12 research outputs found
Definition of the regulatory network of Pseudomonas aeruginosa for adaptation to anaerobic conditions
Das Pathogen Pseudomonas aeruginosa infiziert die Lungen von Mukoviszidose-Patienten. Dort dringt es in den Mukus ein und bildet Mikrokolonien, die eine EntzĂŒndungsreaktion verursachen. Durch die P. aeruginosa-Besiedelung sinkt der Sauerstoffpartialdruck des Mukus, wodurch eine mikroaerobe bis anaerobe Umgebung entsteht. Es ist somit von Bedeutung, die anaeroben Wachstumsbedingungen und das dazugehörige regulatorische Netzwerk von P. aeruginosa zu verstehen. Mittels der vergleichenden Transkriptomanalyse anaerob gegenĂŒber aerob gewachsener P. aeruginosa Zellen konnten 413 differentiell exprimierte Gene identifiziert werden, die den Gruppen Energiemetabolismus, Stressreaktion durch Sauerstoffmangel und Wachstumsstop aufgrund der anaeroben Inkubation aerober Zellen zugeordnet wurden. Zur AufklĂ€rung des regulatorischen Netzwerkes wurden vergleichende Transkriptomanalysen von Negativmutanten des globalen anaeroben Regulators Anr und dem fĂŒr die Denitrifikation notwendigen Regulators Dnr durchgefĂŒhrt. Mittels dieser Analysen konnten dem Anr-Regulon 63 Gene und dem Dnr-Regulon 11 Gene zugeordnet werden. Zur weiteren Charakterisierung des Anr-Regulons wurden P. aeruginosa Chemostat-Kulturen unter mikroaeroben und denitrifizierenden Bedingungen kultiviert und diese einer differentiellen Transkriptom- und Proteomanalyse unterzogen. Mit Hilfe der Microarray-Analyse konnten 94 unterschiedlich regulierte Gene und mittels der Proteomanalyse 24 differentiell regulierte Proteine detektiert werden, die dem Energiestoffwechsel, dem Quorum Sensing und dem Typ III-Sekretionssystem zugeordnet werden konnten. Die im Rahmen der vorliegenden Arbeit gewonnenen Daten können die Grundlage fĂŒr die AufklĂ€rung des komplexen Regulationsnetzwerkes fĂŒr die Umstellung von aerober ĂŒber mikroaerophiler zu anaerober Lebensweise von P. aeruginosa bilden und so zentrale Prozesse der Biofilmbildung und Infektion auf molekularer Ebene verstĂ€ndlich machen.The pathogen Pseudomonas aeruginosa is found in the lungs of cystic fibrosis patients, where it enters the mucus and forms micro-colonies, causing an inflammatory reaction. These colonies of P. aeruginosa further decrease the oxygen tension of the mucus environment, resulting in a microaerobic to anaerobic environment. Thus it is of interest to understand the anaerobic growth conditions and regulatory networks of P. aeruginosa. Using transcriptomic analyses of anaerobically and aerobically grown P aeruginosa cells, 413 differentially expressed genes were identified. These belong to the groups of energy metabolism, stress reaction by oxygen depletion and growth stop due to the anaerobic incubation of aerobic cells. As Anr is the global anaerobic regulator and Dnr is the regulator for denitrification, Anr- and Dnr-mutants were used for comparative analyses. This way, 63 genes could be assigned to the Anr- and 11 genes to the Dnr-Regulon. The Anr-Regulon of P. aeruginosa was further characterised by differential transcriptome and proteome analyses using microaerobic and denitrifying chemostat cultures. While 94 differentially expressed genes were identified using Microarray analyses, 24 differentially regulated proteins were identified using 2D-gel analyses. These genes and proteins could be assigned to energy metabolism, quorum sensing and type III secretion system. This work provides the basis for the identification of the complex regulon used by P. aeruginosa for the adaptation to aerobic, microaerophilic and anaerobic environments. These data can be used to improve the understanding of central processes of biofilm formation and infection on the molecular level
A New Front in Microbial WarfareâDelivery of Antifungal Effectors by the Type VI Secretion System
A New Front in Microbial WarfareâDelivery of Antifungal Effectors by the Type VI Secretion System
Microbes typically exist in mixed communities and display complex synergistic and antagonistic interactions. The Type VI secretion system (T6SS) is widespread in Gram-negative bacteria and represents a contractile nano-machine that can fire effector proteins directly into neighbouring cells. The primary role assigned to the T6SS is to function as a potent weapon during inter-bacterial competition, delivering antibacterial effectors into rival bacterial cells. However, it has recently emerged that the T6SS can also be used as a powerful weapon against fungal competitors, and the first fungal-specific T6SS effector proteins, Tfe1 and Tfe2, have been identified. These effectors act via distinct mechanisms against a variety of fungal species to cause cell death. Tfe1 intoxication triggers plasma membrane depolarisation, whilst Tfe2 disrupts nutrient uptake and induces autophagy. Based on the frequent coexistence of bacteria and fungi in microbial communities, we propose that T6SS-dependent antifungal activity is likely to be widespread and elicited by a suite of antifungal effectors. Supporting this hypothesis, homologues of Tfe1 and Tfe2 are found in other bacterial species, and a number of T6SS-elaborating species have been demonstrated to interact with fungi. Thus, we envisage that antifungal T6SS will shape many polymicrobial communities, including the human microbiota and disease-causing infections
Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in <em>Serratia marcescens</em>
Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between Îł-d-glutamic acid and l-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structureâactivity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1âRap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2âRap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effectorâimmunity protein interactions
Depletion of the Cullin Cdc53p Induces Morphogenetic Changes in Candida albicansâż â
Candida albicans is an important opportunistic human fungal pathogen that can cause both mucosal and systemic infections in immunocompromised patients. Critical for the virulence of C. albicans is its ability to undergo a morphological transition from yeast to hyphal growth mode. Proper induction of filamentation is dependent on the ubiquitination pathway, which targets proteins for proteasome-mediated protein degradation or activates them for signaling events. In the present study, we evaluated the role of ubiquitination in C. albicans by impairing the function of the major ubiquitin-ligase complex SCF. This was done by depleting its backbone, the cullin Cdc53p (orf19.1674), using a tetracycline downregulatable promoter system. Cdc53p-depleted cells displayed an invasive phenotype and constitutive filamentation under conditions favoring yeast growth mode, both on solid and in liquid media. In addition, these cells exhibited an early onset of cell death, as judged from propidium iodide staining, suggesting that CDC53 is an essential gene in C. albicans. To identify Cdc53p-dependent pathways in C. albicans, a genome-wide expression analysis was carried out that revealed a total of 425 differentially expressed genes (fold change, â„2; P †0.05) with 192 up- and 233 downregulated genes in the CDC53-repressed mutant compared to the control strain. GO term analysis identified biological processes significantly affected by Cdc53p depletion, including amino acid starvation response, with 14 genes being targets of the transcriptional regulator Gcn4p, and reductive iron transport. These results indicate that Cdc53p enables C. albicans to adequately respond to environmental signals
Proteomic identification of novel secreted anti-bacterial toxins of the <em>Serratia marcescens</em> Type VI secretion system
It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. âAntibacterialâ T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cellâcell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species
New secreted toxins and immunity proteins encoded within the type VI secretion system gene cluster of Serratia marcescens
Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS-secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (âRapâ proteins) included two specifically conferring self-resistance (âimmunityâ) against T6SS-dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate SspâRap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS-secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS-secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches
The Opportunistic Pathogen Serratia marcescens Utilizes Type VI Secretion To Target Bacterial Competitors
The type VI secretion system (T6SS) is the most recently described and least understood of the protein secretion systems of Gram-negative bacteria. It is widely distributed and has been implicated in the virulence of various pathogens, but its mechanism and exact mode of action remain to be defined. Additionally there have been several very recent reports that some T6SSs can target bacteria rather than eukaryotic cells. Serratia marcescens is an opportunistic enteric pathogen, a class of bacteria responsible for a significant proportion of hospital-acquired infections. We describe the identification of a functional T6SS in S. marcescens strain Db10, the first report of type VI secretion by an opportunist enteric bacterium. The T6SS of S. marcescens Db10 is active, with secretion of Hcp to the culture medium readily detected, and is expressed constitutively under normal growth conditions from a large transcriptional unit. Expression of the T6SS genes did not appear to be dependent on the integrity of the T6SS. The S. marcescens Db10 T6SS is not required for virulence in three nonmammalian virulence models. It does, however, exhibit dramatic antibacterial killing activity against several other bacterial species and is required for S. marcescens to persist in a mixed culture with another opportunist pathogen, Enterobacter cloacae. Importantly, this antibacterial killing activity is highly strain specific, with the S. marcescens Db10 T6SS being highly effective against another strain of S. marcescens with a very similar and active T6SS. We conclude that type VI secretion plays a crucial role in the competitiveness, and thus indirectly the virulence, of S. marcescens and other opportunistic bacterial pathogens