In silico design of Mycobacterium tuberculosis multi-epitope adhesin protein vaccines.

Mycobacterium tuberculosis (Mtb) adhesin proteins are promising candidates for subunit vaccine design. Multi-epitope Mtb vaccine and diagnostic candidates were designed using immunoinformatic tools. The antigenic potential of 26 adhesin proteins were determined using VaxiJen 2.0. The truncated heat shock protein 70 (tnHSP70), 19 kDa antigen lipoprotein (lpqH), Mtb curli pili (MTP), and Phosphate transport protein S1 (PstS1) were selected based on the number of known epitopes on the Immune Epitope Database (IEDB). B- and T-cell epitopes were identified using BepiPred2.0, ABCpred, SVMTriP, and IEDB, respectively. Population coverage was analysed using prominent South African specific alleles on the IEDB. The allergenicity, physicochemical characteristics and tertiary structure of the tri-fusion proteins were determined. The in silico immune simulation was performed using C-ImmSim. Three truncated sequences, with predicted B and T cell epitopes, and without allergenicity or signal peptides were linked by three glycine-serine residues, resulting in the stable, hydrophilic molecules, tnlpqH-tnPstS1-tnHSP70 (64,86 kDa) and tnMTP-tnPstS1-tnHSP70 (63,96 kDa). Restriction endonuclease recognition sequences incorporated at the N- and C-terminal ends of each construct, facilitated virtual cloning using Snapgene, into pGEX6P-1, resulting in novel, highly immunogenic vaccine candidates (0,912-0,985). Future studies will involve the cloning, recombinant protein expression and purification of these constructs for downstream applications.

Reverse vaccinology approach to design a multi-epitope vaccine construct based on the Mycobacterium tuberculosis biomarker PE_PGRS17.

Mycobacterium tuberculosis (Mtb) is responsible for high mortality rates in many low- and middle-income countries. This infectious disease remains accountable for around 1.4 million deaths yearly. Finding effective control measures against Mtb has become imperative. Vaccination has been regarded as the safe and lasting control measure to curtail the impact of Mtb. This study used the Mtb protein biomarker PE_PGRS17 to design a multi-epitope vaccine. A previous study predicted a strong antigenic property of PE_PGRS17. Immunogenic properties such as antigenicity, toxicity, and allergenicity were predicted for the PE_PGRS17 biomarker, specific B- and T-cell epitope sequences, and the final multiple epitope vaccine (MEV) construct. Algorithmic tools predicted the T- and B-cell epitopes and those that met the immunogenic properties were selected to construct the MEV candidate for predicted vaccine development. The epitopes were joined via linkers and an adjuvant was attached to the terminals of the entire vaccine construct. Immunogenic properties, and physicochemical and structural predictions gave insight into the MEV construct. The assembled vaccine candidate was docked with a receptor and validated using web-based tools. An immune simulation was performed to imitate the immune response after exposure to a dosed administrated predicted MEV subunit. An in silico cloning and codon optimisation gave insight into optimal expression conditions regarding the MEV candidate. In conclusion, the generated MEV construct may potentially emit both cellular and humoral responses which are vital in the development of a peptide-based vaccine against Mtb; nonetheless, further experimental validation is still required.

Virulence and Inoculation Route Influence the Consequences of Mycoplasma hyorhinis Infection in Bama Miniature Pigs.

Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. Although the majority of M. hyorhinis-colonized pigs have no apparent clinical disease, the pathogen can induce diseases such as polyserositis, arthritis, and eustachitis in some cases. To explore the mechanisms for the occurrence of these diseases, we challenged 4 groups of Bama miniature pigs with M. hyorhinis isolated from pigs without clinical symptoms (non-clinical origin [NCO] strain) or with typical clinical symptoms (clinical origin [CO] strain) and investigated the impacts of different strains and inoculation routes (intranasal [IN], intravenous [IV] + intraperitoneal [IP], and IV+IP+IN) on disease induction. Another group of pigs was set as a negative control. Pigs inoculated with the CO strain through a combined intravenous and intraperitoneal (IV+IP) route showed a significant decrease in average daily weight gain (ADWG), serious joint swelling, and lameness compared with the pigs in the negative-control group. Furthermore, this group developed moderate-to-severe pericarditis, pleuritis, peritonitis, and arthritis, as well as high levels of IgG and IgM antibodies. Pigs inoculated IV+IP with the NCO strain developed less marked clinical, pathological changes and a weaker specific antibody response compared with the pigs inoculated with the CO strain. The challenging results of the NCO strain via different routes (IV+IP, IV+IP+IN, and IN) indicated that the combined route (IV+IP) induced the most serious disease compared to the other inoculation routes. Intranasal inoculation induced a smaller decrease in ADWG without obvious polyserositis or arthritis. These data suggest that differences in both strain virulence and inoculation route affect the consequences of M. hyorhinis infection. IMPORTANCE Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. The mechanisms or conditions that lead to the occurrence of disease in M. hyorhinis-infected pigs are still unknown. The objective of this study was to evaluate the impact of differences in the virulence of strain and the inoculation route on the consequences of M. hyorhinis infection.

A multifunctional enolase mediates cytoadhesion and interaction with host plasminogen and fibronectin in Mycoplasma hyorhinis.

Mycoplasma hyorhinis may cause systemic inflammation of pigs, typically polyserositis and arthritis, and is also associated with several types of human cancer. However, the pathogenesis of M. hyorhinis colonizing and breaching the respiratory barrier to establish systemic infection is poorly understood. Glycolytic enzymes are important moonlighting proteins and virulence-related factors in various bacteria. In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread of M. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. Recombinant M. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (KD) values of 1.4 nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collectively, these results imply that enolase moonlights as an important adhesin of M. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities.

Exploring the Use of Medicinal Plants and Their Bioactive Derivatives as Alveolar NLRP3 Inflammasome Regulators during Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a successful intracellular pathogen that is responsible for the highest mortality rate among diseases caused by bacterial infections. During early interaction with the host innate cells, M. tuberculosis cell surface antigens interact with Toll like receptor 4 (TLR4) to activate the nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) canonical, and non-canonical inflammasome pathways. NLRP3 inflammasome activation in the alveoli has been reported to contribute to the early inflammatory response that is needed for an effective anti-TB response through production of pro-inflammatory cytokines, including those of the Interleukin 1 (IL1) family. However, overstimulation of the alveolar NLRP3 inflammasomes can induce excessive inflammation that is pathological to the host. Several studies have explored the use of medicinal plants and/or their active derivatives to inhibit excessive stimulation of the inflammasomes and its associated factors, thus reducing immunopathological response in the host. This review describes the molecular mechanism of the NLRP3 inflammasome activation in the alveoli during M. tuberculosis infection. Furthermore, the mechanisms of inflammasome inhibition using medicinal plant and their derivatives will also be explored, thus offering a novel perspective on the alternative control strategies of M. tuberculosis-induced immunopathology.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights as an adhesin in Mycoplasma hyorhinis adhesion to epithelial cells as well as a plasminogen receptor mediating extracellular matrix degradation.

Mycoplasma hyorhinis infects pigs causing polyserositis and polyarthritis, and has also been reported in a variety of human tumor tissues. The occurrence of disease is often linked with the systemic invasion of the pathogen. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), one of the key enzymes of glycolysis, was reported as a surface multifunctional molecule in several bacteria. Here, we investigated whether GAPDH could manifest binary functions; as an adhesin to promote colonization as well as a plasminogen receptor functioning in extracellular matrix (ECM) degradation to promote systemic invasion. The surface localization of GAPDH was observed in M. hyorhinis with flow cytometry and colony blot analysis. Recombinant GAPDH (rGAPDH) was found to be able to bind porcine-derived PK-15 and human-derived NCI-H292 cells. The incubation with anti-GAPDH antibody significantly decreased the adherence of M. hyorhinis to both cell lines. To investigate its function in recruiting plasminogen, firstly, the interaction between rGAPDH and plasminogen was demonstrated by ELISA and Far-Western blot assay. The activation of the rGAPDH-bound plasminogen into plasmin was proved by using a chromogenic substrate, and furtherly confirmed to degrade extracellular matrix by using a reconstituted ECM. Finally, the ability of rGAPDH to bind different ECM components was demonstrated, including fibronectin, laminin, collagen type IV and vitronectin. Collectively, our data imply GAPDH as an important adhesion factor of M. hyrohinis and a receptor for hijacking host plasminogen to degrade ECM. The multifunction of GAPDH to bind both plasminogen and ECM components is believed to increase the targeting of proteolysis and facilitate the dissemination of M. hyorhinis.

Immunoscreening of the M. tuberculosis F15/LAM4/KZN secretome library against TB patients' sera identifies unique active- and latent-TB specific biomarkers.

Tuberculosis (TB) protein biomarkers are urgently needed for the development of point-of-care diagnostics, new drugs and vaccines. Mycobacterium tuberculosis extracellular and secreted proteins play an important role in host-pathogen interactions. Antibodies produced against M. tuberculosis proteins before the onset of clinical symptoms can be used in proteomic studies to identify their target proteins. In this study, M. tuberculosis F15/LAM4/KZN strain phage secretome library was screened against immobilized polyclonal sera from active TB patients (n = 20), TST positive individuals (n = 15) and M. tuberculosis uninfected individuals (n = 20) to select and identify proteins recognized by patients' antibodies. DNA sequence analysis from randomly selected latent TB and active TB specific phage clones revealed 118 and 96 ORFs, respectively. Proteins essential for growth, virulence and metabolic pathways were identified using different TB databases. The identified active TB specific biomarkers included five proteins, namely, TrpG, Alr, TreY, BfrA and EspR, with no human homologs, whilst latent TB specific biomarkers included NarG, PonA1, PonA2 and HspR. Future studies will assess potential applications of identified protein biomarkers as TB drug or vaccine candidates/targets and diagnostic markers with the ability to discriminate LTBI from active TB.

Evaluation of a synthetic peptide for the detection of anti-Mycobacterium tuberculosis curli pili IgG antibodies in patients with pulmonary tuberculosis.

Tuberculosis (TB) remains a serious threat in underdeveloped areas. Mycobacterium tuberculosis curli pili (MTP), a virulence factor, is a potential biomarker for a reliable point of care (POC) test and was evaluated for its ability to react with Immunoglobulin G (IgG) in TB patients. An MTP synthetic peptide in a slot blot assay was used to screen serum/plasma samples (n = 65) in 3 separate cohorts, including 40 TB positive (16 HIV co-infected), 20 TB negative/HIV negative patients and 5 healthy volunteers. Forty samples were true positives (HIV positive, n = 16), 23 true negatives (HIV negative) and 2 false positives (HIV negative). The McNemar test demonstrated a 3.08% accuracy estimate (CI: -2.1% - 3.08%). This confirms that MTP is expressed during infection, including HIV-TB co-infection, is likely to be suitable for the design of a POC test and supports the validation of MTP for TB detection in larger patient populations.

Identification of unique essential proteins from a Mycobacterium tuberculosis F15/LAM4/KZN phage secretome library.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis disease (TB), the leading cause of death from bacterial infection worldwide. Although treatable, the resurgence of multidrug-resistant and extensively drug-resistant TB is a major setback for the fight against TB globally. Consequently, there is an urgent need for new Mtb-derived biomarkers for use in the design of new drugs and rapid point-of-care diagnostic or prognostic tools for the management of TB transmission. Therefore, the present study aimed to identify unique Mtb-secreted proteins from the extensively drug-resistant Mtb F15/LAM4/KZN phage secretome library. A whole genome library was constructed using genomic DNA fragments of the Mtb F15/LAM4/KZN strain. A phage secretome sub-library of 8 × 103 clones was prepared and phage DNA was sequenced from 120 randomly selected clones. DNA sequence BLAST analysis identified 86 open reading frames. Using bioinformatics tools and databases, 10 proteins essential for in vivo growth and survival of Mtb (Nrp, PssA, MmpL5, SirA, GatB, EspA, TopA, EccCa1, Rv1634 and Rv3103c) were identified. Proteins essential for the growth and survival of Mtb during infection have potential application in the development of diagnostic tools, new drugs and vaccines. Further studies will be conducted to evaluate their potential application in the fight against TB.

Single-chain antibody fragments from a display library derived from chickens immunized with a mixture of parasite and viral antigens.

Phage-displayed chicken single-chain antibody fragment libraries can provide useful diagnostic and research reagents. Using avian immunoglobulin genes simplifies the construction of such repertoires since far fewer primer sets are required to access the avian antibody repertoire than is the case with mice or humans. Libraries constructed using mRNA from an immune source are enriched in affinity-matured sequences and consequently need not be as large as "universal" non-immune repertoires to have a reasonable probability of yielding high-affinity binders. Repertoires focused on a number of defined targets can be constructed using lymphocyte mRNA from chickens immunized with a mixture of several different antigens. This approach was evaluated with the aim of economically and rapidly deriving immunodiagnostic reagents for malaria, trypanosomiasis, and malignant catarrhal fever, all of which are important to health or food security in Africa. Two chickens were each immunized with a mixture comprised of recombinantly expressed histidine-rich protein, the aldolase and the lactate dehydrogenase of Plasmodium falciparum, the variant surface glycoprotein of Trypanosoma sp., and purified malignant catarrhal fever virus, a herpesvirus that causes an economically important disease of cattle and other ruminants. Immune responses to each of the individual antigens were determined by extracting egg-yolk IgY and testing for antigen-specific antibodies in ELISA. The chicken splenocytes were then recovered, RNA was extracted, and after reverse transcription, the immunoglobulin VH and VL regions were amplified by PCR and joined via a single glycyl residue for surface expression on a collection of filamentous bacteriophages. The resulting display library was then screened by panning to isolate binders. The immunized chickens did not, however, respond equally well to all the different antigens, nor was it possible to derive antibody fragments against all the targets. These limitations notwithstanding, several useful binders with the potential to be used in malaria diagnosis were obtained.