ABSTRACT In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N- and C-terminal domains, MOSP N and MOSP C . Biophysical analysis of constructs refolded in vitro demonstrated that MOSP C , previously shown to possess porin activity, forms amphiphilic trimers, while MOSP N forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSP C is OM-embedded and surface-exposed, while MOSP N resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the β-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli .

Introduction Outer membrane proteins (OMPs) play critical roles in disease pathogenesis and are vaccinogens. Topologic characterisation of surface-exposed β-barrels of Treponema pallidum (Tp) Nichols rare OMPs enabled a novel strategy to assess sequence diversity and evolution of Tp in geographically diverse locations. Methods Through early 2017, sequences encoding TprC (TP0117), TprD (TP0131), and BamA (TP0326) β-barrels were amplified from secondary syphilis patients from Cali (n=16) and swabs from patients in San Francisco (SF, n=6) and Czech Republic (CZ, n=9). Strains were assigned to the Nichols or SS14 clade based on tp0548 and/or tp0558 sequences. Results 23 assignable CZ and Cali strains belonged to either the SS14 or Nichols clade (SS14 predominant), while all 6 SF strains belong to the SS14 clade. Sequence diversity at the three OMP loci was greatest in Cali, with evidence of recombination within tprC and bamA alleles, as well as between strains and clades at all 3 genetic loci. SF strains contained nearly identical sequences at all 3 genetic loci. The SS14 and Mexico A reference strains, both belonging to the SS14 clade, have identical tprDs (tprD2) but different tprC and bamA alleles. Mexico A tprCs were common at all three geographic locations, including Nichols clade strains from Cali. Mexico A bamAs were prevalent in Cali and SF, while CZ SS14 clades contained only SS14 bamAs. OMP sequences were obtained from all three loci in 7 of 8 Nichols clade strains. Of these 7, only 1 matched the Nichols reference strain, while the other 6 contained Mexico A alleles in at least 1 OMP locus. Of the 21 SS14 clade strains, 10 contained Mexico A alleles at all 3 loci; 2 contained Mexico A trpCs and Nichols bamAs; and 9 contained Mexico A tprCs and SS14 bamAs. Conclusion OMP loci are evolving independently within Tp. Recombination of OMP sequences appears to be occurring between Tp strains and clades within patients. Mexico A OMP alleles are circulating widely among Tp strains. These findings have major ramifications for syphilis vaccine development.

Introduction For treatment of Mycoplasma genitalium (Mg) infection, azithromycin is first line initial treatment but persistent NGU caused by Mg may not respond to repeat azithromycin treatment; quinolones, in particular moxifloxcin, have been shown to be effective. The molecular epidemiology of mutations in macrolide- and quinolone-resistance determining regions (RDRs) of 23S rRNA, parC and gyrA genes remains unclear in China. Methods From April 2011 to August 2015, male subjects with clinical and microscopic evidence of urethritis provided a first-voided urine specimen and the mgpA gene was partially amplified and sequenced to diagnose Mg infection. RDRs of the 23s rRNA, parC and gyrA genes were sequenced. Results Among 1816 male-patients, the overall prevalence of M. genitalium was 19.7% (358/1816). N. gonorrhoeae prevalence was 46.6% (847/1816). Among 969 NGU-cases, prevalence of: C. trachomatis was 42.3% (409/968), M. genitalium 27.9% (271/969), U. urealyticum 21.3% (182/856), T. vaginalis1.8% (17/969) and M. hominis 0.7% (6/855). Based on data available from partial (5.4%) sequencing of the MgPa gene (mgpA) an evolutionary tree was constructed that divided the 358 Mg mgpA sequences into five major clusters. Among 358 Mg positive samples, successful sequencing was accomplished for: the 23s rRNA gene (341 specimens), the parC gene (344 specimens) and the gyrA gene (339 specimens). 88.9% (303/341) had mutations in 23s rRNA, 89.5% (308/344) had sense mutations in parC, and 12.4% (42/339) had sense mutations in gyrA.The most common single base pair mutation in the 23s rRNA gene was A2059G (211/303; 69.6%) followed by A2058G (60/303; 19.8%) and A2059T (30/303; 9.9%). The most common mutation in the parC gene was G248T (289/344; 84%) and in the gyrA gene G285A (22/339, 6.5%). Conclusion The high mutation rate in 23s rRNA and parC in Mg strains in Nanjing are a harbinger of resistance to macrolides and quinolones that may follow. Molecular surveillance of indigenous strains of Mg is warranted to anticipate the development of antimicrobial resistance.

BACKGROUND:Similar to Gram-negative organisms, Borrelia spirochetes are dual-membrane organisms with both an inner and outer membrane. Although the outer membrane contains integral membrane proteins, few of the borrelial outer membrane proteins (OMPs) have been identified and characterized to date. Therefore, we utilized a consensus computational network analysis to identify novel borrelial OMPs.RESULTS:Using a series of computer-based algorithms, we selected all protein-encoding sequences predicted to be OM-localized and/or to form β-barrels in the borrelial OM. Using this system, we identified 41 potential OMPs from B. burgdorferi and characterized three (BB0838, BB0405, and BB0406) to confirm that our computer-based methodology did, in fact, identify borrelial OMPs. Triton X-114 phase partitioning revealed that BB0838 is found in the detergent phase, which would be expected of a membrane protein. Proteolysis assays indicate that BB0838 is partially sensitive to both proteinase K and trypsin, further indicating that BB0838 is surface-exposed. Consistent with a prior study, we also confirmed that BB0405 is surface-exposed and associates with the borrelial OM. Furthermore, we have shown that BB0406, the product of a co-transcribed downstream gene, also encodes a novel, previously uncharacterized borrelial OMP. Interestingly, while BB0406 has several physicochemical properties consistent with it being an OMP, it was found to be resistant to surface proteolysis. Consistent with BB0405 and BB0406 being OMPs, both were found to be capable of incorporating into liposomes and exhibit pore-forming activity, suggesting that both proteins are porins. Lastly, we expanded our computational analysis to identify OMPs from other borrelial organisms, including both Lyme disease and relapsing fever spirochetes.CONCLUSIONS:Using a consensus computer algorithm, we generated a list of candidate OMPs for both Lyme disease and relapsing fever spirochetes and determined that three of the predicted B. burgdorferi proteins identified were indeed novel borrelial OMPs. The combined studies have identified putative spirochetal OMPs that can now be examined for their roles in virulence, physiology, and disease pathogenesis. Importantly, the studies described in this report provide a framework by which OMPs from any human pathogen with a diderm ultrastructure could be cataloged to identify novel virulence factors and vaccine candidates.

The past two decades have seen a worldwide resurgence in infections caused by Treponema pallidum subsp. pallidum, the syphilis spirochete. The well-recognized capacity of the syphilis spirochete for early dissemination and immune evasion has earned it the designation 'the stealth pathogen'. Despite the many hurdles to studying syphilis pathogenesis, most notably the inability to culture and to genetically manipulate T. pallidum, in recent years, considerable progress has been made in elucidating the structural, physiological, and regulatory facets of T. pallidum pathogenicity. In this Review, we integrate this eclectic body of information to garner fresh insights into the highly successful parasitic lifestyles of the syphilis spirochete and related pathogenic treponemes.

ABSTRACT We recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses characteristic BamA bipartite topology. Herein, we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP_0326 contains surface-exposed epitopes in intact T. pallidum . Using the solved structure of Neisseria gonorrhoeae BamA, we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold, the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops, loop 4 (L4), L6, and L7, covers the barrel's extracellular opening. L4, the dome's major surface-accessible loop, contains mainly charged residues, while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the β-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions, immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop in T. pallidum -infected rabbits and humans with secondary syphilis. Antibody capture experiments using Escherichia coli expressing OM-localized TP_0326 as a T. pallidum surrogate further established the surface accessibility of L4. Lastly, we found that a naturally occurring substitution (Leu 593 → Gln 593 ) in the L4 sequences of T. pallidum strains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a “structure-to-pathogenesis” approach to map the surface topology of a T. pallidum OMP within the context of syphilitic infection. IMPORTANCE Previously, we reported that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses the bipartite topology characteristic of a BamA ortholog. Using a homology model as a guide, we found that TP_0326 displays unique features which presumably relate to its function(s) in the biogenesis of T. pallidum 's unorthodox OM. The model also enabled us to identify an immunodominant epitope in a large extracellular loop that is both an opsonic target and subject to immune pressure in a human population. Ours is the first study to follow a structure-to-pathogenesis approach to map the surface topology of a T. pallidum rare OMP within the context of syphilitic infection.

The noncultivable spirochete Treponema pallidum subspecies pallidum (T. pallidum) is the etiological agent of venereal syphilis. In contrast to the outer membranes (OMs) of gram-negative bacteria, the OM of T. pallidum lacks lipopolysaccharide, contains a paucity of integral membrane proteins, and is extremely labile. The lability of the T. pallidum OM greatly hinders efforts to localize the bacterium's rare outer membrane proteins (OMPs). To circumvent this problem, we developed the gel microdroplet method in which treponemes are encapsulated in porous agarose beads and then probed with specific antibodies in the absence or presence of low concentrations of the non-ionic detergent Triton X-100. To demonstrate the general utility of this method for surface localization of any T. pallidum antigen, herein we describe a protocol for immunolabeling of encapsulated treponemes using antibodies directed against the β-barrel and POTRA domains of TP0326, the spirochete's BamA ortholog.

Background:Treponema pallidum contains a paucity of outer membrane proteins. Results: The C-terminal domains of TprC/D and TprI are -barrels with porin function; their N-terminal domains provide periplasmic anchors for the -barrels. Conclusion: Full-length TprC subfamily proteins possess a dual domain topology. Significance: TprC/D are bona fide rare outer membrane proteins and a new class of dual function, bipartite, bacterial outer membrane protein.We previously identified Treponemapallidumrepeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSPN and MOSPC) of Treponema denticola and that TprC(C) is solely responsible for -barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSPC-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSPN-like domains are tethered within the periplasm. TprF, which does not contain a MOSPC-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSPN and MOSPC-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSPN-like domains enhance the structural integrity of the cell envelope by anchoring the -barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP.

Volume 195, no. 9, pages [2060–2071][1], 2013. Page 2066: Figure 4C should appear as shown below. An image containing the two rightmost lanes from [Fig. 4B][2] was inadvertently used to generate the top left panel in [Fig. 4C][2] (Intact T. denticola −/+ PK treatment immunoblotted with anti-Msp

Volume 194, no. 9, pages [2321–2333][1], 2012. Page 2330: The legend to Fig. 6E should read as follows. “Immunoblot of unboiled, recombinant TprCN and TprCC separated by SDS-PAGE and then probed with rabbit anti-TprCFl polyclonal antiserum (asterisk indicates TprCC trimer).” Page 2331: The

Background: Presence of elevated serum HsCRP is strongly associated with morbidity and mortality in diverse populations with sepsis and is a potentially useful biomarker to risk stratify patients with severe sepsis. Aims & Objective: We aimed to study whether the serum HsCRP level at presentation in elderly patients with sepsis correlate with stage of sepsis and ultimate outcome. Material and Methods: In a single centre prospective, observational study conducted in geriatric ward of tertiary care centre, 200 elderly patients (age>60 yrs) with sepsis were included. The primary outcome was 14 days in hospital mortality. Risk factor variable was venous serum HsCRP (mg/l) at presentation. Results: Mortality at 14 days in elderly patients with sepsis was 20%. Mean serum HsCRP in survivor group was 33.42 ± 21.56 while 57.28 ± 25.31 in mortality group (p value

Japanese encephalitis, an inflammatory brain disease prevalent in Southeast Asia, usually presented with fever, headache, convulsions, brain stem signs with pyramidal and extrapyramidal features, and altered sensorium. Acute transverse myelitis, as the initial manifestation of Japanese encephalitis, is an unusual manifestation and is seldom reported. We hereby report a case of 13-year-old adolescent boy who presented to us with fever and acute onset paraparesis with urinary retention initially, progressing to quadriparesis and then followed by headache and altered sensorium. Brain MRI revealed bilateral basal ganglia that were grossly swollen with vasogenic edema tracking along internal capsule and midbrain. Adjacent ventrolateral thalamus and internal capsule also showed mild abnormal intensities. Spinal screening showed abnormal cord intensities in entire cord with gross edema in cervical and conus regions. He had elevated IgM titres against JE virus in cerebrospinal fluid. The patient was treated conservatively along with intravenous methyl prednisolone for 5 days. He regained near normal power at 3 months in followup, but hesitancy, dysarthria, and slowness of movement still persisted. To conclude, a young boy presenting with ATM in an endemic region of JE, then a possibility of Japanese encephalitis, should be sought by clinicians as early use of immunomodulator shows survival benefit.

Takayasu's arteritis (TA) is a chronic, idiopathic, inflammatory disease, that is more common in females and Asian countries. A 38-year-old female presented with recurrent vertigo. Detailed examination revealed discrepancies in peripheral pulses and raised blood pressure in bilateral lower limbs. Possibility of vasculitis involving arch of aorta or its branches was kept. Investigations were suggestive of Takayasu's arteritis, and noncontrast tomographic scanning (NCCT) of head showed B/L parietal infarcts. The disease is itself uncommon, and the presentation with vertigo only is rare. In this case vertigo may be due to Takayasu's arteritis itself or due to bilateral parietal infarcts.

Remitting Seronegative Symmetrical Synovitis with Pitting Edema is a rare clinical entity described as an acute onset polyarthritis with edema affecting geriatric population particularly males. The etiopathogenesis of disease is still elusive but clinical, radiological and immunological characteristics separate it from the more common disease like Rheumatoid arthritis and Polymyalgia rheumatica. Disease has an excellent prognosis with low dose steroids and patients undergo long-term remission even after withdrawal of drugs. We hereby describe a case of 90 year old female presented to us with polyarthritis and pitting edema of hands that we diagnosed as RS3PE.

Site-directed mutants of the gene encoding wild-type Vitreoscilla hemoglobin were made that changed Tyr29 (B10) of the wild-type Vitreoscilla hemoglobin (VHb) to either Phe or Ala. The wild-type and the two mutant hemoglobins were expressed in Escherichia coli and purified to homogeneity. The binding of the two mutants to CO was essentially identical to that of wild-type VHb as determined by CO-difference spectra. Circular-dichroism spectra also showed the two mutants to be essentially the same as wild-type VHb regarding overall helicity. All three VHbs were crystallized and their structures were determined at resolutions of 1.7-1.9 Å, which are similar to that of the original wild-type structure determination. The Tyr29Phe mutant has a structure that is essentially indistinguishable from that of the wild type. However, the structure of the Tyr29Ala mutant has significant differences from that of the wild type. In addition, for the Tyr29Ala mutant it was possible to determine the positions of most of the residues in the D region, which was disordered in the originally reported structure of wild-type VHb as well as in the wild-type VHb structure reported here. In the Tyr29Ala mutant, the five-membered ring of proline E8 (Pro54) occupies the space occupied by the aromatic ring of Tyr29 in the wild-type structure. These results are discussed in the context of the proposed role of Tyr29 in the structure of the oxygen-binding pocket.

The major outer sheath protein (Msp) is a primary virulence determinant in Treponema denticola, as well as the parental ortholog for the Treponema pallidum repeat (Tpr) family in the syphilis spirochete. The Conserved Domain Database (CDD) server revealed that Msp contains two conserved domains, major outer sheath protein(N) (MOSP(N)) and MOSP(C), spanning residues 77 to 286 and 332 to 543, respectively, within the N- and C-terminal regions of the protein. Circular dichroism (CD) spectroscopy, Triton X-114 (TX-114) phase partitioning, and liposome incorporation demonstrated that full-length, recombinant Msp (Msp(Fl)) and a recombinant protein containing MOSP(C), but not MOSP(N), form amphiphilic, β-sheet-rich structures with channel-forming activity. Immunofluorescence analysis of intact T. denticola revealed that only MOSP(C) contains surface-exposed epitopes. Data obtained using proteinase K accessibility, TX-114 phase partitioning, and cell fractionation revealed that Msp exists as distinct OM-integrated and periplasmic trimers. Msp(Fl) folded in Tris buffer contained slightly less β-sheet structure than detergent-folded Msp(Fl); both forms, however, partitioned into the TX-114 detergent-enriched phase. CDD analysis of the nine Tpr paralogs predicted to be outer membrane proteins (OMPs) revealed that seven have an Msp-like bipartite structure; phylogenetic analysis revealed that the MOSP(N) and MOSP(C) domains of Msp are most closely related to those of TprK. Based upon our collective results, we propose a model whereby a newly exported, partially folded intermediate can be either processed for OM insertion by the β-barrel assembly machinery (BAM) or remain periplasmic, ultimately forming a stable, water-soluble trimer. Extrapolated to T. pallidum, our model enables us to explain how individual Tprs can localize to either the periplasmic (e.g., TprK) or OM (e.g., TprC) compartments.

Congenital erythropoietic porphyria (CEP), also called Gunthers is the rarest of the porphyrias , with a prevalence estimated at 1 in 1,000,000 or less. Only approximately 200 cases of CEP have been reported till now worldwide. CEP affects males and females equally, and occurs in all ethnic groups. Clinically it is a subacute to chronic type of porphyria, defect is expressed in infancy and clinical features such as extreme cutaneous photosensitivity, blistering, scarring , hyper- and hypo pigmentation of photo exposed parts. Haemolytic anaemia with splenomegaly, and retarded growth may also be present. Due to its rarity we are presenting Congenital erythropoietic porphyria with haemolytic anaemia.