Loa loa, the African eyeworm, is a major filarial pathogen of humans. Unlike most filariae, Loa loa does not contain the obligate intracellular Wolbachia endosymbiont. We describe the 91.4 Mb genome of Loa loa, and the genome of the related filarial parasite Wuchereria bancrofti, and predict 14,907 Loa loa genes based on microfilarial RNA sequencing. By comparing these genomes to that of another filarial parasite, Brugia malayi, and to several other nematode genomes, we demonstrate synteny among filariae but not with non-parasitic nematodes. The Loa loa genome encodes many immunologically relevant genes, as well as protein kinases targeted by drugs currently approved for humans. Despite lacking Wolbachia, Loa loa shows no new metabolic synthesis or transport capabilities compared to other filariae. These results suggest that the role played by Wolbachia in filarial biology is more subtle than previously thought and reveal marked differences between parasitic and non-parasitic nematodes.

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Hap s , from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Hap s . Purified Hap s binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Hap s to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract.Extracellular matrix (ECM) consists of a diverse group of proteins that form the scaffolding responsible for the development, growth, and maintenance of mammalian tissues. In epithelial tissues, certain ECM proteins function together with cells to form barriers intended to prevent penetration of these tissues by microorganisms. Both pathogenic and commensal bacteria have evolved mechanisms designed to subvert epithelial barriers (3,5,14,23,28). These mechanisms include bacterial surface proteins called adhesins that bind to ECM and bacterial proteases that degrade ECM components and permit migration of bacteria to deeper tissue spaces where they may gain easier access to nutrients and safe harbor from the host immune response.Haemophilus influenzae is a gram-negative bacterium that is often found as a commensal inhabitant of the respiratory tract in healthy adults but also represents a common cause of both localized respiratory tract and invasive systemic disease (25). In studies examining interactions between H. influenzae clinical isolates and human respiratory tract tissue, bacteria were often associated with damaged epithelium and exposed ECM (16,17). Furthermore, examination of bronchial biopsies from patients with persistent H. influenzae bronchitis revealed organisms in the subepithelial compartment, suggesting that this pathogen is capable of penetrating the basement membrane (11). Additional analysis of interactions between clinical isolates and purified ECM components demonstrated that many H. influenzae strains were capable of binding to fibronectin, laminin, and various collagens (2, 27). In recent work, Virkola and coworkers found that H. influenzae hemagglutinating pili mediated attachment to both fibronectin and heparin-binding growth-associated molecule, alt...

SummaryThe pathogenesis of non-typable Haemophilus influenzae disease begins with colonization of the nasopharynx and is facilitated by bacterial adherence to respiratory mucosa. The H. influenzae Hap autotransporter is a non-pilus adhesin that promotes adherence to epithelial cells and selected extracellular matrix proteins and mediates bacterial aggregation and microcolony formation. In addition, Hap has serine protease activity. Hap contains a 110 kDa internal passenger domain called Hap S and a 45 kDa Cterminal translocator domain called Hap b . In the present study, we sought to define the structural basis for Hap adhesive activities. Based on experiments using a panel of monoclonal antibodies against Hap S , a deletion derivative lacking most of Hap S and a purified fragment of Hap S , we established that adherence to epithelial cells is mediated by sequences within the C-terminal 311 residues of Hap S . In additional experiments, we discovered that bacterial aggregation is also mediated by sequences within the C-terminal 311 residues of Hap S and occurs via Hap S -Hap S interaction between molecules on neighbouring organisms. Finally, we found that adherence to fibronectin, laminin and collagen IV is mediated in part by sequences within the C-terminal 311 residues of Hap S and in full by sequences within the C-terminal 511 residues of Hap S . Taken together, these results demonstrate that all Hap adhesive activities reside in the C-terminal portion of Hap S . Coupled with earlier observations, the current results establish that Hap S adhesive activities and Hap S protease activity are contained in separate modules of the protein.

BackgroundThe current antibody tests used for monitoring in lymphatic filariasis (LF) elimination programs suffer from poor specificity because of the considerable geographical overlap with other filarial infections such as Loa loa (Ll), Onchocerca volvulus (Ov), and Mansonella perstans (Mp).MethodsUsing bioinformatics to assemble into contigs 2048 expressed sequence tags (ESTs) from the L3 infective larvae of W. bancrofti (Wb), these were next assessed for homology to known proteins and nucleotides and to similar assemblies of L3 larval ESTs of B. malayi (Bm – n = 5068), Ov (n = 4166), and Ll (n = 3315). Nineteen potential L3- and Wb- and/or Bm-specific antigens were identified. Sixteen of the 19 antigens could be expressed as fusion proteins with Renilla luciferase (Ruc); these were used in a rapid Luciferase Immunopreciptation System (LIPS) assay.ResultsOne of the 16 expressed antigens (Wb123) was both highly immunogenic and specific for Wb. Using Wb123-based IgG and IgG4 LIPS assays on well-defined sera from normal North Americans and those infected exclusively with intestinal helminths, we could detect all of the Wb-infected individuals (from diverse geographic regions) with 100% sensitivity and 100% specificity. Using sera from exclusively Ll-infected, Ov-infected Mp-infected or Bm-infected subjects as the negative comparator, the sensitivities were between 98–100% and the specificities ranged between 84–100% (for IgG anti-Wb123) and between 98–100% (for IgG4 anti-Wb123). Blinded assessments using panels of sera from various Wb-, Bm- or non-Wb helminth-infected subjects demonstrated equally high degrees of sensitivity and specificity.SignificanceWe have identified a Wb-encoded antigen that can be used both as a rapid, high throughput tool to diagnose individual Wb infections and as a sensitive method for early detection of recrudescent infections in areas of control and for mapping new areas of Wb transmission.

BackgroundAccurate diagnosis of Loa loa infection is essential to the success of mass drug administration efforts to eliminate onchocerciasis and lymphatic filariasis, due to the risk of fatal encephalopathic reactions to ivermectin occurring among highly microfilaremic Loa-infected individuals living in areas co-endemic for multiple filarial species.Methodology/Principal FindingsFrom a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269). The qPCR assays were highly specific for L. loa compared with related filariae and also highly sensitive, with detection limits of 0.1 pg genomic DNA, or 1% of DNA extracted from normal blood spiked with a single L. loa microfilaria. Using various DNA extraction methods with dried blood spots obtained from Cameroonian subjects with parasitologically proven loiasis, the LLMF72 qPCR assay successfully estimated mf burden in 65 of 68 samples (50–96,000 mf/mL by microscopy), including all 12 samples subjected to a simple 10-minute boiling extraction. Additionally, the assay detected low-level microfilaremia among 5 of 16 samples from patients thought to be amicrofilaremic by microscopy.Conclusions/SignificanceThis novel, rapid, highly sensitive and specific qPCR assay is an important step forward in the laboratory diagnosis of L. loa infection.

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SummaryLactoferrin is a member of the lactotransferrin family of non-haem, iron-binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N-terminal lobe of the protein, adjacent to a 12-15 Å cleft that separates the N-lobe and the C-lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine-rich region defined by amino acids 1379-1386 (RRSRRSVR) and digests Hap at an arginine-rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine-rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine-rich sequences in a variety of microbial virulence proteins, contributing to its longrecognized antimicrobial properties.

Cupriavidus gilardii is a Gram-negative bacterium that has rarely been associated with human infections. We report a fatal case of sepsis caused by C. gilardii in a previously healthy 12-year-old female. CASE REPORTWhile vacationing in Europe, a previously healthy 12-yearold female was diagnosed with severe idiopathic aplastic anemia. One week after presentation, the patient was transferred to a hospital in the United States near her home. Laboratory evaluation at the time of admission was significant for a total white blood cell count of 2,590/l with a differential of 98.8% lymphocytes, 0.4% monocytes, and 0.8% neutrophils, hemoglobin of 7 g/dl, and platelets of 72,000/l. On hospital day (HD) 1, following placement of a double-lumen Hickman catheter, the patient became febrile to 39.7°C and was started on piperacillin-tazobactam. She remained febrile through HD 12, prompting empirical addition of amikacin and then liposomal amphotericin B to her antibiotic regimen. A contrast computed tomography (CT) scan of the chest, abdomen, and pelvis showed diffuse perirectal thickening, prompting initiation of metronidazole for improved anaerobic coverage. She subsequently defervesced and remained afebrile from HD 15 to 24, though all antimicrobials were continued due to profound neutropenia and persistent perirectal pain.A repeat CT scan on HD 25 revealed progression of moderate to marked perirectal inflammation associated with the appearance of new fluid collection not amenable to surgical drainage. On HD 27, a blood culture from the previous day became positive for vancomycin-resistant enterococcus (VRE), and her central line was removed after initiation of linezolid. By HD 30, persistent fevers and perirectal pain resulted in discontinuation of piperacillin-tazobactam in favor of meropenem, and from HD 32 to 38, the patient clinically improved. During this time, amikacin and liposomal amphotericin B were discontinued, and central venous access was reestablished with a peripherally inserted catheter (PICC line).Low-grade fever (38.0 to 38.3°C) and abdominal pain recurred on HD 40 and persisted through HD 53, when the patient developed high spiking fevers. A repeat CT scan revealed worsening of perirectal inflammation and progression of bowel wall inflammation to involve the cecum, most of the ascending colon, and a portion of the transverse colon. Stool testing for Clostridium difficile antigen was negative. However, a stool surveillance culture was positive for Cupriavidus gilardii, resistant to piperacillin-tazobactam, aztreonam, imipenem, and meropenem, and susceptible to cefepime, trimethoprim-sulfamethoxazole, and ciprofloxacin. Consequently, meropenem was discontinued in favor of cefepime, and amikacin was added. Fever subsided, and linezolid was discontinued. On HD 60, the patient became febrile again, and blood cultures were positive for VRE and C. gilardii, now susceptible only to trimethoprim-sulfamethoxazole and ciprofloxacin.Ciprofloxacin and linezolid were added, and after removal of the PICC line, the pat...