Identification of genes associated with persistence in Mycobacterium smegmatis.

The prevalence of bacterial persisters is related to their phenotypic diversity and is responsible for the relapse of chronic infections. Tolerance to antibiotic therapy is the hallmark of bacterial persistence. In this study, we have screened a transposon library of Mycobacterium smegmatis mc2155 strain using antibiotic tolerance, survival in mouse macrophages, and biofilm-forming ability of the mutants. Out of 10 thousand clones screened, we selected ten mutants defective in all the three phenotypes. Six mutants showed significantly lower persister abundance under different stress conditions. Insertions in three genes belonging to the pathways of oxidative phosphorylation msmeg_3233 (cydA), biotin metabolism msmeg_3194 (bioB), and oxidative metabolism msmeg_0719, a flavoprotein monooxygenase, significantly reduced the number of live cells, suggesting their role in pathways promoting long-term survival. Another group that displayed a moderate reduction in CFU included a glycosyltransferase, msmeg_0392, a hydrogenase subunit, msmeg_2263 (hybC), and a DNA binding protein, msmeg_2211. The study has revealed potential candidates likely to facilitate the long-term survival of M. smegmatis. The findings offer new targets to develop antibiotics against persisters. Further, investigating the corresponding genes in M. tuberculosis may provide valuable leads in improving the treatment of chronic and persistent tuberculosis infections.

Antibody engineering and its therapeutic applications.

As a natural function, antibodies defend the host from infected cells and pathogens by recognizing their pathogenic determinants. Antibodies (Abs) gained wide acceptance with an enormous impact on human health and have predominantly captured the arena of bio-therapeutics and bio-diagnostics. The scope of Ab-based biologics is vast, and it is likely to solve many unmet clinical needs in future. The majority of attention is now devoted to developing innovative technologies for manufacturing and engineering Abs, better suited to satisfy human needs. The advent of Ab engineering technologies (AET) led to phenomenal developments leading to the generation of Abs-/Ab-derived molecules with desirable functional properties proportional to their expanding requirements. Evolution brought by AET, from the naturally occurring Ab forms to several advanced Ab formats and derivatives, was much needed as it is of great interest to the pharmaceutical industry. Thus, numerous advancements in AET have propelled success in therapeutic Ab development, along with the potential for ever-increasing improvements. Unique characteristics of Abs, such as its diversity, specificity, structural integrity and an array of possible applications, together inspire continuous innovation in the field. Overall, the AET could assist in conquer of several limitations of Abs in terms of their applicability in the field of therapeutics, diagnostics and research; AET has so far led to the production of next-generation Abs, which have revolutionized these arenas. Here in this review, we discuss the various distinguished engineering platforms for Ab development and the progress in modern therapeutics by the so-called "next-generation Abs."

Crp/fnr family protein binds to promoters of atxA and sodmn genes that regulate the expression of exotoxins in Bacillus anthracis.

Bacillus anthracis produces a tripartite exotoxin, which is regulated by AtxA. Sodmn is constitutively expressed during invasion. Crp/Fnr family transcriptional regulators are known to bind promoters of toxin regulators as well as constitutively expressed genes during pathogenesis. B. anthracis fnr gene was cloned, over-expressed in E. coli and recombinant protein was purified. Oligomeric nature of recombinant rFnr protein was studied by diamide treatment and DTT reduction. DNA binding of rFnr protein was studied by EMSA. We observed that rFnr exists in both monomeric and oligomeric forms. It was found that rFnr was able to oligomerize after diamide treatment which was reversible through DTT reduction. Promoter regions of atxA and sodmn show binding to monomeric form of rFnr, however, dimeric form was unable to bind. Fnr might be playing a role in regulation of toxin gene expression via regulation of atxA gene. It can also be involved in regulation of pathogenesis by regulating the sodmn expression. Oligomerization can act as an ON/OFF switch for the Fnr mediated regulation.

Biosensors for the detection of Mycobacterium tuberculosis: a comprehensive overview.

Tuberculosis (TB) infection is one of the leading causes of death in the world. According to WHO reports 2019, the average rate of decrease in global TB incidences was only 1.6% per year from 2000 to 2018, besides that the global decline in TB deaths was just 11%. Therefore, the dire need for early detection of the pathogen for the successful diagnosis of TB seems justified. Mycobacterium tuberculosis secretory proteins have gained more attention as TB biomarkers, for the early diagnosis and treatment of TB. Here in this review, we elaborate on the recent advancements made in the field of piezoelectric, magnetic, optical, and electrochemical biosensors, in addition to listing their merits and setbacks. Additionally, this review also discusses the construction of biosensors through modern integrated technologies, such as combinations of analytical chemistry, molecular biology, and nanotechnology. Integrated technologies enhance the detection for perceiving highly selective, specific, and sensitive signals to detect M. tuberculosis. Furthermore, this review highlights the recent challenges and scope of improvement in numerous biosensors developed for rapid, specific, selective, and sensitive detection of tuberculosis to reduce the TB burden and successful treatment.

Recombinant full-length Bacillus Anthracis protective antigen and its 63 kDa form elicits protective response in formulation with addavax.

Introduction: Bacillus anthracis is the causative agent for the lethal disease anthrax, primarily affecting animals and humans in close contact with an infected host. The pathogenicity of B. anthracis is attributed to the secreted exotoxins and their outer capsule. The host cell-binding exotoxin component "protective antigen" (PA) is reported to be a potent vaccine candidate. The aim of our study is to produce several PA constructs and analyze their vaccine potential. Methods: We have designed the various subunit, PA-based recombinant proteins, i.e., full-length Protective antigen (PA-FL), C-terminal 63 kDa fragment (PA63), Protective antigen domain 1-domain 4 chimeras (PA-D1-4) and protective antigen domain 4 (PA-D4) and analyzed their vaccine potential with different human-compatible adjuvants in the mouse model. We have optimized the process and successfully expressed our recombinant antigens as soluble proteins, except full-length PA. All the recombinant antigen formulations with three different adjuvants i.e., Addavax, Alhydrogel, and Montanide ISA 720, were immunized in different mouse groups. The vaccine efficacy of the formulations was analyzed by mouse serum antigen-specific antibody titer, toxin neutralization assay, and survival analysis of mouse groups challenged with a lethal dose of B. anthracis virulent spores. Results: We have demonstrated that the PA-FL addavax and PA63 addavax formulations were most effective in protecting spore-challenged mice and serum from the mice immunized with PAFL addavax, PA-FL alhydrogel, PA63 addavax, and PA63 alhydrogel formulations were equivalently efficient in neutralizing the anthrax lethal toxin. The higher levels of serum Th1, Th2, and Th17 cytokines in PA-FL addavax immunized mice correspond to the enhanced protection provided by the formulation in challenged mice. Discussion: We have demonstrated that the PA-FL addavax and PA63 addavax formulations exhibit equivalent efficiency as vaccine formulation both in a mouse model of anthrax and mammalian cell lines. However, PA63 is a smaller antigen than PA-FL and more importantly, PA63 is expressed as a soluble protein in E. coli, which imparts a translational advantage to PA63-based formulation. Thus, the outcome of our study has significant implications for the development of protective antigen-based vaccine formulations for human use against the lethal disease anthrax.

Insights into the molecular determinants involved in Mycobacterium tuberculosis persistence and their therapeutic implications.

The establishment of persistent infections and the reactivation of persistent bacteria to active bacilli are the two hurdles in effective tuberculosis treatment. Mycobacterium tuberculosis, an etiologic tuberculosis agent, adapts to numerous antibiotics and resists the host immune system causing a disease of public health concern. Extensive research has been employed to combat this disease due to its sheer ability to persist in the host system, undetected, waiting for the opportunity to declare itself. Persisters are a bacterial subpopulation that possesses transient tolerance to high doses of antibiotics. There are certain inherent mechanisms that facilitate the persister cell formation in Mycobacterium tuberculosis, some of those had been characterized in the past namely, stringent response, transcriptional regulators, energy production pathways, lipid metabolism, cell wall remodeling enzymes, phosphate metabolism, and proteasome protein degradation. This article reviews the recent advancements made in various in vitro persistence models that assist to unravel the mechanisms involved in the persister cell formation and to hunt for the possible preventive or treatment measures. To tackle the persister population the immunodominant proteins that express specifically at the latent phase of infection can be used for diagnosis to distinguish between the active and latent tuberculosis, as well as to select potential drug or vaccine candidates. In addition, we discuss the genes engaged in the persistence to get more insights into resuscitation and persister cell formation. The in-depth understanding of persistent cells of mycobacteria can certainly unravel novel ways to target the pathogen and tackle its persistence.

Zur: Zinc-Sensing Transcriptional Regulator in a Diverse Set of Bacterial Species.

Zinc (Zn) is the quintessential d block metal, needed for survival in all living organisms. While Zn is an essential element, its excess is deleterious, therefore, maintenance of its intracellular concentrations is needed for survival. The living organisms, during the course of evolution, developed proteins that can track the limitation or excess of necessary metal ions, thus providing survival benefits under variable environmental conditions. Zinc uptake regulator (Zur) is a regulatory transcriptional factor of the FUR superfamily of proteins, abundant among the bacterial species and known for its intracellular Zn sensing ability. In this study, we highlight the roles played by Zur in maintaining the Zn levels in various bacterial species as well as the fact that in recent years Zur has emerged not only as a Zn homeostatic regulator but also as a protein involved directly or indirectly in virulence of some pathogens. This functional aspect of Zur could be exploited in the ventures for the identification of newer antimicrobial targets. Despite extensive research on Zur, the insights into its overall regulon and its moonlighting functions in various pathogens yet remain to be explored. Here in this review, we aim to summarise the disparate functional aspects of Zur proteins present in various bacterial species.

Common garlic (Allium sativum L.) has potent Anti-Bacillus anthracis activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal anthrax, a disease caused by Bacillus anthracis, remains an important but relatively neglected endemic disease of animals and humans in remote areas of the Indian subcontinent and some parts of Africa. Its initial symptoms include diarrhea and stomachache. In the current study, several common plants indicated for diarrhea, dysentery, stomachache or as stomachic as per traditional knowledge in the Indian subcontinent, i.e., Aegle marmelos (L.) Correa (Bael), Allium cepa L. (Onion), Allium sativum L. (Garlic), Azadirachta indica A. Juss. (Neem), Berberis asiatica Roxb. ex DC. (Daruharidra), Coriandrum sativum L. (Coriander), Curcuma longa L. (Turmeric), Cynodon dactylon (L.) Pers. (Bermuda grass), Mangifera indica L. (Mango), Morus indica L. (Black mulberry), Ocimum tenuiflorum L. (Ocimum sanctum L., Holy Basil), Ocimum gratissimum L. (Ram Tulsi), Psidium guajava L. (Guava), Zingiber officinale Roscoe (Ginger), were evaluated for their anti-Bacillus anthracis property. The usage of Azadirachta indica A. Juss. and Curcuma longa L. by Santals (India), and Allium sp. by biblical people to alleviate anthrax-like symptoms is well documented, but the usage of other plants is traditionally only indicated for different gastrointestinal disturbances/conditions. AIM OF THE STUDY: Evaluate the above listed commonly available edible plants from the Indian subcontinent that are used in the traditional medicine to treat gastrointestinal diseases including those also indicated for anthrax-like symptoms for the presence of potent anti-B. anthracis activity in a form amenable to use by the general population in the endemic areas. MATERIALS AND METHODS: Aqueous extracts made from fourteen plants indicated above were screened for their anti-B. anthracis activity using agar-well diffusion assay (AWDA) and broth microdilution methods. The Aqueous Garlic Extract (AGE) that displayed most potent anti-B. anthracis activity was assessed for its thermostability, stability under pH extremes encountered in the gastrointestinal tract, and potential antagonistic interaction with bile salts as well as the FDA-approved antibiotics used for anthrax control. The bioactive fractions from the AGE were isolated by TLC coupled bioautography followed by their characterization using GC-MS. RESULTS: Garlic (Allium sativum L.) extract was identified as the most promising candidate with bactericidal activity against B. anthracis. It consistently inhibited the growth of B. anthracis in AWDA and decreased the viable colony-forming unit counts in liquid-broth cultures by 6-logs within 6-12 h. The AGE displayed acceptable thermostability (>80% anti-B. anthracis activity retained on incubation at 50 °C for 12 h) and stability in gastric pH range (2-8). It did not antagonize the activity of FDA-approved antibiotics used for anthrax control. GC-MS analysis of the TLC separated bioactive fractions of AGE indicated the presence of previously unreported constituents such as phthalic acid derivatives, acid esters, phenyl group-containing compounds, steroids etc. CONCLUSION: The Aqueous Garlic Extract (AGE) displayed potent anti-B. anthracis activity. It was better than that displayed by Azadirachta indica A. Juss. (Neem) and Mangifera indica L., while Curcuma longa L. (Turmeric) did not show any activity under the assay conditions used. Further work should be undertaken to explore the possible application of AGE in preventing anthrax incidences in endemic areas.

Network analysis of host-pathogen protein interactions in microbe induced cardiovascular diseases.

Large-scale visualization and analysis of HPIs involved in microbial CVDs can provide crucial insights into the mechanisms of pathogenicity. The comparison of CVD associated HPIs with the entire set of HPIs can identify the pathways specific to CVDs. Therefore, topological properties of HPI networks in CVDs and all pathogens was studied using Cytoscape3.5.1. Ontology and pathway analysis were done using KOBAS 3.0. HPIs of Papilloma, Herpes, Influenza A virus as well as Yersinia pestis and Bacillus anthracis among bacteria were predominant in the whole (wHPI) and the CVD specific (cHPI) network. The central viral and secretory bacterial proteins were predicted virulent. The central viral proteins had higher number of interactions with host proteins in comparison with bacteria. Major fraction of central and essential host proteins interacts with central viral proteins. Alpha-synuclein, Ubiquitin ribosomal proteins, TATA-box-binding protein, and Polyubiquitin-C &B proteins were the top interacting proteins specific to CVDs. Signaling by NGF, Fc epsilon receptor, EGFR and ubiquitin mediated proteolysis were among the top enriched CVD specific pathways. DEXDc and HELICc were enriched host mimicry domains that may help in hijacking of cellular machinery by pathogens. This study provides a system level understanding of cardiac damage in microbe induced CVDs.

Delineation of the Residues of Bacillus anthracis Zinc Uptake Regulator Protein Directly Involved in Its Interaction with Cognate DNA.

Zinc uptake regulator (Zur) is a negative transcriptional regulator of bacteria that belongs to the FUR superfamily of proteins and regulates zinc (Zn) homeostasis under extreme Zn conditions. The Zur protein of Bacillus anthracis (BaZur) was though characterized previously, but the residues of this transcriptional regulator, crucial for binding to the consensus Zur box in the cognate DNA, remain unexplored. In this study, we reveal the essential residues of the protein that govern the specific interaction with the cognate DNA, through mutational and binding studies. In silico predicted model of the BaZur protein with the promoter region of one of the regulon candidates was utilized to identify specific residues of the N-terminal domain (NTD), constituting the DNA-binding recognition helix. Our results suggest that two phenylalanine residues, a non-polar aliphatic leucine and a positively charged arginine residue of NTD, are predominantly involved in DNA binding of BaZur. Among these, the arginine residue (Arg58) is conserved among all the Zur proteins and the two Phe residues, namely Phe53 and Phe63, are conserved in the Zur proteins of Staphylococcus aureus and Listeria monocytogenes. Taken together, the current study represents an in-depth investigation into the key DNA-binding residues involved in the BaZur-DNA interaction.

Structure-based drug repurposing to inhibit the DNA gyrase of Mycobacterium tuberculosis.

Drug repurposing is an alternative avenue for identifying new drugs to treat tuberculosis (TB). Despite the broad-range of anti-tubercular drugs, the emergence of multi-drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb) H37Rv, as well as the significant death toll globally, necessitates the development of new and effective drugs to treat TB. In this study, we have employed a drug repurposing approach to address this drug resistance problem by screening the drugbank database to identify novel inhibitors of the Mtb target enzyme, DNA gyrase. The compounds were screened against the ATPase domain of the gyrase B subunit (MtbGyrB47), and the docking results showed that echinacoside, doxorubicin, epirubicin, and idarubicin possess high binding affinities against MtbGyrB47. Comprehensive assessment using fluorescence spectroscopy, surface plasmon resonance spectroscopy (SPR), and circular dichroism (CD) titration studies revealed echinacoside as a potent binder of MtbGyrB47. Furthermore, ATPase, and DNA supercoiling assays exhibited an IC50 values of 2.1-4.7 µM for echinacoside, doxorubicin, epirubicin, and idarubicin. Among these compounds, the least MIC90 of 6.3 and 12 µM were observed for epirubicin and echinacoside, respectively, against Mtb. Our findings indicate that echinacoside and epirubicin targets mycobacterial DNA gyrase, inhibit its catalytic cycle, and retard mycobacterium growth. Further, these compounds exhibit potential scaffolds for optimizing novel anti-mycobacterial agents that can act on drug-resistant strains.

Co-Administration of Aluminium Hydroxide Nanoparticles and Protective Antigen Domain 4 Encapsulated Non-Ionic Surfactant Vesicles Show Enhanced Immune Response and Superior Protection against Anthrax.

Aluminium salts have been the adjuvant of choice in more than 100 licensed vaccines. Here, we have studied the synergistic effect of aluminium hydroxide nanoparticles (AH np) and non-ionic surfactant-based vesicles (NISV) in modulating the immune response against protective antigen domain 4 (D4) of Bacillus anthracis. NISV was prepared from Span 60 and cholesterol, while AH np was prepared from aluminium chloride and sodium hydroxide. AH np was co-administered with NISV encapsulating D4 (NISV-D4) to formulate AHnp/NISV-D4. The antigen-specific immune response of AHnp/NISV-D4 was compared with that of commercial alhydrogel (alhy) co-administered with NISV-D4 (alhydrogel/NISV-D4), NISV-D4, AHnp/D4, and alhydrogel/D4. Co-administration of NISV-D4 with AH np greatly improved the D4-specific antibody titer as compared to the control groups. Based on IgG isotyping and ex vivo cytokine analysis, AHnp/NISV-D4 generated a balanced Th1/Th2 response. Furthermore, AH np/NISV-D4 showed superior protection against anthrax spore challenge in comparison to other groups. Thus, we demonstrate the possibility of developing a novel combinatorial nanoformulation capable of augmenting both humoral and cellular response, paving the way for adjuvant research.

Complement Evasion Strategies of Human Pathogenic Bacteria.

Human pathogens need to overcome an elaborate network of host defense mechanisms in order to establish their infection, colonization, proliferation and eventual dissemination. The interaction of pathogens with different effector molecules of the immune system results in their neutralization and elimination from the host. The complement system is one such integral component of innate immunity that is critically involved in the early recognition and elimination of the pathogen. Hence, under this immune pressure, all virulent pathogens capable of inducing active infections have evolved immune evasive strategies that primarily target the complement system, which plays an essential and central role for host defense. Recent reports on several bacterial pathogens have elucidated the molecular mechanisms underlying complement evasion, inhibition of opsonic phagocytosis and cell lysis. This review aims to comprehensively summarize the recent findings on the various strategies adopted by pathogenic bacteria to escape complement-mediated clearance.

Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191.

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.

Anthrax prevention through vaccine and post-exposure therapy.

INTRODUCTION: Vaccines and therapeutic antibodies are the most crucial components of anthrax prophylaxis (pre- and post-exposure) and treatment. The improvement in the availability and safety profile of vaccines and the therapeutic antibodies has helped immensely in reducing the worldwide burden of anthrax. AREAS COVERED: Current recommendations for anthrax prophylaxis and control, vaccines and therapeutic antibodies, the recent endeavors, particularly, made after 2010 toward making them safer and more efficacious along with our opinion on its future course. Primarily, PubMed and Europe PMC were searched to cover the recent developments in the above-indicated areas. EXPERT OPINION: Some key existing lacunae in our understanding of the working of biologicals-based anthrax-control measures, i.e., vaccines and therapeutic antibodies, should be addressed to improve their overall stability, safety profile, and efficacy. The identification of novel inhibitors targeting different key-molecules and vital-steps contributing to the overall anthrax pathophysiology could make a difference in anthrax control.

Preclinical Evidence of Nanomedicine Formulation to Target Mycobacterium tuberculosis at Its Bone Marrow Niche.

One-third of the world's population is estimated to be latently infected with Mycobacterium tuberculosis (Mtb). Recently, we found that dormant Mtb hides in bone marrow mesenchymal stem cells (BM-MSCs) post-chemotherapy in mice model and in clinical subjects. It is known that residual Mtb post-chemotherapy may be responsible for increased relapse rates. However, strategies for Mtb clearance post-chemotherapy are lacking. In this study, we engineered and formulated novel bone-homing PEGylated liposome nanoparticles (BTL-NPs) which actively targeted the bone microenvironment leading to Mtb clearance. Targeting of BM-resident Mtb was carried out through bone-homing liposomes tagged with alendronate (Ald). BTL characterization using TEM and DLS showed that the size of bone-homing isoniazid (INH) and rifampicin (RIF) BTLs were 100 ± 16.3 nm and 84 ± 18.4 nm, respectively, with the encapsulation efficiency of 69.5% ± 4.2% and 70.6% ± 4.7%. Further characterization of BTLs, displayed by sustained in vitro release patterns, increased in vivo tissue uptake and enhanced internalization of BTLs in RAW cells and CD271+BM-MSCs. The efficacy of isoniazid (INH)- and rifampicin (RIF)-loaded BTLs were shown using a mice model where the relapse rate of the tuberculosis was decreased significantly in targeted versus non-targeted groups. Our findings suggest that BTLs may play an important role in developing a clinical strategy for the clearance of dormant Mtb post-chemotherapy in BM cells.

Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation.

Bacillus anthracis poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial species, the B. anthracis capsule may also inhibit complement-mediated clearance and ensure bacterial survival in the host. Previous reports suggest that B. anthracis spore proteins inhibit complement activation. However, the mechanism through which the B. anthracis capsule imparts a survival advantage to the active bacteria has not been demonstrated till date. Thus, to evaluate the role of the PGA capsule in evading host immunity, we have undertaken the present head-to-head comparative study of the phagocytosis and complement activation of non-encapsulated and encapsulated B. anthracis strains. The encapsulated virulent strain exhibited resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. The non-encapsulated Sterne strain was highly susceptible to phagocytosis by THP-1 macrophages, after incubation with normal human serum (NHS), heat-inactivated serum, and serum-free media, thus indicating that the capsule inhibited both complement-dependent and complement-independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, were observed with the non-encapsulated Sterne strain compared with the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP), and serum amyloid P component (SAP), also bound more prominently with the non-encapsulated Sterne strain. Studies with complement pathway-specific, component-deficient serum demonstrated that the classical pathway was primarily involved in mediating C3b binding on the non-encapsulated bacteria. Both strains equally bound the complement regulatory proteins C4BP and factor H. Importantly, we demonstrated that the negative charge of the PGA capsule was responsible for the differential binding of the complement proteins between the non-encapsulated and encapsulated strains. At lower pH closer to the isoelectric point of PGA, the neutralization of the negative charge was associated with an increased binding of C3b and IgG with the encapsulated B. anthracis strain. Overall, our data have demonstrated that the B. anthracis capsule inhibits complement fixation and opsonization resulting in reduced phagocytosis by macrophages, thus allowing the bacterial pathogen to evade host immunity.

Crystalline and Amorphous Preparation of Aluminum Hydroxide Nanoparticles Enhances Protective Antigen Domain 4 Specific Immunogenicity and Provides Protection Against Anthrax.

INTRODUCTION: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. METHODS AND RESULTS: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150-200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. CONCLUSION: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.

Simultaneous Immunization with Omp25 and L7/L12 Provides Protection against Brucellosis in Mice.

Currently used Brucella vaccines, Brucella abortus strain 19 and RB51, comprises of live attenuated Brucella strains and prevent infection in animals. However, these vaccines pose potential risks to recipient animals such as attenuation reversal and virulence in susceptible hosts on administration. In this context, recombinant subunit vaccines emerge as a safe and competent alternative in combating the disease. In this study, we formulated a divalent recombinant vaccine consisting of Omp25 and L7/L12 of B. abortus and evaluated vaccine potential individually as well as in combination. Sera obtained from divalent vaccine (Omp25+L7/L12) immunized mice group exhibited enhanced IgG titers against both components and indicated specificity upon immunoblotting reiterating its authenticity. Further, the IgG1/IgG2a ratio obtained against each antigen predicted a predominant Th2 immune response in the Omp25+L7/L12 immunized mice group. Upon infection with virulent B. abortus 544, Omp25+L7/L12 infected mice exhibited superior Log10 protection compared to individual vaccines. Consequently, this study recommends that simultaneous immunization of Omp25 and L7/L12 as a divalent vaccine complements and triggers a Th2 mediated immune response in mice competent of providing protection against brucellosis.

ATP-binding cassette (ABC) import systems of Mycobacterium tuberculosis: target for drug and vaccine development.

Nutrient procurement specifically from nutrient-limiting environment is essential for pathogenic bacteria to survive and/or persist within the host. Long-term survival or persistent infection is one of the main reasons for the overuse of antibiotics, and contributes to the development and spread of antibiotic resistance. Mycobacterium tuberculosis is known for long-term survival within the host, and develops multidrug resistance. Before and during infection, the pathogen encounters various harsh environmental conditions. To cope up with such nutrient-limiting conditions, it is crucial to uptake essential nutrients such as ions, sugars, amino acids, peptides, and metals, necessary for numerous vital biological activities. Among the various types of transporters, ATP-binding cassette (ABC) importers are essentially unique to bacteria, accessible as drug targets without penetrating the cytoplasmic membrane, and offer an ATP-dependent gateway into the cell by mimicking substrates of the importer and designing inhibitors against substrate-binding proteins, ABC importers endeavour for the development of successful drug candidates and antibiotics. Alternatively, the production of antibodies against substrate-binding proteins could lead to vaccine development. In this review, we will emphasize the role of M. tuberculosis ABC importers for survival and virulence within the host. Furthermore, we will elucidate their unique characteristics to discover emerging therapies to combat tuberculosis.