198 research outputs found
Implementation of a two-plasmid CRISPR/Cas9 system in Lactobacillus plantarum : a new approach in the development of a novel vaccine against Mycobacterium tuberculosis
Get PDFTuberculosis (TB) is the leading cause of deaths from a single infectious agent, and in 2017 1.7 million people died from TB. To date the only available vaccine against the disease is the bacille Calmette-Guèrin (BCG) vaccine. The BCG vaccine does not ensure full protection against the mature form of TB, and is not recommended to immunocompromised patients. Therefore, a new and more effective vaccine is urgently needed. This study is a part of a larger project with a long-term goal to develop mucosal vaccines, utilizing the lactic acid bacteria (LAB) Lactobacillus plantarum as delivery vectors of antigens. LAB occurs in a wide range of habitats, ranging from food products such as fruit and dairy, but also human mucosal surfaces such as the small intestine and colon. Lactobacilli are natural inhabitants of the human gastro intestinal tract (GIT) and are generally regarded as safe (GRAS). Some Lactobacilli are believed to have probiotic properties and live in close association with the intestinal epithelium and have shown immunomodulatory effects in human. These properties make Lactobacilli, such as L. plantarum, an ideal candidate as a delivery vector of immunogens.
In the present study, pSIP inducible vectors for cytoplasmic and membrane anchoring of the TB fusion antigen Ag85B_TB10.4 in L. plantarum were constructed, and production of surface localized antigen was confirmed. Currently, the production of the antigen is plasmid based. To reduce the number of heterologous genes of the recombinant L. plantarum, it is desirable to integrate the antigen production into genome of the bacteria. In this thesis, methods for utilization of the CRISPR/Cas system was attempted developed for integration of the antigen. To simplify integration, as it is independent of helper genes unlike the pSIP system, construction of vectors for constitutive production of Ag85B_TB10.4 were attempted. Evaluation of the functionality of the CRISPR/Cas system in L. plantarum was performed through experiments of gene editing, replacement and depletion with Cas9, Cas9D10A and dCas9.
In conclusion, Ag85B_TB10.4 was successfully anchored to the cell membrane of L. plantarum by using the pSIP system, while construction of a vector for constitutive production of Ag85B_TB10.4 failed. Conceivably due to toxicity of the constitutive production of the antigen in E. coli. The CRISPR/dCas9 system was successfully developed, and reduction of transcripts of target genes was confirmed by ddPCR. Gene editing and exchange with Cas9 and Cas9D10A gave the expected phenotype, but no mutations were detected from DNA sequencing. These methods require further optimisations
Anchoring of Chlamydia trachomatis Antigens to the Surface of Lactobacillus plantarum
Get PDFDette studiet er en del av et større prosjekt der det langsiktige målet er å utvikle slimhinnevaksiner med melkesyrebakterier som leveringsvektorer. Melkesyrebakterier ansees som gode kandidater til dette formål, da de generelt antas å være trygge for mennesker. Flere arter hevdes å ha helsefremmende og immunmodulerende effekter. De finnes blant annet naturlig i mage-tarmkanalen og vagina hos mennesker, og utviser motstandsdyktighet mot lav pH. Lactobacillus plantarum er en av artene som har fått stort fokus som leveringsvektor av antigener, blant annet fordi den kan overleve i mage-tarmkanalen i opp til en uke. Retting av antigener mot bestemte deler av immunforsvaret, samt ulike ruter for vaksineadministrasjon, har betydning for eventuell vaksineeffekt. Det har tidligere blitt vist at peptider rettet mot dendrittiske celler har økt internaliseringen av leveringsvektoren, og at en slik strategi har medført forsterket immunrespons. Denne oppgaven beskriver studier der L. plantarum modifiseres for bruk som leveringsvektor av overflateankrede Chlamydia trachomatis hybridantigener som er translasjonelt koblet til et dendrittisk cellebindende peptid (DC-pep).
Det foreligger lovende resultater for bruk av hybridantigenene Hirep1- og 2 som vaksinekandidater mot genital klamydiainfeksjon. På bakgrunn av dette ble det konstruert åtte ulike plasmider for ankring av Hirep-baserte antigener til cellemembranen og celleveggen, inkludert versjoner der DC-pep ble translasjonelt koblet til antigenene. Produksjon og overflatelokalisering av antigenene ble stadfestet hos alle de rekombinante L. plantarum. Bakteriene ble karakterisert med tanke på vekst og overlevelse etter ulike lagringsmetoder. Forsøk med dendrittiske celler der effekten av DC-pep ble undersøkt kunne ikke avdekke økt opptak som følge av peptidet. Metodikken som ble benyttet er imidlertid relativt ny, og videre optimalisering vil være aktuelt før endelig konklusjon. Et museforsøk ble gjennomført for deteksjon av rekombinante L. plantarum i nasale- og vaginale slimhinner etter immunisering. Resultatene indikerte god overlevelse i nasale slimhinner i opp til ett døgn etter immunisering, og dårlig overlevelse i vaginale slimhinner.
Arbeidet beskrevet i denne oppgaven viser vellykket ankring av C. trachomatis hybridantigener på overflaten av L. plantarum. Basert på karakterisering av de rekombinante bakteriene i dette studiet, samt tidligere forskning, ble det besluttet å benytte membranankret Hirep2-DC i dyreforsøk.This study is part of a larger project in which the long-term goal is to develop mucosal
vaccines with lactic acid bacteria (LAB) as delivery vectors. LAB are considered good
candidates for this purpose, as they are generally recognized as safe for humans. Several
species are claimed to have health and immunomodulatory effects. LAB are found naturally
in the gastrointestinal tract and the vagina of humans, and exhibit resistance to low pH.
Lactobacillus plantarum is a species that has received great attention as delivery vector of
antigens, partly because it can survive in the gastrointestinal tract for up to one week.
Targeting of antigens to specific parts of the immune system, as well as various routes for
vaccine administration, are important for any vaccine efficacy. It has previously been shown
that peptides targeting dendritic cells have led to increased uptake of the delivery vector, and
that such a strategy has resulted in enhanced immune response. This thesis describes studies
where L. plantarum is modified for use as a delivery vector of surface displayed Chlamydia
trachomatis hybrid antigens that are translationally coupled to a dendritic cell binding peptide
(DC-pep).
Previous studies have indicated that the hybrid antigens Hirep1- and 2 are promising as
vaccine candidates against genital chlamydia infection. With this knowledge, eight plasmids
for anchoring Hirep-based antigens to the cell membrane and cell wall were constructed, and
versions with the DC-pep translationally linked to the antigens were included. Production and
surface display of antigens was successfully determined for all the recombinant strains of L.
plantarum. The bacteria were characterized regarding growth and viability after different
storage methods. Experiments with dendritic cells where the effect of DC-pep was examined
could not reveal increased uptake due to the peptide. The methodology used is relatively new,
and further optimization is applicable to draw a final conclusion. A mouse experiment was
conducted for detection of recombinant L. plantarum in the nasal- and vaginal mucosa after
immunization. The results indicated good survivability in the nasal mucosa for up to 24 hours,
and poor survivability in the vaginal mucosa.
The work described in this thesis demonstrates successful anchoring of C. trachomatis hybrid
antigens to the surface of L. plantarum. Based on characterization of the recombinant bacteria
in this study and previous research, membrane anchored Hirep2-DC was decided to be used in
animal trials.M-BIOTE
Constitutive expression and anchoring of Mycobacterium tuberculosis antigens in Lactobacillus plantarum
Get PDFTuberculosis is the leading cause of death caused by a single agent worldwide. A new and effective vaccine against this infection is therefore imperative. This study is a part of a larger project where the long-term goal is to create an effective vaccine against tuberculosis using LAB as live vectors. Using LAB as a delivery vector for vaccines is highly desirable because of their GRAS status, their non-pathogenicity, probiotic properties, and their ability to deliver functional proteins to mucosal surfaces. These properties make LAB such as L. plantarum an ideal live vector for vaccine delivery.
In this study, a constitutive expression system was constructed by replacing the inducible promoter psppA used in the pSIP vectors with constitutive promoters derived from Lactobacillus spp. Moreover, genes directly related to the inducible system, sppK (HK) and sppR (RR), were removed in an attempt to reduce the fitness cost of the vector. This study reveals the challenges of constructing a constitutive plasmid for heterologous protein production. E. coli TOP10 was utilized as a subcloning vector. The production of AgE6 fusion antigen indicated to elicit a toxic effect in E. coli as most of the constitutive promoter constructs only survived when selected for inactive mutants. The toxic effect in E. coli indicates that most of the Lactobacillus derived promoters were also functional in E. coli.
Plasmids with constitutive protein expression which previously promoted antigen production were immobilized by the removal of the sppK and sppR genes. sppK and sppR were found to most likely be vital for constitutive protein expression utilizing the SIP system. L. plantarum strains harboring the SlpA or PgM promoter produced the most AgE6 anchored on the cell membrane. However, strains harboring the promoter PgM had a significantly higher growth rate. The constitutive AgE6 production is however not comparable to the inducible promoter production of AgE6, and more research is needed. The fluorescent protein mCherry was used to tag the promoters and was successfully cloned downstream of the inducible promoter psppA and the constitutive SlpA promoter. mCherry did not affect the overall fitness cost in L. plantarum and did not lose its ability to fluoresce over time, thus making it a promising candidate for tracking the vaccine through the GIT.Tuberkulose er den største årsaken til dødsfall forårsaket av en singulær infeksjon og en ny og effektiv vaksine mot tuberkulose er derfor betydningsfullt. Denne studien er en del at et større prosjekt der langtidsmålet er å lage en ny og effektiv vaksine mot tuberkulose ved å bruke LAB som levende vektor og leverandør av vaksinen. Bruk av LAB er meget gunstig på grunn av deres GRAS status, de er ikke-patogene, har probiotiske egenskaper og har evne til å levere funksjonelle proteiner til slimhinner. Disse egenskapene gjør at LAB, som L. plantarum, er ideelle som vaksinevektorer.submittedVersionM-BIOTE
Cell wall anchoring of Mycobacterium tuberculosis antigens to the surface of Lactobacillus plantarum
No full textDenne oppgaven er en del av et større forskningsprosjekt hvor det langsiktige målet er å utvikle en slimhinnevaksine mot tuberkulose, hvor melkesyrebakterier benyttes som leveringsvektor av antigener. Melkesyrebakterier er attraktive kandidater for leveringsvektor av vaksineantigener, da de anses som trygge å konsumere av mennesker, og noen hevdes å ha adjuvansegenskaper. Lactobacillus plantarum er spesielt interessant som leveringsvektor av antigener på grunn av sin evne til å overleve i mage-tarmkanalen i opp mot en uke. Ulike ankringsmetoder av antigen på celleoverflaten til L. plantarum har tidligere blitt benyttet, blant annet kovalent, C-terminalt LPxTG-celleveggankring. Tidligere bruk av celleveggankeret cwa2 for ankring av tuberkuloseantigener har gitt immunorespons i mus ved bruk som internasal boostervaksine av BCG. Cwa2 har derimot vist å hemme veksten til verten betydelig. Denne masteroppgaven beskriver studier der L. plantarum modifiseres til å produsere M. tuberculosis-antigen som ankres til celleoverflaten ved bruk av nye LPxTG celleveggankre, og studier som karakteriserer av de rekombinante bakteriene.
Det ble utført en in silico analyse av subcellulære proteiner i L. plantarum WCFS1, for å identifisere LPxTG celleveggankre. Basert på analysen ble syv celleveggankersekvenser med ulike egenskaper valgt. De valgte ankersekvensene ble fusjonert C-terminalt til hybridantigenet Ag85B-ESAT6. Det ble vist en generell hemming av veksten til de rekombinante L. plantarum med celleveggankret hybridantigen. Videre karakterisering ga indikasjoner på noe varierende grad av sekresjon og overflateankring av hybridantigenet, samt reduksjon i levedyktigheten til de ulike stammene med celleveggankre. Stammene med cellevegganker viste indikasjoner på varierende grad av stressrespons. Det ble funnet tegn på forskjeller i transkripsjonsnivået av hybridantigenet i stammene, men disse var ikke signifikante. Resultatet tyder på at variasjonen i stressnivå skyldes faktorer senere i prosessen, etter transkripsjon, som for eksempel translokasjon og ankring.
Ankersekvensen hentet fra genet lp_3001 viste mest lovende resultater, sammenlignet med det originale ankeret cwa2. Imidlertid viste analysene at alle de utvalgte celleveggankrene førte til betydelig stress for verten. Videre analyser for nærmere å identifisere årsaker til stress, og optimalisering av cellevegganker, er nødvendig før cellevegganker kan benyttes i en vaksine mot tuberkulose.This study is part of a lager research project where the long-term goal is to develop a mucosal vaccine against tuberculosis, with lactic acid bacteria (LAB) as delivery vectors of antigens. LAB are good candidates for this purpose, as they are regarded as safe to consume by humans and some are claimed to have adjuvant properties. Lactobacillus plantarum is perticalary interesting as delivery vectors of antigens because they can survive in the gastrointestinal tract for up to one week. Different methods of anchoring antigens to the surface of L. plantarum has been used before, among them C-terminal LPxTG cell wall anchors. A construct using the cell wall anchor cwa2 to anchor tuberculosis antigens has been shown to give an immune response in mouse when used as a booster vaccine to BCG. Cwa2 has been shown to significantly inhibit the growth of the host. This thesis describes studies where L. plantarum is modified to produce M. tuberculosis antigens anchored to the surface by new LPxTG cell wall anchors, and studies that characterise of the recombinant bacteria.
An in silico analysis of the subcellular proteins in L. plantarum WCFS1 was performed to identify LPxTG cell wall anchors. Based on the analysis seven cell wall anchors with different properties were elected. The chosen anchor sequences were then fused C-terminal to the hybrid antigen Ag85B-ESAT6. A general inhibition of growth was shown for the recombinant L. plantarum with cell wall anchored hybrid antigen. Further characterisation indicated some variation in the amount of hybrid antigen secreted and anchored to the surface, as well as a reduction in the viability of the different strains with cell wall anchors. The results indicate varying degrees of stress response in the strains. Signs of variation in transcription levels were found, but not significant. The results point to that variation in stress levels are due to steps later in the production process, for example translocation and anchoring of the hybrid antigen.
The anchor sequence from the lp_3001 gene showed most promise, compared to the original anchor cwa2. However, the analysis showed that use of the cell wall anchors all led to considerable stress for the host. Further research is required to identify the source of stress, and to optimise the cell wall anchors if the anchors are to be used in a vaccine against tuberculosis.M-K
Studies on proteins from Methylococcus capsulatus Bath : expression in Lactobacillus plantarum WCFS1, purification and large-scale fermentation
Get PDFThe work described in this thesis is part of a larger project investigating the non-commensal methanotroph Methylococcus capsulatus Bath. This species has shown anti-inflammatory and obesity-reducing effects in animal models when given as an extract in the diet. In silico studies of the M. capsulatus Bath genome revealed four genes (mam, tir, mif and sim) that encode proteins which are homologous to proteins with known immunomodulatory functions. They might therefore contribute to the observed effects. These proteins (Mam, Tir, Mif and Sim) were the focus of this thesis.
The genetic sequences from M. capsulatus Bath were cloned into the pSIP401 vector of the pSIP inducible gene expression system. They were successfully transformed into the lactic acid bacteria Lactobacillus plantarum WCFS1. Inducible protein expression was successfully accomplished as verified with Western blot. The genetic sequences were also cloned into the pNIC-CH vector for purification of protein. The proteins were successfully produced in Escherichia coli BL21 and purified with immobilized metal ion affinity chromatography.
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After successfully producing purified protein and lysate from L. plantarum harbouring the pSIP401-derivatives, in vitro assays were performed. In the first assay, effects of these bacterial stimuli on interleukin (IL)-1b-induced production of IL-8 was investigated. In the second assay, effects on transepithelial electrical resistance when co-incubated with tumour necrosis factor (TNF)a. Neither of these assays were successful, however.
Finally, L. plantarum harbouring pSIP401_MAM or empty vector were cultured in 12-liter batch fermentations by using a 15-liter bioreactor. The resulting broth was lysed via French pressing and lyophilized before being shipped away to Copenhagen, to be used in a murine study.Arbeidet som ble gjennomført i denne masteroppgave er en del av et større prosjekt som undersøker den metanotrofe bakterien Methylococcus capsulatus Bath. Denne bakterien, som ikke finnes i tarmfloraen, har fått påvist betennelsesdempende og fedmereduserende effekter i dyreforsøk når den blir gitt som et ekstrakt i fôret. In silico-studier av genomet til M.
capsulatus Bath viste at fire gener (mam, tir, mif og sim) koder for proteiner som har
homologi til proteiner med kjente immunomodulerende effecter. Disse proteinene (Mam, Tir,
Mif og Sim) var fokuset I denne oppgaven.
Gensekvensene fra M. capsulatus Bath ble klonet inn i pSIP401-vektoren som er en del av det induserbare genuttrykkssystemet pSIP. De ble deretter transformert inn i melkesyrebakterien
Lactobacillus plantarum WCFS1. Induserbar produksjon av protein ble deretter verifisert ved
bruk av Western blot. Gensekvensene ble også klonet inn i pNIC-CH-vektoren for rensing av
protein. Proteinene ble produsert i Escherichia coli BL21 og renset ved bruk av
kolonnekromatografi.
Etter at renset protein og lysat fra L. plantarum med pSIP401-derivatene var gjennomført ble to in vitro-forsøk utført. I det første forsøket ble effekter av de bakterielle stimuliene på interleukin (IL)-1b-indusert produksjon av IL-8 undersøkt. I det andre forsøket ble effekter av
stimuliene sammen med tumour necrosis factor (TNF)-a på elektrisk motstand over et epitelcellelag undersøkt. Ingen av disse to assayene var vellykket.
Til slutt ble L. plantarum med pSIP401_MAM eller tom vektor dyrket i 12-liters batchfermenteringer
i en 15-liters bioreaktor. Bakteriemassen ble deretter lysert med French press,
før den ble frysetørket og sendt til København for å brukes i et museforsøk.M-MA
Utvikling av en non-GMO slimhinne vaksine mot SARS-CoV-2
No full textLactic acid bacteria (LAB) have been proposed as a good delivery vector of surface-bound antigens, in the development of non-genetically modified organism (non-GMO) mucosal vaccines. LABs are considered good because they have been used in the food industry for centuries and are considered safe for consumption. In addition, LABs can survive, and are found naturally, in the gastrointestinal tract of humans, where they interact with the immune cells in mucosal surfaces. Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum), have been shown to be one of the most promising LAB candidates.
In this study, two different approaches were attempted to express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen RBD. 1) The constitutive expression system pOST in Pichia pastoris, where the expressed protein contained either a monomer or dimer of RBD. 2) The inducible expression system pSIP in L. plantarum. In addition, the antigens were displayed on the surface of L. plantarum using the cell wall binding LysM anchor.
The pOST expression system in P. pastoris was not able to express the SARS-CoV-2 antigen RBD in this study. Multiple attempts of detection and purification showed no positive results. The pSIP expression system was successful in the expression of the SARS-CoV-2 antigen RBD and displayed the antigens on the surface of L. plantarum. Furthermore, binding studies with crude protein extract containing the RBD antigens and L. plantarum cells showed positive results in flow cytometry analysis, indicating successful binding of RBD antigens to the bacterial surface.
The current results show a promising starting point towards the development of a non-GMO mucosal vaccine against SARS-CoV-2, using the pSIP expression system in L. plantarum.Melkesyrebakterier har blitt foreslått som gode kandidater for leveransen av overflate-bundet antigener, i utviklingen av ikke-genetiske modifiserte organisme (non-GMO) slimhinne vaksine. Melkesyrebakterier er ansett som gode kandidater, fordi de har blitt brukt i matindustrien i århundrer og er ansett som trygge å innta. I tillegg kan melkesyrebakterier overleve, og er funnet naturlig, i mage-tarm kanalen hvor de interagerer med immunceller i slimhinnene. Lactiplantibacillus plantarum (tidligere kjent som Lactobacillus plantarum), har vært vist å være en av de mest lovende kandidatene.
I denne studien ble to framgangsmåter for å uttrykke SARS-CoV-2 antigenet RBD, forsøkt. 1) Det konstitutive uttrykningssystemet pOST i Pichia pastoris, hvor det uttrykte proteinet inneholdt enten en monomer eller dimer av RBD. 2) Det induserbare uttrykningssystemet pSIP i L. plantarum. I tillegg ble antigenet eksponert på overflaten av L. plantarum ved bruk av det cellevegg-bindene LysM ankeret.
I denne studien var ikke uttrykningssystemet pOST i P. pastoris istand til å uttrykke SARS-CoV-2 RBD antigenet. Mange forsøk på deteksjon og rensing av antigenet ga ingen positive resultater. pSIP uttrykningssystemet i L. plantarum derimot, ble vist til å kunne uttrykke RBD antigenet, i tillegg til antigenet ble eksponert på overflaten av L. plantarum. Bindingsstudier av rå protein ekstrakt som inneholdt RBD antigenet og L. plantarum celler viste positive resultater etter flow cytometri analyse, noe som indikerer suksessfull binding av RBD antigenet til bakterieoverflaten.
De nåværende resultatene viser en lovende start mot utviklingen av en non-GMO slimhinne vaksine mot SARS-CoV-2, ved bruken av pSIP uttrykningssystemet i L. plantarum.submittedVersionM-BIOTE
Production of Recombinant LysM-Fuison Proteins for Non-GMO Anchoring on Lactic Acid Bacteria
Get PDFLactic acid bacteria (LAB) are traditionally used in the production and preservation of food and are natural inhabitants of the human gastrointestinal tract. This makes LAB generally regarded as safe for consumption. Some LAB exhibit probiotic properties and immunomodulatory effects, which contribute to their potential as ideal candidates as delivery vectors of immunogens. This study is part of a larger research project where the long-term goal is to develop mucosal vaccines by utilizing LAB as delivery vectors for antigens without genetically modifying the bacteria.
The aim of this thesis was to produce, purify, and anchor heterologous proteins to the cell wall of LAB using LysM-domains. In this work, two LysM-domains were used, one derived from Lactiplantibacillus pentosus KW1 and the other from L. pentosus KW2. These domains were fused N-terminally to the HaloTag reporter gene and the NetB W262A vaccine antigen. This antigen is a mutated form of the virulence factor NetB, produced by Clostridium perfringens, which causes necrotic enteritis in poultry. The fusion sequences were cloned into the inducible pBAD/HisB system and expressed in E. coli. Several attempts were made to optimize the production of soluble protein, including varying production conditions and the selection of production strain.
The fusion proteins were purified and bound to LAB under various binding conditions to evaluate their binding properties. Binding reactions were analyzed using SDS-PAGE and/or flow cytometry. Among the tested LAB, L. pentosus KW1 demonstrated the highest binding capacity to KW2_LysM_HaloTag, with minimal influence of temperature and reaction time on the amount of bound protein. However, different washing conditions for the bacterial cells prior to the binding reaction revealed that components in the MRS medium might affect the interaction between the LysM-domain and the peptidoglycan in the cell wall. To investigate the influence of cell wall components on the interaction, KW2_LysM_HaloTag was bound to peptidoglycan from Lactiplantibacillus plantarum WCFS1 and showed binding in PBS at pH 5.4. PBS buffers with varying pH were also used in the binding reactions and indicated that the interaction was pH dependent. The binding strength between the proteins and L. pentosus KW1 was evaluated by washing the bound protein with PBS, and the LysM-anchored proteins exhibited relatively stable binding. The characterization performed in this study demonstrated that the LysM-domain is essential for facilitating binding, and the binding of LysM-anchored proteins to L. pentosus KW1 highlights a promising potential for further development of a non-genetically modified LAB-based vaccine against necrotic enteritis in poultry
Exploring the potential role of lytic polysaccharide monooxygenases from Bacillus licheniformis and Bacillus spizizenii in endospore germination
Get PDFLytic polysaccharide monooxygenases (LPMOs), traditionally recognized for degrading recalcitrant polysaccharides such as cellulose and chitin, constitute a superfamily with diverse substrate specificities and biological roles. This study examines the structural and functional properties of the previously uncharacterized BsLPMO10A from Bacillus spizizenii along with the chitin-active BlLPMO10A from the closely related Bacillus licheniformis, focusing on their potential roles in bacterial cell wall remodeling and endospore germination.
Initially, the study investigated the expression of BlLPMO10A using a reporter gene system, fusing its promoter to mCherry within a vector transformed into B. licheniformis to potentially infer promoter activity. The findings revealed no detectable mCherry signal under various conditions, including growth in different media and during sporulation, suggesting an absence of LPMO expression under the tested conditions.
The activity and affinity of LPMOs for chitin were analyzed by mass spectrometry and binding assays, which confirmed the chitinolytic activity of BsLPMO10A but suggested a relatively low chitin affinity for both LPMOs, which may hint to alternative biological functions.
The potential impact of LPMOs on bacterial cell walls was assessed by monitoring changes in optical density (OD600), indicative of cell lysis. While the data suggested that these LPMOs could potentially enhance lysozyme-mediated peptidoglycan breakdown in vegetative cells, any definitive conclusions were not drawn, and the potential impact of LPMO mediated accumulation of reactive oxygen species (ROS) on cell integrity was considered.
Genomic investigations suggested a potential involvement of family 18 glycoside hydrolases in endospore germination in the parent strains of these LPMOs, substantiating the hypothesis that LPMOs may assist in this process. The potential impact of these Bacillus-derived LPMOs on endospore germination was assessed by monitoring the release of dipicolinic acid (DPA) and OD600. Despite complications in interpreting the results, an observed increase in DPA release in LPMO-treated endospores suggested possible LPMO involvement in endospore germination. Supporting these observations, phase contrast microscopy revealed that LPMO-treated endospores appeared phase bright and with size increases similar to what is observed in initial stages of germination, offering further indication of the involvement of LPMOs in this process.
Further investigations are necessary to explore how these LPMOs might contribute to endospore germination and to determine whether this potential function is widespread
Characterization of the two novel Lactiplantibacillus pentosus strains KW1 and KW2, and exploration of their ability to surface display a Mycobacterium tuberculosis hybrid antigen
Get PDFMany lactic acid bacteria are natural inhabitants of the human gut and are considered safe for human consumption. Food-grade lactic acid bacteria from the genus Lactiplantibacillus are generally tolerant to highly acidic environments, allowing them to survive and colonize the human
gastrointestinal tract. Furthermore, certain species can have vaccine adjuvant effects through direct interactions with the human immune system. Thus, Lactiplantibacilli are promising candidates for the delivery of antigens to mucosal surfaces.
The aim of this study was to characterize the two novel Lactiplantibacillus pentosus strains, KW1 and KW2, isolated from table olives; and explore their capability to surface display a Mycobacterium tuberculosis-derived hybrid antigen. The L. pentosus strains were evaluated by analyzing cell morphology and growth analysis. The microscopy analysis showed that L.
pentosus KW1 and KW2 were morphologically similar and rod-shaped. The optimum growth temperatures were found to be in a range from 33 to 39 °C. In addition, the present study conducted a functional analysis of the inducible gene expression system (pSIP) to evaluate its
applicability for use in L. pentosus. It was shown that the pSIP system is strictly regulated in KW1 and KW2, where the protein production of mCherry, used as a reporter protein, increased more than 20-fold upon full induction. The protein production of mCherry was found to be
highest at 37 °C and L. pentosus KW1 was the most efficient producer.
Eight recombinant bacteria were constructed for surface exposure of the H56 hybrid tuberculosis antigen. Four different anchors derived from the genome of KW1 and KW2 were selected, and translationally fused to the antigen, generating the eight recombinant strains. The selected
anchors were: (1) an N-terminal transmembrane (NTTM) anchor that non-covalently attaches the antigen to the cell membrane, (2) a lipoprotein anchor to covalently attach the antigen to the cell membrane, (3) a LysM anchor for non-covalently anchoring of the antigen to the cell wall, and (4) an LPxTG peptidoglycan anchor to covalently attach H56 to the cell wall. The recombinant strains showed only a slight reduction in growth, except for strains harboring NTTM-anchored antigens. Western blot analysis confirmed antigen production for seven out of eight recombinant strains. Furthermore, flow cytometry analysis detected exposed antigens on the surface for all recombinant strains except for the KW2_LPxTG anchor. The strongest fluorescent shift was observed in L. pentosus KW1, especially with lipoprotein and LysM-anchored antigens.
The successful secretion and surface exposure of the tuberculosis antigen show that these recombinant bacteria are promising candidates for antigen delivery. The analyses demonstrated that L. pentosus KW1 seems to be the most promising strain for further development as a vaccine delivery vehicle
Initial Characterization of WxL Proteins Derived From Lactiplantibacillus pentosus KW1
Get PDFLactic acid bacteria (LAB) are a gram-positive, non-sporing group of bacteria that have shown potential as vaccine delivery vectors by anchoring heterologous antigens on their cell surface. Various anchoring strategies have been developed to achieve surface exposure of these antigens. Proteins harbouring a Trp-X-Leu motif, named WxL proteins, have been suggested to bind non-covalently to peptidoglycan, making them promising candidates for surface display. However, most existing studies are limited to in silico analyses of the WxL proteins, highlighting the need for experimental validation.
In this thesis, the main goal it to provide an initial characterization of selected WxL proteins from Lactiplantibacillus pentosus KW1. The characterization includes determining the conditions in which these proteins are produced, elucidating their potential functions, evaluating possible binding capabilities, and identifying potential interaction partners. To investigate under which conditions these proteins were produced, vectors harbouring the predicted promoters upstream of WxL clusters, were cloned upstream of a reporter gene (mCherry). To investigate binding properties, the sequences of WxL1, WxL2A and WxL3A, were selected and cloned into vectors for overexpression in Escherichia coli. WxL1 was successfully purified, and binding experiments were conducted under different conditions to test external binding affinity to bacterial surface and purified peptidoglycan. Potential binding was assessed using SDS-PAGE, flow cytometry and fluorescence microscopy. In silico analysis of one selected gene cluster (c1) was performed to explore potential cooperative interactions among the encoded proteins in the cluster. Based on the in silico findings, interaction studies were conducted to evaluate potential interactions between WxL1 and the downstream gene, named Tmem1, in the c1 gene cluster.
The results indicate that native WxL production is tightly regulated, possibly influenced by promoters responsive to nutrient availability, suggesting a nutrient-dependent expression profile. WxL proteins show affinity for both intact L. pentosus KW1 cells and purified peptidoglycan, with binding occurring under specific conditions such as defined pH levels and ionic strength, and potentially the presence of calcium ions (CaCl₂). Additionally, WxL1 was found to reduce growth and induce aggregation in E. coli, implying a potential role in cell-cell interactions. WxL1 also indicated a high affinity interaction with Tmem1 encoded in the same cluster, indicating a cooperative function potentially facilitating surface localization and enhancing the binding activity of WxL1
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