The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding.

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

Mesoporous Silica as Sorbents and Enzymatic Nanoreactors for Microbial Membrane Proteomics.

The membrane proteins of microbes are at the forefront of host and parasite interactions. Having a general view of the functions of microbial membrane proteins is vital for many biomedical studies on microbiota. Nevertheless, due to the strong hydrophobicity and low concentration of membrane proteins, it is hard to efficiently enrich and digest the proteins for mass spectrometry analysis. Herein, we design an enzymatic nanoreactor for the digestion of membrane proteins using methylated well-ordered hexagonal mesoporous silica (Met-SBA-15). The material can efficiently extract hydrophobic membrane proteins and host the proteolysis in nanopores. The performance of the enzymatic nanoreactor is first demonstrated using standard hydrophobic proteins and then validated using membrane proteins extracted from Escherichia coli (E. coli) or a mixed bacterial sample of eight strains. Using the nanoreactor, 431 membrane proteins are identified from E. coli, accounting for 38.5% of all membrane proteins of the species, which is much more than that by the widely used in-solution digestion protocol. From the mixed bacterial sample of eight strains, 1395 membrane proteins are identified using the nanoreactor. On the contrary, the traditional in-solution proteolysis workflow only leads to the identification of 477 membrane proteins, demonstrating that the Met-SBA-15 can be offered as an excellent tool for microbial membrane proteome research and is expected to be used in human microbiota studies, e.g. host-microbe interactions.

Separation of Glycosylated OmpA-Like Proteins from Porphyromonas gingivalis and Tannerella forsythia.

OmpA-like proteins located in the outer bacterial membrane are potential virulence factors from the major periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia. Our previous studies have shown that OmpA-like proteins are glycosylated by O-linked N-acetylglucosamine (O-GlcNAc) and are strongly reactive to wheat germ agglutinin (WGA) lectin, which shows sugar specificity to GlcNAc. Utilizing this property, we have developed a separation method for OmpA-like proteins by affinity chromatography using WGA lectin-agarose. The purity of enriched native OmpA-like proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. More importantly, the purified OmpA-like proteins formed a unique trimeric structure keeping their bioactivity intact. In this chapter, we describe a detailed procedure to separate OmpA-like proteins, which may be used to further progress the biological studies of OmpA-like proteins.

Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology.

Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually varied, allowing it to be mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of a specific agent in clinical samples is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody against rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.

Antineoplastic activity of Salmonella Typhimurium outer membrane nanovesicles.

Nano-sized Gram-negative bacterial outer membrane vesicles possess unique structural and immunostimulatory effects that could be exploited to regress tumors by alerting the host immune system and reversing the immunosuppressive tumor microenvironment. The current study was conducted to investigate the antitumor activity of the outer membrane vesicles (ST-OMVs) of Salmonella Typhimurium ATCC 14028, in vitro in human colorectal carcinoma (HTC116), breast cancer (MCF-7), and hepatocellular carcinoma (HepG2) cell lines and in vivo in Ehrlich solid carcinoma-bearing mice model either as a mono-immunotherapy or as an adjuvant to a commonly used conventional chemotherapy. In addition, we investigated the safety of ST-OMVs. Adult Swiss albino female mice with transplanted Ehrlich solid carcinoma were treated with either ST-OMVs, paclitaxel or a combination of both. Tumor volume, growth inhibition rate, quantitative RT-PCR of Bax and VEGF genes expression, histopathology and immune-expression of caspase-3, Beclin-1, CD49b and Ki-67 were all analyzed. Our results showed that ST-OMVs significantly decreased tumor volume, significantly increased tumor growth inhibition rate, up-regulated the immunohistochemical expression of caspase-3, Beclin-1, and CD49b (enhanced recruitment of NK cells). Furthermore, ST-OMVs down-regulated the expression of Ki-67, increased Bax gene expression and decreased VEGF gene expression as detected by qRT-PCR analysis. Histologically, ST-OMVs promoted apoptosis, decreased tumor invasion and mitotic activities. Moreover, ST-OMVs showed a remarkable cytotoxic activity in various investigated in vitro cancer cell lines. Our findings demonstrate potential antitumor activity of ST-OMVs that might be used as a promising safe antitumor immunotherapy or an adjuvant to conventional chemotherapeutic drugs, resolving some of their problems.

Chitoporin from Serratia marcescens: recombinant expression, purification and crystallization.

Serratia marcescens is an opportunistic pathogen that commonly causes hospital-acquired infections and can utilize chitin-enriched nutrients as an alternative energy source. This study reports the identification of a chitoporin (ChiP), termed SmChiP, from the outer membrane of S. marcescens. Sequence alignment with genetically characterized ChiPs suggests that SmChiP is more closely related to the monomeric EcChiP from Escherichia coli than to the trimeric VhChiP from Vibrio campbellii. A single crystal of SmChiP grown under the condition 22%(w/v) PEG 8000, 0.1 M calcium acetate, 0.1 M MES pH 6.0 diffracted X-ray synchrotron radiation to 1.85 Šresolution. SmChiP co-crystallized with chitohexaose under the condition 19%(w/v) PEG 1500, 2 M ammonium phosphate monobasic, 0.1 M HEPES pH 7.0 diffracted X-rays to 2.70 Šresolution. Preliminary crystallographic analysis shows that both SmChiP crystal forms contain one molecule per asymmetric unit and that they belong to the tetragonal space groups P42212 and P41212, respectively. The SmChiP crystal has unit-cell parameters a = 82.97, b = 82.97, c = 189.53 Å, α = ß = γ = 90°, while the crystal of SmChiP in complex with chitohexaose has unit-cell parameters a = 73.24, b = 73.24, c = 213.46 Å, α = ß = γ = 90°. Initial assessment of the complex structure clearly revealed electron density for the sugar ligand. Structure determination of SmChiP in the absence and presence of chitohexaose should reveal the molecular basis of chitin utilization by S. marcescens.

Cats shedding pathogenic Leptospira spp.-An underestimated zoonotic risk?

Shedding of DNA of pathogenic Leptospira spp. has been documented in naturally infected cats in several countries, but urinary shedding of infectious Leptospira spp. has only recently been proven. The climate in Southern Chile is temperate rainy with high annual precipitations which represents ideal preconditions for survival of Leptospira spp., especially during spring and summer. The aims of this study were to investigate shedding of pathogenic Leptospira spp. in outdoor cats in Southern Chile, to perform molecular characterization of isolates growing in culture, and to assess potential risk factors associated with shedding. Urine samples of 231 outdoor cats from rural and urban areas in southern Chile were collected. Urine samples were investigated for pathogenic Leptospira spp. by 4 techniques: qPCR targeting the lipL32 gene, immunomagnetic separation (IMS)-coupled qPCR (IMS-qPCR), direct culture and IMS-coupled culture. Positive urine cultures were additionally confirmed by PCR. Multilocus sequence typing (MLST) was used to molecularly characterize isolates obtained from positive cultures. Overall, 36 urine samples (15.6%, 95% confidence interval (CI) 11.4-20.9) showed positive results. Eighteen (7.8%, 95% CI 4.9-12.1), 30 (13%, 95% CI 9.2-18), 3 (1.3%, 0.3-3.9) and 4 cats (1.7%; 95% CI 0.5-4.5) were positive in qPCR, IMS-qPCR, conventional culture, and IMS-coupled culture, respectively. MLST results of 7 culture-positive cats revealed sequences that could be assigned to sequence type 17 (6 cats) and sequence type 27 (1 cat) corresponding to L. interrogans (Pathogenic Leptospira Subgroup 1). Shedding of pathogenic Leptospira spp. by cats might be an underestimated source of infection for other species including humans. The present study is the first one reporting growth of leptospires from feline urine in culture in naturally infected cats in South-America and characterisation of culture-derived isolates. So far, very few cases of successful attempts to culture leptospires from naturally infected cats are described worldwide.

Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media.

Extracellular membrane vesicles (EMVs) are biogenic secretory lipidic vesicles that play significant roles in intercellular communication related to human diseases and bacterial pathogenesis. They are being investigated for their possible use in diagnosis, vaccines, and biotechnology. However, the existing methods suffer from a number of issues. High-speed centrifugation, a widely used method to collect EMVs, may cause structural artifacts. Immunostaining methods require several steps and thus the separation and detection of EMVs from the secretory cells is time-consuming. Furthermore, detection of EMVs using these methods requires specific and costly antibodies. To tackle these problems, development of a simple and rapid detection method for the EMVs in the cultured medium without separation from the secretory cells is a pressing task. In this study, we focused on the Gram-negative bacterium Shewanella vesiculosa HM13, which produces a large amount of EMVs including a cargo protein with high purity, as a model. Curvature-sensing peptides were used for EMV-detection tools. FAAV, a peptide derived from sorting nexin protein 1, selectively binds to the EMVs even in the presence of the secretory cells in the complex cultured medium. FAAV can fully detect the EMVs within a few minutes, and the resistance of FAAV to proteases enables it to withstand prolonged use in the cultured medium. Fluorescence/Förster resonance energy transfer was used to develop a method to detect changes in the amount of the EMVs with high sensitivity. Overall, our results indicate the potential applicability of FAAV for in situ EMV detection in cultured media.

Slr4, a newly identified S-layer protein from marine Gammaproteobacteria, is a major biofilm matrix component.

S-layers are paracrystalline proteinaceous lattices that surround prokaryotic cells, forming a critical interface between the cells and their extracellular environment. Here, we report the discovery of a novel S-layer protein present in the Gram-negative marine organism, Pseudoalteromonas tunicata D2. An uncharacterized protein (EAR28894) was identified as the most abundant protein in planktonic cultures and biofilms. Bioinformatic methods predicted a beta-helical structure for EAR28894 similar to the Caulobacter S-layer protein, RsaA, despite sharing less than 20% sequence identity. Transmission electron microscopy revealed that purified EAR28894 protein assembled into paracrystalline sheets with a unique square lattice symmetry and a unit cell spacing of ~9.1 nm. An S-layer was found surrounding the outer membrane in wild-type cells and completely removed from cells in an EAR28894 deletion mutant. S-layer material also appeared to be "shed" from wild-type cells and was highly abundant in the extracellular matrix where it is associated with outer membrane vesicles and other matrix components. EAR28894 and its homologs form a new family of S-layer proteins that are widely distributed in Gammaproteobacteria including species of Pseudoalteromonas and Vibrio, and found exclusively in marine metagenomes. We propose the name Slr4 for this novel protein family.

Development of Different Methods for Preparing Acinetobacter baumannii Outer Membrane Vesicles Vaccine: Impact of Preparation Method on Protective Efficacy.

Acinetobacter baumannii (A. baumannii) is becoming a common global concern due to the emergence of multi-drug or pan-drug resistant strains. Confronting the issue of antimicrobial resistance by developing vaccines against the resistant pathogen is becoming a common strategy. In this study, different methods for preparing A. baumannii outer membrane vesicles (AbOMVs) vaccines were developed. sOMV (spontaneously released AbOMV) was extracted from the culture supernatant, while SuOMV (sucrose-extracted AbOMV) and nOMV (native AbOMV) were prepared from the bacterial cells. Three AbOMVs exhibited significant differences in yield, particle size, protein composition, and LPS/DNA content. To compare the protective efficacy of the three AbOMVs, groups of mice were immunized either intramuscularly or intranasally with each AbOMV. Vaccination via both routes conferred significant protection against lethal and sub-lethal A. baumannii challenge. Moreover, intranasal vaccination provided more robust protection, which may be attributed to the induction of significant sIgA response in mucosal sites. Among the three AbOMVs, SuOMV elicited the highest level of protective immunity against A. baumannii infection, whether intramuscular or intranasal immunization, which was characterized by the expression of the most profound specific serum IgG or mucosal sIgA. Taken together, the preparation method had a significant effect on the yield, morphology, and composition of AbOMVs, that further influenced the protective effect against A. baumannii infection.

The emm-Cluster Typing System.

emm-cluster typing system allows to classify most Streptococcus pyogenes variants into 48 different emm clusters. The system correlates nicely with the host serum binding capacities of the M proteins and has been used in epidemiological surveys, strain selection, and vaccine development. Here we describe the allocation of the emm cluster based on the emm-typing defining region.

Sample Preparation and Technical Setup for NMR Spectroscopy with Integral Membrane Proteins.

NMR spectroscopy is a method of choice to characterize structure, function, and dynamics of integral membrane proteins at atomic resolution. Here, we describe protocols for sample preparation and characterization by NMR spectroscopy of two integral membrane proteins with different architecture, the α-helical membrane protein MsbA and the ß-barrel membrane protein BamA. The protocols describe recombinant expression in E. coli, protein refolding, purification, and reconstitution in suitable membrane mimetics, as well as key setup steps for basic NMR experiments. These include experiments on protein samples in the solid state under magic angle spinning (MAS) conditions and experiments on protein samples in aqueous solution. Since MsbA and BamA are typical examples of their respective architectural classes, the protocols presented here can also serve as a reference for other integral membrane proteins.

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors.

Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.

Documentation of the Expansion of the Gulf Coast Tick (Amblyomma maculatum) and Rickettsia parkeri: First Report in Illinois.

The Gulf Coast tick, Amblyomma maculatum, is of public and veterinary health concern, as it is the primary vector of Rickettsia parkeri and Hepatozoon americanum, causative agents of Rickettsiosis and American canine hepatozoonosis. The Gulf Coast tick's range has expanded over the last 50 yr into the mid-Atlantic states, and its expansion is expected to continue northward. We are reporting the presence of A. maculatum for the first time in Illinois, including a total of 18 specimens collected at 6 different sites during surveys in 2013 and 2019. Fourteen of these specimens were screened for Rickettsia parkeri, which resulted in the detection of this bacteria in 8 samples from 4 counties. By depositing these specimens in scientific collections, we provide materialistic evidence of their establishment in 2 counties. We urge health officials to rely on and use scientific collections to document the expansion of these and other vectors across the country. Additionally, we recommend that health practitioners become aware of the clinical similarities between Rocky Mountain Spotted Fever (caused by Rickettsia rickettsii) and "tidewater" fever (caused by R. parkeri).

Changes in emm types and superantigen gene content of Streptococcus pyogenes causing invasive infections in Portugal.

Fluctuations in the clonal composition of Group A Streptococcus (GAS) have been associated with the emergence of successful lineages and with upsurges of invasive infections (iGAS). This study aimed at identifying changes in the clones causing iGAS in Portugal. Antimicrobial susceptibility testing, emm typing and superantigen (SAg) gene profiling were performed for 381 iGAS isolates from 2010-2015. Macrolide resistance decreased to 4%, accompanied by the disappearance of the M phenotype and an increase of the iMLSB phenotype. The dominant emm types were: emm1 (28%), emm89 (11%), emm3 (9%), emm12 (8%), and emm6 (7%). There were no significant changes in the prevalence of individual emm types, emm clusters, or SAg profiles when comparing to 2006-2009, although an overall increasing trend was recorded during 2000-2015 for emm1, emm75, and emm87. Short-term increases in the prevalence of emm3, emm6, and emm75 may have been driven by concomitant SAg profile changes observed within these emm types, or reflect the emergence of novel genomic variants of the same emm types carrying different SAgs.

Membrane directed expression in Escherichia coli of BBA57 and other virulence factors from the Lyme disease agent Borrelia burgdorferi.

Membrane-embedded proteins are critical to the establishment, survival and persistence in the host of the Lyme disease bacterium Borrelia burgdorferi (Bb), but to date, there are no solved structures of transmembrane proteins representing these attractive therapeutic targets. All available structures from the genus Borrelia represent proteins expressed without a membrane-targeting signal peptide, thus avoiding conserved pathways that modify, fold and assemble membrane protein complexes. Towards elucidating structure and function of these critical proteins, we directed translocation of eleven expression-optimized Bb virulence factors, including the signal sequence, to the Escherichia coli membrane, of which five, BBA57, HtrA, BB0238, BB0323, and DipA, were expressed with C-terminal His-tags. P66 was also expressed using the PelB signal sequence fused to maltose binding protein. Membrane-associated BBA57 lipoprotein was solubilized by non-ionic and zwitterionic detergents. We show BBA57 translocation to the outer membrane, purification at a level sufficient for structural studies, and evidence for an α-helical multimer. Previous studies showed multiple critical roles of BBA57 in transmission, joint arthritis, carditis, weakening immune responses, and regulating other Bb outer surface proteins. In describing the first purification of membrane-translocated BBA57, this work will support subsequent studies that reveal the precise mechanisms of this important Lyme disease virulence factor.

Identification of Chlamydia gallinacea in a parrot and in free-range chickens in Australia.

Chlamydia gallinacea is a recently described bacterial species in a genus known to infect and cause disease in animals and humans. Our report describes the identification of C. gallinacea infection in free-range laying chickens (Gallus gallus) in Australia, and the identification of C. gallinacea infection in a parrot, a wild Australian galah (Eolophus roseicapillus). There is currently little knowledge of the effects of C. gallinacea infection on avian hosts, but it has been linked to respiratory disease in humans and could potentially cause similar disease in other species. Our report highlights the need for further study and surveillance of Chlamydia species in both wild and domestic hosts in Australia.

Isolation of Naturally Released Gonococcal Outer Membrane Vesicles as Vaccine Antigens.

The emergence and spread of fully antimicrobial resistant Neisseria gonorrhoeae (GC) highlights a clear need for next-generation antigonococcal therapeutics. A broadly reactive anti-GC vaccine would best address this global public health threat. Polyantigenic outer membrane vesicles (OMVs) derived from GC can overcome the challenges posed by GC's high rate of phase and antigen variation. In fact, GC OMVs have already shown promise as a vaccine antigen; however, all previous studies have utilized vesicles contaminated by RMP, a bacterioprotective antigen known to entirely abrogate vaccine-induced bactericidal activity in vivo. Additionally, these studies primarily utilized vesicles isolated through techniques like membrane disruption with detergents, which are known to increase contamination of cytoplasmic components as compared to naturally released OMVs (nOMVs). This chapter describes the isolation and characterization of naturally released nOMVs through sequential size and weight restrictive filtration. nOMVs are characterized by morphology, proteomics, and bioactivity via various methods. Herein we also describe methods for further evaluation of the innate and induced immunogenicity of rmp-deficient GC nOMVs by cell stimulation and murine vaccination. Per these methods, nOMVs are found to be largely homogenous spherical structures approximately 70 nm in diameter containing a consistent subset of GC outer membrane proteins. The rmp-deficient vesicles demonstrate a morphology and, with the exception of RMP, antigenic profile consistent with that of nOMVs derived from wild time N. gonorrhoeae. Additionally, vesicles lacking RMP are able to engage and strongly activate a diverse array of pattern recognition receptors in vitro. These methods lay the groundwork for future experiments examining the in vivo protective efficacy of the anti-GC response induced by these nOMVs as well as studies examining the mechanism of vaccine induced female genital tract immunity.

Novosphingobium sp. PP1Y as a novel source of outer membrane vesicles.

Outer membrane vesicles (OMVs) are nanostructures of 20-200 nm diameter deriving from the surface of several Gram-negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical "fingerprint", suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.

A Vector Suite for the Overexpression and Purification of Tagged Outer Membrane, Periplasmic, and Secreted Proteins in E. coli.

Outer membrane and secreted proteins in Gram-negative bacteria constitute a high percentage of virulence factors that are critical in disease initiation and progression. Despite their importance, it is often difficult to study these proteins due to challenges with expression and purification. Here we present a suite of vectors for the inducible expression of N-terminally 6His-tagged outer membrane, periplasmic, and secreted proteins in E. coli and show this system to be capable of producing milligram quantities of pure protein for downstream functional and structural analysis. This system can not only be used to purify recombinant virulence factors for structural and functional studies but can also be used to create gain-of-function E. coli for use in phenotypic screens, and examples of each are provided herein.