The TamB ortholog of Borrelia burgdorferi interacts with the ß-barrel assembly machine (BAM) complex protein BamA.

Two outer membrane protein (OMP) transport systems in diderm bacteria assist in assembly and export of OMPs. These two systems are the ß-barrel assembly machine (BAM) complex and the translocation and assembly module (TAM). The BAM complex consists of the OMP component BamA along with several outer membrane associated proteins. The TAM also consists of an OMP, designated TamA, and a single inner membrane (IM) protein, TamB. Together TamA and TamB aid in the secretion of virulence-associated OMPs. In this study we characterized the hypothetical protein BB0794 in Borrelia burgdorferi. BB0794 contains a conserved DUF490 domain, which is a motif found in all TamB proteins. All spirochetes lack a TamA ortholog, but computational and physicochemical characterization of BB0794 revealed it is a TamB ortholog. Interestingly, BB0794 was observed to interact with BamA and a BB0794 regulatable mutant displayed altered cellular morphology and antibiotic sensitivity. The observation that B. burgdorferi contains a TamB ortholog that interacts with BamA and is required for proper outer membrane biogenesis not only identifies a novel role for TamB-like proteins, but also may explain why most diderms harbor a TamB-like protein while only a select group encodes TamA.

Consensus computational network analysis for identifying candidate outer membrane proteins from Borrelia spirochetes.

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 ß-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.

Characterization of the ß-barrel assembly machine accessory lipoproteins from Borrelia burgdorferi.

BACKGROUND: Like all diderm bacteria studied to date, Borrelia burgdorferi possesses a ß-barrel assembly machine (BAM) complex. The bacterial BAM complexes characterized thus far consist of an essential integral outer membrane protein designated BamA and one or more accessory proteins. The accessory proteins are typically lipid-modified proteins anchored to the inner leaflet of the outer membrane through their lipid moieties. We previously identified and characterized the B. burgdorferi BamA protein in detail and more recently identified two lipoproteins encoded by open reading frames bb0324 and bb0028 that associate with the borrelial BamA protein. The role(s) of the BAM accessory lipoproteins in B. burgdorferi is currently unknown. RESULTS: Structural modeling of B. burgdorferi BB0028 revealed a distinct ß-propeller fold similar to the known structure for the E. coli BAM accessory lipoprotein BamB. Additionally, the structural model for BB0324 was highly similar to the known structure of BamD, which is consistent with the prior finding that BB0324 contains tetratricopeptide repeat regions similar to other BamD orthologs. Consistent with BB0028 and BB0324 being BAM accessory lipoproteins, mutants lacking expression of each protein were found to exhibit altered membrane permeability and enhanced sensitivity to various antimicrobials. Additionally, BB0028 mutants also exhibited significantly impaired in vitro growth. Finally, immunoprecipitation experiments revealed that BB0028 and BB0324 each interact specifically and independently with BamA to form the BAM complex in B. burgdorferi. CONCLUSIONS: Combined structural studies, functional assays, and co-immunoprecipitation experiments confirmed that BB0028 and BB0324 are the respective BamB and BamD orthologs in B. burgdorferi, and are important in membrane integrity and/or outer membrane protein localization. The borrelial BamB and BamD proteins both interact specifically and independently with BamA to form a tripartite BAM complex in B. burgdorferi. A working model has been developed to further analyze outer membrane biogenesis and outer membrane protein transport in this pathogenic spirochete.

The utilization of an unconventional approach to introduce basic bacteriology in a medical school bridge program.

Bacteria form an intense portion of reading and learning for students enrolled in microbiology education. As a part of the foundational course outline of bacteriology, bacterial classification is a significant topic of discussion. The purpose of our study was to analyze whether bacterial classification can be taught with a phylogenetic tree approach that might be more engaging and beneficial to student learners of microbiology. This methodology is unique compared to the conventional approach applied in introductory lectures of bacteriology that relies on morphology and Gram-staining to classify bacteria. The participants of this study were students enrolled in a two-semester medical school bridge program that offers a Master's degree in Pre-clinical Sciences. We presented bacterial origin and classification in the light of evolution and used a phylogenetic tree to signify clinically relevant groups of bacteria. Students were also taught the traditional bacterial classification using Gram stains and morphology. Both methods of classification were delivered in a didactic classroom session considering equal time spent and utilizing the same format. An online survey was distributed to the students after the session to collect their feedback. The results from the survey showed that 74% of participants would prefer learning bacterial classification using a combined approach that includes both Gram-staining and morphology as well as the phylogenetic tree. When asked if the study of bacterial classification through an evolutionary tree diagram is a clear and concise way of understanding bacteria, 79% of the students either agreed or strongly agreed with this statement. Interestingly, the alternative phylogenetic tree approach was considered more engaging and regarded as a means to expand the clinical knowledge of bacteria by 78% and 71% of the students, respectively. Overall, our study strongly supports the use of tree-based classification as an additional method to improve the learning of medically important groups of bacteria at varying levels of education.

A Case of Previously Undiagnosed Systemic Lupus Erythematosus and Mycobacterium tuberculosis Infection Presenting as Diffuse Alveolar Hemorrhage.

Diffuse alveolar hemorrhage (DAH) is described as the collection of blood in alveolar spaces caused by damaged pulmonary vasculature. It often presents as a life-threatening medical emergency that requires urgent medical intervention along with timely diagnosis and management of the underlying cause. We hereby report a 19-year-old female who presented with clinical and radiological characteristics consistent with DAH. Laboratory workup studies revealed a diagnosis of systemic lupus erythematosus (SLE) as well as Mycobacterium tuberculosis (MTB) infection. This report describes an extremely unusual case of undiagnosed SLE and coexistent tuberculosis presenting as DAH. This leads to an interesting possibility of risks in patients with immune-mediated vasculitis towards developing severe pulmonary disease in the setting of pulmonary mycobacterial infection.

A classic case of Capnocytophaga induced septic shock with multi-organ failure after a dog bite in an asplenic patient.

We hereby report a case of a 40-year-old male with a recent dog bite, a past history of immune thrombocytopenic purpura (ITP) and therapeutic splenectomy. He presented to the hospital with abdominal pain and shortness of breath, which progressed to sepsis and disseminated intravascular coagulation (DIC). Based on clinical presentation, Capnocytophaga-induced sepsis was suspected, and the diagnosis was confirmed through blood culture. Upon confirmation of the diagnosis, the patient was started on IV ampicillin/sulbactam which improved his condition and led to complete recovery without any long-term effects. Capnocytophaga is a genus of Gram-negative bacteria that are commensal to the oral cavity of common household pets such as dogs and cats. This case highlights the importance of considering Capnocytophaga as a potential pathogen in asplenic patients with recent pet-bites and emphasizes how early recognition and intervention can significantly improve outcomes in these critically-ill patients. It also warrants the need for healthcare providers to consider Capnocytophaga infections from minor pet-bites as a differential diagnosis in immunocompromised as well as immunocompetent individuals.

Co-immunoprecipitation for Identifying Protein-Protein Interactions in Borrelia burgdorferi.

Co-immunoprecipitation can be utilized to study protein-protein interactions from various environments, cell types, or tissues. Herein, we describe a co-immunoprecipitation protocol that can be used to examine protein complexes found in the pathogenic spirochete Borrelia burgdorferi. The method outlined here has successfully identified known and unknown members of borrelial protein complexes and is an efficient method for studying protein interactions in this pathogenic spirochete.

Pancreaticopericardial Fistula: A Case Report and Literature Review.

Purpose. Pancreaticopericardial fistula (PPF) is an extremely rare complication of acute or chronic pancreatitis. This paper presents a rare case of PPF and provides systematic review of existing cases from 1970 to 2014. Methods. A PubMed search using key words was performed for all the cases of PPF from January 1970 to December 2014. Fourteen cases were included in the study. The cases were reviewed for demographic characteristics, diagnostic modalities, and treatment. Descriptive analysis of these variables was performed. Results. Median age was 43 years. 78% were known alcoholics and 73.3% had chronic pancreatitis. Dyspnea was present in 78%. Cardiac tamponade was present in 53%; 75% of patients had known chronic pancreatitis (RR = 0.74). Surgery was associated with best treatment outcomes and 50% of patients who underwent endoscopic treatment survived. Conclusion. PPF is a rare disease. This paper indicates that acute cardiac tamponade in patients with history of alcoholism and chronic pancreatitis could be a sign of an existing pancreaticopericardial fistula and early surgical intervention could be life-saving.

Regression of glioma tumor growth in F98 and U87 rat glioma models by the Nitrone OKN-007.

BACKGROUND: Glioblastoma multiforme, a World Health Organization grade IV glioma, has a poor prognosis in humans despite current treatment options. Here, we present magnetic resonance imaging (MRI) data regarding the regression of aggressive rat F98 gliomas and human U87 glioma xenografts after treatment with the nitrone compound OKN-007, a disulfonyl derivative of α-phenyl-tert-butyl nitrone. METHODS: MRI was used to assess tumor volumes in F98 and U87 gliomas, and bioluminescence imaging was used to measure tumor volumes in F98 gliomas encoded with the luciferase gene (F98(luc)). Immunohistochemistry was used to assess angiogenesis (vascular endothelial growth factor [VEGF] and microvessel density [MVD]), cell differentiation (carbonic anhydrase IX [CA-IX]), hypoxia (hypoxia-inducible factor-1α [HIF-1α]), cell proliferation (glucose transporter 1 [Glut-1] and MIB-1), proliferation index, and apoptosis (cleaved caspase 3) markers in F98 gliomas. VEGF, CA-IX, Glut-1, HIF-1α, and cleaved caspase 3 were assessed in U87 gliomas. RESULTS: Animal survival was found to be significantly increased (P < .001 for F98, P < .01 for U87) in the group that received OKN-007 treatment compared with the untreated groups. After MRI detection of F98 gliomas, OKN-007, administered orally, was found to decrease tumor growth (P < .05). U87 glioma volumes were found to significantly decrease (P < .05) after OKN-007 treatment, compared with untreated animals. OKN-007 administration resulted in significant decreases in tumor hypoxia (HIF-1α [P < .05] in both F98 and U87), angiogenesis (MVD [P < .05], but not VEGF, in F98 or U87), and cell proliferation (Glut-1 [P < .05 in F98, P < .01 in U87] and MIB-1 [P < .01] in F98) and caused a significant increase in apoptosis (cleaved caspase 3 [P < .001 in F98, P < .05 in U87]), compared with untreated animals. CONCLUSIONS: OKN-007 may be considered as a promising therapeutic addition or alternative for the treatment of aggressive human gliomas.