Klasse II bakteriosiner : målcellespesifisitet, resistens og immunitet

Bacteriocins are bacterial antimicrobial peptides which are active against strains often closely related to the producer. Bacteriocins produced by food grade lactic acid bacteria are considered promising for applications in food preservation and infection treatment since many of them kill food-spoiling bacteria and pathogens with high potencies. During the last decades, hundreds of different bacteriocins from lactic acid bacteria have been identified, however, the molecular mechanisms underlying target cell recognition, resistance development and producer cell self-immunity are often poorly characterized. The papers I-IV presented in this thesis all shed light on such mechanisms for class II bacteriocins. In paper I and II the receptor recognition by class IIa bacteriocins and the class IIc bacteriocin lactococcin A was investigated. These bacteriocins bind to the membrane-located protein complex IIC-IID of the mannose phosphotransferase system (man-PTS) to form pores in the membrane of sensitive cells. By phylogenetic analyses and heterologous expression of man-PTSs from different genera, it was demonstrated that class IIa bacteriocins specifically recognize a phylogenetically defined subgroup of man-PTSs. The man-PTSs in this group originated from Gram-positive bacterial genera such as Listeria, Enterococcus and Lactobacillus, which are known to be class IIa bacteriocin sensitive. Moreover, the class IIc bacteriocin lactococcin A was shown to exclusively target the lactococcal man-PTS as receptor, which is in line with the narrow inhibition spectrum observed for this bacteriocin. These results suggest that variation in bacteriocin sensitivity between bacterial species/genera can mainly be ascribed to differences in the sequence/structure of the man-PTS receptor. In order to characterize the species-specific receptor-bacteriocin interaction, the man-PTS genes from the class IIa bacteriocin sensitive Listeria monocytogenes (mpt) and the non-sensitive Lactococcus lactis (ptn) were used to construct a series of chimeric man-PTSs and site-directed mutations. It was demonstrated that class IIa bacteriocins specifically recognize an extracellular loop region in the membrane-located man-PTS IIC protein. In contrast, lactococcin A (class IIc) seemed to possess a much more complex receptor interaction by specifically recognizing regions both in the IIC and IID protein. In paper III the mechanisms by which sensitive bacteria become resistant to the man-PTS targeting bacteriocins (class IIa bacteriocins and lactococcin A) were studied. It has previously been reported that induced resistance to class IIa bacteriocins in Li. monocytogenes is linked with downregulation of man-PTS gene expression. By examining natural isolates of Li. monocytogenes with varying levels of sensitivity to class IIa bacteriocins as well as lactococcin A resistant L. lactis mutants, it was demonstrated that such downregulation of man-PTS gene expression is a general resistance mechanism against different man-PTS targeting bacteriocins. The same resistance mechanism was found both among the natural isolates with low susceptibility and the laboratory induced resistant mutants. Moreover, it was shown that another, yet unknown resistance mechanism is also involved, since a significant number of resistant cells with normal man-PTS expression were identified. While the molecular mechanisms underlying target cell recognition and self-immunity for class IIa bacteriocins now have been studied in detail, very little is known about such mechanisms for other class II bacteriocins. Recently it was found that genes belonging to the family Abi, which encode putative transmembrane proteases, are associated with some bacteriocin loci in Streptococci and Lactobacillus. The Abi genes are thought to encode immunity proteins in these systems, and in paper IV the Abi family was investigated in order to gain more insight into the role of these proteins in selfimmunity to class II bacteriocins. Using a bioinformatics approach it was shown that the Abi family islarger than currently annotated in the databases, and by mining sequenced genomes for Abi genes, seven putative new bacteriocin loci were identified. By heterologous expression, two putative bacteriocin genes in one of these new loci (skkA and skkB of Lactobacillus sakei 23K), were indeed shown to be bacteriocinogenic. The associated Abi gene skkI conferred immunity to the bacteriocins when expressed in a sensitive strain. Correspondingly, the Abi genes plnI and plnLR from the plantaricin locus of Lactobacillus plantarum were also shown to confer immunity to their cognate class IIb bacteriocins (plantaricin EF and JK, respectively). Most known immunity genes act specifically against their cognate bacteriocins. However, cross immunity between skkI, plnI and plnLR was observed, thus suggesting that different Abi immunity proteins might recognize the same target molecule(s). Proteins of the Abi family are characterized by three highly conserved sequence motifs which are thought to constitute the active site of a proteolytic function, and indeed, site-directed mutations in these motifs in SkkI abolished its immunity function. This finding might suggest that the Abi proteins confer bacteriocin immunity via a proteolytic mechanism.Bakteriosiner er antimikrobielle peptider produsert av bakterier som viser aktivitet mot stammer som oftest er nært beslekta med produsenten. Bakteriosiner produsert av melkesyrebakterier har vært spesielt i fokus de siste tiårene. Disse viser antimikrobiell aktivitet mot uønskede bakterier i mat og mot patogene bakterier, og de kan derfor potensielt komme til nytte både innenfor næringsmiddelindustrien og i behandling av infeksjoner. Flere hundre ulike bakteriosiner fra melkesyrebakterier har blitt identifisert, men de molekylære mekanismene som ligger til grunn for antimikrobiell aktivitet, resistensutvikling og produsentimmunitet er for det meste ukjente. De fire arbeidene i denne avhandlinga belyser alle ulike aspekter ved slike mekanismer. I den første delen avhandlinga (artikkel I og II) ble det undersøkt hvordan bakteriosiner som tilhører klasse IIa og IIc spesifikt gjenkjenner en reseptor på målcellene. Det er kjent fra tidligere at disse bakteriosinene binder til det membranlokaliserte proteinkomplekset IIC-IID i mannose fosfotransferasesystemet (man-PTS) og danner porer i membranen til sensitive celler. I den første artikkelen ble det vist ved fylogenetiske analyser og heterologt uttrykk av man-PTS proteiner fra ulike bakterier at klasse IIa bakteriosiner kun gjenkjenner man-PTSer fra en fylogenetisk definert undergruppe. Man-PTSene i denne gruppa stammer fra Gram-positive bakterier som tidligere har vist seg å være sensitive for klasse IIa bakteriosiner. Bakteriosinet lactococcin A fra klasse IIc viste derimot en helt annen reseptorspesifisitet enn bakteriosinene fra klasse IIa og kunne utelukkende benytte man-PTS fra Lactococcus som reseptor. Dette resultatet stemmer overens med at lactococcin A kun er aktiv mot ulike Lactococcus-stammer. Til sammen viser disse funnene at forskjeller i sensitivitet for klasse IIa bakteriosiner og lactococcin A mellom ulike bakteriearter/-slekter i stor grad kommer av ulik sekvens/struktur hos man-PTS reseptorene. For videre å karakterisere den spesifikke interaksjonen mellom man-PTS og klasse IIa bakteriosiner, ble en rekke hybride man-PTS systemer konstruert basert på genene fra den klasse IIa-sensitive Listeria monocytogenes (mpt) og den insensitive Lactococcus lactis (ptn). Denne framgangsmåten gjorde det mulig å identifisere en ekstracellulær loop i man-PTS IIC proteinet som var ansvarlig for den spesifikke interaksjonen med klasse IIa bakteriosiner. På tilsvarende måte ble det vist at lactococcin A har en mer kompleks reseptorinteraksjon som trolig involverer binding til både IIC og IID proteinene. I artikkel III ble det studert hvordan sensitive bakterier utvikler resistens mot klasse IIa bakteriosiner og lactococcin A. Tidligere studier har påvist en sammenheng mellom indusert resistens mot klasse IIa bakteriosiner i Li. monocyotogenes og redusert genuttrykk av man-PTS. I artikkel III ble naturlige isolater av Li. monocytogenes med ulik sensitivitet for klasse IIa bakteriosiner samt lactococcin A resistente L. lactis mutanter undersøkt. Det ble vist at nedregulering av man-PTS genene utgjør en generell resistensmekanisme mot ulike typer bakteriosiner som finnes både i naturlige isolater med lav bakteriosinsensitivitet og i resistente mutanter generert ved bakteriosineksponering. I tillegg ble det også identifisert resistente celler som hadde normalt uttrykk man-PTS. Dette viser at en annen, men hittil ukjent mekanisme også er involvert i resistens mot disse bakteriosinene. De molekylære mekanismene som ligger til grunn for målcellespesifisitet og immunitet hos klasse IIa bakteriosiner har nå blitt grundig studert. For andre bakteriosiner fra klasse II derimot, er slike mekanismer fortsatt lite kjent. Det ble nylig påvist at noen bakteriosinsystemer i streptokokker og laktobasiller inneholder gener som koder for såkalte Abi proteiner. Abi proteiner er transmembrane proteaser som antas å være involvert i bakteriosinimmunitet i disse bakteriene. I den siste delen av avhandlinga (artikkel IV) ble Abi proteinfamilien studert med spesielt fokus på hvordan disse proteinene er involvert i immunitet mot bakteriosiner fra klasse II. Bioinformatiske analyser viste at Abi familien har flere medlemmer enn det som i dag er kjent ut fra annotering i databasene. Ved å søke etter Abi gener i sekvenserte genomer ble sju nye genklustere, som potensielt koder for bakteriosiner, identifisert. Ved heterologt uttrykk av bakteriosingenene fra ett av genklusterene (skkA og skkB fra Lactobacillus sakei 23K) ble det vist at disse har bakteriosinaktivitet. Det korresponderende Abi genet skkI ga immunitet mot bakteriosinene når det ble uttrykt i en sensitiv stamme. På samme måte ble det vist at to andre Abi gener, plnI og plnLR fra plantaricinlokuset i Lactobacillus plantarum ga immunitet mot sine respektive klasse IIb bakteriosiner, plantaricin EF og JK. Kryssimmunitet mellom skkI, plnI and plnLR ble også observert, noe som kan tyde på at de ulike Abi proteinene benytter samme mekanisme for å gi celler immunitet mot disse bakteriosinene. Abi proteiner kjennetegnes ved tre konserverte motiver som antas å utgjøre et aktivt sete med proteolytisk funksjon. Ved å innføre mutasjoner i aminosyrer som er en del av disse motivene, ble det vist at SkkI mister immunitetsfunksjonen. Dette kan tyde på at Abi proteiner gir bakteriosinimmunitet via en proteolytisk mekanisme.Norges Forskningsrå

Class A PBPs: it is time to rethink traditional paradigms

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EloR interacts with the lytic transglycosylase MltG at midcell in Streptococcus pneumoniae R6

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Antibiotic-induced replication stress triggers bacterial competence by increasing gene dosage near the origin

Streptococcus pneumoniae (pneumococcus) kills nearly 1 million children annually, and the emergence of antibiotic-resistant strains poses a serious threat to human health. Because pneumococci can take up DNA from their environment by a process called competence, genes associated with antibiotic resistance can rapidly spread. Remarkably, competence is activated in response to several antibiotics. Here, we demonstrate that antibiotics targeting DNA replication cause an increase in the copy number of genes proximal to the origin of replication (oriC). As the genes required for competence initiation are located near oriC, competence is thereby activated. Transcriptome analyses show that antibiotics targeting DNA replication also upregulate origin-proximal gene expression in other bacteria. This mechanism is a direct, intrinsic consequence of replication fork stalling. Our data suggest that evolution has conserved the oriC-proximal location of important genes in bacteria to allow for a robust response to replication stress without the need for complex gene-regulatory pathways. PAPERCLIP

The ParB-parS Chromosome Segregation System Modulates Competence Development in Streptococcus pneumoniae

ParB proteins bind centromere-like DNA sequences called parS sites and are involved in plasmid and chromosome segregation in bacteria. We previously showed that the opportunistic human pathogen Streptococcus pneumoniae contains four parS sequences located close to the origin of replication which are bound by ParB. Using chromatin immunoprecipitation (ChIP), we found here that ParB spreads out from one of these parS sites, parS(-1.6 degrees), for more than 5 kb and occupies the nearby comCDE operon, which drives competence development. Competence allows S. pneumoniae to take up DNA from its environment, thereby mediating horizontal gene transfer, and is also employed as a general stress response. Mutating parS(-1.6 degrees) or deleting parB resulted in transcriptional up-regulation of comCDE and ssbB (a gene belonging to the competence regulon), demonstrating that ParB acts as a repressor of competence. However, genome-wide transcription analysis showed that ParB is not a global transcriptional regulator. Different factors, such as the composition of the growth medium and antibiotic-induced stress, can trigger the sensitive switch driving competence. This work shows that the ParB-parS chromosome segregation machinery also influences this developmental process. IMPORTANCE Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for more than a million deaths each year. Like all other organisms, S. pneumoniae must be able to segregate its chromosomes properly. Not only is understanding the molecular mechanisms underlying chromosome segregation in S. pneumoniae therefore of fundamental importance, but also, this knowledge might offer new leads for ways to target this pathogen. Here, we identified a link between the pneumococcal chromosome segregation system and the competence-developmental system. Competence allows S. pneumoniae to take up and integrate exogenous DNA in its chromosome. This process plays a crucial role in successful adaptation to-and escape from-host defenses, antibiotic treatments, and vaccination strategies. We show that the chromosome segregation protein ParB acts as a repressor of competence. To the best of our knowledge, this is the first example of a ParB protein controlling bacterial competence

Оголені душі

Рецензія на книги: Слапчук Василь. Осiнь за щокою: Роман. - К.: Факт, 2006. - 280 с. та Сорока Петро. Денники 2004 - 2005. - Тернопiль: Сорока, 2006. - 364 с

A novel proteinaceous molecule produced by Lysinibacillus sp. OF-1 depends on the Ami oligopeptide transporter to kill Streptococcus pneumoniae.

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Expression of Streptococcus pneumoniae Bacteriocins Is Induced by Antibiotics via Regulatory Interplay with the Competence System

Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition

A bet-hedging strategy for denitrifying bacteria curtails their release of N2O

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