Outer Membrane Protein Research Articles

OmpA controls order in the outer membrane and shares the mechanical load

OmpA, a predominant outer membrane (OM) protein in Escherichia coli, affects virulence, adhesion, and bacterial OM integrity. However, despite more than 50 y of research, the molecular basis for the role of OmpA has remained elusive. In this study, we demonstrate that OmpA organizes the OM protein lattice and mechanically connects it to the cell wall (CW). Using gene fusions, atomic force microscopy, simulations, and microfluidics, we show that the β-barrel domain of OmpA is critical for maintaining the permeability barrier, but both the β-barrel and CW-binding domains are necessary to enhance the cell envelope's strength. OmpA integrates the compressive properties of the OM protein lattice with the tensile strength of the CW, forming a mechanically robust composite that increases overall integrity. This coupling likely underpins the ability of the entire envelope to function as a cohesive, resilient structure, critical for the survival of bacteria.

OMP38 of Carbapenem-Resistant Acinetobacter Baumannii-Mediated mtDNA Release Activates the cGAS-STING Signaling to Induce Inflammatory Response.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a major threat in the treatment of bacterial infection, and immunotherapy in a non-antibiotic-dependent manner is an effective way to overcome CRAB infection. However, the role of the innate immune response in CRAB infection is poorly understood. Here, it is reported that CRAB infection induced a cytosolic DNA-sensing signaling pathway and significant IFN-β production in mice post-CRAB infection. The knockout of STING reduced bacterial burden, the production of inflammatory cytokines, and lung injury in mice post CRAB infection. The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) and the adaptor protein stimulator of interferon genes (STING) are required for CRAB-induced IFN-β expression in macrophages. Intriguingly, CRAB utilized outer membrane vesicles (OMVs) to transport outer membrane protein 38 (OMP38) into mitochondria, triggering mitochondrial DNA (mtDNA) release into the cytosol through the mitochondrial permeability transition pore (mPTP) and activating the cGAS-STING signaling. Finally, epigallocatechin gallate (EGCG) is demonstrated to block the activation of the cGAS-STING pathway and ameliorate CRAB-induced excessive inflammatory response. These results demonstrated that the early innate immune response to CRAB infection is activated in a cGAS-STING-dependent manner, which could be a potential therapeutic target for CRAB infection.

Cell-free mitochondria are detected in high concentrations in the plasma of orthopedic trauma patients.

Trauma and surgery can derange inflammatory and hemostasis responses, potentially leading to multiple organ failure. Mitochondrial damage-associated molecular patterns are known to be part of the pathomechanism, but their exact origin remains uncertain. Recently, intact mitochondria were detected in healthy individuals' peripheral blood, which suggested a potential role in inflammation. In this case-control study, we quantitated cell-free mitochondria in the blood of healthy subjects (n = 4) and trauma patients (n = 25) and assessed their relationship with patient demographics, injury and shock severity, markers of tissue injury, inflammation, and blood transfusions. Blood samples were collected before and after major orthopedic trauma surgery, and cell-free mitochondria were quantified using flow cytometry, targeting the outer mitochondrial membrane protein, TOMM70. Mitotracker Deep Red staining was used to assess mitochondrial membrane potential. Trauma patients had significantly more cell-free mitochondria in their plasma compared with healthy controls, with highest counts immediately after surgery. The number of cell-free mitochondria decreased by day 5 postoperatively. Trauma patients exhibited a higher proportion of active cell-free mitochondria compared with healthy controls, especially immediately after surgery, and this proportion correlated with tissue injury markers. Associations were also found with acute thrombocytopenia, Denver multiple organ failure score, and transfusion of fresh frozen plasma and cryoprecipitate. Our findings indicate that the mere high number of cell-free mitochondria in the circulation of trauma patients is not necessarily pro-inflammatory, but their active status is associated with more severe secondary tissue injury. The natural history of cell-free mitochondria in trauma needs to be characterized, including their potential cause-effect relationship with major postinjury complications. Prognostic and Epidemiological; Level III.

Molecular characterization of the permanent outer-inner membrane contact site of the mitochondrial genome segregation complex in trypanosomes

The parasitic protozoan Trypanosoma brucei has a single unit mitochondrial genome linked to the basal body of the flagellum via the tripartite attachment complex (TAC). The TAC is crucial for mitochondrial genome segregation during cytokinesis. At the core of the TAC, the outer membrane protein TAC60 binds to the inner membrane protein p166, forming a permanent contact site between the two membranes. Although contact sites between mitochondrial membranes are common and serve various functions, their molecular architecture remains largely unknown. This study elucidates the interaction interface of the TAC60-p166 contact site. Using in silico, in vitro, and mutational in vivo analyses, we identified minimal binding segments between TAC60 and p166. The p166 binding site in TAC60 consists of a short kinked α-helix that interacts with the C-terminal α-helix of p166. Despite the presence of conserved charged residues in either protein, electrostatic interactions are not necessary for contact site formation. Instead, the TAC60-p166 interaction is driven by the hydrophobic effect, as converting conserved hydrophobic residues in either protein to hydrophilic amino acids disrupts the contact site.

Immunoinformatics design of a multi-epitope vaccine for Chlamydia trachomatis major outer membrane proteins

Chlamydia trachomatis (CT) remains a significant infectious cause of blindness and sexually transmitted infections worldwide. The objective and novelty of this study lie in using different serovars of CT to design a broad-spectrum multi-epitope vaccine that might confer immunity against different CT infections. As the major outer membrane protein in CT has good immunodominance properties and high conservation and also determines the several serotypes of CT, it is selected as an antibody target in this study. T-cell and B-cell epitopes from serovars A, B, D, E, L1, and L2 were predicted and combined into a single construct by incorporating adjuvants and linkers to enhance immunogenicity and stability. Physicochemical characterization confirmed the constructed vaccine’s anti-allergic, immunogenicity, and thermostable characteristics, followed by structural modeling to refine its 3D configuration. The 3D model structure of the vaccine was validated through the Ramachandran plot and ProSA z-score. Molecular docking studies of the vaccine demonstrated stable binding with toll-like receptor 3, along with molecular dynamics simulations and binding free energy calculations supporting the complex’s stability. In silico cloning has indicated a high potential for expression in Escherichia coli. Lastly, immune simulations revealed robust activation of B cells, cytotoxic T cells, and antigen-presenting cells, alongside significant production of IgM, IgG antibodies, and balanced Th1/Th2 cytokine response, which is crucial for effective immunity. These results suggest the multi-epitope vaccine could effectively induce comprehensive immune responses against CT, highlighting the need for further in vivo validation to advance this promising candidate toward clinical use.

Deciphering Cronobacter sakazakii pathogenicity: Exploring virulence factors and host interactions": A short review

Opportunistic human pathogen called Cronobacter spp. can cause severe illnesses in immunocompromised individuals and newborns. These species are mostly responsible for disorders like meningitis, necrotizing enterocolitis, and bacteremia because they can survive in very dry foods like PIF and have also been identified in various environmental niches, including plants. These species' contamination is ascribed to biofilms, which are biotic or abiotic surface communities. Because they build biofilms on a variety of surfaces, identifying and eliminating them is extremely difficult. For the purpose of creating efficient treatment interventions and preventative measures, it is essential to comprehend the processes of virulence and the relationship between this pathogen and its host. A variety of virulence factors, such as adhesion factors, invasion proteins, biofilm formation components, and secreted toxins, contribute to Cronobacter spp. pathogenicity. Genes involved in capsule formation—such as those involved in exopolysaccharide synthesis—are essential to its pathogenicity because they are critical in desiccation resistance and resistance to host immunological defenses. Furthermore, genes that encode flagella, fimbriae, and outer membrane proteins are linked to host cell adherence and invasion. This review also explores the emerging link between Cronobacter spp. and plants, highlighting the bacterium's ability to survive and persist in the environment because Plants may act as reservoirs, contributing to the contamination of raw agricultural products, which can subsequently enter the food supply chain. Developing focused preventative measures and treatment approaches requires knowledge of the various biological as well as genetic factors that contribute to Cronobacter spp. virulence and how it affects the host. This review clarifies the effects of Cronobacter spp. on the host and investigates the genes linked to virulence in Cronobacter.

MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7cells.

V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75mM and 15°C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7cells, with its optimal concentration determined to be 7.5μg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p<0.05) in NO levels. Additionally, the relative mRNA levels of IL-1β, IL-6, and TNF-α in RAW264.7cells were measured by qRT-PCR, showing a remarkable elevation (p<0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.

Whole genome-based characterization of extended-spectrum β-lactamase-producing Enterobacter cloacae from orthopedic patients and environment of a tertiary referral hospital in Tanzania

ObjectivesWe investigated the genomic epidemiology of extended-spectrum β-lactamase-producing Enterobacter cloacae (ESBL-Ec) isolates from patients and hospital environment to better understand their distribution to help devising effective strategies for infection prevention and control. MethodsWe screened ESBL-Ec at Bugando Medical Center (BMC) in Mwanza, Tanzania. Rectal swabs from orthopedic patients on admission and swabs from the neighboring inanimate environment were collected. Following microbial culture, DNA was extracted from pure ESBL-Ec, and whole-genome sequencing was done. Sequence typing (ST), plasmid replicons, drug resistance, and virulence genes were deciphered using the Rapid Microbial Analysis Pipeline (rMAP). ResultsWe obtained 209 ESBL isolates, of which 15 (7.2%) were ESBL-Ec [8 (53.30%) from patients and 7 (46.7%) from the environment]. Seven isolates were novel and eight were diverse each with a unique ST. All isolates harbored two to five β-lactamase genes, with the predominance of blaCTX-M-15(15/15), blaOXA-1(14/15), blaTEM(14/15) and blaACT(12/15). The most common non β-lactam drug resistance genes were aac(3)-IIa (14/15), aac(6’)-Ib-cr (14/15), fosA (14/15), and qnrB1 (12/15), aph(3’’)-Ib (10/15) and aph(6)-Id (10/15). Eleven different types of plasmid replicons were identified in 14/15 of the isolates, harboring one to five plasmids, with the most common plasmids being IncFII (11/15) and IncFIB (10/15). All isolates harbored the outer membrane protein (ompA), and curli protein (csg) was in 14/15 isolates. ConclusionAdmitted orthopedic patients and the hospital environment act as a reservoir of ESBL-Ec with diverse STs and endowed with drug resistance and arsenals of virulence genes, calling for their routine screening on admission for mitigation of potential subsequent infections.

Development and utilization of Treponema pallidum expressing green fluorescent protein to study spirochete-host interactions and antibody-mediated clearance: expanding the toolbox for syphilis research.

Syphilis, a sexually transmitted infection caused by Treponema pallidum (TPA), remains a pressing threat to global public health. TPA has a remarkable and still poorly understood ability to disseminate rapidly from the site of inoculation and establish persistent infection throughout the body. Recent advances in in vitro cultivation and genetic manipulation of syphilis spirochetes enabled the development of fluorescent TPA. In the study, we generated and characterized an infectious TPA strain that constitutively expresses green fluorescent protein and used this strain to visualize the interaction of TPA with host cells and functionally characterize antibodies directed against treponemal outer membrane proteins. Most notably, we assessed the ability of surface-bound antibodies to inhibit the growth of TPA in vitro and/or disrupt the spirochete's fragile outer membrane. Fluorescent TPA strains provide a powerful new tool for elucidating host-pathogen interactions that enable the syphilis spirochete to establish infection and persistent long-term within its obligate human host.

Mutation-based mechanism and evolution of the potent multidrug efflux pump RE-CmeABC in Campylobacter

The resistance-nodulation-cell division (RND) superfamily of multidrug efflux systems are important players in mediating antibiotic resistance in gram-negative pathogens. Campylobacter jejuni, a major enteric pathogen, utilizes an RND-type transporter system, CmeABC, as the primary mechanism for extrusion of various antibiotics. Recently, a functionally potent variant of CmeABC (named RE-CmeABC) emerged in clinical Campylobacter isolates, conferring enhanced resistance to multiple antibiotic classes. Despite the clinical importance of RE-CmeABC, the molecular mechanisms for its functional gain and its evolutionary trajectory remain unknown. Here, we demonstrated that amino acid substitutions in RE-CmeB (inner membrane transporter), but not in RE-CmeA (periplasmic protein) and RE-CmeC (outer membrane protein), in conjunction with a nucleotide mutation in the promoter region of the efflux operon, are responsible for the functional gain of the multidrug efflux system. We also showed that RE-cmeABC is emerging globally and distributed in genetically diverse C. jejuni strains, suggesting its possible spread by horizontal gene transfer. Notably, many of RE-cmeABC harboring isolates were associated with the human host including strains from large disease outbreaks, indicating the clinical relevance and significance of RE-CmeABC. Evolutionary analysis indicated that RE-cmeB likely originated from Campylobacter coli, but its expansion mainly occurred in C. jejuni, possibly driven by antibiotic selection pressure. Additionally, RE-cmeB, but not RE-cmeA and RE-cmeC, experienced a selective sweep and was progressing to be fixed during evolution. Together, these results identify a mutation-based mechanism for functional gain in RE-CmeABC and reveal the key role of RE-CmeB in facilitating Campylobacter adaptation to antibiotic selection.

Isolation, Characterization, and Unlocking the Potential of Mimir124 Phage for Personalized Treatment of Difficult, Multidrug-Resistant Uropathogenic E. coli Strain

Escherichia coli and its bacteriophages are among the most studied model microorganisms. Bacteriophages for various E. coli strains can typically be easily isolated from environmental sources, and many of these viruses can be harnessed to combat E. coli infections in humans and animals. However, some relatively rare E. coli strains pose significant challenges in finding suitable phages. The uropathogenic strain E. coli UPEC124, isolated from a patient suffering from neurogenic bladder dysfunction, was found to be resistant to all coliphages in our collections, and initial attempts to isolate new phages failed. Using an improved procedure for phage enrichment, we isolated the N4-related phage Mimir124, belonging to the Gamaleyavirus genus, which was able to lyse this “difficult” E. coli strain. Although Mimir124 is a narrow-spectrum phage, it was effective in the individualized treatment of the patient, leading to pathogen eradication. The primary receptor of Mimir124 was the O antigen of the O101 type; consequently, Mimir124-resistant clones were rough (having lost the O antigen). These clones, however, gained sensitivity to some phages that recognize outer membrane proteins as receptors. Despite the presence of nine potential antiviral systems in the genome of the UPEC124 strain, the difficulty in finding effective phages was largely due to the efficient, non-specific cell surface protection provided by the O antigen. These results highlight the importance of an individualized approach to phage therapy, where narrow host-range phages—typically avoided in pre-fabricated phage cocktails—may be instrumental. Furthermore, this study illustrates how integrating genomic, structural, and functional insights can guide the development of innovative therapeutic strategies, paving the way for broader applications of phage therapy in combating multidrug-resistant bacterial pathogens.

Study on the biosafety and targeting efficiency of Escherichia coli outer membrane vesicles in breast tumor.

To seek out the targeting of Escherichia coli outer membrane vesicles (E. coli OMVs) in breast tumor-bearing mice and their biosafety in healthy mice. Ultrafiltration in conjunction with ultracentrifugation was utilized to concentrate E. coli OMVs, and characterize them. Subcutaneous breast tumors were induced in BALB/c mice to serve as an experimental model, and the biodistribution of E. coli OMVs in both tumor-bearing and healthy mice was monitored using an in vivo fluorescence imaging system. Utilizing frozen sections, the infiltration of E. coli OMVs in tumor tissues was appraised at the 24-hour post-injection. Healthy BALB/c mice were randomly divided into control group and vesicles group. Following the intravenous injection of E. coli OMVs, monitoring encompassed variations in body weight, blood routine indices, serum levels of AST, ALT, and BUN, organ indices (heart, liver, spleen, lung, and kidney), along with tissue histopathology over a 14-day period. The spherical E. coli OMVs had a diameter of (155.8 ± 3.1) nm and exhibited the expression of outer membrane proteins OmpA and OmpC. Upon assessment, it was evident that the E. coli OMVs persisted in the tumor tissues even 24h post-injection. An evident decrease in the body weight of the vesicles group, distinct from the control group, was observed on the second day after injection (P < 0.001); in contrast, no considerable differences were noted at subsequent time points (P > 0.05). Following the injection, the vesicles group displayed notable reductions in WBC and PLT as relative to the control group (P < 0.0001) on the initial day, however, there were no noteworthy distinctions as opposed to the control group for other hematological indices; No notable variances in hematological indices between the two groups were observed on the seventh and fourteenth day (P > 0.05). Over the 14 days, no substantial differences were observed in the serum levels of BUN, AST, ALT, and organ indices within the vesicles group as opposed to the control group (P > 0.05). Furthermore, there were no obvious abnormal changes in tissue morphology. 0.5mg/kg of E. coli OMVs can safely and effectively target 4T1 breast tumor in mice.

Identification of determinants that allow maintenance of high-level fluoroquinolone resistance in Acinetobacter baumannii.

Acinetobacter baumannii is associated with multidrug-resistant infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates. We, here, identify proteins that contribute to the fitness of fluoroquinolone-resistant (FQR) strains overexpressing three known RND systems using high-density insertion mutagenesis. Overexpression of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced lipooligosaccharide (LOS) biosynthesis and blocked production of the major A. baumannii porin. In contrast, AdeAB pump hyperexpression, in the absence of elevated adeC expression (the outer membrane component of the pump), was relatively tolerant to loss of these functions, consistent with the outer membrane protein being the primary disruptive component. Surprisingly, overexpression of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overexpression, including the activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from oxidative stress. These results indicate that RND efflux pump overproduction is compensated by maintenance of outer membrane integrity in A. baumannii to facilitate fitness of FQR isolates.IMPORTANCEAcinetobacter baumannii is a pathogen that often causes multidrug-resistant infections in healthcare settings, presenting a threat to the efficacy of known therapeutic interventions. Fluoroquinolones such as ciprofloxacin are currently ineffective against a majority of clinical A. baumannii isolates, many of which express pumps that remove this antibiotic class from within the bacterium. Three of these pumps can be found in most clinical isolates, with one of the three often hyperproduced at all times. In this study, we identify proteins that are necessary for the fitness of pump hyperproducers. The identified proteins are necessary to stabilize the outer membrane and allow the cytoplasm to tolerate the accumulation of ions as a consequence of excess pump activity. These results point to strategies for developing therapies that combine known antibiotics with drugs that target proteins important for survival of strains hyper-expressing efflux pumps.

Small intestinal derived Prevotella histicola simulates biologic as a therapeutic agent

A role of gut microbiome in pathogenesis as well as response to treatment is documented in rheumatoid arthritis. Using a novel duodenal derived Prevotella histicola strain MCI 001, we have shown that it suppresses disease progression in a collagen-induced arthritis (CIA), a model for rheumatoid arthritis (RA) using humanized mice expressing HLA-DQ8 gene in the absence of endogenous class II genes. Here we compared efficacy of P. histicola MCI 001 with tumor necrosis factor inhibitor (TNFi) for treating arthritis. DQ8 arthritic mice treated with P. histicola by oral gavage or TNFi, were compared for disease onset, incidence and severity. We demonstrate that oral treatment with P. histicola mimics treatment with TNFi in arthritic DQ8 mice. A pangenome comparison of our P. histicola MCI 001 with its closest available neighbors depicted it as a novel strain with unique gene sequences that may contribute to immune modulatory effects. Notably, it possesses a unique sequence of an outer membrane protein, BtuB, which is involved in vitamin B12 transport. Our data indicate that P. histicola MC001 is an attractive candidate to prevent the progression of disease in RA patients with ongoing disease.

Re-sensitization of imipenem-resistant Pseudomonas aeruginosa and restoration of cephalosporins susceptibility in Enterobacteriaceae by recombinant Esterase B.

Sphingobium sp. SM42 Esterase B (EstB) is an enzyme with a dual function in degrading dibutyl phthalate (DBP) and catalyzing the cleavage of the C-S bond in C3-sidechains of the dihydrothiazine ring of cephalosporins, generating more active β-lactam derivatives. Global prokaryotic genome analysis revealed the existence of a gene identical to estB in Pseudomonas aeruginosa strain PS1 suggesting a horizontal gene transfer event involving estB. To investigate the effect of ectopic expression of EstB in the periplasm of Pseudomonas aeruginosa and several Enterobacteriaceae on antibiotic susceptibility levels, plasmid, pEstB, carrying a recombinant EstB fused with the signal peptide from Escherichia coli outer membrane protein A (OmpA) for periplasmic localization was constructed. The expression of EstB in the periplasm of P. aeruginosa and the Enterobacteriaceae: E. coli, Klebsiella pneumoniae, and Salmonella enterica serovar Typhi, increased susceptibility to carbapenems and cephalosporins. EstB reversed the imipenem resistance of P. aeruginosa ΔmexS and restored the changes in susceptibility to cephalosporins conferred by the downregulation of the outer membrane proteins, OmpK35 and OmpK36, in K. pneumoniae ΔramR-ompK36 to wild-type level. The introduction of EstB to the periplasmic space of Gram-negative bacteria can increase carbapenem and cephalosporin susceptibility.

Design of a multi-epitope vaccine candidate against carrion disease by immunoinformatics approach

Carrion's disease, caused by the bacterium Bartonella bacilliformis, is a serious public health problem in Peru, Ecuador and Colombia. Currently there is no available vaccine against B. bacilliformis. While antibiotics are the standard treatment, resistant strains have been reported, and there is a potential spread of the vector that transmits the bacteria. This study aimed to design a multi-epitope vaccine candidate against the causative agent of Carrion's disease using immunoinformatics tools. Predictions of B-cell epitopes, as well as CD4+ and CD8+T cell epitopes, were performed from the entire proteome of B. bacilliformis KC583 using the most frequent alleles from Peru, Ecuador, Colombia, and worldwide. B-cell epitopes and T-cell nested epitopes from outer membrane and virulence-associated proteins were selected. Epitopes were filtered out based on promiscuity, non-allergenicity, conservation, non-homology and non-toxicity. Two vaccine constructs were assembled using linkers. The tertiary structure of the constructs was predicted, and their stability was evaluated through molecular dynamics simulations. The most stable construct was selected for molecular docking with the TLR4 receptor. This study proposes a vaccine construct evaluated in silico as a potential vaccine candidate against Bartonella bacilliformis.

Adhesive interactions within microbial consortia can be differentiated at the single-cell level through expansion microscopy

Investigating microbe-microbe interactions at the single-cell level is critical to unraveling the ecology and dynamics of microbial communities. In many situations, microbes assemble themselves into densely packed multispecies biofilms. The density and complexity pose acute difficulties for visualizing individual cells and analyzing their interactions. Here, we address this problem through an unconventional application of expansion microscopy, which allows for the "decrowding" of individual bacterial cells within a multispecies community. Expansion microscopy generally has been carried out under isotropic expansion conditions and used as a resolution-enhancing method. In our variation of expansion microscopy, we carry out expansion under heterotropic conditions; that is, we expand the space between bacterial cells but not the space within individual cells. The separation of individual bacterial cells from each other reflects the competition between the expansion force pulling them apart and the adhesion force holding them together. We employed heterotropic expansion microscopy to study the relative strength of adhesion in model biofilm communities. These included mono- and dual-species Streptococcus biofilms and a three-species synthetic community (Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sanguinis) under conditions that facilitated interspecies coaggregation. Using adhesion mutants, we investigated the interplay between F. nucleatum outer membrane protein RadD and different Streptococcus species. We also examined the Schaalia-TM7 epibiont association. Quantitative proximity analysis was used to evaluate the separation of individual microbial members. Our study demonstrates that heterotropic expansion microscopy can "decrowd" dense biofilm communities, improve visualization of individual bacterial members, and enable analysis of microbe-microbe adhesive interactions at the single-cell level.

Computational studies on metabolic pathways of Coxiella burnetii to combat Q fever: A roadmap to vaccine development

Coxiella burnetii (Cbu) is the gram-negative intracellular pathogen responsible for deadly zoonotic infection, Q fever. The pathogen is environmentally stable and distributed throughout the world which is sustained in nature by chronic infection of ruminants. The epidemiological studies on Q fever indicates it as emerging public health problem in various countries and it is imperative to promptly identify an appropriate therapeutic solution for this pathogen. In the current study, metabolic pathways of Cbu were analysed by the combination of multiple computational tools for the prediction of suitable therapeutic candidates. We have identified 25 metabolic pathways which were specific to Cbu containing 287 unique proteins. A total of 141 proteins which were either virulent, essential or resistant were shortlisted that do not show homology with the host proteins and considered as potential targets for drug and vaccine development. The potential therapeutic targets were classified in to seven functional classes, i.e., metabolism, transport, gene expression and regulation, signal transduction, antimicrobial resistance, stress response regulator and unknown. The majority of the proteins were found to be present in metabolism and transport class. The functional annotation showed the predominant presence of proteins containing HATPase_c, Beta-lactamase, GerE, ACR_tran, PP-binding, CsrA domains. We have identified Type I secretion outer membrane protein for the design of multi-epitope subunit vaccine using reverse vacciniology approach. Four B cell epitopes, six MHC-I epitopes and four MHC-II epitopes were identified which are non-toxic, non-allergen and highly antigenic. The multi-epitope subunit vaccine construct was 327 amino acid residues long which include adjuvant, B cell epitopes, MHC-I epitopes and MHC-II epitopes. The Cholera enterotoxin subunit B is included as an adjuvant in the N terminal of vaccine construct which will help to produce a strong immune response to the vaccine. The multi-epitope vaccine construct was non-toxic, non-allergen and probable antigen having molecular weight 35.13954 kDa, aliphatic index 85.50, theoretical PI 9.65, GRAVY -0.001, and instability index of 28.37. The tertiary structure of the vaccine construct was modeled and physiochemical properties were predicted. After validation and refinement of tertiary structure the molecular docking of vaccine exhibited strong binding with TLR2, TLR3, TLR4, TLR5 and TLR8. The TLRs and vaccine construct formed hydrogen bonds, salt bridges and non-bonded contacts with all TLR receptors. The in-silico immune simulations showed the ability to trigger primary immune response as shown by increment in B-cell and T-cell population. The research paves the way for more effective control of zoonotic disease Q fever.

Molecular Analysis of Aggregatibacter actinomycetemcomitans ApiA, a Multi-Functional Protein.

Aggregatibacter actinomycetemcomitans ApiA is a trimeric autotransporter outer membrane protein (Omp) that participates in multiple functions, enabling A. actinomycetemcomitans to adapt to a variety of environments. The goal of this study is to identify regions in the apiA gene responsible for three of these functions: auto-aggregation, buccal epithelial cell binding, and complement resistance. Initially, apiA was expressed in Escherichia coli. Finally, wild-type A. actinomycetemcomitans and an apiA-deleted version were tested for their expression in the presence and absence of serum and genes related to stress adaptation, such as oxygen regulation, catalase activity, and Omp proteins. Sequential deletions in specific regions in the apiA gene as expressed in E. coli were examined for membrane proteins, which were confirmed by microscopy. The functional activity of epithelial cell binding, auto-aggregation, and complement resistance were then assessed, and regions in the apiA gene responsible for these functions were identified. A region spanning amino acids 186-217, when deleted, abrogated complement resistance and Factor H (FH) binding, while a region spanning amino acids 28-33 was related to epithelial cell binding. A 13-amino-acid peptide responsible for FH binding was shown to promote serum resistance. An apiA deletion in a clinical isolate (IDH781) was created and tested in the presence and/or absence of active and inactive serum and genes deemed responsible for prominent functional activity related to A. actinomycetemcomitans survival using qRT-PCR. These experiments suggested that apiA expression in IDH781 is involved in global regulatory mechanisms that are serum-dependent and show complement resistance. This is the first study to identify specific apiA regions in A. actinomycetemcomitans responsible for FH binding, complement resistance, and other stress-related functions. Moreover, the role of apiA in overall gene regulation was observed.

Enabling high-efficiency plasmon-induced visible-light-driven reduction of hexavalent chromium with Au-TiO2/Shewanella biohybrid

The application of microbe-photocatalyst biohybrid (MPB) systems to pollutant removals has drawn considerable attentions due to the high demands on energy shortage and environmental pollution prevention. However, the stability and utilization rate of photoelectrons generated under the photocatalysis of plasmonic metals are still low. Herein, we constructed a new Au-TiO2/Shewanella biohybridsystem by combining photocatalyst and electrogenic bacteria to realize the plasmon-inducedvisible-light-driven reduction of hexavalent chromium. The highly hydrophilic Au-TiO2 and the outer membrane protein (OmcA) of Shewanella were effectively complexed to form a tight composite. The irradiation of visible light increases the expression level of extracellular polymeric substances (EPS) in the MPB system and upregulates the function gene of OmcA and MtrC, suggesting that the photoelectrons are absorbed by the conductive protein and deposited into the microbes to realize high efficiency chromium removal (68.9%). This study successfully utilize the photogenerated electrons under the catalysis of plasmonic gold nanoparticles and opens up a new avenue to the application of MPB system in water treatment.