8 research outputs found
AmiA and AliA peptide ligands are secreted by Klebsiella pneumoniae and inhibit growth of Streptococcus pneumoniae
Streptococcus pneumoniae colonizes the human nasopharynx, a multi-species microbial niche. Pneumococcal Ami-AliA/AliB oligopeptide permease is an ABC transporter involved in environmental sensing with peptides AKTIKITQTR, FNEMQPIVDRQ, and AIQSEKARKHN identified as ligands of its substrate binding proteins AmiA, AliA, and AliB, respectively. These sequences match ribosomal proteins of multiple bacterial species, including Klebsiella pneumoniae. By mass spectrometry, we identified such peptides in the Klebsiella pneumoniae secretome. AmiA and AliA peptide ligands suppressed pneumococcal growth, but the effect was dependent on peptide length. Growth was suppressed for diverse pneumococci, including antibiotic-resistant strains, but not other bacterial species tested, with the exception of Streptococcus pseudopneumoniae, whose growth was suppressed by the AmiA peptide ligand. By multiple sequence alignments and protein and peptide binding site predictions, for AmiA we have identified the location of an amino acid in the putative binding site whose mutation appears to result in loss of response to the peptide. Our results indicate that pneumococci sense the presence of Klebsiella pneumoniae peptides in the environment
Meningitis-associated pneumococcal serotype 8, ST 53, strain is hypervirulent in a rat model and has non-haemolytic pneumolysin which can be attenuated by liposomes
Introduction: Streptococcus pneumoniae bacteria cause life-threatening invasive pneumococcal disease (IPD), including meningitis. Pneumococci are classified into serotypes, determined by differences in capsular polysaccharide and both serotype and pneumolysin toxin are associated with disease severity. Strains of serotype 8, ST 53, are increasing in prevalence in IPD in several countries.
Methods: Here we tested the virulence of such an isolate in a rat model of meningitis in comparison with a serotype 15B and a serotype 14 isolate. All three were isolated from meningitis patients in South Africa in 2019, where serotype 8 is currently the most common serotype in IPD.
Results and Discussion: Only the serotype 8 isolate was hypervirulent causing brain injury and a high mortality rate. It induced a greater inflammatory cytokine response than either the serotype 15B or 14 strain in the rat model and from primary mixed-glia cells isolated from mouse brains. It had the thickest capsule of the three strains and produced non-haemolytic pneumolysin. Pneumolysin-sequestering liposomes reduced the neuroinflammatory cytokine response in vitro indicating that liposomes have the potential to be an effective adjuvant therapy even for hypervirulent pneumococcal strains with non-haemolytic pneumolysin
Carbon source–dependent capsule thickness regulation in Streptococcus pneumoniae
BackgroundThe polysaccharide capsule of Streptococcus pneumoniae plays a major role in virulence, adherence to epithelial cells, and overall survival of the bacterium in the human host. Galactose, mannose, and N-acetylglucosamine (GlcNAc) are likely to be relevant for metabolization in the nasopharynx, while glucose is the primary carbon source in the blood. In this study, we aim to further the understanding of the influence of carbon sources on pneumococcal growth, capsule biosynthesis, and subsequent adherence potential.MethodsWe tested the growth behavior of clinical wild-type and capsule knockout S. pneumoniae strains, using galactose, GlcNAc, mannose, and glucose as carbon source for growth. We measured capsule thickness and quantified capsule precursors by fluorescein isothiocyanate (FITC)–dextran exclusion assays and 31P-nuclear magnetic resonance measurements, respectively. We also performed epithelial adherence assays using Detroit 562 cells and performed a transcriptome analysis (RNA sequencing).ResultsWe observed a reduced growth in galactose, mannose, and GlcNAc compared to growth in glucose and found capsular size reductions in mannose and GlcNAc compared to galactose and glucose. Additionally, capsular precursor measurements of uridine diphosphate-(UDP)-glucose and UDP-galactose showed less accumulation of precursors in GlcNAc or mannose than in glucose and galactose, indicating a possible link with the received capsular thickness measurements. Epithelial adherence assays showed an increase in adherence potential for a pneumococcal strain, when grown in mannose compared to glucose. Finally, transcriptome analysis of four clinical isolates revealed not only strain specific but also common carbon source-specific gene expression.ConclusionOur findings may indicate a careful adaption of the lifestyle of S. pneumoniae according to the monosaccharides encountered in the respective human niche
Klebsiella pneumoniae peptide hijacks a Streptococcus pneumoniae permease to subvert pneumococcal growth and colonization.
Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis
Meningitis-associated pneumococcal serotype 8, ST 53, strain is hypervirulent in a rat model and has non-haemolytic pneumolysin which can be attenuated by liposomes
IntroductionStreptococcus pneumoniae bacteria cause life-threatening invasive pneumococcal disease (IPD), including meningitis. Pneumococci are classified into serotypes, determined by differences in capsular polysaccharide and both serotype and pneumolysin toxin are associated with disease severity. Strains of serotype 8, ST 53, are increasing in prevalence in IPD in several countries.MethodsHere we tested the virulence of such an isolate in a rat model of meningitis in comparison with a serotype 15B and a serotype 14 isolate. All three were isolated from meningitis patients in South Africa in 2019, where serotype 8 is currently the most common serotype in IPD.Results and DiscussionOnly the serotype 8 isolate was hypervirulent causing brain injury and a high mortality rate. It induced a greater inflammatory cytokine response than either the serotype 15B or 14 strain in the rat model and from primary mixed-glia cells isolated from mouse brains. It had the thickest capsule of the three strains and produced non-haemolytic pneumolysin. Pneumolysin-sequestering liposomes reduced the neuroinflammatory cytokine response in vitro indicating that liposomes have the potential to be an effective adjuvant therapy even for hypervirulent pneumococcal strains with non-haemolytic pneumolysin
Screening og karakterisering av bakteriociner produsert av melkesyrebakterier mot Lactococcus garvieae
Infectious diseases caused by bacteria have ravaged humankind multiple times in history until the discovery of antibiotics. Now, several decades later, bacteria resistant to nearly all antibiotics have been reported due to the overuse and misuse of the latter. To remediate the alarming situation and avoid a disastrous future, novel approaches to antibiotics must be investigated.
Bacteriocins are good candidates for the treatment of bacterial infections for several reasons. First, they have a narrow spectrum of activity compared to antibiotics, which limit the selective pressure for resistance to the pathogens instead of all the commensal bacteria. Bacteriocins are also non-toxic to humans since many are already used as food preservatives. Finally, they could be effective to fight antibiotic resistant bacteria due to their different killing mechanism.
The purpose of this study was to isolate and characterize lactic acid bacteriocins that could inhibit the fish and emerging human pathogen Lactococcus garvieae. To accomplish that goal, 50 samples of fermented fruits and vegetables were screened against L. garvieae by using a “sandwich overlay” method. The potential bacteriocin-producers were then identified by 16S rRNA gene sequencing and REP PCR profiling. To characterize the antimicrobials produced by the isolates, proteinase K and heat stability tests were conducted, and the inhibition spectrum was determined. Based on the results of these experiments, seven different strains were selected, and their genomes were sequenced on an Illumina Miseq system. The sequenced genomes were then uploaded on BAGEL4 to look for bacteriocin genes. The results showed that each genome contained putative bacteriocin genes and in some cases, more than one bacteriocin was identified. In addition, the observation that the identified bacteriocins belonged to different classes illustrates well the diversity of lactic acid bacteriocins.
The last experiment was the purification of the most relevant bacteriocin in respect to the purpose of the study. In that context, the bacteriocin from Enterococcus thailandicus was purified by ammonium sulfate precipitation followed by one reverse-phase chromatography step. The molecular mass was determined to be 6312 Da by MALDI-TOF MS, which confirms that the purified bacteriocin was the circular thaiocin 1.
The results of the study suggest the use of thaiocin 1 from E. thailandicus to control the growth of L. garvieae in fish farming.Infeksjonssykdommer forårsaket av bakterier har herjet menneskeheten flere ganger i historien, til oppdagelsen av antibiotika. Nå, flere tiår senere ser det ut som at bakteriene har kontrollen igjen grunnet observasjoner av bakterier som er resistente mot alle typer antibiotika. Overforbruk og misbruk av sistnevnte førte til den resistens-krisen i dag. For å rette opp den alarmerende situasjonen og unngå en katastrofal fremtid, må nye tilnærminger til antibiotika undersøkes.
Bakteriociner er gode kandidater for behandling av bakterielle infeksjoner av flere grunner. For det første har de et smalt spekter av antimikrobiell aktivitet sammenlignet med antibiotika, noe som begrenser det selektive trykket for resistens mot patogener istedenfor alle kommensale bakterier. Bakteriociner er også ikke-giftige for mennesker, ettersom mange er allerede brukt som mat konserveringsmidler. Til slutt kan de være effektive for å bekjempe antibiotikaresistente bakterier på grunn av deres ulike drepemekanisme.
Hovedmålet med denne oppgaven var å isolere og karakterisere bakteriociner produsert av melkesyrebakterier, som kunne hemme veksten av fiskepatogenen og fremvoksende humanpatogenen Lactococcus garvieae. For å oppnå målet ble 50 prøver av fermentert frukt og grønnsaker screenet mot L. garvieae ved å bruke en "sandwich overlay" metode. De potensielle bakteriocin-produsentene ble deretter identifisert med 16S rRNA-gen-sekvensering og REP-PCR-profilering. For å karakterisere de antimikrobielle forbindelsene produsert av isolatene ble proteinase K og varmestabilitet tester utført, og inhiberingsspekteret ble undersøkt. Basert på resultatene av disse undersøkelsene ble det valgt syv forskjellige stammer, og deres genom ble sekvensert på et Illumina Miseq-system. De sekventerte genomene ble deretter lastet opp på BAGEL4 for å lete etter bakteriocin-gener. Resultatene viste at hvert genom inneholdt putative bakteriocin-gener, og i noen tilfeller ble mer enn ett bakteriocin identifisert. I tillegg tilhørte de ulike bakteriocinene forskellige klasser, noe som illustrerer mangfoldet av bakteriociner fra melkesyrebakterier. Det siste forsøket var rensingen av det mest relevante bakteriocinet i forhold til formålet med studien. I den sammenhengen ble bakteriocinet fra E. thailandicus renset ved ammoniumsulfatutfelling etterfulgt av et omvendt-fase kromatografi trinn. Molekylmassen ble bestemt til å være 6312 Da ved MALDI-TOF MS, som bekrefter at det rensede bakteriocinet var den sirkulære thaiocin 1. Resultatene av studien oppfordrer bruken av thaiocin 1 fra E. thailandicus for å hemme veksten av L. garvieae i fiskeoppdrett.submittedVersionM-BIOTE
Garvicin Q: characterization of biosynthesis and mode of action
Bacteriocins are ribosomally synthesized antimicrobial peptides, that either kill target bacteria or inhibit their growth. Bacteriocins are used in food preservation and are of increasing interest as potential alternatives to conventional antibiotics. In the present study, we show that Lactococcus petauri B1726, a strain isolated from fermented balsam pear, produces a heat-stable and protease-sensitive compound. Following genome sequencing, a gene cluster for production of a class IId bacteriocin was identified consisting of garQ (encoding for the bacteriocin garvicin Q), garI (for a putative immunity protein), garC, and garD (putative transporter proteins). Growth conditions were optimized for increased bacteriocin activity in supernatants of L. petauri B1726 and purification and mass spectrometry identified the compound as garvicin Q. Further experiments suggest that garvicin Q adsorbs to biomass of various susceptible and insusceptible bacteria and support the hypothesis that garvicin Q requires a mannose-family phosphotransferase system (PTS(Man)) as receptor to kill target bacteria by disruption of membrane integrity. Heterologous expression of a synthetic garQICD operon was established in Corynebacterium glutamicum demonstrating that genes garQICD are responsible for biosynthesis and secretion of garvicin Q. Moreover, production of garvicin Q by the recombinant C. glutamicum strain was improved by using a defined medium yet product levels were still considerably lower than with the natural L. petauri B1726 producer strain. Collectively, our data identifies the genetic basis for production of the bacteriocin garvicin Q by L. petauri B1726 and provides insights into the receptor and mode of action of garvicin Q. Moreover, we successfully performed first attempts towards biotechnological production of this interesting bacteriocin using natural and heterologous hosts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01952-9
Garvicin Q-Characterization of biosynthesis and mode of action
Bacteriocins are ribosomally synthesized antimicrobial peptides, that either kill target bacteria or inhibit their growth. Bacteriocins are used in food preservation and are of increasing interest as potential alternatives to conventional antibiotics. In the present study, we show that Lactococcus petauri B1726, a strain isolated from fermented balsam pear, produces a heat-stable and protease-sensitive compound. Following genome sequencing, a gene cluster for production of a class IId bacteriocin was identified consisting of garQ (encoding for the bacteriocin garvicin Q), garI (for a putative immunity protein), garC, and garD (putative transporter proteins). Growth conditions were optimized for increased bacteriocin activity in supernatants of L. petauri B1726 and purification and mass spectrometry identified the compound as garvicin Q. Further experiments suggest that garvicin Q adsorbs to biomass of various susceptible and insusceptible bacteria and support the hypothesis that garvicin Q requires a mannose-family phosphotransferase system (PTS(Man)) as receptor to kill target bacteria by disruption of membrane integrity. Heterologous expression of a synthetic garQICD operon was established in Corynebacterium glutamicum demonstrating that genes garQICD are responsible for biosynthesis and secretion of garvicin Q. Moreover, production of garvicin Q by the recombinant C. glutamicum strain was improved by using a defined medium yet product levels were still considerably lower than with the natural L. petauri B1726 producer strain. Collectively, our data identifies the genetic basis for production of the bacteriocin garvicin Q by L. petauri B1726 and provides insights into the receptor and mode of action of garvicin Q. Moreover, we successfully performed first attempts towards biotechnological production of this interesting bacteriocin using natural and heterologous hosts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01952-9