciferase reporter constructs demonstrated that the 3' untranslated region (UTR) and intergenic region (IR) following the coding sequence of FL-160 was sufficient to generate 8-fold higher luciferase expression in trypomastigotes compared with epimastigotes. Transfection of 3' UTR/IR deletion constructs revealed cis-acting elements which conferred a trypomastigote-specific expression pattern similar to that of FL-160. Parasites treated with translation and transcription inhibitors, cyclohexamide and Actinomycin D, respectively, displayed a stage-specific pattern of FL-160 mRNA degradation. Epimastigotes, but not trypomastigotes, treated with the inhibitors accumulated a 1.4 Kb FL-160 cleavage product. The cleavage site mapped to a 31 base poly-purine tract in the FL-160 coding region. The first 526 aa of FL-160, containing the 31 base poly-purine tract and several smaller tracts, were fused to green fluorescent protein (GFP) and expressed from the T. cruzi tubulin locus. Stable transformants expressed 4-fold more FL-160:GFP fusion mRNA and 12-fold more fusion protein in the trypomastigote stage than in the epimastigote stage suggesting post-transcriptional and translational control elements. These data reveal at least two distinct control mechanisms for trypomastigote-specific expression of FL-160 surface glycoproteins, one involving the 3' UTR/IR and one involving the coding region of FL-160.

T-Cell responses to Treponema pallidum subsp. pallidum antigens during the course of experimental syphilis infection.

In this study we describe the development of the T-cell response to a panel of Treponema pallidum antigens over the course of syphilis infection in the rabbit and determine whether these antigens induce the expression of Th1 cytokines. It was determined that the membrane proteins TpN17 and TpN47, as well as the endoflagellar sheath protein TpN37, induce strong proliferation responses through most of syphilis infection; Tromp1 induced only weak proliferative responses. An unexpected drop in proliferative response to these antigens at day 90 of infection, followed by a dramatic increase in response at day 180, suggests that there may be a secondary dissemination of T. pallidum which induces a recall response. Crude epitope mapping of TpN17 and TpN37 showed that multiple epitopes may be present on both antigens, which is likely a contributing factor in the immunodominance of these antigens. The T-cell response to the TpN37 molecule shows acquisition of newly recognized epitopes during the course of infection. Sonicated T. pallidum was found to induce the expression of interleukin-2 (IL-2) and gamma interferon and not IL-10 mRNA, showing that the general T-cell response to T. pallidum antigens in syphilis infection is biased towards the Th1 phenotype. Of the antigens tested, TpN37 appears to contribute the most to the Th1 cytokine response and therefore may play a key role in the clearance of T. pallidum from lesions.

Sequence conservation of glycerophosphodiester phosphodiesterase among Treponema pallidum strains.

Previous investigations have demonstrated that immunization with Treponema pallidum subsp. pallidum glycerophosphodiester phosphodiesterase significantly protects rabbits from subsequent treponeme challenge. In this report, we show that the glycerophosphodiester phosphodiesterase amino acid sequence is conserved among 12 strains from a total of five pathogenic treponemes. The invariant nature of this immunoprotective antigen makes it an attractive candidate for inclusion in a universal subunit vaccine against T. pallidum infection. In addition, these studies show a silent nucleotide substitution at position 579 of the gpd open reading frame which is consistently observed in the non-T. pallidum subsp. pallidum strains. This sequence alteration introduces a PleI restriction site in the nonsyphilis strains and thus allows genetic differentiation from T. pallidum subsp. pallidum strains.

Structure-based design of submicromolar, biologically active inhibitors of trypanosomatid glyceraldehyde-3-phosphate dehydrogenase.

The bloodstream stage of Trypanosoma brucei and probably the intracellular (amastigote) stage of Trypanosoma cruzi derive all of their energy from glycolysis. Inhibiting glycolytic enzymes may be a novel approach for the development of antitrypanosomatid drugs provided that sufficient parasite versus host selectivity can be obtained. Guided by the crystal structures of human, T. brucei, and Leishmania mexicana glyceraldehyde-3-phosphate dehydrogenase, we designed adenosine analogs as tight binding inhibitors that occupy the pocket on the enzyme that accommodates the adenosyl moiety of the NAD+ cosubstrate. Although adenosine is a very poor inhibitor, IC50 approximately 50 mM, addition of substituents to the 2' position of ribose and the N6-position of adenosine led to disubstituted nucleosides with micromolar to submicromolar potency in glyceraldehyde-3-phosphate dehydrogenase assays, an improvement of 5 orders of magnitude over the lead. The designed compounds do not inhibit the human glycolytic enzyme when tested up to their solubility limit (approximately 40 microM). When tested against cultured bloodstream T. brucei and intracellular T. cruzi, N6-(1-naphthalenemethyl)-2'-(3-chlorobenzamido)adenosine inhibited growth in the low micromolar range. Within minutes after adding this compound to bloodstream T. brucei, production of glucose-derived pyruvate ceased, parasite motility was lost, and a mixture of grossly deformed and lysed parasites was observed. These studies underscore the feasibility of using structure-based drug design to transform a mediocre lead compound into a potent enzyme inhibitor. They also suggest that energy production can be blocked in trypanosomatids with a tight binding competitive inhibitor of an enzyme in the glycolytic pathway.

Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response.

We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.